Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Vascular endothelial growth factor-induced permeability in the pathogenesis of cardiac allograft vasculopathy Wong, Brian Wing Chi 2011

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2011_fall_wong_brian.pdf [ 6.44MB ]
Metadata
JSON: 24-1.0105116.json
JSON-LD: 24-1.0105116-ld.json
RDF/XML (Pretty): 24-1.0105116-rdf.xml
RDF/JSON: 24-1.0105116-rdf.json
Turtle: 24-1.0105116-turtle.txt
N-Triples: 24-1.0105116-rdf-ntriples.txt
Original Record: 24-1.0105116-source.json
Full Text
24-1.0105116-fulltext.txt
Citation
24-1.0105116.ris

Full Text

VASCULAR ENDOTHELIAL GROWTH FACTOR-INDUCED PERMEABILITY IN THE PATHOGENESIS OF CARDIAC ALLOGRAFT VASCULOPATHY  by  BRIAN WING CHI WONG  B.M.L.Sc., The University of British Columbia, 2001  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Pathology and Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  July 2011  © Brian Wing Chi Wong, 2011  Abstract Rationale: Endothelial dysfunction can lead to increased permeability, and this may contribute to the pathogenesis of cardiac allograft vasculopathy (CAV). This dissertation focuses on vascular endothelial growth factor (VEGF), a protein that can mediate angiogenesis and is a potent inducer of vascular permeability. It was my goal to characterize the expression and localization of VEGF in CAV and to elucidate the mechanisms which may relate to its role in the pathogenesis of CAV. Central hypothesis: VEGF plays a significant role in the pathogenesis of CAV by inducing endothelial cell hyperpermeability to low-density lipoproteins (LDL). Methods: Immunohistochemistry and in situ hybridization for VEGF were performed on coronary artery segments from patients with native atherosclerosis (NA), diabetes mellitus with atherosclerosis (DM) and CAV. Human coronary artery endothelial cell (HCAEC) and human cardiac microvascular endothelial cell (HCMEC) primary cultures were used to investigate VEGFinduced permeability using transendothelial electrical resistance (TER) measurements, immunocytochemistry for tight junction proteins and LDL permeability. A mouse model of heterotopic cardiac transplantation was used to assess the therapeutic potential of abrogation of VEGF function on CAV using soluble VEGF receptor-1 (sVEGFR1) administration. Key results: There was significant upregulation of VEGF-A in the intima and media of coronary arteries in CAV, NA and DM. As well, there was significant upregulation of VEGF-D in the media of coronary arteries in CAV and in the intima and media of coronary arteries in DM. Treatment with VEGF-A121, VEGF-A165 and VEGF-D significantly decreased TER, significantly increased LDL permeability, and induced the formation of intercellular gaps and decreased immunoreactivity ii | P a g e  of the tight junctional protein zonula occludens-1 (ZO-1) along adjacent endothelial membranes in confluent monolayers. Co-incubation with the mitogen-activated protein kinase kinase (MAPKK/MEK1) inhibitor U0126 prevented the formation of intercellular gaps and maintained regularity of ZO-1 immunoreactivity along endothelial membranes. Administration of sVEGFR1 in a mouse model of heterotopic cardiac transplantation resulted in a significant decrease in luminal narrowing in transplanted hearts at 21 days post-transplantation. Conclusion: Taken together, this body of work clearly demonstrates that VEGF plays a significant role in the pathogenesis of CAV.  iii | P a g e  Preface The following dissertation includes chapters which are based on published manuscripts. Portions of Chapter 1 are based on a review article published in the journal Cardiovascular Pathology [Wong BW, Rahmani M, Rezai N, McManus BM. Progress in heart transplantation. Cardiovasc Pathol. 2005; 14: 176-80]. I wrote this review article and the coauthors assisted in critical review and revision. Portions of Chapter 3 and 4 are based on two manuscripts published in the Journal of Heart and Lung Transplantation [Wong BW, Rahmani M, Luo Z, Yanagawa B, Wong D, Luo H, McManus BM. Vascular endothelial growth factor increases human cardiac microvascular endothelial cell permeability to low-density lipoproteins. J Heart Lung Transplant. 2009; 28: 950-7] and [Wong BW, Wong D, Luo H, McManus BM. Vascular endothelial growth factor-D is overexpressed in human cardiac allograft vasculopathy and diabetic atherosclerosis and induces endothelial permeability to low-density lipoproteins in vitro. J Heart Lung Transplant. 2011; 30: 955-62]. I performed all of the writing and the majority of the experiments presented in these manuscripts. Dr. Donald Wong was instrumental in the initial conception of some of the experiments. Ms. Zongshu Luo helped in some of the experiments. Drs. Maziar Rahmani, Bobby Yanagawa, Honglin Luo and Bruce McManus made intellectual contributions to the design and final editing of the manuscripts.  iv | P a g e  Portions of Chapter 5 are based on a manuscript submitted to the journal Circulation Research which is currently in revision [Wong BW, Williams SJ, Tao K, West LJ, Luo H, Bernatchez PN, McManus BM. Soluble vascular endothelial growth factor receptor-1 reduces luminal narrowing in mouse cardiac allograft vasculopathy]. I performed all of the writing on the manuscripts and assisted in care of the animals post-transplantation, administration of the drugs, sacrifice and tissue harvesting. As well, I performed the morphometric assessment of luminal narrowing and the in vitro aortic ring angiogenesis assays co-cultured with bone marrow-derived cells. Dr. Sarah Williams assisted in care of the animals, sacrifice and tissue harvesting, blood chemistry analyses, immunohistochemistry for VEGF and some image analysis of aortic ring outgrowth. Dr. Kesheng Tao was the skilled microsurgeon who performed all of the heterotopic heart transplants. Drs. Lori West, Honglin Luo, Pascal Bernatchez and Bruce McManus made intellectual contributions to the design and final editing of the manuscript. The procedures detailed in Chapter 5 were reviewed and approved by the University of British Columbia Animal Care Committee (Protocol #A08-0509).  v|Page  Table of contents Abstract ................................................................................................................................................................. ii Preface ................................................................................................................................................................. iv Table of contents .................................................................................................................................................. vi List of tables .......................................................................................................................................................... x List of figures ........................................................................................................................................................ xi List of symbols .................................................................................................................................................... xiii List of abbreviations ........................................................................................................................................... xiv Acknowledgements ............................................................................................................................................ xix Dedication .......................................................................................................................................................... xxi Chapter 1 – Background ......................................................................................................................................... 1 1.1 – HEART FAILURE .....................................................................................................................................................1 1.1.1 – Primary causes of heart failure ................................................................................................................1 1.1.2 – Current treatment strategies ...................................................................................................................3 1.1.3 – End-stage heart failure .............................................................................................................................3 1.2 – OVERVIEW OF HEART TRANSPLANTATION ...................................................................................................................4 1.2.1 – Understanding allo-immunity ..................................................................................................................4 1.2.2 – Surgical milestones ...................................................................................................................................5 1.2.3 – Successful heart transplants .....................................................................................................................6 1.2.4 – Pathological diagnosis..............................................................................................................................7 1.2.5 – Past and present immunosuppressive regimens ......................................................................................8 1.2.6 – Etiology of cardiac allograft vasculopathy ...............................................................................................8 1.2.7 – Current treatment strategies .................................................................................................................10 1.3 – CURRENT CONCEPT OF THE PATHOGENESIS OF CAV....................................................................................................11 1.4 – DYSREGULATION OF ENDOTHELIAL PERMEABILITY IN VASCULAR DISEASE..........................................................................14 1.4.1 – Endothelial dysfunction in native atherosclerosis ..................................................................................15 1.4.2 – Altered endothelial permeability in cardiac allograft vasculopathy ......................................................16 1.5 – ENDOTHELIAL BARRIER FUNCTION AND PERMEABILITY .................................................................................................20 1.5.1 – Pathways through the endothelium .......................................................................................................20 1.5.1.1 – Intracellular passage ........................................................................................................................................ 20 1.5.1.1.1 – Endocytosis .............................................................................................................................................. 21  vi | P a g e  1.5.1.1.2 – Transcytosis ............................................................................................................................................. 23 1.5.1.2 – Intercellular permeability ................................................................................................................................. 24 1.5.1.2.1 – Tight junctions ......................................................................................................................................... 25 1.5.1.2.2 – Adherens junctions .................................................................................................................................. 27 1.5.1.3 – Focal adhesion complexes and the extracellular matrix .................................................................................. 30  1.5.2 - Modulation of permeability ....................................................................................................................33 1.5.3 – Second messengers and signal transduction pathways .........................................................................35 1.5.3.1 – Calcium ............................................................................................................................................................. 36 1.5.3.2 – Phospholipases ................................................................................................................................................. 37 1.5.3.3 – Tyrosine kinases ............................................................................................................................................... 39 1.5.3.4 – cAMP and cGMP ............................................................................................................................................... 40  1.6 – VASCULAR ENDOTHELIAL GROWTH FACTOR ...............................................................................................................41 1.6.1 – Search for a tumor angiogenic factor.....................................................................................................41 1.6.2 – Vascular endothelial growth factor family members and receptors ......................................................42 1.6.3 – The role of vascular endothelial growth factor ......................................................................................43 1.6.4 – Vascular endothelial growth factor in disease .......................................................................................43 Chapter 2 – Hypothesis and specific aims ............................................................................................................ 47 2.1 – RATIONALE.........................................................................................................................................................47 2.2 – CENTRAL HYPOTHESIS ...........................................................................................................................................48 2.3 – SPECIFIC AIMS .....................................................................................................................................................48 Chapter 3 – The role of vascular endothelial growth factor in cardiac allograft vasculopathy .............................. 50 3.1 – RATIONALE.........................................................................................................................................................50 3.2 – MATERIALS AND METHODS ....................................................................................................................................51 3.2.1 – Case materials ........................................................................................................................................51 3.2.2 – Immunohistochemistry ...........................................................................................................................52 3.2.3 – In situ hybridization ................................................................................................................................53 3.2.4 – Color segmentation and image analysis ................................................................................................54 3.2.5 – Statistical analysis ..................................................................................................................................55 3.3 – RESULTS ............................................................................................................................................................56 3.3.1 – Aberrant VEGF-A expression in human cardiac allograft vasculopathy .................................................56 3.3.2 – Aberrant VEGF-A expression in native atherosclerosis and diabetes mellitus........................................58 3.3.3 – VEGF-D in cardiac allograft vasculopathy ..............................................................................................60 3.3.4 – VEGF-D in native atherosclerosis and diabetes mellitus.........................................................................60 3.4 – DISCUSSION .......................................................................................................................................................63  vii | P a g e  Chapter 4 – Vascular endothelial growth factor induces endothelial hyperpermeability to low-density lipoproteins in vitro ............................................................................................................................................. 68 4.1 – RATIONALE.........................................................................................................................................................68 4.2 – MATERIALS AND METHODS ....................................................................................................................................69 4.2.1 – Reagents and antibodies ........................................................................................................................69 4.2.2 – Cell cultures ............................................................................................................................................70 4.2.3 – Transendothelial electrical resistance experiments ...............................................................................71 4.2.4 – Immunocytochemistry ............................................................................................................................72 4.2.5 – Immunofluorescent microscopy .............................................................................................................72 4.2.6 – Cell lysates, electrophoresis, and Western blotting................................................................................73 4.2.7 – Low-density lipoprotein permeability experiments ................................................................................73 4.2.8 – Statistical analysis ..................................................................................................................................74 4.3 – RESULTS ............................................................................................................................................................75 4.3.1 –Effect of VEGF in HCAEC and HCMEC on TER ..........................................................................................75 4.3.1.1 – The effect of VEGF-A165 on TER ........................................................................................................................ 76 4.3.1.2 – The effect of VEGF-A121 on TER ........................................................................................................................ 78 4.3.1.3 – The effect of VEGF-D on TER ............................................................................................................................ 81  4.3.2 – VEGF increases LDL permeability through confluent HCMEC monolayers in vitro .................................83 4.3.3 – VEGF-induced alterations to endothelial tight junctions ........................................................................87 4.3.3.1 – Treatment with VEGF results in the formation of intercellular gaps ................................................................ 87 4.3.3.2 – VEGF increases cytoplasmic immunoreactivity of ZO-1 ................................................................................... 88  4.3.4 – Profiling of the VEGF-induced signaling pathways in endothelial cells. .................................................93 4.4 – DISCUSSION .......................................................................................................................................................98 Chapter 5 – Administration of soluble vascular endothelial growth factor receptor-1 in a mouse model of heterotopic cardiac transplantation .................................................................................................................. 106 5.1 – RATIONALE.......................................................................................................................................................106 5.2 – MATERIALS AND METHODS ..................................................................................................................................109 5.2.1 – Animals .................................................................................................................................................109 5.2.2 – Heterotopic cardiac transplant model..................................................................................................110 5.2.3 – Administration of soluble VEGFR1 and monitoring of mice .................................................................111 5.2.4 – Tissue harvesting and histopathological examination .........................................................................112 5.2.5 – Blood chemistry ....................................................................................................................................113 5.2.6 – Morphometry .......................................................................................................................................113 5.2.7 - In vitro aortic ring angiogenesis assay ..................................................................................................115  viii | P a g e  5.2.8 – Statistical analysis ................................................................................................................................115 5.3 – RESULTS ..........................................................................................................................................................116 5.3.1 – Heterotopic cardiac transplantation and response to soluble VEGFR1 ................................................116 5.3.2 – Treatment with soluble VEGFR1 does not affect lipid levels ................................................................117 5.3.3 – Treatment with soluble VEGFR1 significantly increases plasma levels of VEGF ...................................118 5.3.4 – Treatment with soluble VEGFR1 significantly reduces luminal narrowing ...........................................119 5.3.5 – Treatment with soluble VEGFR1 reduces edema in transplanted hearts .............................................121 5.3.6 – Treatment with soluble VEGFR1 reduces capillary growth induced by bone marrow..........................124 5.4 – DISCUSSION .....................................................................................................................................................126 Chapter 6 – Closing remarks .............................................................................................................................. 131 References ......................................................................................................................................................... 139 Appendix – Supplemental information for case materials from Chapter 3 ......................................................... 177  ix | P a g e  List of tables Table 1 – Summary of cases used for immunohistochemical profiling of VEGF in atheromatous disease. ......................................................................................................................................... 52 Table 2 – Grading criteria for monitoring heterotopic heart beat function by abdominal palpation. .................................................................................................................................... 111 Table 3 – Case listing for Normal group (Pathobiological Determinants of Atherosclerosis in Youth study). ............................................................................................................................... 177 Table 4 – Case listing for Native Atherosclerosis (NA) group. .................................................... 178 Table 5 – Case listing for Diabetes Mellitus (DM) group. ........................................................... 178 Table 6 – Case listing for Cardiac Allograft Vasculopathy (CAV) group. ..................................... 179  x|Page  List of figures Figure 1 – Representative micrographs of coronary arteries with native atherosclerosis and cardiac allograft vasculopathy. ....................................................................................................... 9 Figure 2 – Structural progression of cardiac allograft vasculopathy. ........................................... 12 Figure 3 – Assessment of endothelial permeability under transmission electron microscopy (TEM) using horseradish peroxidase (HRP) as a tracer. ................................................................ 15 Figure 4 – Examination of endothelial perturbations in a rat model of heterotopic cardiac transplantation. ............................................................................................................................ 19 Figure 5 – Regulation of endocytosis by signal transduction pathways. ...................................... 22 Figure 6 – Regulation of tight junctions by signal transduction pathways................................... 25 Figure 7 – Regulation of adherens junctions by signal transduction pathways. .......................... 28 Figure 8 – Regulation of focal adhesion complexes by signal transduction pathways. ............... 31 Figure 9 – Vascular endothelial growth factor family and their receptors. ................................. 44 Figure 10 – Diagrammatic representation of the specific aims of this thesis. ............................. 49 Figure 11 – Diagrammatic representation of color segmentation analysis using “areas of interest” (AOI) in ImagePro Plus®. ................................................................................................ 55 Figure 12 – Immunohistochemical profiling of VEGF-A in human CAV. ....................................... 57 Figure 13 – Immunohistochemical profiling of VEGF-A in native atherosclerosis (NA) and diabetes mellitus with atherosclerosis (DM). ............................................................................... 59 Figure 14 – Immunohistochemical profiling of VEGF-D in human CAV. ....................................... 61 Figure 15 – Immunohistochemical profiling of VEGF-D in human native atherosclerosis (NA) and diabetes mellitus with atherosclerosis (DM). ............................................................................... 62 Figure 16 – Diagrammatic representation of the key findings from Chapter 3. .......................... 67 Figure 17 – VEGF-A165 significantly decreases TER in HCAEC and HCMEC. .................................. 77 Figure 18 – VEGF-A121 significantly decreases TER in HCAEC and HCMEC. .................................. 80 Figure 19 – VEGF-D significantly decreases TER in HCAEC and HCMEC. ...................................... 82 Figure 20 – VEGF significantly increases LDL permeability through HCMEC monolayers............ 84 Figure 21 – LDL permeability is greater than acLDL permeability. ............................................... 86 Figure 22 – VEGF induces intercellular gap formation. ................................................................ 88 xi | P a g e  Figure 23 – VEGF induces changes in ZO-1 immunoreactivity. .................................................... 89 Figure 24 – Non-conventional localization of occludin in HCAEC and HCMEC............................. 90 Figure 25 – Occludin does not localize to cultured endothelial cell membranes. ....................... 92 Figure 26 – Profiling of signal transduction molecules activated by VEGF. ................................. 94 Figure 27 – Treatment with VEGF induces ERK1/2 phosphorylation in endothelial cells. ........... 95 Figure 28 – Inhibition of ERK1/2 prevents VEGF-induced intercellular gap formation and changes in ZO-1 immunoreactivity. .............................................................................................. 97 Figure 29 – Diagrammatic representation of the key findings from Chapter 4. ........................ 105 Figure 30 – Determination of luminal narrowing in a mouse model of heterotopic cardiac transplantation. .......................................................................................................................... 114 Figure 31 – Treatment with soluble VEGFR1 does not result in a change in mouse body weight. ..................................................................................................................................................... 116 Figure 32 – Treatment with soluble VEGFR1 does not change plasma lipid levels. ................... 117 Figure 33 – Treatment with soluble VEGFR1 significantly increases plasma VEGF concentrations. ..................................................................................................................................................... 118 Figure 34 – Quantitation of luminal narrowing in intramyocardial arteries. ............................. 120 Figure 35 – Quantitation of ventricular cross-sectional area. .................................................... 122 Figure 36 – Wet heart weight is significantly reduced in soluble VEGFR1-treated transplanted hearts. ......................................................................................................................................... 123 Figure 37 – In vitro aortic ring angiogenesis assay co-culture with bone marrow (BM)-derived cells. ............................................................................................................................................ 125 Figure 38 – Diagrammatic representation of the key findings from Chapter 5. ........................ 130 Figure 39 – Revised concept of the early pathogenic mechanisms in cardiac allograft vasculopathy. .............................................................................................................................. 137 Figure 40 – Revised concept of the later events in the pathogenesis of cardiac allograft vasculopathy. .............................................................................................................................. 138  xii | P a g e  List of symbols α – alpha β – beta γ – gamma  – delta ε – epsilon  – zeta  – eta  – theta  – lambda μ – micron [mu]  xiii | P a g e  List of abbreviations ABC/AP – streptavidin-biotin complex conjugated with alkaline phosphatase ACE – angiotensin converting enzyme acLDL – acetylated low-density lipoprotein AgNO3 – silver nitrate ANOVA – analysis of variance AP-2 – activating protein-2 apo – apolipoprotein ATP – adenosine triphosphate AV – allograft vasculopathy β-catenin – beta-catenin Ca2+ – calcium CaM – calmodulin cAMP – cyclic adenosine monophosphate CAS – Crk-associated substrate CAV – cardiac allograft vasculopathy cGMP – cyclic guanosine monophosphate CRAM – cysteine-rich acidic transmembrane protein CVB3 – coxsackievirus B3 DAG – 1, 2- diacylglycerol DI – diabetes insipidous diI – 1, 1’-dioctadecyl-3, 3, 3’, 3’-tetramethylindocarbocyanine perchlorate xiv | P a g e  DM – diabetes mellitus E-cadherin – epithelial-cadherin EC – endothelial cell ECM – extracellular matrix EGF – epithelial growth factor EGFR – epithelial growth factor receptor eNOS – endothelial nitric oxide synthase ER – endoplasmic reticulum ERK – extracellular signal-regulated kinase FAK – focal adhesion kinase FITC – fluorescein isothiocyanate flk-1 – fetal liver kinase-1 flt-1 – fms-like tyrosine kinase-1 flt-4 – fms-like tyrosine kinase-4 GEF – guanine nucleotide exchange factor GRAF – GTPase regulator associated with FAK GSK3β – glycogen synthase kinase-3 beta GTPase – guanosine triphosphate hydrolase enzyme H2O2 – hydrogen peroxidase HCAEC – human coronary artery endothelial cell HCASMC – human coronary artery smooth muscle cell HCMEC – human cardiac microvascular endothelial cell xv | P a g e  HLA – human leukocyte antigen HRP – horseradish peroxidase HSPG – heparan sulphate proteoglycans HUVEC – human umbilical vein endothelial cell IFN – interferon iNOS – inducible nitric oxide synthase IP3 – inositol 1, 4, 5- triphosphate IP3R – inositol triphosphate receptor ISH – in situ hybridization ISHLT – International Society for Heart and Lung Transplantation IVUS – intravascular ultrasound JAM – junctional adhesion molecule kDa – kilodalton KDR – kinase domain receptor LDL – low-density lipoprotein MDCK – Madin-Darby canine kidney [cells] MI – myocardial infarction MLC – myosin light chain MLCK – myosin light chain kinase MMP – matrix metalloproteinase NA – native atherosclerosis NBT/BCIP – nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate xvi | P a g e  N-cadherin – neural-cadherin oxLDL – oxidized low-density lipoprotein PAF – platelet-activating factor PBS – phosphate-buffered saline PDAY – Pathobiological Determinants of Atherosclerosis in Youth [study] PDE – phosphodiesterase PDGF – platelet-derived growth factor PDGFR – platelet-derived growth factor receptor PECAM-1 – platelet and endothelial cell adhesion molecule-1; CD31 PI3K – phosphotidylinositol-3 kinase PIP2 – phosphotidylinositol 4, 5-bisphosphate PKA – protein kinase A PKB – protein kinase B; Akt PKC – protein kinase C PL – phospholipase PLA2 – phospholipase A2 PLC – phospholipase C PLD – phospholipase D PlGF – placental growth factor PMA – phorbol 12-myristate 13-acetate PMNL – polymorphonuclear leukocyte Rap1 – Ras-proximate-1; Ras-related protein-1 xvii | P a g e  SE – standard error [of the mean] SEM – scanning electron microscopy SMC – smooth muscle cell SM-α actin – smooth muscle-alpha actin SOS – Son of sevenless SPSS – Statistical Package for the Social Sciences [software] SSC – saline sodium citrate [buffer] TEM – transmission electron microscopy TER – transendothelial electrical resistance TGF-β – transforming growth factor-beta TNF-α – tumor necrosis factor-alpha U0126 – 1, 4-diamino-2, 3-dicyano-1, 4-bis[2-aminophenylthio] butadiene VE-cadherin – vascular endothelial-cadherin VEGF / VPF – vascular endothelial growth factor / vascular permeability factor VEGFR1 / flt-1 – vascular endothelial growth factor receptor-1 / fms-like tyrosine kinase-1 VEGFR2 / flk-1 / KDR – vascular endothelial growth factor receptor-2 / fetal liver kinase-1 / kinase domain receptor VEGFR3 / flt-4 – vascular endothelial growth factor receptor-3 / fms-like tyrosine kinase-4 VLDL – very low-density lipoprotein VVO – vesiculo-vacuolar organelle ZO – zonula occludens  xviii | P a g e  Acknowledgements My research career began under the direct tutelage of Dr. Donald Wong, a post-doctoral fellow in the laboratory of my supervisor, Dr. Bruce McManus. Through the many personal and professional conversations we have engaged in, Donald has instilled solid academic foundations of diligence, persistence and honesty. As well, Donald was instrumental in establishing the basis for the grant-funded VEGF research program in the laboratory. I first met Dr. Honglin Luo when she began as a research associate under the supervision of Dr. McManus. Honglin has always provided unbiased and straightforward assessment of my work and her serendipitous rise to associate professor is a constant reminder that strong scientific bases and productivity in publication prevails. It is of particular satisfaction that I was able to collaborate with her and through that interaction, expand my scientific basis. Ms. Zongshu Luo has been one of my closest friends and colleagues within the laboratory. Of particular note, after unforeseen interruptions in my studies, Zongshu went above and beyond in ensuring that I ‘kept at it’ despite my personal setbacks. My interactions with Ms. Huifang (Mary) Zhang and Dr. Decheng Yang were one of my first collaborative interactions in research and re-enforced the central idea that the most important thing in science is the science. Through numerous collaborations and publications, I have been afforded the opportunity to elevate my level of scientific writing and technique while assisting in their research focuses. I would like to acknowledge the personal and professional contributions of Dr. Maziar Rahmani, Dr. Bobby Yanagawa, Dr. Hubert Walinski, and Dr. Jonathan Choy – through our many  xix | P a g e  conversations, debates and heated arguments, you have always pushed me to do more and be better. There have been many dedicated and skilled individuals who have helped me with various portions of my graduate work and training, including Ms. Agripina Suarez (Pining), Ms. Sylvia Loo, Mr. Nathanael Kuipers, Dr. Thomas Podor, Mr. Albert Lee, Mr. Dean English, Ms. Elizabeth Walker, Ms. Elaine Humphreys, Ms. Kris Gillespe, Dr. Kesheng Tao, Ms. Tatjana Bozin, and Ms. Claire Smits. I would like to also like to specifically acknowledge the tutelage, support and friendship of Mr. Stuart Greene and Dr. Alexandra (Sasha) Kerjner. During my studies I have been fortunate to be supported by awards from the Heart and Stroke Foundation, the Canadian Institutes of Health Research and the Michael Smith Foundation for Health Research. As well, the research program has been generously supported by grants from the Heart and Stroke Foundation and the Canadian Institutes of Health Research. The guidance and direction provided by my doctoral thesis committee members, Dr. Edward Pryzdial (chair), Dr. Urs Steinbrecher and Dr. Wan Lam, have helped to refine and develop the scientific structure and foundation of my doctoral thesis project. Finally, I would like to acknowledge the patience, support and mentorship provided by my doctoral thesis supervisor, Dr. Bruce McManus. It is impossible to summarize everything I have learned in my time under his tutelage but his unwavering enthusiasm and dedication towards research and science reminds me to always “Read More” and “Go for the Gold.”  xx | P a g e  Dedication  xxi | P a g e  Chapter 1 – Background 1.1 – Heart failure Heart failure is characterized as an abnormality of the structure or function of the heart which impairs its ability to provide sufficient blood flow to the body. Heart failure is a highly prevalent condition with significant economic, social and personal costs. In Western society, approximately 2% of adults suffer from heart failure.1,2 The lifetime risk of developing heart failure is approximately 20% for individuals over 40 years of age.3,4 This percentage increases by 6-10% in those over the age of 65.2,5 About 30-40% of patients die from heart failure within one year after receiving the diagnosis.2 Over time, prognosis of heart failure generally worsens, and this progressive disease is associated with an overall annual mortality rate of approximately 10%.6 As well, heart failure is associated with significant reductions in physical and mental health, resulting in a striking decrease in quality of life.2  1.1.1 – Primary causes of heart failure Heart failure results from abnormalities in cardiac structure, function, rhythm or conduction. It can be caused by a wide variety of conditions including myocardial infarction (MI), hypertension and cardiomyopathy. As well, degenerative valvular diseases are becoming more common. Over time, increased cardiac demand results in reduced contractility due to ventricular overload, reduced stroke volume, reduced reserve capacity, increased heart rate, hypertrophy and enlargement or dilation of the ventricles.2  1|Page  Native atherosclerosis is a condition where a combination of environmental, genetic and lifestyle factors lead to the gradual change in architecture of arteries and accumulation of lipids and lipoproteins.7,8 Factors such as oxidative stress,9,10 hypercholesterolemia,11,12 diabetes mellitus (DM)13 and others contribute to intimal hyperplasia – the proliferation and expansion of the intimal layer of arteries. As a result of thickening of the intimal layer of the vessel wall, the area of the lumen of the blood vessel is reduced as the intima encroaches on the lumen resulting in a reduction in blood flow. This process results in blood vessel dilation to compensate for this reduction in luminal area, and ultimately triggers a compensatory thinning or dissolution of the medial layer of smooth muscle cells (SMC) when the vessel is no longer able to dilate further to increase blood flow. Changes in the intima and media of the blood vessel also result in an increased propensity for the insudation and retention of lipids and lipoproteins within the deep intimo-medial layer.8,14 Progressive occlusion of the coronary arteries within the heart may result in either complete blockage or obstruction, resulting in downstream myocardial infarction (MI) or an increased propensity for thrombolytic events, where, in the face of a thinning fibrous cap overlying the pro-thrombotic atherosclerotic lesion may result in plaque rupture and intravascular thrombosis, which may eventually lead to downstream embolism and ischemia. Atherosclerosis can lead to heart failure through the reduction of blood flow in the coronary arteries, resulting in increased strain on the heart to maintain cardiac output and perfusion. Progressive advancement of atherosclerosis can ultimately result in MI, with ischemic damage to the myocardium, thereby further reducing the ability of the heart to function.8  2|Page  1.1.2 – Current treatment strategies Treatment for heart failure consists of lifestyle changes (such as decreased dietary intake of salt; increased physical activity) and medications such as beta-blockers,15 angiotensin converting enzyme (ACE) inhibitors16,17 or angiotensin receptor blockers,18,19 vasodilators, and in cases of severe cardiomyopathy, aldosterone receptor antagonists.17 Treatment for heart failure focuses on improving symptoms and preventing progression of the disease. Despite all of these methodologies for intervention, heart failure is still associated with an annual mortality rate of 10%.20  1.1.3 – End-stage heart failure At maturity, adult cardiomyocytes are largely terminally differentiated, and as such, the heart is unable to replace damaged or dead myocytes. Instead, compensatory changes such as cardiomyocyte hypertrophy and polyploidy and cardiac fibrosis results, and dilation of the ventricular chamber initially helps to maintain cardiac output. Despite significant advances in our ability to treat heart disease and heart failure, currently, there are only two long-term solutions for end-stage heart failure, the main one being organ transplantation. The advent of ventricular assist devices and other supportive technologies may allow for the maintenance and extension of the length of time a patient may live with end-stage heart failure; however, ultimately the only long-term solution is heart transplantation.  3|Page  1.2 – Overview of heart transplantation To appreciate the significance of heart transplantation as both a life-saving medical procedure and also the accumulative understanding of the efforts to modify and manipulate the cardiovascular and immune systems, one must appreciate the many milestone advancements which have been cornerstones for transplantation medical science and heart transplantation in particular.  1.2.1 – Understanding allo-immunity In 1901, Karl Landsteiner published a paper demonstrating that clumping of the donor’s red blood cells was responsible for the clinical manifestations of the transfusion reaction. 21 This paper revealed that the clumping was due to the presence of three different types of isoagglutinins, which formed the basis for his blood group classification known initially as A, B and C. His suggestions received little attention until 1909, when he classified the human blood into the A, B, AB and O groups and showed that catastrophic reactions could occur when a person receives blood from a different group.21 Compatibility was later found to be not only a requirement for transfusion, but also for transplantation. Peter Medawar was the first to demonstrate that the immune system was responsible for the rejection of transplanted organs, and later went on to show that it could be “tricked” into tolerating transplanted tissues. Medawar started his pioneering work in Glasgow on skin grafting burns. He found that skin grafted from a donor lasted about 10 days, but a second graft was rejected immediately. It was as though the immune system remembered what the intruder looked like, and promptly rejected it.22 Medawar suggested that the rejection was an 4|Page  immunological process. In the early 1950s, Medawar inoculated mouse embryos with the cells of mice from another strain. After their birth they were grafted with skin taken from the strain of mice to which they had been exposed in utero. Remarkably, these grafts were not rejected, introducing the concept of acquired immunological tolerance.23 Frank Burnet suggested that the body's immune cells learn very early on to accept whatever tissues are there as part of the body and only attack and reject material that shows up later. This theory later developed the notion of clonal selection and the recognition of self and non-self by vertebrate immune systems.24 In 1958, Jean Dausset described the first leukocyte antigen, MAC (now known as human leukocyte antigen-A2 (HLA-A2)).25,26 The discovery allowed for tissue matching beyond blood types.  1.2.2 – Surgical milestones Vascular surgery, preceding solid organ transplantation, did not emerge as a specialty until the end of the 19th century. At that time, Alexis Carrel introduced a “leak-proof” technique for the anastomosis of blood vessels without constricting the lumen or causing thrombosis. Anastomosis of blood vessels was a crucial advance required for the field of solid organ transplantation to become tenable. Carrel demonstrated the feasibility of grafting veins to arteries, and arteries to arteries using his innovative anastomotic approach.27-29 His work detailed the refinement and perfection of vascular anastomotic techniques, the usage of vein grafts in the arterial system, the development of tissue preservation techniques, and organ and limb transplantation.29-31 Alexis Carrel performed the first heterotopic canine heart transplant with Charles Guthrie in 1905.29 At the time, he clearly recognized the difference in the survival 5|Page  times between autografts and allografts in experimental animals, but he did not conceptualize rejection as distinct from other graft-destroying processes. Twenty years later, the concept of cardiac allograft rejection was proposed by Frank Mann at the Mayo Clinic to explain the eventual failure of heterotopic canine allografts. He described the rejection process as a “biologic incompatibility between donor and recipient” manifested by an impressive leukocytic infiltration of the rejecting myocardium.32  1.2.3 – Successful heart transplants On January 23rd, 1964, the first heart transplant of a non-human primate into a human was performed by James Hardy at the University of Mississippi Medical Center in Jackson. The heart of a chimpanzee was transplanted into the 68-year-old, Boyd Rush; however, the heart was too small to maintain independent circulation and functioned only for 90 minutes before failing.33 On December 3rd, 1967, Christiaan Barnard successfully transplanted the heart of Denise Darvell, a young woman who had died in a car crash, into 54-year-old Louis Washkansky at the Groote Schuur Hospital in Cape Town, South Africa. He died of pneumonia 18 days later.34 On January 6th, 1968, Norman Shumway performed the first adult human-to human heart transplant in North America at the Stanford University School of Medicine in Palo Alto, California.35 In 1969, Denton Cooley implanted the first total artificial heart (the Liotta Total Artificial Heart) at the Texas Heart Institute in Houston. The heart was implanted into the 47year-old Haskell Karp, but was not intended to be permanent. It was used as a bridge to transplant until he could receive a donor heart, which he did 64 hours later. 36  6|Page  1.2.4 – Pathological diagnosis The detection of allograft rejection is one of the most important yet unsettled areas of cardiac transplantation. The investigation of the transvascular endomyocardial bioptome by Sakakibara and Konno in 1963,37 and the introduction of transvenous endomyocardial biopsy by Philip Caves in 1973 finally provided a reliable means for monitoring allograft rejection. 38 Throughout the 1980s, various grading scales emerged from different centers, causing much confusion. The International Society for Heart & Lung Transplantation (ISHLT) commissioned the development of a common grading scale in 1990, in an attempt to develop uniform description and grading criteria of various transplant histologies to refine communication and comparison of treatment regimens and outcomes between transplant centers. Due to the insight and diligence of cardiac pathologists such as Margaret Billingham, the ISHLT grading system for cellular rejection was developed in 1990.39 In 2004, a new grading scale was commissioned by the ISHLT to address the challenges and inconsistencies in the use of the old grading system.40 Although the current grading system has allowed for better consistency in assessment of rejection severity, there are inherent limitations to its usage: variability in assessment of rejection severity, particularly regarding grade 2 lesions, the presence of Quilty lesions (endocardial infiltrates) tends to cause overestimation of rejection severity, and humoral (antibody-mediated) rejection is just beginning to be addressed. Hemodynamic change in the absence of acute cellular rejection is termed biopsynegative rejection, and occurs in 10 to 20% of cardiac allograft recipients.41 In the precyclosporine era, biopsy-negative rejection was not apparently an important phenomenon. It is suggested that immunologic pathways other than lymphocytic infiltration are important in 7|Page  mediating cardiac allograft dysfunction and injury, and humoral rejection may be the primary mediator.42,43 Humoral rejection is associated with increased graft loss, accelerated coronary allograft vasculopathy and increased mortality.42,43  1.2.5 – Past and present immunosuppressive regimens With advances in immunosuppression and surgical techniques, the pathologies seen post-transplantation have changed. Over the last 20 years, the rates of acute rejection and infection leading to graft failure have greatly declined owing to refined immunosuppressive drug regimens, better diagnosis of ischemic injury, and improved monitoring of immune status. As such, chronic rejection and cardiac allograft vasculopathy (CAV) have become more prevalent as major expressions of transplant rejection. CAV is an accelerated form of atherosclerosis which occurs in 30-60% of transplant recipients within the first 5 years posttransplantation.44 Studies using intravascular coronary ultrasound (IVUS) techniques have demonstrated intimal thickening in 75% of cardiac allograft recipients by the end of the first year post-transplantation.45  1.2.6 – Etiology of cardiac allograft vasculopathy CAV is a rapidly progressing form of atherosclerosis, whereby the heightened allogeneic immune response between donor and recipient, along with classical risk factors for atherosclerosis and ischemic factor contribute to the rapid narrowing of blood vessels. Although there are many similarities between CAV and native atherosclerosis, they are clinically and pathologically separate entities (Figure 1). 8|Page  Native atherosclerosis  Cardiac allograft vasculopathy  i  L  Early disease  L  m  i  Late disease  L *  i m m  Figure 1 – Representative micrographs of coronary arteries with native atherosclerosis and cardiac allograft vasculopathy. Cardiac allograft vasculopathy is characterized by a largely immune-driven etiology, with concentric matrix and lipid deposition and rapid progression, whereas native atherosclerotic lesions are generally focal, eccentric, proliferative and degenerative lesions in the intima of proximal coronary vessels, mostly fibroinflammatory, fatty plaques with ultimate necrotic cores and progressively thinned fibrous caps. Scale bars represent 1mm. L – lumen  i – intima  m – media  * – atheromatous core  9|Page  As acute rejection is better controlled by tailored immunosuppressive regimens, the central role of the immune system in the pathogenesis of CAV is also complemented by other factors which may augment its etiology. These factors include peri-transplant injury, generally resulting in epicardial fibrosis and myocardial infarction, graft denervation, and infection. It is important to note that allograft vasculopathy (AV) occurs to a significant degree in all solid organ transplants.  1.2.7 – Current treatment strategies Over the last 20 years, rates of acute rejection and infection leading to graft failure have greatly declined owing to refined immunosuppressive drug regimens, better diagnosis of ischemic injury and improved monitoring of immune status.45-49 Chronic rejection and AV have become a major focus. As noted, studies employing IVUS have revealed intimal thickening in 75% of cardiac allograft recipients by one year post-transplantation.45,50 Current treatment strategies primarily focus on immunosuppression. Targeted therapies against CAV have been investigated, primarily as adjuncts to existing therapies used in native atherosclerotic disease, such as “statins”, low-dose acetylsalicylic acid (Aspirin®)51 and others.52 Currently, there is no effective treatment for CAV and AV in other solid organ transplants, and it remains the primary cause of graft loss beyond one year post-transplantation.  10 | P a g e  1.3 – Current concept of the pathogenesis of CAV My current concept of the pathogenesis of CAV is fundamentally rooted to structural observations of histopathological sections of CAV (Figure 2), coupled with specific observations from the literature, these guide my experimental direction. Endothelial insults, which may arise from oxidative stress,53-55 hemodynamic changes to blood flow,56-58 direct physical or mechanical injury,59 are coupled with an ongoing allogeneic immune response against the donor vasculature.60 These insults and injuries result in both an endothelial and immune response. Vascular endothelial cells (EC) produce pro-inflammatory cytokines and chemokines,61 as well as protective anti-apoptotic and pro-survival growth factors.53,62,63 Growth factors and pro-inflammatory mediators facilitate the preservation and repair of the endothelium. Congruent to their effects on the endothelium, these growth factors and pro-inflammatory cytokines also act on the vascular SMC underlying the basal lamina.64-66 This milieu results in excess intimal SMC proliferation and extracellular matrix (ECM) remodeling, termed neointimal formation or intimal hyperplasia. Concomitant with inflammation and re-endothelialization is an increase in endothelial permeability, and coupled with the remodeling of the ECM, results in the insudation and retention of circulating lipids and lipoproteins from the blood stream. An important publication established the relationship between hyperlipidemia and post-transplant obesity with luminal narrowing in human heart allografts.67 Subsequent investigations demonstrated profound lipid accumulation in coronary arteries of many grafts begins very early post-transplant and appears to contribute substantially to intimal thickening.68,69  11 | P a g e  I IEL intimomedial layer  I I  M M  * M  EEL  Normal  Mild CAV  Severe CAV  Figure 2 – Structural progression of cardiac allograft vasculopathy. Human vessels were obtained from the Cardiac Registry at the Institute for Heart + Lung Health and slides were stained with Movat’s pentachrome. In a normal coronary artery from a 20-year-old male, the intimal layer (I) is minimal, and consists primarily of the endothelium and its supporting basal lamina and is bounded by the internal elastic lamina (IEL); there is an intimo-medial layer; and the media (M) itself, which is bounded by the IEL and the external elastic lamina (EEL). In normal vessels the media is thick, smooth muscle- and elastin-rich, and exhibits normal vascular function. In mild CAV, represented here by a coronary artery from a 60-year-old-male transplant recipient whose graft failed 102 days post-transplantation, there is already a notable thickening of the intimal layer. This thickening is largely comprised of smooth muscle cells and matrix. Conversely, the medial layer is thinned. In severe CAV, represented here by a coronary artery from a 67-year-old female transplant recipient whose graft failed 360 days post-implantation, there is marked thickening of the intimal layer. As well, at the intimo-medial junction there is abundant accumulation of lipids, lipoproteins, and infiltrating macrophages (*). The medial layer in this vessel with advanced AV is further diminished, damaged by intimal processes as well as adventitial inflammatory processes. Scale bars represent 50 microns.  12 | P a g e  This accumulation of lipids, in concert with inflammatory mediators, results in the chemoattraction of monocytes, which differentiate into macrophages within the vessel wall, as well as the chemoattraction of other inflammatory cells. Concurrently, oxidative and cytotoxic mediators released by macrophages and other inflammatory cells result in the modification of low-density lipoproteins (LDL) to the more pernicious oxidized low-density lipoproteins (oxLDL) within the vessel wall.70-72 Macrophages and SMC are able to uptake oxLDL but are unable to extravasate or breakdown the lipid. This leads to excess lipid accumulation within the cells and resultant lipid overload.73,74 Pro-survival effects from oxLDL itself, as well as other growth factors and cytokines in the extracellular milieu of the evolving lesion lead to the transformation into lipid-laden foam cells.72,75-77 Death of these foam cells results in an acellular, necrotic lipid-rich core. SMC apoptosis and ECM breakdown in the superficial intima covering the atheromatous core is mediated by infiltrating and now-resident inflammatory cells, resulting in the thinning of the fibrous cap containing the atheromatous lesion. A thin fibrous cap in native atherosclerosis increases the propensity for plaque rupture and thrombotic events, leading to acute MI or death.78-80 Less is known about the precipitants of acute coronary occlusion in CAV.  13 | P a g e  1.4 – Dysregulation of endothelial permeability in vascular disease Alterations in endothelial permeability in vascular disease are often concomitants of inflammation. Inflammation is characterized in part by edema due to increased endothelial permeability to fluid and other blood components, and such leakage plays a key role in the pathogenesis of many diseases. Inflammatory mediators increase vascular permeability by inducing the retraction or contraction of endothelial cells and the formation of gaps between adjacent endothelial cells in post-capillary venules.81-84 Since the 1980s, it has been generally held that diverse classes of inflammatory mediators produce this type of gap formation exclusively at the post-capillary venules, allowing fluids and macromolecules to enter the tissue and cause potentially massive edema,85 affecting vascular beds proximate or remote to the inflammatory process. Capillary leak subsequent to prolonged inflammation can be achieved. The modeling in vitro using endothelial cell cultures treated with pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) has been visualized using light microscopy and silver nitrate (AgNO3) staining or transmission electron microscopy (TEM) and horseradish peroxidase (HRP) tracers (Figure 3; panels b,d from Dr. Donald Wong). A number of clinical disorders are associated with aberrant vascular permeability.  14 | P a g e  a  b  c  d  Figure 3 – Assessment of endothelial permeability under transmission electron microscopy (TEM) using horseradish peroxidase (HRP) as a tracer. Human endothelial cells were cultured and received either 100ng/mL tumor necrosis factor-alpha (TNF-α; c,d) or no treatment (a,b). Silver nitrate staining (AgNO3; a,c), and captured by light microscopy allowing for the visualization of intercellular gaps at the structural level [arrow]. Use of HRP tracer molecules in conjunction with visualization by TEM allows not only for the ultrastructural examination of junctional integrity, but also for demonstrating the permeability of small molecule tracers and their deposition in the junctions and beneath the monolayer (b,d).  1.4.1 – Endothelial dysfunction in native atherosclerosis Inflammation has been clearly associated with the pathogenesis of human atherosclerosis as illustrated by the presence of infiltrating monocytes-macrophage and Tlymphocytes in the subendothelial tissue and in the shoulders of lipid rich plaques.8,86,87 Atherosclerosis was initially viewed as a disease of lipid accumulation in the vessel wall, with the endothelium being affected secondary to the underlying cell death within the atheroma. It is now clear that the endothelium is intimately involved in the initiating processes, with early endothelial dysfunction being a hallmark in the pathogenesis of atherosclerosis. As well, associated vascular hyperpermeability results in the insudation of lipids, lipoproteins, and other 15 | P a g e  plasma components.56,88-91 Increased permeability of the arterial wall to lipoproteins such as LDL leads to lipid accumulation or retention, atheromatous plaque formation and subsequent progression of atherosclerosis.92 Many factors contribute to increased arterial wall permeability, including changes in flow dynamics and shear stress (i.e., at areas of bifurcation or luminal narrowing),93,94 and expression of vasoactive factors such as TNF-α,95-98 histamine99-101 and vascular endothelial growth factor (VEGF).77,102  1.4.2 – Altered endothelial permeability in cardiac allograft vasculopathy Although CAV and native atherosclerosis are clinically and pathologically distinct entities, these diseases share certain pathogenic mechanisms. Both processes may be regarded as responses to injury, including participation of the immune system in the injury and the response, early endothelial dysfunction and early atheromatous plaque formation are cornerstones in both conditions.103 The major mechanisms of injury in organ transplantation rest with the immune reactions involving allogeneic lymphocytes and infiltrating macrophages. The endothelial antigens of the transplanted organ are among the first to be recognized by the host’s immune system and thus plays a pivotal role in both acute and chronic responses to injury that lead to vasculopathy following solid organ transplantation. Interactions between donor endothelial cells and the recipient’s immune system initiate a series of inflammatory responses with repetitive injury to the endothelium, resulting in endothelial dysfunction and destruction, increased permeability with enhanced influx of blood components into the sub-endothelial 16 | P a g e  space, exposure of vascular ECM, and subsequent intimal hyperplasia.104 This pathobiological series of events leads to rapidly accumulating, concentric lipid deposits as prominent features within the transplanted vessel.69,105 Using a rat model of heterotopic cardiac transplantation, we have observed insults to the donor endothelium, both functional and structural.106-109 The overexpression of endothelial nitric oxide synthase (eNOS) early and inducible nitric oxide synthase (iNOS) late within intramural allograft coronary vessels was associated with a progressive and profound loss of endothelial agonist responses and myogenic tone.107 The loss of normal tone in resistance vessels may convey an increase in hydrostatic force favoring the development of interstitial edema in the transplanted heart.109 In related studies, we observed intercellular gap formation and transcellular disruptions of endothelial integrity in allograft aorta and epicardial coronary arteries. These changes clearly result in increased endothelial permeability (Figure 4; data from Mr. John Lai; complementary to106). It has been suggested that intercellular gap formation may begin at tricellular corners in the endothelium.110 In addition, endothelial exfoliation and denudation in allografts, as a result of cell death by apoptosis and/or necrosis, may also result in increased endothelial permeability.106,111 Endothelial disruptions may allow macromolecules, particularly larger lipoproteins such as very low-density lipoprotein (VLDL) particles, and inflammatory cells to readily enter the subendothelial intimal compartment, where entrapment can occur in the ECM. In the pathogenesis of native atherosclerosis, LDL is the predominant lipoprotein particle, particularly small and dense LDL particles – VLDL particles do not play a crucial role, in part due to their large size. The 17 | P a g e  endothelial damage and increased vascular permeability in the allograft setting, perhaps being more profound and progressive than in native atherosclerosis, may allow larger numbers of lipoprotein particles (both LDL and VLDL) to enter the sub-endothelial compartment at a higher influx rate. Indeed, we have observed marked, early accumulation of apolipoprotein (apo) B-, apo (a)- and apo E-containing lipoproteins in human allograft arteries.112 We also observed increased permeability using 131I-radiolabelled sucrose, albumin, and LDL in allograft rat hearts as compared to syngraft hearts as early as four days post-transplantation (unpublished data). These entrapped lipoproteins may then undergo oxidative modification and be taken up by macrophages through scavenger receptors.71,113 These stages of vasculopathy are further accelerated due to increased oxidative stress and the dyslipidemia associated with transplantation.114-116  18 | P a g e  a  b  c  d  e  f  Figure 4 – Examination of endothelial perturbations in a rat model of heterotopic cardiac transplantation. Right coronary arteries from explanted donor rat hearts were perfused with glutaraldehyde and prepared en face for silver nitrate staining (AgNO3; a,b) or scanning electron microscopy (SEM; c-f). At the structural level, coronaries from non-transplanted control rats demonstrate a classic cobblestone arrangement without apparent intercellular gaps or endothelial cell loss (a). In comparison, non-immunosuppressed allografts as early as one day post-transplantation demonstrate abundant cell loss or presence of intercellular gaps, as demonstrated by the black AgNO 3 precipitation as a result of interactions with the exposed basal lamina (b). SEM of a segment of native recipient aorta from a rat syngraft 42 days posttransplantation (c) demonstrates an intact, largely unaltered endothelium, mirroring the cobblestone pattern seen in the AgNO3-stained controls. In stark comparison, SEM of a rat aorta (d) or septal artery (e) from a non-immunosuppressed allograft four days post-transplantation visualizes endothelial inflammation and activation, as well as the presence of interendothelial gaps (arrow). At 42 days post-transplantation, SEM of a rat aorta from a cyclosporine-treated allograft (f) demonstrates preserved endothelial morphology; however, there are still numerous intercellular gaps (arrows).  19 | P a g e  1.5 – Endothelial barrier function and permeability The endothelium separates the blood from underlying tissue, and is well situated to modulate the physiology of both compartments. The classical view of the endothelium as a static barrier has been replaced in face of the identification of a large number of inducible endothelial functions that reflect adaptation to changing conditions and pathophysiological processes. The following section focuses on the cellular mechanisms that regulate endothelial permeability and particularly govern its modulation in vascular disease.  1.5.1 – Pathways through the endothelium Two basic routes exist across the endothelium: intracellular (through the endothelium) and intercellular. The former can be mediated by the process of endocytosis; the latter, by opening of the intercellular junctions. The first endothelial structure at the luminal endothelial surface is the glycocalyx, a layer made up of glycated proteins. The composition and thickness of the glycocalyx varies with the location of the vessel and treatments/stimuli given. 117,118 A thicker glycocalyx is associated with lower permeability of the endothelium.119 Removal, in part, of the sugar or protein components by gentle enzymatic digestion with heparanase, hyaluronidase or pronase invariably increases permeability.79,120-122  1.5.1.1 – Intracellular passage Vesicular transport via receptor-mediated endocytosis and transcytosis is important for the movement of substances such as lipoproteins, albumin, insulin and transferrin across the endothelium. Albumin binds to receptors in vesicles of a limited number of capillaries, including 20 | P a g e  heart, lung, skeletal muscle and adipose tissue.123 In other types of endothelium (artery, arteriole, venule, vein, endocardial, fenestrated and sinusoidal), it is taken up by fluid phase endocytosis, while the endothelium of macrovessels (aorta, large arteries, veins) utilize both pathways.124  1.5.1.1.1 – Endocytosis The uptake of macromolecules usually occurs by receptor-mediated endocytosis in clathrin-coated vesicles. After binding the appropriate receptor on the cell surface, macromolecules are collected into a localized area where the plasma membrane becomes coated on the cytoplasmic side by the protein clathrin. The membrane invaginates and fuses to form a vesicle containing the ligand-receptor complex. This vehicle is subsequently moved to another compartment for processing (i.e., the lysosomal compartment) or to the opposite end of the cell where the contents are released into the extracellular milieu. A short list of substances utilizing this pathway includes iron,125,126 insulin127,128 and lipoproteins.129,130 Uncoated vesicles may non-specifically take up a portion of the extracellular milieu in fluid phase endocytosis. Actin and microtubules appear to be involved in the initiation of endocytosis and the movement of endocytotic vesicles in the cytoplasm, respectively.131,132 Endocytosis is coupled to a number of signal transduction pathways (Figure 5). Cell surface receptors, such as epithelial growth factor receptor (EGFR), platelet derived growth factor receptor (PDGFR), and vascular endothelial growth factor receptor-2 (VEGFR2/flk-1/KDR), possess intrinsic tyrosine kinase activity, which regulates the internalization and downregulation of the receptors themselves. Tyrosine phosphorylation of the regulatory 21 | P a g e  regions in the cytoplasmic domain of these receptors recruits the receptors into clathrin-coated pits133 by binding to the clathrin-associated adaptor complex activating protein-2 (AP-2).134,135 Inhibition of phosphatidylinositol-3 kinase (PI3K) prevents transport of PDGF-R from early to late endosomes and their translocation to the nucleus but the initial internalization of the receptors is unaffected.136,137 Further, inhibition also extends to non-clathrin-dependent fluidphase endocytosis but not clathrin-dependent endocytosis.138 Other signal transduction pathways implicated in endocytosis include protein kinase C (PKC)139 and protein kinase A (PKA),140 depending on the cell type.  2+  3  3  2+  2+  Figure 5 – Regulation of endocytosis by signal transduction pathways. Ligand-receptor interactions such as vascular endothelial growth factor (VEGF) interaction with vascular endothelial growth factor receptor-2 (VEGFR2), a tyrosine kinase receptor, can modify the endocytosis of other molecules, such as low density lipoproteins, through signal transduction pathways downstream of VEGFR2. Signaling through inositol triphosphate (IP 3) receptors in the endoplasmic reticulum (ER) can 2+ facilitate calcium (Ca ) release, which may directly or indirectly (for example through 1,2- diacylglycerol (DAG) and protein kinase C (PKC) affect other endocytotic processes.  22 | P a g e  1.5.1.1.2 – Transcytosis Caveolae at the cell membrane and vesicles in the cytoplasm with a diameter of less than 70nm can potentially take up substances at one side of the endothelium by endocytosis and shuttle them to the opposite side, a process called transcytosis. Like endocytotic vesicles, they are also coated invaginations of the cell membrane, but their coating is distinct from those mentioned above with the presence of receptors, signal transduction molecules, other integral membrane proteins and lipids such as caveolin-1, eNOS, Ras, PDGF-R, interferon (IFN)-α/β receptor,  IFN-γ  receptor,  glycosylphosphatidylinositol-anchored  membrane  proteins,  cholesterol and sphingomyelin.141-143 These vesicles are made at the Golgi apparatus, shipped to specific areas of the cell membrane and do not interact with other vesicles. 144 They appear to open and close but do not leave their general locale. These caveolae can open to receive extracellular material such as folate145 and simian virus 40,146 then close to process, concentrate or store these substances that can subsequently be transported into the cell by endocytosis, or by carriers or channels, the latter process called potocytosis. In contrast to endocytosis, potocytosis is inhibited by PKC.145 It has also been shown that caveolae can be induced to swell, fuse together and form transcellular channels called vesiculo-vacuolar organelles (VVO), which allow the passage of molecules across the endothelium. The most prominent molecule able to induce this function is VEGF.147,148  23 | P a g e  1.5.1.2 – Intercellular permeability The cytoskeleton, especially actin and myosin, plays a dynamic role in the regulation of vascular permeability. Intercellular gap formation is dependent on the rearrangement of F-actin and myosin, resulting in a reversible loss of the peripheral actin band and increased F-actin stress fiber density.149-151 As noted above, structural components of the tight junction and adherens junction are linked to the cytoskeleton; individual actin fibers terminate at the plasma membrane.152 Pharmacological studies using agents that disrupt actin such as cytochalasin B, cytochalasin D, and latrunculin B increase tracer flux, decrease resistance and decrease the complexity of tight junctions.153-155 It has been observed that agents that increase the contractility of the cytoskeleton, especially affecting the dense peripheral band consisting of actin and myosin adjacent to junctions, lead to an increase in permeability. Thus, the tension generated by the perijunctional cytoskeleton may regulate permeability.156 In addition to actin, myosin is a major component of the endothelial cytoskeleton. The mechanism of contraction in the peripheral band is comparable to that of the SMC. It is regulated by the phosphorylation of the myosin light chains (MLC) by the myosin light chain kinase (MLCK), which activates myosin to slide along actin. In addition, a decrease in the action of MLC phosphatase has the same effect. 157 A large number of studies have demonstrated that inflammatory mediators induce myosin phosphorylation mainly through MLCK, thereby leading to endothelial cell retraction and hyperpermeability.158-160 Tyrosine phosphatases can, in turn, regulate the activity of MLCK.161  24 | P a g e  1.5.1.2.1 – Tight junctions Tight junctions are regions where the outer leaflets of the cell membrane of adjacent cells are fused together. They are formed by the phosphorylation-dependent assembly of zonula occludens (ZO)-1, ZO-2, and ZO-3 proteins to the plasma membrane, where they interact with transmembrane occludin and claudin proteins, and attach the tight junction to the cellular cytoskeleton (Figure 6). Immunohistochemical staining has localized occludin to tight junction strands in EC and epithelial cells,162-164 and its expression may be modulated by the ECM composition.165 It has been shown that occludin is involved in conferring low permeability at the tight junction. This concept was supported by an observed decrease in permeability due to overexpression of occludin in cultured Madin-Darby canine kidney (MDCK) cells.166 Reciprocally, a reduction in occludin expression is correlated with an increase in endothelial permeability.167  Figure 6 – Regulation of tight junctions by signal transduction pathways. Ligand-receptor interactions such as vascular endothelial growth factor (VEGF) interaction with vascular endothelial growth factor receptor-2 (VEGFR2) can also impact tight junctional integrity and organization. Signaling downstream of tyrosine kinase receptors can modify the phosphorylation status of zonula occludens (ZO), such as ZO-1, which serve to bind the transmembrane tight junctional proteins such as occludin and claudin to the actin cytoskeleton.  25 | P a g e  Occludin is also involved in the recruitment of a number of proteins in the family of membrane-associated guanylate kinase homologues, ZO-1, ZO-2 and ZO-3, to the cytoplasmic side of the tight junction. ZO-1 is a ~200kDa protein. It binds via its amino terminus to the carboxy terminus of occludin and claudin.168-170 Actin and -catenin binding sites are present at their carboxy termini.171,172 Partly because of these specific binding sites, ZO-1 is present at the tight junction, but not at the adherens junction of epithelial cells. ZO-2 is a 160kDa protein with occludin, claudin, β-catenin and actin binding sites. It also binds ZO-1 to form a heterodimer.170,172,173 ZO-3 is a 130kDa protein that binds to occludin, claudin, actin and ZO-1, but not to ZO-2.170,172,174 ZO-3 is also targeted to tight junctions in epithelial cells. ZO-1, ZO-2 and ZO-3 can cross-link the cytoskeleton to the tight junction and adherens junction, potentially regulating their involvement in activities such as vascular permeability.171,175 Claudins are 22kDa proteins with four transmembrane domains.176 When fibroblasts were transfected with these genes, they formed tight junctional strands in extensive networks.177,178 This is in contrast to cells transfected with occludin, which formed only a small number of short strands.179 Claudins appear to form the backbone of tight junctions. Different sets of these proteins seem to make up the tight junction in a tissue dependent fashion, perhaps related to the level of permeability of the junctions.180 It has been proposed that the number, type and ratio of the different species of claudin in the tight junction may determine permeability. Some combinations are able to form heterodimers and thus a tight seal in the junction, while other combinations cannot adhere to each other. Thus, when they are present in the junction, a “pore” is formed through which material can pass across the tight junction. 181 The current view holds that tight junction strands are composed of several species of claudin 26 | P a g e  polymerized to form a backbone with occludin co-polymerized into the strands. Tight junctions can be seen as dynamic structures that control vascular permeability in response to extracellular and intracellular stimuli, thereby regulating vascular integrity and function.  1.5.1.2.2 – Adherens junctions Another junctional complex that is present between adjacent endothelial cells is the adherens junction (Figure 7). This structure does not form barriers to molecular movement, but rather forms areas where adjacent cells attach to each other and are held together through the homophilic calcium-dependent interactions of transmembrane proteins called cadherins. Adherens junctions regulate the integrity of the tight junctions. Endothelial cells express at least two types of cadherins. N-cadherins are distributed diffusely, while VE-cadherins are located at the junctions.182 VE-cadherin, also called cadherin-5, is a 150kDa protein specific for endothelial cells, with β-catenin and plakoglobin-binding domains at its carboxy-terminal region.183 The tyrosine phosphorylation status of adherens junction proteins has been implicated in the regulation of vascular permeability. Overexpression of VE-cadherin increases the recruitment of β-catenin into adherens junctions and subsequently reduces its nuclear translocation for transactivation. Transfection of VE-cadherin promotes adhesion and contact inhibition of growth. The use of a truncated form of the protein abolishes the latter effects as well as control of paracellular permeability and stabilization of junction.184,185 Subconfluent and migrating cells exhibit high levels of tyrosine phosphorylation of this protein. Phosphorylation is correlated with binding to β-catenin and a low level of adhesiveness as compared to other members of the cadherin family, such as E- and N-cadherin in other cell types. The dynamic nature of this 27 | P a g e  interaction is illustrated by a reduction in the level of phosphorylation when cells become confluent and the junctions mature and stabilize. Binding to β-catenin is replaced by plakoglobin and actin at this time.186  2+  2+  plakoglobin  Figure 7 – Regulation of adherens junctions by signal transduction pathways. Similar to their modification of tight junctions, signal transduction molecules such as protein kinase C (PKC) downstream of tyrosine kinase receptors can affect barrier properties maintained by adherens junctions. 2+ Typically, this may occur through calcium (Ca ) and/or calmodulin (CaM)-dependent modulation of myosin light chain kinases, which can affect cell contraction through myosin light chains. This may also occur through modifications in phosphorylation status of adherens junction molecules by tyrosine phosphatases or kinases.  Thrombin, histamine, VEGF, TNF-α, IFN-γ and polymorphonuclear leukocytes (PMNL) are all known to increase endothelial permeability and have been shown to affect the adherens junction, especially VE-cadherin. VEGF induces tyrosine phosphorylation of VE-cadherin 15 minutes to one hour after treatment.187 TNF-α and IFN-γ cause disorganization of VE-cadherin in focal areas.188 Thrombin reduces plakoglobin binding to VE-cadherin, while PMNL reduce βcatenin binding.184,186,189,190 Anti-VE-cadherin antibodies induce a redistribution of VE-cadherin and reorganization of the cytoskeleton along with an increase vascular permeability, as 28 | P a g e  illustrated by interstitial edema, inflammatory cell accumulation, endothelial gap formation and exposure of basement membrane to the circulation.191-193 At the same time, a rapid production of VEGF and re-synthesis of VE-cadherin occurs to restore cell-cell contact.194 The importance of the cytoskeletal interaction (especially actin) for the function of VE-cadherin is illustrated by the redistribution of the latter after treatment with isoproterenol or an inhibitor of small GTPase, p21 Rho.192 N-cadherin, the other major cadherin in endothelial cells, is a 140kDa protein that is cytoplasmically distributed rather than localized to the adherens junction. It has been proposed that whereas VE-cadherin promotes homotypic interactions between adjacent endothelial cells, N-cadherin anchors endothelial cells to surrounding N-cadherin-expressing cells such as SMC and pericytes.182 Cadherins are connected to actin through the linking proteins catenins, vinculin, vimentin, talin, α-actinin, zyxin and moesin. The contractile tension generated by the cytoskeleton modulates the barrier function. Members of the catenin family include α-catenin, β-catenin and plakoglobin/γ-catenin.195,196 β-catenin, plakoglobin and p120, members of the Armadillo family, bind to VE-cadherin. Plakoglobin and β-catenin also binds α-catenin, which is homologous to vinculin. In turn, α-catenin bind to α-actinin, actin, vinculin and talin.197 This association strengthens cell-cell binding via VE-cadherin to withstand shear stress.198 Overexpression of vinculin decreases cell motility, while disruption of vinculin expression results in decreased adhesion and enhanced motility.199,200 Binding of actin to vinculin induces the dimeric conformation of the latter different from that formed by phosphotidylinositol 4, 5bisphosphate (PIP2). It has been proposed that actin may thus function directly in signal 29 | P a g e  transduction.201 Vinculin phosphorylation by PKC and tyrosine kinases has been correlated to hyperpermeability during energy depletion in coronary endothelial cells (Figure 7).202 Since p120 does not bind α-catenin, its presence leads to relatively weaker adhesive strength and a more dynamic junction. In recently confluent cells, VE-cadherin is bound to p120 and the adherens junction is weak and immature. After 48-72 hours, p120 is replaced by plakoglobin and the adherens junction stabilizes.183,186 VEGF induces the tyrosine phosphorylation of βcatenin, plakoglobin and p120 15 minutes to one hour after treatment.187 A number of other growth factors such as PDGF, epithelial growth factor (EGF) and transforming growth factorbeta (TGF-β) also cause phosphorylation of the catenins, either directly through tyrosine kinase receptors or indirectly through Src tyrosine kinases. Phosphorylation of β-catenin and plakoglobin leads to their dissociation from cadherin and disrupts cell-cell adhesion. Conversely, a number of protein tyrosine phosphatases associated with the adherens junction dephosphorylate catenins and increase adhesion.203,204  1.5.1.3 – Focal adhesion complexes and the extracellular matrix Complex structures called focal adhesions link the cytoskeleton of the cell to the ECM. They are composed of a cytoplasmic focal adhesion plaque, transmembrane integrins and ECM molecules. The focal adhesion plaque is a complex structure composed of α-actinin, vinculin, talin, paxillin and tyrosine kinases (Figure 8), most notably focal adhesion kinase (FAK). Similar to the adherens junction, α-actinin links vinculin and actin. This is linked to integrins by the binding of talin to vinculin and the β subunit of integrins. 205 Alternatively, α-actinin can directly link actin to the β subunit of integrin.206 A number of key enzymes localized to the focal 30 | P a g e  adhesion plaque participate in the regulation of permeability through this structure. The major regulators include FAK,207,208 c-Src, PKC, and Ca2+-dependent proteases. Vinculin and talin are substrates for these enzymes (Figure 8). FAK itself is tyrosine phosphorylated in the presence of inflammatory mediators209 and in subsequent vascular hyperpermeability.210 However, the function of these kinases varies between different cell types. In endothelial cells, vinculin and talin are redistributed and the cytoskeleton is rearranged by thrombin treatment, but the phosphorylation state of these proteins remains unchanged. 211 Other focal adhesion proteins that are substrates for these kinases include paxillin,212,213 GTPase regulator associated with FAK (Graf) and Crk-associated substrate (Cas).214  Figure 8 – Regulation of focal adhesion complexes by signal transduction pathways. The integrity of focal adhesion complexes can be modulated through signal transduction pathways such as protein kinase C (PKC) or tyrosine kinases downstream of tyrosine kinase receptors, such as vascular endothelial growth factor receptor-2 (VEGFR2). These may act on focal adhesion complex molecules such as vinculin or talin, respectively, which facilitate the binding of integrin complexes joined to the extracellular matrix at the abluminal side of the cell to the actin cytoskeleton through intermediates such as α-actinin.  31 | P a g e  Talin is a homodimer of 230kDa polypeptides.215 It cross-links actin and potentiates the function of α-actinin.216,217 Paxillin is a 68kDa protein that binds vinculin, FAK and Src. It is phosphorylated by inflammatory mediators and growth factors212 as well as during subsequent increases in vascular permeability.210 Graf is a regulatory protein that can stimulate the intrinsic GTPase activity of RhoA and cdc42. It may serve as an effector or negative regulator of these signals.218 Cas regulates the Ras family of GTP binding proteins by binding the adaptor protein Crk and CrkL which then binds the guanidine nucleotide exchange factors (GEF) Son of sevenless (SOS) and C3G, which in turn regulate the activity of Ras and Ras-proximate-1 (Rap1).219 Inhibition of RhoA activity reduces endothelial permeability and stress fiber linkage to focal adhesions.220 FAK itself can be regulated by tyrosine phosphorylation at six sites and serine phosphorylation in multiple sites. These events appear to be associated with cell attachment, recruitment of kinases and activation of Ras.214 Integrins are heterodimeric transmembrane proteins. Their β-subunit non-covalently binds to the α-subunit, which confers the specificity of binding. Of the eight β-subunits, β1, β2, β3, β4 and β5 are present in endothelial cells, along with α1, α2, α3, α5, α6 and αv from the 16 α subunits in different combinations.205,206,221,222 Inhibition of their function by antibodies leads to altered permeability as well as migration and attachment.223 Increased permeability in diseased states also involves integrins. TNF-α induces internalization of the α5β1 integrin224 and reduces its co-localization with fibronectin.225 The ECM provides points for cell binding and components include fibronectin, laminin, vitronectin, collagen and proteoglycans. The basement membrane and ECM also affect permeability. The diffusion of albumin is decreased by the presence of matrix. Components of the vessel wall – elastin, collagen and 32 | P a g e  proteoglycans – in isolation can variably retain molecules such as LDL.226 It is not known how this translates to the blood vessel wall. Interactions between endothelial cells and ECM are required to maintain low permeability. Endothelial cells bind to the ECM via integrins on their abluminal surface, which in turn, bind talin, vinculin, α-actinin and actin (Figure 8). Digestion of the extracellular matrix by protease treatment or endogenous metalloproteinases significantly increases permeability. The mode of action for this proteolytic effect is uncertain. It may involve changes in force transduction between the extracellular matrix and the cytoskeleton or of “outside in” signal transduction through the integrins.227  1.5.2 - Modulation of permeability The transport pathways used across different endothelium vary, even within the same organ. The permeability of different vascular beds can vary over several orders of magnitude under normal conditions.228 As well, numerous substances, both exogenous and endogenous, affect the permeability of the endothelium. As early as the 1960s, Majno et al had shown that inflammatory mediators act on venular endothelium and increase vascular permeability to colloidal carbon, thus establishing the endothelium as an important functional component of the vasculature.82,83 Both histamine and serotonin induce the formation of gaps 0.1-0.8μm in width in venules, visible under the electron microscope. It was believed that these are not holes through the endothelial cells but rather intercellular gaps. Later, these investigators hypothesized that such gaps formed due to histamine, serotonin and bradykinin are a results of focal disruption of endothelial cell junctions following endothelial contraction. 81,84 This view is supported by the demonstration of contractile elements (actin, myosin, and microfilaments) in 33 | P a g e  endothelial cells. The gaps produced permit fluid and macromolecules to flood the tissue – one of the cardinal signs of inflammation. The dynamics of these processes were studied in the hamster cheek pouch model using intra-vital light microscopy and direct measurements of tracer (dextran-conjugated fluorescein isothiocyanate (FITC)) movement into the tissue.229 Using this model with a combination of electron and light microscopy, Arfors et al showed that sites of leakage correspond to gaps in the endothelium.230 The number of gaps increases with the dose of the inflammatory agent and, importantly, this event is reversible. The number of known permeability-altering agents has grown steadily. In addition to histamine, serotonin and bradykinin discussed above, there are prostaglandins, thromboxane, leukotrienes, thrombin, platelet-activating factor (PAF), cytokines, complement, substance P, vascular permeability factor (VPF), free radicals, endotoxin, various inorganic and organic salts, hypoxia and many others.196,231,232 Permeability changes were historically demonstrated mainly in venules. However, due to the polymorphic structural and functional nature of the endothelium in different categories of vessels and different organ systems, the action of these agents can vary considerably in different vascular beds.231 In this regard, it has also been important to compare studies utilizing different vessels in vivo and in vitro. Thrombin and histamine cause an increase in cytoplasmic calcium. In the presence of adenosine triphosphate (ATP) and calcium, myosin light chain kinase MLCK rapidly and transiently phosphorylate myosin light chains, allowing actin to interact with myosin, leading to contraction. The nature and identity of endothelial (MLCK) is not fully known. A number of candidates have been found in different vascular beds.233 In addition to contraction, some authors have also described retraction. Such retraction may be a passive process independent 34 | P a g e  of MLCK, but requiring phosphorylation of cytoskeleton linking proteins such as vinculin, talin, caldesmon and vimentin, by PKC and/or tyrosine kinases, thus leading to reduced cell-cell and cell-extracellular matrix contact and increased gap formation.227 Vinculin connects the cadherins and catenins to α-actinin and is involved in retraction of endothelial cells from adjacent cells. Talin bridges the integrins, vinculin and α-actinin at the abluminal cell membrane and is involved in retraction from the extracellular matrix. Endothelial cells from different vascular beds possess different abilities to contract. Pulmonary arterial endothelial cells do not develop enough tension to deform silicon supports when stimulated, whereas microvascular endothelial cells readily cause deformation.234 Thrombin and histamine has also been shown to cause the redistribution of VE-cadherin at the adherens junction. This is associated with a reduction in intracellular free calcium and an increase in permeability, but with no detectable gap formation. 235 It has been demonstrated that neutrophil adhesion to rat brain endothelial cells, leading to blood-brain barrier breakdown, causes an increase in phosphotyrosine, loss of occludin and ZO-1, and induces vinculin redistribution.236 With human umbilical vein endothelial cells, neutrophils disrupt adherens junctions and reduce the amount of VE-cadherin and β-catenin.189  1.5.3 – Second messengers and signal transduction pathways The signal transduction pathway involved in the regulation of endothelial permeability has been the focus of many studies. Second messengers implicated include calcium, G proteins, phospholipases, PKC, tyrosine kinases, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Many of the inflammatory mediators noted above utilize 35 | P a g e  these second messengers. Thrombin increases intracellular calcium and activates PKC. Hypoxia decreases cAMP levels. Prostaglandins increase cAMP. However, the same second messenger can also have diverse actions on endothelial cells of different origins. Cyclic AMP can increase the permeability of bovine pulmonary artery endothelial monolayers, but prevent the permeability increases due to thrombin in bovine aortic endothelial cells.231 The targets of these second messengers and how these different pathways interact with each other are less well characterized.  1.5.3.1 – Calcium Calcium is one of the most studied messengers involved in permeability changes. Histamine treatment causes intracellular free calcium to increase to a peak level within 15-30 seconds, and then fall to 30-50% of this level within 1-2 minutes. This calcium first originates from intracellular pools, then by way of influx from the extracellular milieu. These changes are consistent between intact microvessels and cultured cells, and upon treatment with different inflammatory agents such as thrombin and bradykinin.237 The increased level of free calcium can activate calcium-calmodulin dependent kinases, as well as calcium and phospholipiddependent PKC, leading to phosphorylation of actin-binding proteins and myosin light chains, eventually leading to endothelial contraction.238 However, it has been reported that increases in intracellular calcium alone do not increase the permeability of microvessels, in contrast to effects in larger vessels such as arteries.239  36 | P a g e  1.5.3.2 – Phospholipases Another early step in signal cascades is the activation of the phospholipases. Shear stress and oxygen radicals can directly activate phospholipases. On the other hand, thrombin, histamine and other agents first bind to and activate their receptors, leading to G protein activation, which in turn activates the phospholipases. There are four classes of G proteins (G s, Gi, Gq and G12) each formed from one α, β and γ subunit and coupled to specific receptors. Each class of G protein can be activated by the binding of a ligand to a number of different receptors, and each receptor can activate a number of classes of G proteins. These proteins regulate the activity  of  phospholipase  C  (PLC),  phospholipase  A2  (PLA2),  adenylyl  cyclase,  phosphodiesterases and ion channels. Ligand binding to the H2 histamine receptor causes the level of intracellular cAMP to rise, probably via Gs, which activates adenylyl cyclase. Binding to the H1 receptor causes phosphoinositide turnover and calcium mobilization via the activation of PLC by Gi and Gq.227 There are five types of phospholipases (A1, A2, B, C and D). The most important one relative to permeability is PLC. It is a membrane-bound enzyme that mediates the hydrolysis of phosphotidylinositol 4, 5-bisphosphate (PIP2) to inositol 1, 4, 5-trisphosphate (IP3) and 1, 2diacylglycerol (DAG). IP3 binds its receptor on the endoplasmic reticulum to induce mobilization of calcium. This event allows the initial rapid and maximal increase in concentration of internal calcium discussed above. Thrombin increases IP3 levels in pulmonary artery endothelial cells within 10 seconds and intracellular calcium levels within 17 seconds. This is followed by cytoskeletal reorganization and an increase in permeability at about two minutes. 240 Both the increase in intracellular calcium and DAG activates PKC.241 The rise in intracellular calcium can 37 | P a g e  also induce the formation of DAG by PLC and arachidonic acid by PLA2 in most systems. Arachidonic acid can go on to activate PKC and ion channels, including calcium channels. Phospholipase D (PLD) may also be involved. It also generates DAG, but does not require a rise in intracellular calcium. Bradykinin induces an increase in permeability dependent on PKC, but not on increased intracellular calcium. This pathway has been suggested as a mechanism for persistent increases in permeability.227 These pathways appear to converge on PKC. There are ten PKC isoenzymes, all of which phosphorylate serine and threonine residues. The isoenzymes α, βI, βII and γ are activated by calcium, phosphatidylserine and DAG, while , ,  and  do not require calcium, and  and  do not require either DAG or calcium.227 Although it is not clear which isoenzyme is involved in permeability changes, it is known that PLC and DAG are involved. PKC-α and PKC-β are the prime candidates since they are most abundant in endothelial cells. Moreover, overexpression of transfected PKC-β1 in endothelial cells increases the permeability due to phorbol 12-myristate 13-acetate (PMA) treatment.242 However, PKC activation by different agents can have contradictory effects on permeability. Depletion of PKC, treatment with PKC inhibitors or inhibitors of PKC activation all prevent the increase in permeability due to thrombin241 and H2O2243,244 in pulmonary microvascular endothelial cells. Although activation of PKC increases PAF-induced permeability, PKC activation by phorbol esters reduces permeability. Moreover, MLC were not phosphorylated. Indeed, thrombin causes MLC phosphorylation by a mechanism independent of PKC.233 In addition to the cytoskeleton, PKC also affects components of the adherens junctions. Thrombin-mediated disruption of the VE-cadherin-catenin complex is inhibited by PKC and tyrosine kinase inhibitors.245 Other PKC targets proposed include MLCK, vinculin and vimentin.246 How PKC is 38 | P a g e  involved in the regulation of the tight junction components and endocytosis/transcytosis is less clear. One study indicates that an increase in endocytosis due to angiotensin II is also mediated by PLC and PLD production of IP3 and a rise in intracellular calcium levels, but not PKC.247  1.5.3.3 – Tyrosine kinases Tyrosine kinases are another family of kinases implicated in the regulation of permeability. Tyrosine kinases are located at the adherens junctions.248 The components of these junctions such as vinculin, talin and β-catenin are substrates for these kinases. Tyrosine phosphorylation of β-catenin is associated with an increase in permeability.249 These enzymes have also been implicated in endocytosis in one study.250 gp60 is a cell surface albumin binding protein present in caveolae. Activation of this receptor by albumin or by cross-linking leads to the phosphorylation of its tyrosine residues, which in turn phosphorylates the Src family tyrosine kinases. Tyrosine kinase inhibitors prevent the uptake of albumin and gp60 activation. Increases in permeability due to inflammatory mediators are reversible. Permeability returns to basal levels 15-60 minutes after the removal of the agent.227 Many negative feedback mechanisms have been proposed, including receptor desensitization by proteolysis, internalization or phosphorylation, downregulation of G proteins, uncoupling of PLC and G proteins by PKC, activation of phosphatases, or inhibition of protein kinases. 227 For example, thrombin causes the degradation of its receptor after binding. 251 Bradykinin causes its receptor to be internalized.252 PKC uncouples PLC from its G protein, but not from PLD. 253,254 Dephosphorylation of myosin returns permeability to basal levels.255  39 | P a g e  1.5.3.4 – cAMP and cGMP The cyclic nucleotides cAMP and cGMP affect the permeability of the endothelium and modulate the effects of inflammatory mediators. Increases in cAMP levels by activation of adenylate cyclase, inhibition of phosphodiesterase or direct increases of intracellular cAMP by incubation with membrane permeable analogs of cAMP not only decrease the basal permeability of the endothelium,238 but also prevent the increase in permeability due to thrombin, histamine and other agents in the micro- and macrovasculature.233 The mechanism involves cAMP dependent protein kinases, reduction in MLC phosphorylation, loss of actinmyosin interactions, and redistribution of F-actin. In addition, the restoration of junctional complexes and inhibition of PL and PKC have also been proposed. 233,238 Cyclic GMP can be increased by similar mechanisms and this can produce similar effects on permeability in many cases, including reduction of thrombin and H2O2-induced permeability and basal permeability.233 This effect seems to involve the reduction of intracellular calcium by cGMP dependent protein kinases.256 However, differing results have also been reported. Elevation of cGMP levels does not affect the basal permeability of aortic endothelial cells. 257 In stark contrast, one group working with animals in vivo has reported that cAMP and cGMP increase albumin penetration of brain microvessel consistently.258 Other groups working with human material in vitro has found the opposite.259 One possible explanation is that different vascular beds and species may have different amounts of phosphodiesterases (PDE) that break down these cyclic nucleotides.233 In human umbilical vein endothelial cells (HUVEC), cGMP reduces the effect of thrombin by inhibiting a cGMP-inhibited PDE III. A PDE III inhibitor has the same effect on HUVEC, but not on human aorta or pulmonary artery endothelial cells. 256 Another 40 | P a g e  PDE, cGMP stimulated PDE II, may also be involved in permeability. Activation of this PDE by elevated cGMP levels can reduce both the concentration of cAMP and cGMP. Unfortunately, the level of PDE in different vascular beds has rarely been studied. Another cGMP dependent mechanism of interest is its reduction of intracellular calcium concentrations.233 Increases in calcium activate eNOS to produce nitric oxide (NO), which in turn increases cGMP production by guanylyl cyclase. This increased level of cGMP then reverses the change in calcium levels and the increase in permeability. Indeed, NO generated by thrombin counters the increase in aortic and pulmonary artery endothelial cell permeability. 233 Through this mechanism, NO also modulates its own calcium-dependent formation.  1.6 – Vascular endothelial growth factor 1.6.1 – Search for a tumor angiogenic factor More than a century ago, increased vascularization was observed alongside tumor growth. In 1939, Ide et al suggested the existence of a tumor-derived blood vessel growth stimulating factor.260 In 1945, Algire et al suggested that the rapid growth of tumor transplants is dependent on the development of a rich vascular supply after observing that the growth of tumor xenografts was preceded by local increases in vascular density.261 In 1968, Greenblatt and Shubick provided early evidence that tumor angiogenesis may be mediated by diffusible molecules.262 In 1971, Folkman proposed that anti-angiogenesis might be a strategy to treat human cancer.263 This key hypothesis fueled particular interest in the field of angiogenesis research and began the search for regulators of blood vessel growth. However, the identification and isolation of such factors proved elusive. 41 | P a g e  In 1983, Senger et al identified a protein which induces vascular leakage in the supernatant of a guinea pig tumor cell line and named it vascular permeability factor (VPF).264 It was proposed that VPF may be a mediator of the increased leakiness or permeability in tumor blood vessels. In 1989, VEGF was sequenced and described as an angiogenesis-inducing factor.265 Another group at Monsanto Company led by Daniel Connolly had also reported the cloning of VPF at approximately the same time and sequence comparison revealed that these two groups had independently identified the protein.266 This report described a human clone which encoded a protein identical to vascular endothelial growth factor (VEGF)-A189.266 Inactivation of the VEGF gene in mice provided definitive evidence for a key role in angiogenesis. In 1996, groups led by Carmeliet and Ferrara reported that VEGF is required for normal embryonic vasculogenesis and angiogenesis.267,268 Inactivation of even a single VEGF allele in mice resulted in developmental abnormalities and early embryonic lethality.267,268  1.6.2 – Vascular endothelial growth factor family members and receptors VEGF belongs to a gene family that also includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PlGF).269 Multiple isoforms of VEGF-A (121, 145, 165, 189, 206 amino acids) can be generated by alternative exon splicing. These isoforms differ in their ability to bind heparan sulphate, which determines its bioavailability and may play distinct roles in angiogenesis during development.270 In addition, extracellular proteolysis can regulate VEGF activity. Early studies showed that plasmin is able to cleave heparan sulphate-binding VEGF isoforms at the COOH-terminus to generate bioactive and diffusible fragments.271,272 More recently, it has been reported that matrix metalloproteinase-3 (MMP-3) is able to generate 42 | P a g e  VEGF proteolytic fragments which are biologically and biochemically similar to those resulting from plasmin cleavage.273 All VEGF isoforms can bind to VEGF receptor-1 (VEGFR1; fms-like tyrosine kinase-1, flt-1) and VEGF receptor-2 (VEGFR2; kinase insert domain receptor, KDR; fetal liver kinase-1, flk-1) (Figure 9). Despite the finding that VEGF binds to VEGFR1 with an approximately 10-fold higher affinity than VEGFR2, VEGFR2 primarily mediates VEGF signaling in endothelial cells.274 It is believed that VEGFR1 may act as a decoy receptor in some respects, preventing interaction with VEGFR2.  1.6.3 – The role of vascular endothelial growth factor Research conducted over the last two decades has established that VEGF plays an essential role in the regulation of embryonic,267,275 postnatal physiological angiogenesis processes,276,277 including normal development.278,279 A variety of animal models have generated much information on the biology of VEGF and the therapeutic potential of VEGF or VEGF inhibitors.280-286 VEGF has many effects on endothelial cells – it is a potent endothelial growth factor, inducer of vascular permeability,287 and vasodilator.288 VEGF can cause the migration of endothelial cells, new vessel formation,289 can affect pericytes and vessel maturation,290 and can impact the matrix metalloproteinases secreted by endothelial cells. 291  1.6.4 – Vascular endothelial growth factor in disease VEGF has been reported to play a role in a variety of diseases, including cancer, 292-303 diabetic retinopathy,304-307 age-related macular degeneration,308 renal failure,309,310 and native atherosclerosis,311-313 among others. 43 | P a g e  Heparan sulfate proteoglycan  (VEGF related factor, VRF)  (VPF)  (c-fos-induced (VEGF related growth factor, protein, VRP) FIGF)  VEGF-A121, 145, 165, 189, 206 VEGF-B167, 186  Neuropilin-2  PlGF-2  VEGFR-1 (flt-1)  PlGF-1  VEGF-C  VEGFR-2 (flk-1)  VEGF-D  VEGFR-3 (flt-4)  VEGF-E (Orf virus derived)  Figure 9 – Vascular endothelial growth factor family and their receptors. There are numerous members of the VEGF family and a number of receptors, including VEGF-A (VPF), VEGFB, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PlGF). There are 5 splice variants of VEGF-A (121, 145, 165, 189, 206 in humans) – the lowest molecular weight form is free in the circulation while the others are immobilized by binding to heparan sulphate proteoglycans. There are also 2 splice variants of VEGF-B (167, 186). VEGF-A binds VEGFR1 and VEGFR2, also called flt-1 and flk-1, respectively. VEGF-B binds VEGFR1. VEGF-C and VEGF-D bind VEGFR2 and VEGFR3. VEGF-E is from the Orf (pox) virus and binds to VEGFR2. Both PlGF-1 and PlGF-2 bind to VEGFR1. Neuropilins are also co-receptors for splice variants of VEGF-A and PlGF.  44 | P a g e  Pathological VEGF expression is driven by hypoxia, and in cancer, is one of the primary regulators of tumor angiogenesis. VEGF produced in this pathological state drives not only angiogenesis, but also increases vascular permeability, resulting in edema. In several models, inhibition of VEGF in ovarian carcinoma,314 brain tumors,315 and vestibular Schwannomas316 result in decreased edema around the tumors, resulting in reduced morbidity. Similarly, increased VEGF expression results in the neovascularization of the choroidal vasculature underlying the retina.278 When these choroidal vessels invade the retina, increased vascular permeability results in sub-retinal edema, causing loss of sight.317 In diabetes, there is systemic endothelial dysfunction, resulting in loss of endothelial cells from the retina and localized areas of ischemia.318 As a result, hypoxic induction of VEGF acts on the vasculature surrounding the periphery of the ischemic area, resulting in proliferation and increased leakage of vessels, ultimately leading to hemorrhage, edema and neovascularization.319 Since the first characterization of VEGF expression in human coronary atherosclerotic lesions,311 the possible pathophysiological significance of VEGF in the progression of atherosclerosis was introduced. Key concepts that have developed along these lines, including the discovery of VEGF-induced chemotactic response in monocytes mediated by VEGFR1 (flt1),320,321 suggesting a new role for VEGF in monocyte/macrophage chemotaxis, a crucial inflammatory process in wound repair. As well, the interplay of VEGF gene expression and its induction by hypoxia,322-324 transforming growth factor-β (TGF-β),325 angiotensin II,326 basic fibroblast growth factor327 and interleukin-1,328,329 among others, present a collection of factors that are all known to be expressed in atherosclerotic lesions.  45 | P a g e  Another primary role for VEGF in atherogenesis is the neovascularization of plaque tissue, often driven by the development of a hypoxic region within the developing neointima and fueled by the emerging lipid-rich core.8,330-332 In addition, VEGF plays a significant role in the induction of vascular permeability. Within the context of atheromatous disease and CAV, this role has been one that has been less focused upon. In contrast to VEGF-mediated effects in physiological angiogenesis where excess edema is uncommon or physiological VEGF-induced increases in permeability where angiogenesis is rarely seen, VEGF expression and actions in pathological states often results in aberrant angiogenesis coupled with excess permeability.  46 | P a g e  Chapter 2 – Hypothesis and specific aims 2.1 – Rationale Cardiac allograft vasculopathy (CAV) is the leading expression of chronic rejection and the major cause of heart transplant failure beyond one year post-transplantation. This chronic vascular disease results in the partial or complete obstruction of blood vessels, particularly macrovessels in transplanted organs, and results in tissue ischemia and heart failure. As such, CAV invokes substantial personal, social and financial costs in our society. In physiological and pathological settings, the endothelium serves as a key structural and functional regulator of vascular health, guiding leukocyte traffic, modulating transport of micro- and macromolecules and ions, and regulating homeostatic vascular function. Our laboratory has shown through a long-running series of investigations that pathogenesis of CAV in transplanted hearts involves endothelial injury and dysfunction, smooth muscle perturbations, inflammation, accumulation of extracellular matrix, and insudation of lipids and lipoproteins within affected vessels. As well, the abundance of lipids, lipoproteins and proteoglycans within the coronary arteries of human heart allografts occurs with an apparent lack of endothelial damage, death or denudation, suggesting the possibility of a permeability-inducing agent facilitating the insudation of plasma lipids and lipoproteins into the vessel wall post-transplantation. Dysfunction of the endothelium results in hyperpermeability to these factors and may contribute to the pathogenesis of CAV. Particularly relevant is the mechanism of increased low density lipoprotein (LDL) insudation and deposition in CAV arteries.  47 | P a g e  Among all growth factors known to date, vascular endothelial growth factor (VEGF) is the only one capable of inducing inflammation. VEGF increases vascular permeability, leukocyte adhesion and transmigration, and platelet aggregation via the synthesis of various paracellular signaling molecules such as platelet-activating factor and tissue factor, and may be potentially deleterious to grafts. On the other hand, VEGF induces endothelial cell proliferation, migration, and angiogenesis as well as bone marrow-derived cell mobilization and re-endothelialization, events that may be potentially beneficial to grafts. This doctoral dissertation focuses on the characterization of VEGF expression in CAV, the investigation of VEGF-induced endothelial hyperpermeability to LDL, and whether VEGF abrogation in vivo can reduce the number or severity of CAV lesions. These concepts are summarized in Figure 10.  2.2 – Central hypothesis Vascular endothelial growth factor plays a significant role in the pathogenesis of CAV by inducing endothelial cell hyperpermeability to low-density lipoproteins.  2.3 – Specific aims 1. To characterize the expression and localization of VEGF in CAV. 2. To elucidate the mechanisms of VEGF-induced endothelial hyperpermeability to LDL. 3. To examine the effect of abrogation of VEGF on the pathogenesis of CAV.  48 | P a g e  2  VEGF - ?  3  sVEGFR1  lumen  ?  % luminal narrowing  1  VEGF VEGF -- ??  VEGF VEGF -- ??  intima  media  Figure 10 – Diagrammatic representation of the specific aims of this thesis. 1.  To characterize the expression and localization of VEGF in CAV, immunohistochemistry was performed for VEGF. Digital micrographs were analyzed using ImagePro Plus® image analysis software to allow for direct comparison of VEGF immunoreactivity in the intima and media of coronary arteries from human heart allografts as compared to age-matched and sex-matched normal, non-atherosclerotic controls from the Pathobiological Determinants of Atherosclerosis in Youth study.  2.  To elucidate the mechanisms of VEGF-induced endothelial hyperpermeability to LDL, in vitro investigation using human coronary artery endothelial cells (HCAEC) and human cardiac microvessel endothelial cells (HCMEC) was performed. Transendothelial electrical resistance (TER) was used as an indirect measure of endothelial barrier properties in confluent endothelial monolayers. Immunocytochemistry was performed to examine the localization of tight junction protein components. A modified transwell system and fluorescently conjugated low-density lipoprotein (LDL) were used to determine whether VEGF can increase LDL passage through the endothelium.  3.  To examine the effect of abrogation of VEGF on the pathogenesis of CAV, a minor histocompatibility complex-mismatched mouse model of heterotopic cardiac transplantation was used to assess the role of VEGF in the pathogenesis of CAV in a hypercholesterolemic environment. Mice received intraperitoneal injections of either soluble VEGF receptor-1 (sVEGFR1) or vehicle control (PBS) every two days for 21 days, alongside an immunosuppressive regimen of FK506 (tacrolimus). The primary endpoint of this investigation was frequency and severity of CAV lesions.  49 | P a g e  Chapter 3 – The role of vascular endothelial growth factor in cardiac allograft vasculopathy 3.1 – Rationale The major cause of morbidity and mortality in human cardiac allograft recipients is the development of an accelerated form of atherosclerosis termed cardiac allograft vasculopathy (CAV).333,334 CAV is characterized by diffuse, intimal hyperplasia which occurs soon after transplantation and affects the entire arterial vasculature.45,335 Our laboratory and others have shown that lipid accumulation is an important early and persistent phenomenon in the development of CAV.68,69 Several growth factors are believed to be involved in various aspects of vessel growth, remodeling, and physiology. Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a multifunctional angiogenic cytokine first reported to increase the permeability of tumor microvessels to plasma and plasma proteins.336,337 VEGF is a potent and specific mitogen for vascular endothelial cells that is capable of stimulating angiogenesis,265,266 enhancing vascular permeability,162,338-340 and modulating thrombogenicity.341,342 VEGF is expressed by smooth muscle cells (SMC),343 macrophages77 and endothelial cells.344-346 VEGF-A has been previously been associated with animal models and human CAV.347,348 Native atherosclerosis (NA) is a chronic vascular disease which results in the hardening of the arteries. It is a multifactorial and multifaceted disease where fibro-fatty deposits accumulate in the inner lining of elastic and medium to large muscular arteries. These plaques are comprised of a lipid-rich core containing a heterogeneous milieu of fibrous tissue, vascular and inflammatory cells and an abundant and dynamic matrix composition. Congruently, diabetes mellitus (DM) is an independent risk factor for the development of atherosclerosis. It 50 | P a g e  is believed that a combination of hypertension, impaired vascular function, and increased glucose levels, among other factors; contribute to the development of atherosclerosis. Patients with DM often develop atherosclerosis at a more rapid pace than those with atherosclerosis but without DM. In this chapter, I characterize the differential immunoreactivity and localization of VEGFA and VEGF-D within coronary arteries from patients with either NA, DM, CAV, or “normal” patients consisting of individuals under the age of 35 who died of acute trauma unrelated to the heart, with less than 25% vessel occlusion. The purpose of investigating VEGF immunoreactivity in NA and DM alongside CAV was to gain insight into the localization with respects to etiology and atherogenesis. The results from this study characterize the differential expression of VEGF isoforms and suggest possible roles in the pathogenesis of CAV.  3.2 – Materials and methods 3.2.1 – Case materials Normal, non-atherosclerotic coronary artery tissues were obtained from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study. The vessels were from male and female individuals between the ages of 15-34 years and who died as the result of acute trauma. These cases from the PDAY study provided a baseline for investigation of VEGF expression in coronary arteries which did not have significant atherosclerosis (<25% luminal narrowing). PDAY cases were age- and sex-matched with the donors from the CAV cases. All of the pathological materials for study of CAV were obtained from the Cardiovascular Registry of the Institute for Heart + Lung Health (Vancouver, BC). Patients with native atherosclerosis were 51 | P a g e  selected on the criterion of greater than 25% luminal narrowing, as examined at the time of autopsy. Patients in the diabetic group were selected based on diagnosis of both diabetes mellitus and presence of atherosclerosis. All of the diabetic patients were diagnosed with noninsulin-dependent diabetes mellitus with a duration ranging from 3 to 23 years and all had their glucose levels under control by a combination of diet, anti-hypoglycemic drugs, or subclinical doses of insulin. Cases of native atherosclerosis and diabetes mellitus with atherosclerosis were also age-matched and sex-matched with each other. Refer to Table 1 for a summary of case details. A full listing of all of the cases used for immunohistochemical profiling can be found in the Appendix in Tables 3-6.  Group  n  Males  Females  Mean Age +/- SD (years)  Range of Ages (years)  Mean Implant Duration +/- SD (days)  Implant Duration (days)  Mean Age of Donor +/- SD (years)  Range of Donor Ages (years)  Normal  16  8  8  22.0 +/- 5.9  17 - 34  —  —  —  —  Native Atherosclerosis  17  11  6  65.2 +/- 17.0  24 - 87  —  —  —  —  Diabetes Mellitus with Atherosclerosis  15  8  6  64.6 +/- 9.6  39 - 85  —  —  —  —  Cardiac Allograft Vasculopathy  21  12  8  41.7 +/- 15.4  16-67  355.1 +/- 329.8  13 - 1432  26.8 +/- 10.8  16 - 47  Table 1 – Summary of cases used for immunohistochemical profiling of VEGF in atheromatous disease.  3.2.2 – Immunohistochemistry Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections for VEGF-A, and VEGF-D using streptavidin-biotin amplification. Polyclonal rabbit antihuman antibodies against VEGF-A463 (a kind gift from Dr. Harold F. Dvorak, Harvard Medical School; Boston, MA) and VEGF-D (Santa Cruz) were used. Antibodies used in immunohistochemical profiling of VEGF-A and VEGF-D were verified by Western blot using 52 | P a g e  recombinant VEGF-A and VEGF-D (R&D Systems), and found to be specific, recognizing the correct molecular weight bands with no observed non-specific reactivity. Briefly, sections were dewaxed and rehydrated in xylene and graded ethanols, then incubated with primary antibody overnight. Biotinylated goat anti-rabbit IgG (Vector Laboratories, Inc.; Burlingame, CA) and StreptABComplex/AP (Dako; Mississauga, ON) were incubated sequentially at room temperature. Antibodies were localized using the chromagen Vector Red (Vector), followed by counterstaining with hematoxylin. The positive control tissues consisted of placenta and normal-appearing kidney tissue adjacent to a kidney carcinoma. Negative controls included isotype-matched rabbit IgG and omission of primary antibody.  3.2.3 – In situ hybridization In situ hybridization (ISH) was performed as previously described.349 Formalin-fixed, paraffin-embedded tissue sections were dewaxed, rehydrated and permeabilized with proteinase K (Sigma-Aldrich Canada Ltd.; Oakville, ON). Tissues were subsequently acetylated, dehydrated, air-dried then hybridized overnight at 55°C using digoxigenin-labeled VEGF-A and VEGF-D antisense riboprobes or irrelevant probe (CVB3 antisense riboprobe used as a negative control). Placental tissue was used as a positive control. The VEGF-A and VEGF-D riboprobes were prepared from VEGF-A and VEGF-D cDNA, respectively (a kind gift from Dr. Steve Charnock-Jones, University of Cambridge, UK) by in vitro transcription according to manufacturer’s (Promega-Fisher; Nepean, ON) instructions. Following stringent washing in 50% formamide in 2x SSC, detection of hybridization was performed by incubating the tissue in alkaline phophatase-conjugated anti-digoxigenin antibody followed by an overnight incubation 53 | P a g e  in the color substrate 5-bromo-4-chloro-3-indolyl phosphate with nitro blue tetrazolium chloride (BCIP/NBT).  3.2.4 – Color segmentation and image analysis The area and intensity of positive staining were quantified using the ImagePro Plus® software. Briefly, an image of the tissue was digitally captured and an “area of interest” (AOI) was traced (Figure 11a). The AOI separated areas in the structure for assessment of immunoreactivity intensity. Adjacent sections stained with hematoxylin & eosin (H&E) and Movat’s pentachrome were used to determine the location of the internal and external elastic laminae, which were used to define the intima, media and adventitia within the vessel segment. A segmentation file was then created using positive and negative control slides and stains to define hue, saturation and intensity ranges considered positive immunoreactivity (Figure 11b). The segmentation file was then applied to the image, allowing the software program to report the percentage area of the staining intensities within the AOI (Figure 11c). The numerical data was exported to Microsoft Excel® for statistical analysis and further confirmed using SPSS statistical software (Figure 11d).  54 | P a g e  a) Capture image and construct AOI  b) Prepare segmentation file  c) Apply segmentation file  d) View statistics  Figure 11 – Diagrammatic representation of color segmentation analysis using “areas of interest” (AOI) in ImagePro Plus®. (a) Digital micrographs were captured using identical capture settings and AOI were defined using the internal and external elastic laminae. (b) A color segmentation profile was established using positive and negative control stains and slides to define positive immunoreactivity based on hue, saturation and intensity. (c) This color segmentation profile was used to determine positive immunoreactivity within AOI. (d) These data were then exported to Microsoft Excel® for further analyses.  3.2.5 – Statistical analysis Analysis of variance (ANOVA) was first performed between all the groups for each structure of interest. Where a significant difference was found between the groups, Student’s t-test was performed to further determine the significance of particular differences between specific disease groups and the normal group, with p < 0.05 considered statistically significant.  55 | P a g e  3.3 – Results 3.3.1 – Aberrant VEGF-A expression in human cardiac allograft vasculopathy Staining in control cases (PDAY) was diffuse and had minimal immunoreactivity (Figure 12a), while the positive controls of these antibodies differentially stain the decidual trophoblasts of the placenta [inset]. Computer-assisted image analysis demonstrated a significant increase in VEGF immunoreactivity in the intima and media of coronary arteries from cardiac allografts when compared to healthy arteries (Figure 12b). The average percentage VEGF-A positive area in the intima of CAV vessels was significantly increased as compared to age- and sex-matched controls from the PDAY study (12.18±5.84% vs 3.98±0.69%, p = 0.0036). As well, the average percentage VEGF-A positive area in the media of CAV was also significantly increased compared with controls (27.13±13.78% vs 5.21±1.13%). ISH in adjacent sections demonstrated increased VEGF transcript within cells of the superficial intima and in smooth muscle cells in the intimo-medial layer, as well as within the media of diseased vessels (Figure 12c).  56 | P a g e  A a)  Allograft Cardiac allograft vasculopathy  Normal  vasculopathy  c)C B b)  Intima of coronary arteries  CAV AV  Media of coronary arteries  CAV AV  ISH  Figure 12 – Immunohistochemical profiling of VEGF-A in human CAV. (a) Healthy arteries had weak, diffuse immunoreactivity for VEGF, while the positive controls of these antibodies differentially stain the decidual trophoblast cells of the placenta [inset]. The representative micrograph of a coronary artery from a cardiac allograft clearly illustrates VEGF immunoreactivity (red), largely localized to the superficial intima, the intimo-medial region, and strikingly within smooth muscle cells in the media. (b) Color segmentation analysis of the IHC reaction product was performed using ImagePro Plus® imaging software as described in the Materials and Methods. Computer-assisted image analysis demonstrated a significant increase in VEGF immunoreactivity in the intima and media of coronary arteries from cardiac allografts when compared to healthy arteries. Data are represented as mean ± SE; *, p < 0.01. (c) In situ hybridization in adjacent sections demonstrated increased VEGF transcript within cells of the superficial intima and in smooth muscle cells in the intimo-medial layer, as well as within the media of diseased vessels.  57 | P a g e  3.3.2 – Aberrant VEGF-A expression in native atherosclerosis and diabetes mellitus Staining was moderate in NA cases and further increased in DM (Figure 13). The percentage of VEGF-A positive area in the intima of coronary arteries is significantly higher in DM as compared to normal controls (9.75±4.61% vs 3.98±0.69%, p = 0.0085) and also significantly increased in NA (6.54±2.61% vs 3.98±0.69%, p = 0.0347). There was also a significant increase in VEGF immunoreactivity in the media of coronary arteries from both NA and DM cases compared with controls (25.23±6.16% and 32.83±8.90%, respectively, vs 5.21±1.13%).  58 | P a g e  a)  b)  Normal  Native atherosclerosis  Intima of coronary arteries  NA  Diabetes mellitus with atherosclerosis  Media of coronary arteries  NA  Figure 13 – Immunohistochemical profiling of VEGF-A in native atherosclerosis (NA) and diabetes mellitus with atherosclerosis (DM). VEGF protein localization was characterized using immunohistochemical staining. (a) Healthy arteries had weak, diffuse immunoreactivity for VEGF. The representative micrograph of a coronary artery from a patient with native atherosclerosis illustrates VEGF immunoreactivity primarily localized to the media, as well as the superficial and deep intima. Comparatively, the representative micrograph of a coronary artery from a patient with diabetes mellitus as well as atherosclerosis demonstrates a largely similar localization pattern to that of native atherosclerosis; however, with an apparent increase in immunoreactivity or staining intensity. (b) Color segmentation analysis of the IHC reaction product was performed using ImagePro Plus® imaging software as described in the Materials and Methods. Computer-assisted image analysis demonstrated a significant increase in VEGF immunoreactivity in the intima and media of coronary arteries in both native atherosclerosis and diabetes mellitus with native atherosclerosis as compared to healthy arteries. Data are represented as mean ± SE; *, p < 0.01.  59 | P a g e  3.3.3 – VEGF-D in cardiac allograft vasculopathy VEGF-D immunoreactivity was also significantly increased in coronary arteries from patients with CAV when compared with controls (Figure 14). Pseudocolored images generated by ImagePro Plus® imaging software help to visually highlight the lower intensity immunoreactivity detected. There was no significant difference in VEGF-D immunoreactivity in the intima of CAV vessels (9% vs 4%). The percentage VEGF-D positive area was, however, significantly increased in the media of CAV vessels as compared to controls from the PDAY study (15% vs 1%).  3.3.4 – VEGF-D in native atherosclerosis and diabetes mellitus The percentage of VEGF-D positive area in the intima of coronary arteries is significantly higher in DM compared with controls (Figure 15). Interestingly, there was an observed significant decrease in VEGF-D immunoreactivity in the intima of coronaries from patients with NA. Within the medial layer of coronary arteries measured, there was a significant increase in VEGF-D immunoreactivity only in cases of DM. VEGF-D was significantly increased in the intima of DM vessels (19% vs 4%). Interestingly, in NA vessels, the percentage VEGF-D positive area was significantly decreased as compared to controls (<1% vs 4%). Within the media, VEGF-D was significantly increased in DM vessels compared with normal cases from the PDAY study (22% vs 1%). There was no observed significant difference in VEGF-D immunoreactivity between NA and control vessels (2% vs 1%).  60 | P a g e  a)  Cardiac allograft vasculopathy  Normal  VEGF-D  Pseudocolor  VEGF-D  of staining: Highest Intensity ofIntensity staining: Highest Intensity of staining: Highest  30  % VEGF-D positive area  % VEGF-D positive area  b)  Intima of coronary arteries  20 10  0  Normal CAV  40  Pseudocolor  Media of coronary arteries  Lowest Lowest  30 *  20 10 0  Normal CAV  Figure 14 – Immunohistochemical profiling of VEGF-D in human CAV. (a) Healthy arteries had weak, diffuse immunoreactivity for VEGF-D as compared to CAV cases, which had abundant, albeit low intensity immunoreactivity for VEGF-D. Using pseudocolor overlay derived from color segmentation analysis of VEGF-D immunoreactivity intensity helps to highlight lower intensity immunoreactivity, as seen by the yellowish-green color over the black pseudocolored tissue. (b) Computerassisted image analysis demonstrated a significant increase in VEGF-D immunoreactivity only in the media of coronary arteries from cardiac allografts when compared to healthy arteries. Data are represented as mean ± SE; *, p < 0.01.  61 | P a g e  Lowes  a)  Normal  Native Diabetes mellitus atherosclerosis with atherosclerosis  VEGF-D  Pseudocolor  Lowest  Intima of coronary arteries  *  * Normal NA DM  % VEGF-D positive area  b)  % VEGF-D positive area  Intensity of staining: Highest Media of coronary arteries  *  Normal NA DM  Figure 15 – Immunohistochemical profiling of VEGF-D in human native atherosclerosis (NA) and diabetes mellitus with atherosclerosis (DM). (a) Healthy arteries had weak, diffuse immunoreactivity for VEGF-D. In NA cases, there was no apparent difference as compared to normal controls; however, in DM cases there is an abundant increase in lower intensity immunoreactivity throughout the vessel wall. (b) Computer-assisted image analysis demonstrated a significant increase in VEGF-D immunoreactivity in the intima and media of coronary arteries from patients with DM, compared with normal controls. Interestingly, patients with NA actually had significantly less VEGF-D within the intimal layer of the vessel wall. Data are represented as mean ± SE; *, p < 0.01.  62 | P a g e  3.4 – Discussion My immunohistochemical profiling and subsequent computer-assisted image analysis clearly illustrates the specific overexpression of VEGF-A within both the intima and media of coronary arteries from patients with NA, DM, and CAV (Figures 12 and 13). These observations are commensurate with studies examining VEGF-A polymorphisms which confer high or low VEGF-A protein expression. Although genotypes which conferred high VEGF-A expression did not impact the risk of repeated or late rejection in cardiac transplantation, when data was combined with high IL-6 (pro-inflammatory) and low IL-10 (regulatory cytokine) phenotypes, there was a significant increase in risk of late rejection irrespective of age and race/ethnicity.350 As well, phenotypes leading to decreased VEGF-A expression has been shown to have a protective effect in atherogenesis.351 Conversely, polymorphisms which confer high VEGF-A expression are significantly associated with improved graft survival in multiple transplantation models, as compared to low expression genotypes.352-356 Complementary to the above observations, IHC on adjacent sections for smooth musclealpha (SM-α) actin and CD68, a human macrophage surface marker confirmed my histological observations of apparent immunoreactivity of SMC and macrophages within the deep intimomedia layer and SMC within the media of affected vessels. Revisiting my current concepts of the pathogenesis of CAV overviewed in Subsection 1.3, these results implicate infiltrating cells within the intima and SMC within the media as the primary producers of VEGF-A in atheromatous disease.  63 | P a g e  A role for VEGF-A in heart transplantation and rejection was initially sought in biopsies from transplanted human hearts and this report suggested VEGF-A may impact the cardiac microvasculature during myocardial damage.347 VEGF-A was observed in cardiomyocytes and the ECM, and rarely on endothelial cells and vascular SMC.347 A follow-up study by the same group also demonstrated the relationship between fibrin deposition and VEGF-A immunoreactivity in biopsies from cardiac allografts, suggesting that cardiomyocyte-derived VEGF-A production following microvascular fibrin deposition may act in a paracrine manner to promote changes in the microvasculature that provide a survival advantage for heart allografts.357 As well, a relationship between increased VEGF-A expression in rejecting human cardiac allografts and the development of CAV has been suggested.358 Reinders et al demonstrated the relationship between VEGF-A expression in human cardiac allografts and mononuclear cell infiltrates and acute rejection, and suggested that chronic overexpression of VEGF during the first year post-transplantation identifies patients likely to develop CAV.358 My work complements these reports by demonstrating aberrant VEGF-A overexpression in the intima and media of coronary arteries from patients with CAV, as well as in NA and DM. Consolidating these other results with my own, I believe that early, high VEGF-A expression may reduce the risk of acute rejection episodes through pro-survival/proliferation signaling in endothelial cells and inducing progenitor cell differentiation to endothelial cells.359,360 Conversely, chronic, high VEGF-A expression in CAV is significantly associated with increased risk of repeated or late rejection, suggesting that this overexpression may lead to lipid and lipoprotein insudation, driving atherogenesis in CAV. Thus, promotion of early VEGF-A 64 | P a g e  expression with inhibition of late VEGF-A expression may provide the best therapeutic approach in the evolving allogeneic environment of transplantation as it progresses from one of mainly acute rejection to one plagued by CAV. I also demonstrated significant, lower-intensity immunoreactivity for VEGF-D within the media of coronary arteries from patients with CAV and significant overexpression of VEGF-D within both the intima and media of coronaries from patients with DM with atherosclerosis (Figures 14 and 15). Interestingly, quantitative analysis of coronary arteries from patients with NA demonstrated a significant decrease in VEGF-D immunoreactivity within the intimal layer, and no significant difference when compared with controls within the medial layer of the vessel (Figure 15). This result correlates with previous reports from Rutanen et al, who demonstrated a reduction in VEGF-D staining in intima of complicated human atherosclerotic lesions. 361 My immunohistochemical profiling and computer-assisted image analysis clearly illustrates the specific overexpression of VEGF-D in the media of coronary arteries in CAV and in both the intima and media of coronary arteries in DM. This result differs from our observations, and those of others, in NA, and this may be due to the relative stage within the time-course of atherogenesis between the diseases. Both DM and CAV are accelerated forms of atherosclerosis, and the overexpression we observed in these disease settings may reflect an earlier time-point in atherogenesis. This is the first characterization of aberrant overexpression of VEGF-D in CAV and diabetic atherosclerosis in humans. In the literature, VEGF-D has been primarily been characterized as a member of the VEGF family which is able to induce lymphangiogenesis. The pathobiological implications of this finding require more specific experiments tailored to elucidate a role related to this function; 65 | P a g e  however, VEGF-D has also been reported in the literature to be able to induce vascular EC permeability, and as such, I chose to include the investigation of this isoforms alongside VEGF-A in their ability to induce endothelial hyperpermeability to LDL in Chapter 4. Concurrent with my profiling of VEGF-A and VEGF-D in atheromatous disease, other relevant VEGF family members were investigated in the same cases using IHC, including VEGFB, PlGF, VEGFR1 and VEGFR2. Semi-quantitative assessment of immunoreactivity for these proteins using a 0-4 scale did not demonstrate significant increases in graded immunoreactivity. As such, the subsequent in vitro verification studies detailed in Chapter 4 focuses on the investigation of the effects of VEGF-A and VEGF-D on endothelial permeability. Taken together, it is likely that not only is VEGF-A aberrantly regulated in CAV, but also VEGF-D and possibly other VEGF family members and receptors. Synthesizing my work with the literature, it appears that multiple VEGF family members and receptors may be dysregulated in atherogenesis in the vessel wall in light of numerous pathogenic insults. In continuing this line of investigation, it is necessary to consider the regulation of the entire VEGF family and related receptors and co-receptors to determine the balance of physiological and pathological stimuli dictating vascular permeability, angiogenesis, lymphangiogenesis and ultimately atherogenesis. The work from this chapter has clearly validated my original hypothesis by verifying that, indeed, VEGF is overexpressed in the coronary arteries of not only CAV vessels, but also those from NA and DM vessels. The implications of the key findings of this chapter are discussed in Figure 16.  66 | P a g e  Injury eg. alloimmune, ischemia/reperfusion hypoxia  lumen  Response to injury  Cytokines eg. IFN-  ↑VEGF-A Migration Proliferation  ↑VEGF-A  Cytokines eg. TNF-, IL-1  intima media  ↑VEGF-D Figure 16 – Diagrammatic representation of the key findings from Chapter 3. There are numerous injurious stimuli and factors which contribute to endothelial injury in transplantation, including alloimmune injury, mediated through Fas and granzyme B, ischemia/reperfusion injury, and hypoxia, among others. In response to injury, and other stimulatory factors such as cytokines induced by the alloimmune response against the graft, vascular endothelial growth factor (VEGF)-A and VEGF-D are expressed by smooth muscle cells in the media. I believe this response to injury occurs initially as a physiological response to repair the endothelium and maintain endothelial integrity; however, chronic overexpression of VEGF within the vessel wall results in endothelial hyperpermeability, to factors such as low-density lipoproteins (LDL), and also contributes to vascular remodeling. The next chapter in this thesis examines one aspect of this response. I believe that VEGF overexpression within the vessel wall increases endothelial permeability to LDL, and this may, in part, contribute to increased LDL insudation within the vessel wall post-transplantation. Other factors such as increased proteoglycan expression and reorganization of the extracellular matrix may increase lipid and LDL retention within the vessel wall, resulting in continued stimuli for vascular remodeling and increased propensity of oxidative modification of LDL.  67 | P a g e  Chapter 4 – Vascular endothelial growth factor induces endothelial hyperpermeability to lowdensity lipoproteins in vitro 4.1 – Rationale Tight junctions form an impermeable seal between adjacent endothelial cells, and prevent the lateral migration of substances through the endothelium. They are formed by the phosphorylation-dependent assembly of a tight junctional complex, with the transmembrane occludin and claudin proteins associating with the cytoplasmic zonula occludens (ZO) proteins ZO-1, ZO-2 and ZO-3, which attach the tight junctional complex to the cellular cytoskeleton.104,169 Vascular endothelial growth factor (VEGF) was first discovered in tumor cells as a potent inducer of vascular permeability.264,337 It is a potent and specific mitogen for vascular endothelial cells that is capable of stimulating angiogenesis and is one of the most potent inducers of vascular permeability known.264-266,337 Previous reports have suggested that VEGF may enhance vascular permeability by affecting tight junction protein expression and assembly via various signaling pathways such as protein kinase B (PKB/Akt),362,363 endothelial nitric oxide synthase (eNOS),363-365 Src kinase366-370 and protein kinase C (PKC),304,338,371 among others. Our laboratory and others have characterized the aberrant expression of VEGF-A311,372 and VEGFD361 in the coronary arteries of patients with native atherosclerosis, as well in human heart allografts with transplant-associated atherosclerosis.347 The goal of my experiments was to determine whether VEGF-A121, VEGF-A165 or VEGF-D disrupts tight junctions in primary human cardiac microvessel endothelial cell cultures. I hypothesized that VEGF induces the disassembly of tight junctions between adjacent 68 | P a g e  endothelial cells, resulting in the formation of intercellular gaps. It is likely the presence of these intercellular gaps will result in a loss of endothelial integrity and create a hyperpermeable state, facilitating increased low-density lipoprotein (LDL) permeability through confluent endothelial monolayers.  4.2 – Materials and methods 4.2.1 – Reagents and antibodies Endothelial basal medium (EBM) and endothelial growth medium - microvascular (EGMMV) SingleQuots® (BBE 3mg/mL, 2mL; hEGF, 0.5mL; hydrocortisone, 0.5mL; FBS, 25mL; GA1000, 0.5mL) were obtained from Clonetics (San Diego, CA). Cellagen® solution (0.5% type I collagen, pH 3.0) used to coat various cultureware was obtained from ICN Biomedicals (Costa Mesa, CA). BioCoat® Collagen I coated flasks and plates used to expand cell cultures, and BioCoat® Collagen I coated 8-well chamber slides used for immunocytochemical staining experiments were obtained from Becton Dickinson (Mississauga, ON). BD Falcon™ HTS FluoroBlok™ 1.0µm inserts and 1.0µm transparent cell culture inserts for 24-well plates used for LDL permeability assays were also purchased from Becton Dickinson. 1, 1’-dioctadecyl-3, 3, 3’, 3’-tetramethylindocarbocyanine perchlorate (diI)-labeled LDL and diI-labeled acetylated lowdensity lipoprotein (acLDL) used for permeability assays were obtained from Intracel Corp. (Frederick, MD). Recombinant human VEGF-A121, VEGF-A165 and VEGF-D protein was obtained from R&D Systems Inc. (Minneapolis, MN). Mouse anti-von Willebrand Factor and anti-smooth muscle-α actin antibodies were obtained from Dako Canada, Inc. (Burlington, ON). Mouse antiplatelet and endothelial cell adhesion molecule-1 (PECAM-1; CD31) and mouse anti-β-actin 69 | P a g e  antibodies were obtained from Sigma-Aldrich Canada Ltd. (Oakville, ON). Rabbit anti-ZO-1 antibodies were obtained from Zymed Laboratories Inc. (San Francisco, CA). Rabbit antiphospho extracellular signal-regulated kinase (ERK) 1/2, p38, glycogen synthase-3 beta (GSK3β) and pan-protein kinase C (PKC) antibodies were obtained from New England Biolabs (Pickering, ON). The mitogen-activated protein kinase kinase (MAPKK/MEK1) inhibitor 1, 4-diamino-2, 3dicyano-1, 4-bis[2-aminophenylthio] butadiene (U0126) was obtained from was purchased from Promega-Fisher (Nepean, ON). U0126 acts as a selective, non-competitive inhibitor of MEK1 and MEK2, two MAPK upstream of ERK1/2 which function in signal transduction pathways involved in cell proliferation and differentiation.373-375 MEK1 and MEK2 are activated by Raf-like molecules through the specific phosphorylation at serine residues 217 and 221, which are located in the activation loop of subdomain VIII.376  4.2.2 – Cell cultures Primary human coronary artery endothelial cells (HCAEC) and human cardiac microvessel endothelial cells (HCMEC) were obtained from Clonetics. HCAEC and HCMEC were used for in vitro investigations of VEGF-induced effects as they represent site- and tissuespecific endothelial cells relevant to the heart and CAV. HCAEC represent macrovascular endothelial cells that line the coronary arteries affected by CAV, whereas HCMEC represent cardiac-specific endothelial cells which populate the cardiac capillary network, and also may be representative of the vasa vasora, and possibly the microvasculature that supports the neovascularization of CAV and atherosclerotic lesions. 70 | P a g e  Cells were grown in EGM-MV at 37°C under 5% CO2-95% air. Cells were passaged by trypsinization with 0.25% trypsin-EDTA and were seeded onto various type I collagen coated cultureware. All experiments used cells between the fourth and sixth passage, and cell phenotype was routinely monitored by phase contrast microscopy, as well as occasionally by positive immunocytochemical staining for von Willebrand Factor (vWF) and negative immunoreactivity for smooth muscle-α actin.  4.2.3 – Transendothelial electrical resistance experiments The barrier properties of tight junctions can be directly measured by transendothelial electrical resistance (TER).166,377,378 Briefly, TER measurements are the gold standard for endothelial and epithelial barrier integrity, and TER values have shown to be indirectly correlated with the number of tight junction strands within a confluent monolayer. Endothelial cells used for the TER experiments were grown on Cellagen® discs (ICN Biomedicals). TER measurements were performed using a System EVOM voltohmmeter and Endohm-12 tissue resistance measurement chamber obtained from World Precision Instruments, Inc. (Sarasota, FL). Briefly, cells were grown to 100% confluence as verified using phase contrast microscopy and TER measurements. Once confluent, cells were serum starved for 24 hours before being treated with 0.01, 1 or 100pg/mL of VEGF-A121, VEGF-A165, or VEGF-D. TER measurements were recorded at 2, 4 and 20 hours post-treatment. Six independent experiments from three donors were performed, with four replicates per treatment group.  71 | P a g e  4.2.4 – Immunocytochemistry HCMEC used for immunocytochemical staining were grown to confluence on type I collagen coated BioCoat® 8-well chamber slides, as verified by phase contrast microscopy. Cells were serum starved for 24 hours before treatment with 100pg/mL of VEGF-A121, VEGF-A165 or VEGF-D. Cells were fixed with Clark’s solution (90% ethanol, 10% acetic acid) at 30, 60, and 120 minutes post-treatment. Briefly, cells were permeabilized using 0.1% Triton X-100 (Sigma), blocked using normal goat serum, then incubated with primary antibody overnight. Biotinylated goat anti-rabbit IgG (Vector Laboratories, Inc., Burlingame, CA) and StreptABComplex/AP (Dako) were incubated sequentially at room temperature. Antibodies were visualized using the chromagen Vector Red (Vector), followed by counterstaining with hematoxylin. Negative controls included isotype-matched IgG and omission of primary antibody.  4.2.5 – Immunofluorescent microscopy HCMEC used for confocal microscopy were grown to confluence on type I collagen coated BioCoat® 8-well chamber slides. Cells were fixed with Clark’s solution (90% ethanol, 10% acetic acid) at 120 minutes post-treatment. Briefly, cells were permeabilized using 0.1% Triton X-100 (Sigma), blocked for 1 hour using normal goat serum, then incubated with rabbit anti-ZO1 and mouse anti-PECAM-1 antibodies overnight. AlexaFluor® 594-conjugated goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR) were used to detect rabbit anti-ZO-1 antibodies. AlexaFluor® 488-conjugated goat anti-mouse IgG (Molecular Probes) was used to detect mouse anti-PECAM-1 antibody. Hoechst 33342 (Molecular Probes) was used to stain nuclei. Negative controls included isotype-matched IgG and omission of primary antibody. Slides were mounted 72 | P a g e  using Prolong® antifade reagent (Molecular Probes) and visualized using a Leica TCS SP2 AOBS confocal microscope (Leica Microsystems (Canada), Inc., Richmond Hill, ON).  4.2.6 – Cell lysates, electrophoresis, and Western blotting Cells were washed twice in cold PBS then suspended in 1mL of cold lysis buffer (20mM Tris pH 8; 137mM NaCl; 10% glycerol; 1% Nonidet P-40; 1mM phenylmethylsulfonyl fluoride; 10μg/mL aprotinin) per 100-mm2 culture area. After 15 minutes on ice, supernatant was collected, followed by centrifugation at 10,000g at 4°C. Cell lysate protein concentration was determined by the BCA method (Pierce Chemical Co., Rockford, IL). Samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and then proteins were transferred to nitrocellulose (Hybond ECL; Amersham Pharmacia Biotech, Uppsala, Sweden). Following blocking with 1% skim milk and incubation with primary and secondary antibodies, horseradish peroxidase conjugated secondary immunoglobulins were detected using the enhanced chemiluminescence (ECL) method (Amersham Pharmacia Biotech) and exposed to Hyperfilm (Amersham Pharmacia Biotech). Densitometry results were obtained using NIH Image J software.  4.2.7 – Low-density lipoprotein permeability experiments HCMEC used for the LDL permeability experiments were grown to confluence on Cellagen® coated HTS Fluoroblok® inserts, as determined by TER measurements using a System EVOM voltohmmeter and Endohm-6 tissue resistance measurement chamber (World Precision Instruments). As well, cells were subcultured onto one transparent cell culture insert per 2473 | P a g e  well plate to allow for direct visualization using phase contrast microscopy and correlation to TER values. At confluence, cells were serum starved for 24 hours before treatment with 100pg/mL of VEGF-A121, VEGF-A165 or VEGF-D. Concomitant with treatment, 25μg/mL of diI-LDL or diI-acLDL were added to the insert. Cells were incubated at 37°C in the cell culture incubator and serial fluorometric measurements were taken at 5, 15, 30, 60, 120, 180, 240, 300 and 360 minutes post-treatment using a GENios Plus multi-label reader (Tecan Instruments, Inc., Durham, NC). Fluorometric measurements were normalized to an empty Fluoroblok® insert containing only medium and standardized to a standard curve of fluorescent substrate alone for each independent experiment.  4.2.8 – Statistical analysis Analysis of variance (ANOVA) was performed on all treatment groups at each time point. Student’s t-test (p < 0.05) was used to make individual comparisons between untreated and VEGF-treated groups at each time point for TER measurements and fluorometric measurements to determine significance.  74 | P a g e  4.3 – Results 4.3.1 –Effect of VEGF in HCAEC and HCMEC on TER To optimize culture and treatment conditions for my in vitro investigations on the effects of VEGF on endothelial permeability to LDL, numerous factors were tested, including variations in culture media and growth supplements, composition of basal matrix coating on cultureware, the effects of various VEGF isoforms at a range of concentrations and the duration of treatment. It was determined that HCAEC and HCMEC grew most uniformly when cultured in EBM supplemented by EGM-MV Singlequot® supplements (which contain BBE, hydrocortisone and FBS). All cells were cultured on either BioCoat® Collagen I coated cultureware, or cultureware custom-coated with a type I collagen solution (Cellagen®). Endothelial phenotype was regularly verified by a combination of microscopic examination of cell morphology, TER measurements, vWF-positive  immunocytochemical  staining  and  smooth  muscle-α  actin-negative  immunocytochemical staining. It was observed that cells maintained these endothelial phenotypes until at least 7th passage, and as such, all experiments were performed between passages 4-6. As well, serum-reduction using EBM supplemented with 0.5% FBS was used as an overnight pre-treatment to synchronize cells before treatment. This pre-treatment was chosen as it best-maintained TER, while allowing to synchronize cells in serum-reduced conditions before experimental treatment with VEGF, without changing cell number as verified by microscopic examination of cell morphology and cell number, and MTS assay.  75 | P a g e  4.3.1.1 – The effect of VEGF-A165 on TER To begin with, I wanted to confirm and extend the basis of the hypothesis from observations from Chapter 3, so, I examined the effects of VEGF-A165 on TER over time in HCAEC and HCMEC. HCAEC primary cultures were performed in quadruplicate from cells originating from two independent donors. At 2 hours post-treatment, there was a significant decrease in TER in HCAEC treated with 0.01, 1 or 100pg/mL VEGF-A165 compared with untreated controls (-26.73±2.82%, -35.30±3.97% and -41.55±3.45%, respectively). This significant decrease was also observed at 4 hours post-treatment in HCAEC treated with either 0.01, 1, or 100pg/mL VEGF-A165 (-26.73±2.82%, -46.55±5.64% and -41.55±3.45%, respectively, compared with untreated controls) and this significant decrease persisted at 20 hours posttreatment with 0.01, 1 or 100pg/mL VEGF-A165 (-35.30±3.97%, -54.29±2.54% and -46.55±5.64%, respectively) (Figure 17a). HCMEC primary cultures were performed from cells originating from three independent donors. Treatment with 0.01, 1 or 100pg/mL VEGF-A165 significantly reduced TER, compared with controls, at 2 hours (-39.48±4.83%, -46.48±3.34% and -45.56±6.48%, respectively) and at 4 hours (-39.90±3.56%, -49.07±2.74% and -50.00±2.58%, respectively). Of note, the untreated HCMEC controls at 20 hours post-treatment had significantly decreased resistance as compared to untreated HCMEC controls at 2 and 4 hours; however, despite this decrease, samples treated with either 0.01, 1 or 100pg/mL VEGF-A165 were still significantly decreased compared with untreated controls at 20 hours post-treatment (-43.96±6.90%, -52.59±1.74% and -51.67±3.07%, respectively) (Figure 17b). Overall, all doses of VEGF-A165 tested in both HCAEC and HCMEC significantly decreased TER at 2, 4 and 20 hours post-treatment. 76 | P a g e  a)  n = 4 from 2 donors  *  *  *  *  * *  * * *  b)  n = 6 from 3 donors  *  * *  *  * * *  * *  Figure 17 – VEGF-A165 significantly decreases TER in HCAEC and HCMEC. HCAEC and HCMEC were grown to confluence on Cellagen® inserts, and then treated with 0.01, 1, or 100pg/mL VEGF-A165. (a) Treatment of HCAEC with VEGF-A165 significantly decreased TER at 2, 4 and 20 hours post-treatment. (b) Similarly, VEGF-A165 significantly decreased TER in HCMEC at 2, 4 and 20 hours post-treatment. Data are represented as mean±SE; *, p < 0.0005.  77 | P a g e  4.3.1.2 – The effect of VEGF-A121 on TER After observing the ability of VEGF-A165 to decrease TER in HCAEC and HCMEC, I then examined the effects of VEGF-A121 on TER over time in HCAEC and HCMEC. VEGF-A121 differs from VEGF-A165 in that it lacks the heparan sulphate binding regions found on VEGF-A165 through alternative exon splicing. In HCAEC at 2 hours post-treatment, there was a significant decrease in TER only in HCAEC treated with 1pg/mL VEGF-A121, compared with untreated control (-26.88±10.87%; p < 0.05). In samples treated with either 0.01 or 100pg/mL VEGF-A121 at 2 hours post-treatment, there was a trend towards decreased TER; however, this was not significantly different (-21.07±10.52%; p = 0.0920 and -29.58±12.37%; p = 0.0539, respectively). At 4 hours post-treatment, only samples treated with 100pg/mL VEGF-A121 resulted in a significant decrease in TER (-29.58±12.37%; p < 0.05). At 20 hours post-treatment, 0.01, 1 and 100pg/mL VEGF-A121 all significantly decreased TER in HCAEC (-32.98±6.31%, -31.04±7.80% and -28.75±5.06%, respectively; p < 0.005) (Figure 18a). Treating HCMEC with doses of 1 or 100pg/mL VEGF-A121 significantly decreased TER at 2 hours post-treatment (-31.98±7.05% and –31.77±4.11%, respectively; p < 0.001). At 4 hours post-treatment, 0.01, 1 and 100pg/mL VEGF-A121 all significantly decreased TER (-12.96±2.75%, -15.60±3.53%, and -22.28±3.34%, respectively; p < 0.01). This significant decrease was only maintained in HCMEC treated with 100pg/mL VEGF-A121 at 20 hours (-32.56±5.13%; p < 0.05) (Figure 18b).  78 | P a g e  Overall, VEGF-A121 significantly decreased TER at 20 hours post-treatment at all doses tested in HCAEC. As well, 1pg/mL VEGF-A121 at 2 hours and 100pg/mL at 4 hours also significantly decreased TER, compared with control. In HCMEC, VEGF-A121 significantly decreased TER at all doses at 4 hours post-treatment. Additionally, both 1 and 100pg/mL VEGFA121 at 2 hours and 100pg/mL at 20 hours resulted in significant decreases in TER in HCMEC. The high degree of variability (larger standard deviation/error values) may account for the dissimilar results to VEGF-A165, and this may be due, at least in part, to the fact that VEGF-A121 is “more soluble,” lacking heparan sulphate binding regions.  79 | P a g e  a)  n = 4 from 2 donors  *  b)  *  * * *  n = 6 from 3 donors  * * * *  *  *  Figure 18 – VEGF-A121 significantly decreases TER in HCAEC and HCMEC. HCAEC and HCMEC were grown to confluence on Cellagen® inserts, and then treated with 0.01, 1, or 100pg/mL VEGF-A121. (a) Treatment of HCAEC with VEGF-A121 significantly decreased TER at 2 hours with a 1pg/mL dose, at 4 hours with a 100pg/mL dose, and at 20h at 0.01, 1, and 100pg/mL doses. (b) In HCMEC, VEGF-A121 significantly decreased TER in HCMEC at 2h at 1 and 100pg/mL doses, at 4 hours at 0.01, 1, and 100pg/mL doses, and at 20 hours only at the 100pg/mL dose. Data are represented as mean±SE; *, p < 0.05.  80 | P a g e  4.3.1.3 – The effect of VEGF-D on TER Treatment of HCAEC using 0.01, 1, or 100pg/mL of VEGF-D significantly decreased TER at 2 hours post-treatment (-35.00±8.66%, -25.83±8.09% and -29.58±4.43%, respectively; p < 0.05). At 4 hours post-treatment, this significant decrease was maintained when treated with 0.01, 1, or 100pg/mL VEGF-D (-47.50±15.88%, -36.25±13.41% and -34.58±8.91%, respectively; p < 0.05). At 20 hours post-treatment, 0.01, 1 and 100pg/mL VEGF-D maintained significant decreases in TER in HCAEC (-41.25±13.29%, -30.00±7.58% and -35.83±6.29% respectively; p < 0.05) (Figure 19a). When 0.01, 1 or 100pg/mL VEGF-D were used to treat HCMEC cultures, there was a significant decrease in TER at 2 hours (-21.94±2.60%, -28.97±6.25% and -38.26±7.00%, respectively; p < 0.001). At 4 hours, treatment of HCMEC with 0.01, 1 or 100pg/mL all significantly decreased TER (-26.34±5.47%, -28.04±9.24% and -34.46±6.08%, respectively; p < 0.005). At 20 hours post-treatment, only the 100pg/mL dose VEGF-D significantly decreased TER in HCMEC (-40.77±7.40%; p < 0.05) (Figure 19b). Overall, in HCAEC, all doses of VEGF-D tested significantly decreased TER at 2, 4 and 20h. In HCMEC, all doses significantly decreased TER at 2 and 4 hours post-treatment; however, only 100pg/mL VEGF-D significantly decreased TER at 20 hours post-treatment.  81 | P a g e  a)  n = 4 from 2 donors  *  b)  *  *  *  * *  *  *  *  n = 6 from 3 donors  * *  *  * *  *  *  Figure 19 – VEGF-D significantly decreases TER in HCAEC and HCMEC. HCAEC and HCMEC were grown to confluence on Cellagen® inserts, and then treated with 0.01, 1, or 100pg/mL VEGF-D. (a) Treatment of HCAEC with VEGF-D significantly decreased TER at 2, 4 and 20 hours with 0.01, 1, or 100pg/mL VEGF-D. (b) In HCMEC, VEGF-D significantly decreased TER in HCMEC at 2 and 4 hours with 0.01, 1, and 100pg/mL VEGF-D. At 20 hours, only 100pg/mL VEGF-D significantly decreased TER in HCMEC. Data are represented as mean±SE; *, p < 0.05.  82 | P a g e  4.3.2 – VEGF increases LDL permeability through confluent HCMEC monolayers in vitro Based on my experiments from 4.3.1, I decided to perform subsequent experiments using the 100pg/mL dose for VEGF-A121, VEGF-A165 and VEGF-D, as it provided consistent TER decreases in all VEGFs tested. It is important to note that this 100pg/mL dose is much lower than the standard 10-100ng/mL doses routinely used in the literature to investigate VEGFinduced effects. As peak TER decreases were observed at 2 hours post-treatment, the following time points were chosen for investigation: 5, 15, 30, 60, 120, 180, 240, 300 and 360 minutes. The lipophilic fluorescent dye 1, 1’-dioctadecyl-3, 3, 3’, 3’-tetramethylindocarbocyanine perchlorate (diI) conjugated to either LDL or acLDL was co-incubated with VEGF on the luminal side of HCMEC grown to 100% confluence on Fluoroblok® transwell inserts. Passage of LDL and acLDL was measured using a fluorometer over the time-course of the experiment. After stimulation with VEGF-A165, I observed significant increases in LDL permeability as early as 5 minutes post-treatment (30% increase as compared with control). This increased permeability peaked at 60 minutes post-treatment at around 40%, as compared with control) and remained significantly increased up to 6 hours post-treatment. When confluent HCMEC grown on Fluoroblok® inserts were co-incubated with VEGF-A121 and LDL, significant increases in permeability were measured beginning at 60 minutes post-treatment (25%, as compared with control) and was sustained to 6 hours post-treatment. Treatment with VEGF-D significantly increased LDL permeability, peaking at 60 minutes post-treatment (48%, as compared to control) (Figure 20a).  83 | P a g e  a)  LDL  *  *  *  **  * ** * * **  b)  * * *  ** *  *  *  *  *  *  * *  * * *  * * *  * * *  acLDL  *  *  *  *  * *  *  *  *  *  *  *  *  *  *  *  Figure 20 – VEGF significantly increases LDL permeability through HCMEC monolayers. (a) All treatments significantly increased LDL permeability through endothelial monolayers as early as 5 minutes post-treatment. VEGF-A121 showed modest (10-20%) increases in LDL passage, whereas, VEGF-A165 and VEGF-D showed greater increases, which peaked at 60 minutes post-treatment (as compared with control). These increases in LDL were sustained for up to 6 hours. (b) Interestingly, only VEGF-A165 and VEGF-D significantly increased acLDL permeability (~40% and 20%, respectively). VEGF-A121 actually induced significant decreases in acLDL permeability, as compared with control, at some time points. Experiments were performed in triplicate, with four replicates per treatment group per independent experiment. Data are represented as mean±SE; *, p < 0.05.  84 | P a g e  When cells were treated with 100pg/mL VEGF-A165 and co-incubated with diI-acLDL, peak increases in acLDL permeability were observed at 15 minutes post-treatment (52%, as compared to control) and was maintained to 6 hours post-treatment. When VEGF-A121 was coincubated with acLDL, permeability was actually decreased (as compared with control (approximately 15% decrease through the 6 hour time-course of the experiment). When coincubated with acLDL, VEGF-D significantly increased permeability, peaking at 15 minutes (23%), and maintained to 6 hours post-treatment (Figure 20b). It is important to note that Figure 20 represents experimental data as a percentage of control through the time-course of the experiment. When calculating total protein permeability (in μg) based on relative fluorescence readings from a standard curve of LDL or acLDL substrate only, total quantity of LDL permeability is clearly greater than acLDL permeability for untreated samples and also those treated with VEGF-A121, VEGF-A165 or VEGF-D (Figure 21a). When re-plotting the sum of LDL and acLDL protein permeability as a percentage of control, this derivation more clearly illustrates the early VEGF-A165-induced increase in permeability at 5 minutes. At 60 minutes post-treatment, a common peak of increased permeability induced by VEGF-A121, VEGF-A165 and VEGF-D, to varying degrees, was also evident (Figure 21b). Of note, and consistent with previous derivations, it is apparent that VEGF-A165 and VEGF-D induced the greater changes in lipoprotein permeability measured.  85 | P a g e  a)  LDL LDL acLDL  acLDL  b)  LDL + acLDL  Figure 21 – LDL permeability is greater than acLDL permeability. (a) Plot of quantity of lipoprotein permeability (μg) measured, as derived from standard curve of substrate only. (b) Plot of LDL + acLDL totals as a percentage of control. Data are represented as mean±SE.  86 | P a g e  4.3.3 – VEGF-induced alterations to endothelial tight junctions After demonstrating the ability of VEGF to significantly increase LDL permeability in endothelial cells, I performed immunocytochemistry for junctional proteins to observe changes in endothelial cell morphology and tight junctional integrity and protein localization.  4.3.3.1 – Treatment with VEGF results in the formation of intercellular gaps To determine whether the disassembly of tight junctions in confluent endothelial monolayers would result in the formation of intercellular gaps, as we have observed in vivo,106 we used immunocytochemistry for platelet and endothelial cell adhesion molecule-1 (PECAM1/CD31) to highlight the membranes of adjacent endothelial cells. In untreated cells, CD31 immunoreactivity highlighted adjacent endothelial borders, with no apparent disruptions in endothelial cell contact (Figure 22a,e). Two hours after treatment with 100pg/mL of VEGF-A165 (Figure 22c,g) or VEGF-D (Figure 22d,h), I observed the appearance of interendothelial cell gaps; whereas the presence of these gaps was less apparent in VEGF-A121-treated monolayers (Figure 22b,f). Isotype matched IgG and primary antibody omission negative controls showed no non-specific immunoreactivity (Figure 22 insets).  87 | P a g e  a  b  c  d  e  f  g  h  Figure 22 – VEGF induces intercellular gap formation. HCMEC (a-d) and HCAEC (e-h) were stained using immunocytochemistry for CD31. In untreated monolayers, CD31 was localized to cell membranes of adjacent untreated ECs (a,e). An increase in the number of intercellular gaps (arrows), as well as an decrease in the continuity and increase in immunoreactivity was shown as a result of VEGF-A121 (b,f), VEGF-A165 (c,g), and VEGF-D (d,h) at 2 hours post-treatment. Isotype-matched mouse IgG was used as a negative control [insets]. Pictures taken at 400x.  4.3.3.2 – VEGF increases cytoplasmic immunoreactivity of ZO-1 To further examine the effect of VEGF on the structure of the tight junctions, I performed immunocytochemistry for the tight junction protein ZO-1. I observed diffuse ZO-1 immunoreactivity in the cytosol as well as along adjacent cell membranes (Figure 23a,e). As early as 30 minutes post-treatment with VEGF-A121 (Figure 23b,f), VEGF-A165 (Figure 23c,g) and VEGF-D (Figure 23d,h). I observed an increase in both cytoplasmic and membrane ZO-1 immunoreactivity, with maximal increases observed at 2 hours post-treatment. Isotypematched IgG and primary antibody omission negative controls had no immunoreactivity (Figure 88 | P a g e  23 insets). This result suggests that the formation of intercellular gaps in confluent monolayers may be due to tight junction disruption.  a  b  c  d  e  f  g  h  Figure 23 – VEGF induces changes in ZO-1 immunoreactivity. HCMEC (a-d) and HCAEC (e-h) were stained using immunocytochemistry for ZO-1. ZO-1 was localized to cell membranes, with some diffuse cytoplasmic staining in untreated ECs (a,e). An increase cytoplasmic immunoreactivity as well as decrease in the continuity of ZO-1 immunoreactivity along the cellular membrane was seen as a result of VEGF-A121 (b,f), VEGF-A165 (c,g), and VEGF-D (d,h) at 2 hours posttreatment. Isotype-matched rabbit IgG was used as a negative control [insets]. Pictures taken at 400x.  4.3.3.3 – Non-conventional localization of occludin in HCAEC and HCMEC Occludin has previously been characterized to be a transmembrane protein which associates with ZO-1 in the tight junctions of human epithelial cells and human umbilical vein endothelial  cells.163,169,179,379,380  My  profiling  for  occludin  expression  using  immunocytochemistry revealed perinuclear occludin immunoreactivity in HCAEC and HCMEC. I 89 | P a g e  performed Western blot analysis and confirmed that the antibody recognized both α and β forms of occludin, at 62kDa and 60kDa, respectively (data not shown). Interestingly, VEGF-A121, VEGF-A165 and VEGF-D all increased occludin protein levels in comparison to untreated cells, which correlated with an apparent increase in occludin immunoreactivity. However, occludin localization was not altered by VEGF stimulation (Figure 24).  a  b  c  d  e  f  g  h  Figure 24 – Non-conventional localization of occludin in HCAEC and HCMEC. HCMEC (a-d) and HCAEC (e-h) were stained using immunocytochemistry for occludin. Occludin was unconventionally localized exclusively to the cytosol in untreated ECs (a,e). A slight increase in perinuclear, cytoplasmic immunoreactivity was observed in samples treated with VEGF-A121 (b,f), VEGF-A165 (c,g), and VEGF-D (d,h); however, occludin localization was not altered by VEGF treatment. Isotype-matched rabbit IgG was used as a negative control [insets]. Pictures taken at 400x.  90 | P a g e  Immunocytochemical staining for occludin revealed an atypical localization in untreated, confluent HCAEC and HCMEC monolayers. Occludin immunoreactivity was exclusively perinuclear, with increased intensity of immunoreactivity upon treatment with VEGF-A121, VEGF-A165 or VEGF-D; however, the localization of occludin immunoreactivity did not change upon treatment. To verify the atypical localization of occludin in my endothelial cell cultures, I performed double-immunofluorescence staining for PECAM-1 and occludin (Figure 25a-d), as well as staining for F-actin and occludin (Figure 25e), simultaneously. Congruent with my immunocytochemistry using colorimetric substrate and brightfield microscopy, dual immunofluorescent labeling and visualization with confocal microscopy confirmed the lack of membrane localization of occludin immunoreactivity in cultured endothelial cells.  91 | P a g e  a  d  b  c  e  Figure 25 – Occludin does not localize to cultured endothelial cell membranes. Dual immunofluorescent labeling and visualization with confocal microscopy demonstrate the exclusively cytoplasmic localization of occludin. In the top panel, cells were stained for nuclei (a; blue), CD31 (b; green) and occludin (c; red). (d) Color overlay of the three color channels demonstrates the lack of co-localization between the membrane-localized CD31 and occludin. (e) In the bottom panel, cells were stained for nuclei (blue), F-actin (green) and occludin (red). Orthogonal reconstruction from confocal stacks helps to illustrate the XZ and YZ planes, clearly demonstrating the lack of membrane localization of occludin.  92 | P a g e  4.3.4 – Profiling of the VEGF-induced signaling pathways in endothelial cells. Previous reports have suggested that VEGF may enhance vascular permeability by affecting tight junction protein expression and assembly via various signaling pathways such as PKB/Akt, eNOS, Src, and PKC, among others. I sought to profile for signal transduction molecule expression and phosphorylation after VEGF-treatment. Initial profiling for a variety of signal transduction pathways, including ERK1/2, p38, GSK3β and PKC revealed that only the ERK1/2 pathway was markedly activated by VEGF-A121, VEGF-A165 and VEGF-D in my model system at the time points investigated (Figure 26a,b). As the apparent disruption of endothelial tight junctions was observed as early as 30 minutes post-treatment with VEGF, I sought to determine the relevant signal transduction pathways that may be mediating this effect. Initially, I treated endothelial cells with 100pg/mL VEGF-A165 and profiled signal transduction pathway activation at 0, 5, 10, 15 and 30 minutes post-treatment. VEGF-A165 treatment resulted in the phosphorylation of ERK1/2, peaking at 10 minutes post-treatment (Figure 27a). After establishing the 10 minute time-point as the peak ERK1/2 phosphorylation posttreatment with VEGF-A165, I examined ERK1/2 activation in response to VEGF-A121, VEGF-A165 and VEGF-D. VEGF-A165 and VEGF-D resulted in an approximately 2-fold increase in ERK1/2 phosphorylation; VEGF-A121 also increased ERK1/2 phosphorylation, albeit to a lesser extent (Figure 27b).  93 | P a g e  VEGF-D  VEGF-A165  VEGF-A121  TNF-α  Control  a)  30 minutes  p-ERK1/2 p-p38  VEGF-D  VEGF-A165  VEGF-A121  Control  VEGF-D  VEGF-A165  Control  b)  VEGF-A121  p-GSK-3β  p-PKC  15 minutes  60 minutes  Figure 26 – Profiling of signal transduction molecules activated by VEGF. (a) Western blot analysis was performed for phosphorylation of ERK1/2, p38 and GSK3β in endothelial cells treated with VEGF-A121, VEGF-A165 and VEGF-D at 30 minutes post-treatment. Only ERK1/2 appeared to be activated by treatment with VEGF. TNF-α was used as a positive control for signal transduction pathway activation. (b) As well, a pan PKC phosphorylation marker was used to profile VEGF-induced activation at 15 and 60 minutes, and no increase was observed over baseline.  94 | P a g e  Relative densitometry  a)  0.14 0.12 0.1 0.08 0.06 0.04 0.02 0  30 C  30’ Control  0  0’  5  5’  10  15  10’  15’  30  30’  VEGF  b)  Relative densitometry  p-ERK β-actin 2.5 2  1.5 1  0.5 0 p-ERK  Control  VEGF-A121  VEGF-A165  VEGF-D  ControlVEGF-A121 VEGF-A165 VEGF-D  β-actin Figure 27 – Treatment with VEGF induces ERK1/2 phosphorylation in endothelial cells. (a) Western blot analysis was performed for endothelial cells treated with VEGF-A165 at 0, 5, 10, 15 and 30 minutes post-treatment. Peak ERK1/2 phosphorylation was seen at 10 minutes. (b) Western blot analysis was then performed on lysates from endothelial cells treated with VEGF-A121, VEGF-A165 and VEGF-D for 10 minutes. All treatments with VEGF resulted in ERK1/2 activation at 10 minutes post-treatment, although VEGF-A121 induction of ERK1/2 phosphorylation occurred to a lesser extent. Blots from this figure are representative of three independent experiments.  95 | P a g e  To determine whether the ERK1/2 activation observed in Figure 27 was responsible for changes in tight junction organization and the formation of intercellular gaps, I used the MEK1 inhibitor U0126 and performed immunocytochemistry for ZO-1. Treatment of endothelial cells with either DMSO or U0126 compound alone without the addition of VEGF had no apparent effect on ZO-1 immunoreactivity or localization (Figure 28a,e). The addition of U0126 appeared to preserve tight junctional integrity 30 minutes after treatment with 100pg/mL of VEGF-A121, VEGF-A165 or VEGF-D. At this 30 minute time point, the presence of intercellular gaps was less noticeable compared to the 2 hour time point from Figure 22. Of note, this earlier time point illustrates the early disruption of endothelial tight junctions induced by VEGF, as evidenced by the ZO-1 immunoreactivity highlighting the increased irregularity along adjacent endothelial cell membranes (Figure 28b-d). The disruption of tight junction regularity appeared to be restored in samples treated with VEGF and co-incubated with U0126 (Figure 28f-h).  96 | P a g e  Figure 28 – Inhibition of ERK1/2 prevents VEGF-induced intercellular gap formation and changes in ZO-1 immunoreactivity. Endothelial cells treated with DMSO (control; a-d) or the MEK1 inhibitor U0126 (e-h) were stained using immunocytochemistry for ZO-1. As before, ZO-1 was localized to cell membranes in cells treated with DMSO or U0126 alone (a,e). Cells treated with DMSO as well as either VEGF-A121 (b), VEGF-A165 (c) or VEGFD (d) resulted in the formation of intercellular gaps (blue arrows), as well as an increased irregularity of ZO1 immunoreactivity along adjacent endothelial cells membranes (white triangles). When cells treated with U0126 as well as either VEGF-A121 (f), VEGF-A165 (g) or VEGF-D (h), there were no observable intercellular gaps, and the regularity of ZO-1 immunoreactivity along endothelial cell membranes was restored.  97 | P a g e  4.4 – Discussion Taken together, the results from this aim demonstrate the ability of VEGF to disrupt HCAEC  and  HCMEC tight  junctions, as determined  by TER  measurements  and  immunocytochemical staining for tight junctional proteins. This tight junctional disruption resulted in the formation of intercellular gaps, which is correlated to the increase in LDL permeability through endothelial monolayers. However, there is also a possible contribution of endocytotic transport induced by VEGF treatment. Previous work has suggested a role for VEGF in the induction of endothelial cell hyperpermeability. These reports have been primarily completed in human umbilical vein endothelial cells or bovine aortic endothelial cells and make no distinction between the different splice variants of VEGF-A or whether other VEGF family members may have similar effects. My initial profiling attempts in native and transplant-associated atherosclerotic coronary arteries reveal aberrant VEGF expression within the intima and media of diseased vessels. As VEGF was first described as a permeability-inducing agent in tumor cells, I sought to determine whether aberrant VEGF expression in atherosclerotic arteries resulted in a hyperpermeable endothelium. I first hypothesized that VEGF-A121, VEGF-A165 and VEGF-D disrupt tight junctions between HCAEC and HCMEC. I sought to determine the relative efficacy of VEGF-A121, VEGF-A165 and VEGF-D in human coronary-derived endothelial cells. TER measurements were used as an indirect measure of the number of tight junction strands between adjacent endothelial cells in a confluent monolayer, which is an important factor in determining the barrier properties of tight junctions. I have demonstrated that VEGF-A121, VEGF-A165 and VEGF-D can all induce significant 98 | P a g e  decreases in TER (Figures 17-19). Comparing the TER changes upon VEGF-A121, VEGF-A165 or VEGF-D treatment, VEGF-A165 caused the greatest reduction of TER at all doses tested. As well, the effects of VEGF-A165 were sustained up to 20 hours post-treatment, and extended time points from these experiments demonstrate VEGF-A165-induced TER changes remain significantly decreased as long as 72 hours post-treatment (data not shown). VEGF-A121 and VEGF-A165 have been reported to interact with VEGFR1 and VEGFR2. Comparing the TER reducing effects of VEGF-A165 to VEGF-A121, VEGF-A165 appears more potent across all doses and time points, especially in HCAEC monolayers. This may be due to the difference in the splice variants, as VEGF-A121 lacks a heparan sulphate binding region, rendering it “soluble”, while its presence may allow for retention of VEGF-A165 protein on heparan sulphate proteoglycans present on the endothelial glycocalyx or extracellular matrix produced in culture. 381,382 Treatment of endothelial monolayers with VEGF-D resulted in decreases in TER of similar magnitude to VEGF-A165. VEGF-D has been reported to interact with VEGFR2 and VEGFR3.383 It has previously been reported that VEGFR2 is responsible for VEGF-induced permeability effects and it seems that the permeability-inducing actions of VEGF-A121, VEGF-A165 and VEGF-D in this model are acting primarily through this receptor, as it is the common receptor between the different VEGF family members tested. The hyperpermeability-inducing effects of VEGF on endothelial monolayers has been well described in the literature using tracers such as horseradish peroxidase, 384,385 FITCdextran,362,386-390 and other small molecular weight compounds.385,391,392 It has been suggested that these tracers cross the endothelium through intercellular gaps or possibly through transcytosis or vesiculo-vacuolar organelles (VVO);147,345,393,394 however, the effect of VEGF on 99 | P a g e  LDL permeability through endothelial monolayers has not been examined. I demonstrated that VEGF-A121, VEGF-A165 and VEGF-D are all able to significantly increase diI-conjugated LDL permeability through confluent endothelial monolayers (Figure 20a). This effect was observed as early as 5 minutes post-treatment, peaked around one hour post-treatment, and was sustained for up to 6 hours post-treatment. Correlated with my observations of decreased TER and the formation of intercellular gaps, it may be likely that VEGF-induced LDL permeability may be through an intercellular route. When the lysine residues of the LDL apoprotein have been acetylated, the LDL complex no longer binds to the LDL receptor, but instead, is taken up by scavenger receptors specific for modified LDL. I used diI-conjugated acLDL to determine whether the VEGF-induced LDL hyperpermeability observed may be mediated through specific LDL receptor interactions, or whether it may simply be due to the size of the large intercellular gaps formed between endothelial cells. Surprisingly, when examining diI-conjugated acLDL permeability through endothelial monolayers, only VEGF-A165 and VEGF-D significantly increased acLDL permeability. The pattern of response to treatment did not appear to be time-dependent, with a consistent increase of approximately 40 and 20%, respectively, for VEGF-A165 and VEGF-D (as compared to control) (Figure 20b). VEGF-A121 may not have induced significant increases in acLDL permeability because once the acLDL complexes accumulate within the cells; diI-acLDL conjugates are covalently bound to the modified apoprotein portion of the LDL complex, and are not extracted during subsequent manipulations of the cells. As a result, the majority of diIacLDL may be retained within endothelial cell, and thus, would not be detected through the opaque Fluoroblok® inserts by the fluorometer. The increase in acLDL permeability observed in 100 | P a g e  VEGF-A165 and VEGF-D treated cells may be a result from acLDL passage through the intercellular gaps formed through VEGF-induced transcytosis of acLDL. When comparing absolute amounts of lipoprotein passage through endothelial monolayers, there is significantly more LDL passage as compared to acLDL passage, suggesting that both pathways may be involved in VEGF-induced endothelial cell hyperpermeability to lipoproteins; however, possibly through different routes of passage. After verifying that VEGF-A121, VEGF-A165 and VEGF-D all significantly decrease TER in HCAEC and HCMEC, I wanted to determine whether this decrease in TER and number of tight junction strands resulted in the formation of intercellular gaps whether the localization of the tight junctional proteins was affected by VEGF treatment. Using immunocytochemistry for PECAM-1, I visualized intact cell contacts between adjacent endothelial cells in confluent, untreated monolayers, and did not observe the presence of any intercellular gaps (Figure 22). However, upon stimulation with VEGF-A121, VEGF-A165 or VEGF-D, I observed an increase in the number of intercellular gaps between adjacent endothelial cells. As well, in some cases, there was an apparent increase in PECAM-1 immunoreactivity, which may be attributed to increased epitope availability when gaps are formed in endothelial cell junctions. As evident in the micrographs, VEGF-treated monolayers displayed heterogeneity in cell shape and size, however, cell number between treatment groups within 2h was not significantly different (as determined by MTS assay and manual cell counting; data not shown). Increased vascular permeability has been linked to angiogenesis as early as 1935, where Clark and Clark demonstrated that dyes could leak out of growing capillaries.395 It is recognized that a cardinal feature of pathological angiogenesis is increased vascular permeability, and growing evidences 101 | P a g e  demonstrate that a regulated increase in vascular permeability to both solute and water can occur as capillaries grow and form new vessels,396,397 a phenomenon which may be happening during plaque neovascularization. It has been demonstrated that exposure to tumor necrosis factor-alpha (TNF-α) led to PECAM-1 surface redistribution and disruption of cytoskeletal contacts, accompanied by increased permeability to macromolecules.398 Indeed, my own observations of TNF-α-induced changes to PECAM-1 in HCAEC and HCMEC mirrored those of previous reports (data not shown).398 Immunocytochemical staining for ZO-1 in untreated HCAEC and HCMEC monolayers demonstrated localization along adjacent endothelial membranes, with faint cytoplasmic immunoreactivity. Upon treatment with VEGF-A121, VEGF-A165 or VEGF-D, there was an increase in the cytoplasmic localization of ZO-1, suggesting dissociation of tight junctional complexes at the membrane. Interestingly, ZO-1 immunoreactivity in treated groups appeared strongest in the perinuclear region of the cytosol. To confirm the localization of ZO-1 and occludin immunoreactivity observed using immunocytochemistry in my HCAEC and HCMEC cultures, I performed double-immunofluorescent staining for PECAM-1 and ZO-1 and verified the membrane localization of ZO-1 (Figure 23). Immunocytochemical staining for occludin revealed an atypical localization of occludin in untreated, confluent HCAEC and HCMEC monolayer. Occludin immunoreactivity was exclusively perinuclear, with increased intensity of immunoreactivity upon treatment with VEGF-A121, VEGF-A165 or VEGF-D; however, the localization of occludin immunoreactivity did not change upon treatment (Figure 24). These results are different from previous reports in the literature, which have demonstrated exclusive membrane localization of occludin in different 102 | P a g e  endothelial cultures such as HUVEC and BAEC. My results suggest that occludin may not be an important protein in the formation and regulation of tight junctional complexes in HCAEC and HCMEC in culture. To verify the atypical localization of occludin in my HCAEC and HCMEC cultures, I performed double immunofluorescent staining for PECAM-1 and occludin, as well as staining for F-actin and occludin (Figure 25). These two approaches verified that occludin immunoreactivity was cytoplasmic and not localized along the membrane. This observed difference of occludin localization may be as a result of cell-specific expression and localization in HCAEC and HCMEC, as other reports utilized endothelial cells from other tissue sites or other species. Another possibility may be due to the specific culture conditions in my experimental investigations; however, the basal media and growth supplements used in these studies was certified and purchased from Cambrex Corporation and TER measurements indirectly demonstrated the formation of tight junction strands, despite the lack of membrane localization of occludin. Many signaling molecules have been implicated in VEGF-induced permeability effects in endothelial cells, including PKB/Akt,362,363 eNOS,363-365 Src kinase,366-370 and PKC,304,338,371,399 among others. I demonstrated ERK1/2 activation 30 minutes post-treatment with 100pg/mL of VEGF-A121, VEGF-A165 and VEGF-D (Figure 27b). Inhibition using the MEK1 inhibitor U0126 abrogated VEGF-induced tight junctional disruption 30 minutes post-treatment (Figure 28). At 30 minutes post-stimulation with VEGF, I did not observe activation of p38, GSK3β (downstream of PKB) or PKC in my model system (Figure 26). The differences between my results and others may be as a result of the lower dosage used for my experiments, the time 103 | P a g e  points examined, or as a result of species- or tissue-specific differences in endothelial cells. These results suggest that in my model system, VEGF-induced disruption of endothelial tight junctions occurs through an ERK1/2-dependent pathway, and inhibition of ERK1/2 signaling using the MEK1 inhibitor U0126 can preserve tight junctional protein organization and thus tight junction and endothelial cell morphology. In conclusion, these studies have demonstrated that VEGF-A121, VEGF-A165 and VEGF-D can all induce significant decreases in TER, resulting in increased ZO-1 immunoreactivity within the cytosol, and the formation of intercellular gaps. As well, treatment with VEGF-A121, VEGFA165 or VEGF-D significantly increases LDL permeability through confluent endothelial monolayers. Interestingly, only VEGF-A165 and VEGF-D significantly increased acLDL permeability through endothelial monolayers, suggesting that VEGF may be inducing transcytosis of internalized acLDL, although to a lesser extent than intercellular passage of LDL. Inhibition of ERK1/2 signaling using the MEK1 inhibitor U0126 prevented VEGF-induced disruption of endothelial tight junctions. These key concepts are represented in Figure 29.  104 | P a g e  a)  VEGF  VEGF-R tight junctions  b)  VEGF VEGF-R ERK1/2  tight junctions  ZO-1  c)  VEGF VEGF-R ERK1/2  intercellular passage tight junctions  ZO-1  Figure 29 – Diagrammatic representation of the key findings from Chapter 4. (a) Vascular endothelial growth factor (VEGF) disrupts endothelial barrier function at the level of the tight junction. (b) This tight junctional disruption is ERK1/2-dependent, and results in the formation of intercellular gaps and relocation of zonula occludens-1 (ZO-1) protein from the tight junctions at the cell membrane to the cytoplasm. (c) Concurrently, this endothelial barrier disruption and intercellular gap formation results in endothelial hyperpermeability to low-density lipoproteins (LDL). This chapter affirms my original hypothesis that VEGF can increase endothelial permeability to LDL and supports the notion that this may contribute to the prominent lipid and lipoprotein accumulation observed in CAV. The next chapter in this thesis investigates the role of VEGF in the pathogenesis of CAV using a proof-of-principle study focusing on the abrogation of VEGF in a hypercholesterolemic mouse model of heterotopic cardiac transplantation.  105 | P a g e  Chapter 5 – Administration of soluble vascular endothelial growth factor receptor-1 in a mouse model of heterotopic cardiac transplantation 5.1 – Rationale Cardiac allograft vasculopathy (CAV) is an occlusive vascular disease which occurs in approximately 70% of transplant patients and is the leading cause of organ rejection/failure one year after solid organ transplantation.400 The concentric vascular atherogenesis characteristic of CAV occurs, and progresses rapidly, within almost all solid organ transplants. Although the etiology is poorly defined, it is widely accepted that the pathology is initiated by a combination of allogeneic and ischemia/reperfusion injury to the graft, which result in endothelial damage post-revascularization.103,330,401-404 Progression of CAV involves a multifactorial process, strongly related to increased adhesiveness and permeability of the endothelium of blood vessels. Plasma components including fibrinogen, lipids69 and apolipoproteins B, (a) and E112 enter and accumulate in vessel walls, causing further injury. This results in diffuse, concentric intimal thickening which progressively occludes blood flow. CAV is the leading expression of chronic organ rejection and the major cause of graft failure beyond one year post-transplantation. This chronic vascular disease results in the partial or complete obstruction of blood vessels, particularly macrovessels in transplanted organs, resulting in tissue ischemia and organ failure. In physiological and pathological settings, the endothelium serves as a key structural and functional regulator of vascular health, guiding leukocyte traffic, modulating transport of micro- and macromolecules and ions, and regulating homeostatic smooth muscle function. My work, and that of our laboratory, has shown through a long-running series of investigations that pathogenesis of CAV in transplanted hearts involves 106 | P a g e  endothelial injury106 and dysfunction,107,108 smooth muscle perturbations,405 aberrant expression of inflammatory cytokines,406 immune-mediated cell death,402,407,408 accumulation of extracellular matrix,112,409 and insudation of lipids and lipoproteins410,411 within affected vessels. One intriguing observation from my work is the documentation of endogenous overexpression of vascular endothelial growth factor (VEGF) within coronary arteries from human heart allografts.412 Among all growth factors known to date, VEGF is the only one capable of inducing inflammation. VEGF increases vascular permeability, leukocyte adhesion and transmigration, and platelet aggregation via the synthesis of various paracellular signaling molecules such as platelet-activating factor and tissue factor which could potentially be deleterious to grafts. On the other hand VEGF induces endothelial cell proliferation, migration, and angiogenesis as well as bone marrow-derived cell mobilization and re-endothelialization. In light of these possible roles, coupled with my findings in Chapter 4, demonstrating the ability of VEGF to increase endothelial permeability to LDL in coronary artery and cardiac-specific endothelial cells, determining the role of VEGF in the pathogenesis of CAV is an interesting prospect. Endothelial cell injury, which can arise through a variety of stimuli during transplantation such as mechanical damage, hypoxia, ischemia and reperfusion, contributes to the initiation and progression of CAV.47,413 Moreover, alloimmune injury to the graft endothelium also contributes to endothelial dysfunction.47 While current immunosuppressive regimens are typically effective in managing acute rejection, they have not diminished the prevalence of CAV accordingly.46,414 Once activated, the endothelial response can promote atherogenesis via numerous mechanisms, including platelet adhesion, release of growth factors 107 | P a g e  and donor antigens, major histocompatibility complex (MHC) class I and II expression, adhesion molecule expression and promotion of vascular smooth muscle cell proliferation. 415 Endothelial damage can promote CAV development by increasing vascular permeability, and increasing intimal smooth muscle cell proliferation. Concurrently, some factors produced by the damaged endothelium initiate the physiological process of re-endothelialization, or repair of the endothelium, including via bone marrow-derived cell recruitment.359,360 It is thought that a specific subpopulation416 of bone marrow-derived cells may be endothelial progenitor cells, whose role is to specifically repair the endothelium.416-420 With these concepts in mind, it is likely that the observed overexpression of VEGF in injured arteries within the allograft, produces both beneficial and adverse effects, and their respective contribution to the overall outcome is dependent on timing and related pathogenic factors. Since VEGF is expressed endogenously in CAV, it is therapeutically relevant to assess methods of inhibiting its pathological activity, while leaving its healing potential intact. It is my intent to investigate the biological or pathological functions of endogenous VEGF overexpression in CAV by abrogation of the VEGF axis using treatment with soluble VEGF receptor-1 (sVEGFR1; soluble fms-like tyrosine kinase-1, sFlt-1). Soluble VEGFR1 contains the extracellular ligand-binding domain of the full-length, membrane-bound VEGFR1, and is generated physiologically by alternative splicing of the same pre-mRNA that encodes the VEGFR1 gene.421 Soluble VEGFR1 can bind VEGF, preventing its interaction with VEGFR2, and thus inhibiting its downstream signaling and action.422 It has been used in several contexts and has been shown that usage of it can inhibit intraplaque angiogenesis and suppress the development of atherosclerotic plaque.423 As well, Onoue et al 108 | P a g e  demonstrated that administration of sVEGFR1 reduced atherosclerotic plaque formation while significantly reducing infiltration of macrophages into aortic tissues.424 Thus, the investigation of the use of sVEGFR1 in the context of interfering with VEGF-mediated actions in the pathogenesis of CAV is an attractive prospect and the focus of this chapter.  5.2 – Materials and methods 5.2.1 – Animals The C57Bl/6 (C57; Jackson Laboratories, Bar Harbor, ME) mouse was chosen as the recipient because it is the most common background for transgenic mice. The 129X1/SvJ (129J; Jackson Laboratories) mouse was chosen as the donor because its antigenic profile creates a minor histocompatibility antigen-mismatch between donor and recipient mice species. Mice received Western diet (TD.88137; Harlan Teklad, Madison, WI) with water ab libitum and were acclimatized for a minimum of one week before surgery. Hearts from 12 female 129J mice were used as donors into 12 C57 male recipients (6 transplants per group). The number of animals per treatment group was determined using power calculations based on our previous experience with the mouse model of heterotopic cardiac transplantation, the expected variability of our primary experimental endpoint, percentage luminal narrowing, and accounted for expected morbidity and mortality. All procedures were reviewed and approved by the University of British Columbia Animal Care Committee (Protocol #A08-0509).  109 | P a g e  5.2.2 – Heterotopic cardiac transplant model Cardiac transplantation was performed as previously described.359,360,425,426 Hearts from female 129J donors were implanted into the abdomen of 5- to 7-week old male C57 mice (six transplants were performed for each group). Animals were anesthetized with 4% halothane and anesthesia maintained with 1-2% halothane (Halocarbon Laboratories, River Edge, NJ). Donor mice were infused with heparinized saline and their hearts excised. The recipient’s abdominal aorta and inferior vena cava were located and clamped. The donor’s aorta and pulmonary artery were anastomosed to the recipient’s abdominal aorta and inferior vena cava, respectively, in an end-to-side manner. Transplantation was performed within 30 to 40 minutes of removal of the donor heart. One dose of 0.01mg/kg buprenorphine (Buprenex Injectable; Reckitt and Colman Pharmaceuticals, Richmond, VA) was administered sub-cutaneously after surgery. Mice were given intraperitoneal injections of 6mg/kg/day FK506 (Prograf®, tacrolimus; Fujisawa Canada, Markham, ON) as the primary immunosuppressive agent in this study. FK506 was chosen as the primary immunosuppressive agent in this study after pilot investigation in the murine model of heterotopic cardiac transplantation revealed unexpected bleeding complications leading to significant mortality in apolipoprotein E-deficient mouse recipients treated with cyclosporine at multiple doses. Multiple doses of FK506, and oral and injectable forms of sirolimus were also tested, and the 6mg/kg/day dose of FK506 was found to completely prevent acute rejection while allowing the development of CAV within 30 days posttransplantation. Gross examination of all organs, including kidney, liver, lung, pancreas and spleen, as well as histological examination were used in all animals to ensure no non-specific drug toxicity. 110 | P a g e  5.2.3 – Administration of soluble VEGFR1 and monitoring of mice Mice were monitored regularly by the animal care staff in the Genetically Engineered Models (GEM) facility in the UBC James Hogg Research Centre. In conjunction, at 10am daily, I weighed each mouse individually to determine the exact dosing for daily injections of FK506 and bi-daily injections of either 5µg/kg soluble VEGFR1 (R&D Systems, Minneapolis, MN) or vehicle (PBS). As well, during this time, observations of mouse health and well-being were noted and abdominal palpations were performed and recorded for assessment of heterotopic heart function according to Table 2.  Grade  Description  A  Strong, regular heart beat  B  Regular heart beat  C  Weak and/or irregular heart beat  D  No heart beat detectable  Table 2 – Grading criteria for monitoring heterotopic heart beat function by abdominal palpation.  The following criteria were followed to determine premature sacrifice of animals in the experiment: a) no detectable heart beat (D grade) which persisted for greater than two consecutive days; b) >10% weight loss (excluding the first three days); c) bi-lateral hind limb paralysis persisting >3 days or if in conjunction with significant body weight changes; and/or d) steady decline in general signs of animal well-being, including grooming, socialization, nesting,  111 | P a g e  appearance of stool and urine, eating and drinking, among others, in accordance with GEM facility and UBC Animal Care Committee guidelines. For mice that did not reach experimental endpoint, autopsy or necropsy was performed to assess gross organ pathology and all tissue were harvested according to regular specified parameters for this study for archiving, and in some cases, histopathological review by cardiovascular pathologist experienced in transplantation pathology.  5.2.4 – Tissue harvesting and histopathological examination Mice were fasted at least 12 hours prior to euthanization for tissue and blood collection. At 21 days post-transplantation, mice were anesthetized by injection with ketamine/xylazine. After anaesthetization, the abdominal cavity was opened for visual verification of transplant heart function and gross examination. The native and transplanted hearts were perfused with sterile saline followed by 10% formalin. Subsequent to perfusion-fixation, hearts were rapidly removed, photographed, weighed and dissected, then immersion-fixed in 10% formalin overnight before tissue processing. Ventricular transverse sections were embedded in paraffin. As well, liver, lung, kidney, spleen and pancreas were harvested for tissue processing and archival. Paraffin-embedded sections were cut serially (4µm) and stained with hematoxylin and eosin (H&E) and Movat’s pentachrome. Blinded assessment of vasculitis, rejection and CAV were performed in H&E section by trained cardiovascular pathologist (0-4+ scale). Luminal narrowing in all observed medium to large-size coronary arteries was also evaluated in Movat’s  112 | P a g e  pentachrome-stained sections. These histopathological assessments by cardiovascular pathologist guided my further morphometric evaluation of CAV.  5.2.5 – Blood chemistry Mice were injected with 100µL Hepalean (heparin sodium solution; Eli Lily and Company, Indianapolis, IN) to prevent coagulation 10 minutes before sacrifice. Blood was aspirated by direct cardiac puncture of the native heart and immediately placed on ice. Subsequently, the whole blood was centrifuged at 10,000 rpm for 10 minutes at 4°C. Plasma was aspirated and stored at -80°C. High-density lipoprotein (HDL), LDL, and triglyceride levels were assayed using BioVision quantification kits (Mountain View, CA). ELISA was also performed to quantify mouse plasma VEGF-A levels (R&D Systems, Minneapolis, MN) as per manufacturer’s instructions.  5.2.6 – Morphometry To evaluate CAV, all visible medium to large arteries from both native and donor hearts were photographed at 400x magnification using a Spot digital camera. Using ImagePro Plus® software, “areas of interest” (AOI) were created by tracing the endothelium, internal elastic lamina and external elastic lamina in digital micrographs of Movat’s pentachrome-stained sections. The area bounded by the endothelium was defined as the lumen. The area bounded by the internal elastic lamina and endothelium was defined as the intima. The area bounded by the external elastic lamina and internal elastic lamina was defined as the media. Luminal narrowing was the primary measure of CAV used in this study (Figure 30). 113 | P a g e  endothelium  I  IEL L  L  I  M  M EEL  vessel from PBS-treated native heart % luminal narrowing =  intima (lumen + intima)  vessel from PBS-treated transplant heart Native 12.26%  Transplant 85.56%  Figure 30 – Determination of luminal narrowing in a mouse model of heterotopic cardiac transplantation. Digital micrographs were captured of all visible arteries and arterioles in the cross-section of native and transplant hearts. Using ImagePro Plus® software, “areas of interest” (AOI) are defined by tracing the endothelium, internal elastic lamina (IEL) and external elastic lamina (EEL). The areas bounded by these AOI define the lumen (L), intima (I) and media (M).  114 | P a g e  5.2.7 - In vitro aortic ring angiogenesis assay Segments from 129J mouse aortas were collected and cultured in Matrigel (BD Biosciences, Mississauga, ON). Whole bone marrow was harvested from C57 mice by flushing femurs and tibias with cold PBS/2% fetal bovine serum (FBS; Hyclone, Logan, UT) solution. To mimic the allogeneic mismatch in our transplant model, co-incubation of the aortic segments with bone marrow was performed using the transwell inserts. Co-cultures were maintained in endothelial basal medium (EBM-2; Lonza, Guelph, ON) supplemented with EGM-MV SingleQuots®. To analyze microvascular outgrowth from the aortic segment, NIH ImageJ imaging software was used to measure outgrowth length and area. Briefly, measurements of outgrowth length were obtained at 0°, 90° and 180° from the aortic ring segment. Measurements of outgrowth area were obtained by tracing the circumferential area of outgrowth to determine the total area and subtracting that from the area of the aortic ring segment alone.  5.2.8 – Statistical analysis Analysis of variance (ANOVA) was first performed between all the groups for each dataset of interest. When a significant difference was found within the groups, Student’s t-test (p < 0.05) was performed to determine the significance between specific data points.  115 | P a g e  5.3 – Results 5.3.1 – Heterotopic cardiac transplantation and response to soluble VEGFR1 Through the course of this study, 6/6 vehicle-treated transplants and 4/6 sVEGFR1treated transplants reached the predetermined experimental endpoint of 21 days. Of the two mice in the sVEGFR1 group which did not reach endpoint, one was sacrificed the morning after transplant due to post-surgical complications, while the other was sacrificed on day three posttransplant due to no detectable heartbeat. Alongside daily observations of animal health and well-being, transplant recipient body weight changes were used as a primary measure of recovery from the heterotopic transplantation surgical procedure and toxicity to administration of sVEGFR1. Over the time course of the experiment, there was no apparent significant difference between vehicle- and sVEGFR1-treated animals (Figure 31). vehicle sVEGFR1  Figure 31 – Treatment with soluble VEGFR1 does not result in a change in mouse body weight. Intraperitoneal injections of soluble VEGFR1 (sVEGFR1) does not significantly affect mouse body weight after heterotopic implantation of 129J mouse donor hearts as compared to vehicle control injections (PBS).  116 | P a g e  5.3.2 – Treatment with soluble VEGFR1 does not affect lipid levels Triglyceride levels trended to a decrease in sVEGFR1-treated animals when compared to vehicle-treated animals (11.74±1.81mg/dL vs 36.97±11.37mg/dL), although this difference was not statistically significant (p = 0.15) (Figure 32). There were no significant differences in HDL serum concentration (201.34±3.02mg/dL vs 197.41±3.07mg/dL; p = 0.4122) or LDL serum concentration (114.33±12.77mg/dL vs 102.66±12.58mg/dL; p = 0.5541) when comparing vehicle-treated animals and those treated with sVEGFR1 (Figure 32).  vehicle PBS  Plasma concentration (mg/dL)  200  sVEGFR1 sR1  150  100  NS 50  0 TG  HDL  LDL  Figure 32 – Treatment with soluble VEGFR1 does not change plasma lipid levels. No significant difference in plasma levels of triglycerides (TG), high-density lipoprotein (HDL) or low-density lipoprotein (LDL) was observed when comparing vehicle-treated (PBS) and soluble VEGFR1 (sVEGFR1)treated animals. Data are represented as mean±SE.  117 | P a g e  5.3.3 – Treatment with soluble VEGFR1 significantly increases plasma levels of VEGF Interestingly, animals treated with sVEGFR1 had significantly greater serum concentrations of VEGF as compared to vehicle-treated controls (56.61±4.45pg/mL vs 36.16±4.93pg/mL; p = 0.0316) (Figure 33).  Plasma  *  vehicle  sVEGFR1  Figure 33 – Treatment with soluble VEGFR1 significantly increases plasma VEGF concentrations. Plasma levels of VEGF protein were measured using ELISA. Soluble VEGFR1 (sVEGFR1)-treated animals had significantly increased plasma VEGF concentrations as compared to vehicle-treated (PBS) animals. Data are represented as mean±SE; *, p < 0.05.  118 | P a g e  5.3.4 – Treatment with soluble VEGFR1 significantly reduces luminal narrowing Micrographs were digitally captured of all visible intramyocardial arteries within native and transplanted hearts in vehicle- and sVEGFR1-treated animals (Figure 34a). When comparing the percentage luminal narrowing between transplant and native hearts in both treatment groups, transplanted hearts had significantly increased percentage luminal narrowing in both groups, despite being on 6mg/kg FK506 daily immunosuppression. In vehicletreated animals, transplanted hearts had significantly greater luminal narrowing as compared to native hearts (51.15±3.92% vs 19.16±1.47%; p = 1.75 x 10-5) (Figure 34b). As well, in sVEGFR1-treated animals, transplanted hearts had significantly greater percentage luminal narrowing as compared to native hearts (36.56±1.70% vs 14.11±1.95%; p = 0.0010) (Figure 34b). This serves to validate this mouse model, which is able to present with significant development of luminal narrowing in arteries of the transplanted heart, which is a hallmark of CAV, in a minor MHC-mismatched allogeneic transplant model in the presence of the immunosuppressant FK506. Animals treated with sVEGFR1 had significantly reduced degrees of luminal narrowing as compared to vehicle-treated animals (36.56±1.70% vs 51.15±3.92%; p = 0.0413) (Figure 34b). Although percentage luminal narrowing trended to be decreased in native hearts of sVEGFR1treated animals as compared to vehicle-treated animals (14.11±1.95% vs 19.16±1.47%), this difference was not statistically significant (p = 0.828) (Figure 34b).  119 | P a g e  native  a)  transplant  vehicle  sVEGFR1  b)  *  Figure 34 – Quantitation of luminal narrowing in intramyocardial arteries. (a) Representative micrographs of Movat’s pentachrome-stained intramyocardial arteries from native and transplant hearts in vehicle- and sVEGFR1-treated animals. (b) Treatment with sVEGFR1 resulted in a significant decrease in luminal narrowing in transplanted hearts as compared with vehicle-treated controls -5 (36.56±1.70% vs 51.15±3.92%, respectively; *, p = 1.75x10 ). Data are represented as mean±SE. Scale bar = 50µm.  120 | P a g e  5.3.5 – Treatment with soluble VEGFR1 reduces edema in transplanted hearts Micrographs were digitally captured of ventricular cross-sections of native and transplanted hearts in vehicle- and sVEGFR1-treated animals (Figure 35a). The average wet heart weight in sVEGFR1-treated transplanted hearts was significantly less than vehicle-treated transplanted hearts (0.15±0.01g vs 0.25±0.05g; p = 0.0423). There was no significant difference between the weights of vehicle- and sVEGFR1-treated native hearts (0.13±0.01g vs 0.14±0.01g, respectively), nor between sVEGFR1-treated transplanted hearts and vehicle- or sVEGFR1treated native hearts (Figure 35b). Digital micrographs of mid-ventricular cross-sections of native and transplant hearts from vehicle- and sVEGFR1-treated mice were captured and ImagePro Plus® software was used to quantitate cross-sectional area of histological sections. When comparing the mean crosssectional area of vehicle- and sVEGFR1-treated transplant hearts, no significant difference was measured (11.96±1.50µm2 vs 10.43±0.67µm2; p = 0.4843) (Figure 36).  121 | P a g e  native  transplant  a)  vehicle  sVEGFR1  b)  Figure 35 – Quantitation of ventricular cross-sectional area. (a) Representative Movat’s pentachrome-stained micrographs of ventricular cross-section of native and transplant hearts from vehicle- and sVEGFR1-treated mice. (b) There was no significant difference between native heart or transplant heart ventricular cross-sectional area in vehicle- or sVEGFR1-treated animals. Data are represented as mean±SE. Scale bar = 500µm.  122 | P a g e  *  Figure 36 – Wet heart weight is significantly reduced in soluble VEGFR1-treated transplanted hearts. Wet heart weight (g) was significantly reduced in transplanted hearts from sVEGFR1-treated animals as compared to vehicle-treated animals (0.15±0.01g vs 0.25±0.05g; p = 0.0423). Data represent mean±SE.  Consolidating the results from Figures 35 and 36, transplant hearts from vehicle-treated animals had significantly greater wet heart weights compared with transplanted hearts from sVEGFR1-treated animals. This result, coupled with the measurement of no significant difference in cross-sectional area of either transplant or native hearts from vehicle- or sVEGFR1treated animals suggests that treatment with sVEGF1 reduces edema in transplanted hearts.  123 | P a g e  5.3.6 – Treatment with soluble VEGFR1 reduces capillary growth induced by bone marrow Aortic segments were cultured for 5 days alone, or co-incubated with BM and either vehicle or sVEGFR1 (Figure 37a). When aortic ring segments were incubated with whole bone marrow from C57 male mice, the average length of outgrowth increased 123% as compared to control aortic rings which were co-cultured with an empty transwell insert (p = 0.000463). For aortic ring segments cultured with only bone marrow, the average outgrowth area was not significantly different as compared to control (104%; p = 0.4037) (Figure 37b). When aortic ring segments were co-incubated with bone marrow and sVEGFR1, there was a significant decrease in the average outgrowth length as compared to control (51%, p = 5.35 x 10-5), and this difference was also significantly decreased as compared to bone marrow co-culture alone (51% vs 123%; p = 5.92 x 10-8). As well, samples treated with sVEGFR1 had significantly decreased outgrowth area (relative to aortic ring area) compared with control (43%; p = 4.69 x 10-6) and compared with bone marrow co-culture alone (43% vs 104%; p = 2.56 x 10-6) (Figure 37b).  124 | P a g e  a)  control  BM + vehicle  +  b)  BM + sVEGFR1  ++  *  + vehicle  ** ***  Figure 37 – In vitro aortic ring angiogenesis assay co-culture with bone marrow (BM)-derived cells. (a) Representative phase contrast micrographs of aortic ring segments without co-culture or cultured with BM-derived cells and either vehicle or sVEGFR1. (b) Co-culture with BM significantly increased average length of outgrowth (120% as compared with control; *, p = 0.000463), while addition of sVEGFR1 with BM -5 significantly reduced average length of outgrowth (51% as compared with control; **, p = 5.36x10 ). sVEGFR1-treated groups also had significantly shorter outgrowth length than vehicle-treated groups co-8 cultured with BM (+, p = 5.92x10 ). Co-culture with BM did not significantly increase average outgrowth area; however, co—culture with BM and sVEGFR1 significantly reduced average outgrowth area (***, p = -6 4.69x10 ). sVEGFR1-treated groups also had significantly smaller outgrowth area as compared with vehicle-6 treated groups co-cultured with BM (++, p = 2.56x10 ). Data are represented as mean±SE; n = 3 per group.  125 | P a g e  5.4 – Discussion During the conceptualization of this proof-of-principle in vivo verification of my doctoral dissertation thesis, the model system and potential therapeutic avenues explored have evolved to change from a rat to a mouse model of heterotopic cardiac transplantation, has included much troubleshooting and optimization of appropriate immunosuppressive regimens to balance inhibition of acute rejection while producing significant CAV, and a fundamental shift in my approach to better understand the role of VEGF in the pathogenesis of CAV. I moved from administration of VEGF protein in vivo, to the use of antisense deoxynucleotide molecules directed against the VEGF internal ribosomal entry sequence, to the use of signal transduction pathway inhibitors, to more specific VEGF receptor tyrosine phosphorylation inhibitors and anti-VEGF neutralizing antibodies. In this chapter, I focus on the utilization of soluble VEGFR1, which not only had a significant effect in reducing luminal narrowing in CAV, but also was welltolerated by the transplant recipients and resulted in no unexpected post-surgical complications or any evident drug toxicity. One of the primary reasons for my shift from a rat model to a mouse model of heterotopic cardiac transplantation was the desire for a model system which was capable of sustaining a pro-atherogenic hyperlipidemic environment. This particular point was tantamount as our laboratory was the first to demonstrate the presence and involvement of lipids in the pathogenesis of CAV.69 In my experiments, animals were placed on a Western diet which resulted in significantly increased LDL cholesterol levels in transplanted mice as compared to untransplanted C57 and 129J mice on normal rodent chow diet (data not shown). In the examination of the effect of administration of soluble VEGFR1, I observed no statistically 126 | P a g e  significant difference in triglyceride, LDL cholesterol or HDL cholesterol levels (Figure 32). This suggests that the role VEGF plays in CAV may not directly relate to lipids, at least in this model at the time point investigated; however, this hypothesis needs to be further explored. To clearly rule out the contribution of lipids to the soluble VEGFR1-mediated reduction in CAV, a series of further experiments is required to specifically examine the changes in lipid profile during the pathogenesis of CAV using greater animal numbers, more numerous time points (especially early post-transplantation) and tail vein collections of blood through the time course of pathogenesis; however, these investigations are beyond the scope of my thesis. One interesting observation I made was the significant increase in plasma VEGF concentrations in animals treated with soluble VEGFR1 (Figure 33). Initially, this result seemed counter-intuitive, as my entire VEGF-centered focus in my doctoral dissertation project began with the observation of increased VEGF expression in human heart allografts with CAV, whereas in this circumstance, we have increased plasma VEGF concentrations coupled with a reduction in CAV. It may be possible that the addition of soluble VEGFR1, an agent which can bind circulating VEGF, may result in a compensatory increase in the expression or secretion in a feedback-like manner. It should be noted that the particular antibody used in the ELISA to profile VEGF in mouse plasma samples can also function in vivo as an anti-VEGF neutralizing antibody, and thus, the observed increased in VEGF plasma levels is not likely as a result of measurement of VEGF-sVEGFR1 complexes. My immunohistochemical profiling of the transplant heart tissues from this chapter for VEGF expression revealed no significant difference in VEGF immunoreactivity between vehicleand soluble VEGFR1-treated animals (data not shown). Overall, the time point chosen for this 127 | P a g e  experiment produces a significant degree of luminal narrowing and CAV; however, in comparison to the cases from human heart allografts used in Chapter 3, the human CAV samples were from archival tissue from explanted heart transplants or those obtained at autopsy, suggesting severe CAV and/or end-stage heart failure. To mirror the results obtained in my immunohistochemical investigations in human heart allografts, it would be necessary to significantly extend the experimental time point. However, I believe that as this research direction continues in the laboratory, it is more vital to examine the changes in VEGF secretion, expression and localization during the early transplant time course before significant luminal narrowing or CAV may be detected. To truly elucidate the role of VEGF in CAV, it is necessary to separate  the  potentially  beneficial  effects  which  may  be  involved  in  healing  ischemia/reperfusion and/or alloimmune injury initially post-transplantation from the potentially deleterious effects of persistent pathological expression of VEGF may have in the long term. Irregardless, my experiments clearly demonstrate that abrogation of the VEGF axis with soluble VEGFR1 has an overall positive effect in reducing luminal narrowing and CAV at 21 days post-transplantation (Figure 34). Further optimization of dosing regimen to enhance beneficial VEGF effects while minimizing deleterious ones may, in the future, provide an even greater degree of prevention or reduction of CAV. In exploring the potential mechanisms of the soluble VEGFR1-mediated reduction in CAV, there are two emerging directions from my work. One, related to the overarching hypothesis of Chapter 4 and this thesis, relates to VEGF-induced permeability – in vivo, this permeability can be directly observed as edema. I have demonstrated that in comparison to vehicle-treated transplants, transplants which received soluble VEGFR1 have a significant 128 | P a g e  reduction in wet heart weight (Figure 35). Coupled with my morphometric assessment of no change in heart cross-sectional area (i.e. change in size or hypertrophy) (Figure 36), this suggests that abrogation of the VEGF axis in CAV reduces edema, and this may be a mechanism related to the significant reduction of CAV in animals treated with soluble VEGFR1. The other emerging direction is the relationship of the VEGF axis to the bone marrow response post-transplantation. Research performed in our laboratory has demonstrated a significant increase in bone marrow mobilization to the transplanted heart. 359,360 The results from my in vitro aortic ring angiogenesis assays, which were co-cultured with bone marrow, suggest that the bone marrow plays a role in at least directing microvascular growth. Usage of soluble VEGFR1 in aortic rings co-cultured with bone marrow significantly reduced both outgrowth length and overall outgrowth area (Figure 37). This result suggests that this may, in part, be another potential mechanism of action when attempting to decipher the mechanism of soluble VEGFR1-mediated reduction of CAV. This in vitro model also serves to verify that the dose and nature of the soluble VEGFR1 used in my in vivo investigations does, indeed, inhibit classic VEGF functions of induced endothelial migration, proliferation and angiogenesis. Ultimately, further investigation into the evolving role of VEGF in CAV will allow for better manipulation of the VEGF axis to promote beneficial physiological functions while reducing deleterious pathological ones. The key findings from this chapter are reviewed in Figure 38.  129 | P a g e  BM-derived cells  VEGF  sVEGFR1  lumen  VEGF VEGF-R % luminal narrowing ↑ permeability  intimal hyperplasia  intima  media Figure 38 – Diagrammatic representation of the key findings from Chapter 5. The proof-of-principle series of experiments detailed in this chapter provide the first characterization of the ability of administration of soluble VEGFR1 (sVEGFR1) to significantly reduce percentage luminal narrowing (intimal area / luminal area + intimal area) in a hypercholesterolemic mouse model of heterotopic cardiac transplantation. Percentage luminal narrowing is the primary measure of cardiac allograft vasculopathy (CAV) in humans and animal models. As well, administration of sVEGFR1 also reduced edema in transplanted hearts, suggesting that abrogation of the VEGF axis can reduce permeability in the allogeneic transplant setting. A complementary investigation using an in vitro aortic ring angiogenesis assay cocultured with bone marrow cells demonstrated that sVEGFR1 can inhibit bone marrow-mediated microvascular growth. Taken together, these provide two possible mechanisms for the reduction in CAV observed after application of sVEGFR1. Moving forward, the results from this thesis provide significant new knowledge with respect to the role of VEGF in the pathogenesis of CAV. These prospects, a summary of my work, and a revised concept diagram which includes the role of VEGF in the pathogenesis of CAV are provided in the final chapter of this thesis.  130 | P a g e  Chapter 6 – Closing remarks The clear conclusions from the summation of my doctoral studies are: i) VEGF is aberrantly expressed in the coronary arteries of human heart allografts with CAV, and other atheromatous diseases; ii) VEGF is able to induce significant increases in endothelial permeability to LDL in human coronary or cardiac-specific macro- and microvascular endothelial cells, likely through the disruption of tight junctions via an ERK1/2-dependent pathway; and iii) Alteration of the balance of the VEGF axis in a hyperlipidemic model of heterotopic cardiac transplantation in mice significantly reduces the severity of CAV. These main points solidify a role for VEGF in the pathogenesis of CAV. Continued diligence and investigation along these lines will undoubtedly further our understanding of the pathogenesis of CAV such that therapeutic avenues such as the use of soluble VEGFR1 may one day be utilized to significantly reduce or even prevent the pathogenesis of CAV and AV in all solid organ transplants. Specifically related to the concepts covered in my doctoral dissertation thesis, the highest priority hypothesis to test would be to delineate the expression and localization of VEGF family members and receptors during the time course of progression of CAV. Optimally, this investigation could be performed in an expanded version of the studies performed in Chapter 3, where a significantly larger sample size for CAV cases may allow the delineation of VEGF expression and localization during different time periods of the pathogenesis of CAV, possibly to delineate “early” and “late” disease. Complementary to this approach, larger sample sizes will allow for the correlation of VEGF expression and CAV pathogenesis with lipid and lipoprotein insudation and retention. The main challenge to this approach, however, remains 131 | P a g e  the acquisition of sufficient numbers of CAV case materials to adequately power this investigation. As well, implant duration does not directly correlate with the time course of progression of CAV, as many adjacent factors such as donor- and host-specific risk factors and variable levels of immunosuppression post-transplantation may serve as determinants for the onset and progression of CAV. As such, this hypothesis could be more appropriately addressed in the murine model of heterotopic cardiac transplantation used in Chapter 5, as it provides a controlled model of CAV where significant CAV develops within 3-4 weeks post-transplantation with a minor MHCmismatch in the presence of a common immunosuppressive agent to inhibit acute rejection. With this time course of VEGF expression and localization in relation to progression of CAV, one could appropriately inhibit VEGF signaling and action using soluble VEGFR1, or other inhibition strategies such as anti-VEGF neutralizing antibodies or chemical inhibitors of VEGFR2 tyrosine kinase phosphorylation within specific time periods post-transplantation (i.e., within 1 week; between 1-2 weeks; after 2 weeks; etc.) to determine whether VEGF may play beneficial or deleterious roles within different time periods during the pathogenesis of CAV. As well, one of the primary reasons for transitioning from a rat model of CAV to a mouse model of CAV was to be able to take advantage of hyperlipidemic models in the mouse, be it through diet or transgene modification, or both. It would be interesting to examine the direct relationship between VEGF expression and localization with lipid and lipoprotein insudation in the vessel wall in the pathogenesis of CAV, and whether augmentation of VEGF through the aforementioned inhibition strategies may also modify lipid and lipoprotein permeability.  132 | P a g e  Overall, continued investigation is warranted to determine the predominant mechanism for the reduction of luminal narrowing by administration of sVEGFR1. During my doctoral thesis studies, I have been fortunate to be directly or peripherally involved with a variety of transplant-related programs. This began with my involvement in Program Project Grant awarded to multiple investigators from the Heart and Stroke Foundation of BC and Yukon. In this project, the primary model of cardiac allograft vasculopathy was performed in the Fisher-to-Lewis rat model of heterotopic cardiac transplantation. One of the primary observations that arose from this series of experiments was the disruption of the endothelium, both functionally and structurally. A key observation was, at 21 and 42 days posttransplantation, the presence of intercellular gaps in cyclosporine-treated animals.106 This observation, along with others, solidified the hypothesis that a hyperpermeability-inducing agent may play a role in the pathogenesis of CAV. Coupled with my observations in human heart allografts of the aberrant expression of VEGF, this line of thinking lead to the exploration of the role of VEGF in the permeability or hyperpermeability of human cardiac-specific primary endothelial cells in culture, explored in Chapter 4. In addition, as our animal transplant program and model evolved from the rat to the mouse, I was fortunate to have the opportunity to be involved with the development of the model and the optimization of immunosuppressive and transplant regimens to produce a murine model of heterotopic cardiac transplantation rooted in a C57/Bl6 recipient which would facilitate vertical expansion in the future for application of transgenic mouse models. As well, the transition to a mouse model of transplantation allowed for the augmentation of lipid profiles, either by diet, transgene, or both, to more closely resemble that of humans. 133 | P a g e  Expositing, several studies have observed the clinical benefit statin therapy posttransplantation.427-430 It has also been demonstrated by other investigators that greater change in serum LDL cholesterol levels during the first year post-transplantation is associated with more severe vasculopathy.431 Most recently, there has been an intriguing case report describing the use of dextran sulphate cellulose LDL adsorption apheresis in a 50-year-old male orthotopic heart transplant recipient with familial hyperlipidemia resulting in stabilization and reversal of cardiac allograft vasculopathy.432 I believe that in the context of CAV, and other atheromatous diseases, VEGF may contribute to their pathogenesis, in part, through increased LDL permeability. Taken together with my previous report of a similar role for VEGF-A in CAV,412 it is likely that there may be disruption of several VEGF family members and receptors which augment the balance of physiological healing and pathological atherogenic outcomes. In one of our laboratory’s transplant-related focuses, examining the role of granzyme B and perforin in CAV, I was able to contribute technically and conceptually, and we observed a reduction of CAV and endothelial damage in perforin knockout mice. 426 This line of experimentation served to further highlight the immune contribution to CAV, and specifically, its relationship to endothelial damage. Our laboratory has demonstrated a role for apoptosis,111 and both the Fas-mediated407 and granzyme B-mediated402,425,426,433 pathways in the pathogenesis of CAV. This immune-mediated damage to the graft may be another initiating stimulus to early endothelial damage and dysfunction resulting in the overexpression of VEGF in CAV characterized in Chapter 3. Another transplant-related focus I was peripherally involved in was the investigation of the role of bone marrow-derived cells in transplantation and CAV.359,360 This work highlighted 134 | P a g e  an increased mobilization of the bone marrow to the transplanted heart, and revealed an interesting possibility of relevance to my doctoral thesis focus. As the field of stem cells emerged, the role of VEGFR1 and VEGF in the mobilization of bone marrow-derived cells became more apparent. The revelations from our work, and that in the literature highlighted an additional possibility for the role of VEGF in the pathogenesis of CAV. Finally, a disparate line of investigation which occurred in our laboratory was the investigation of a differentially regulated proteoglycan, versican, which had previously been shown by our laboratory to be increased in human heart allografts. Work performed by Dr. Maziar Rahmani delineated the role of the Wnt-signaling pathway in the transcriptional regulation of versican.405 An interesting complement I was fortunate to be involved in was the investigation of Wnt-related transcriptional regulation of the VEGF gene as well. We determined the ability of T cell factors (TCF) to increase the transcriptional activity of the VEGF gene. This result, coupled with my observations of both increased beta-catenin in arteries from a rat model of aortic stenosis, introduced another possible role for VEGF in the pathogenesis of atheromatous disease. Specifically, it is hypothesized that a Wnt-related gene program may regulate a host of downstream genes including VEGF, versican and matrix metalloproteinase-9, among others, to be a relevant axis determining neovascularization in the plaque. Taken together, the convergence of what began as several disparate lines of investigation serves to highlight the interplay between all networks, be it within a cell, tissue, organ or organ system, or throughout the body. What began in my thesis with the simple observation of aberrant VEGF expression in CAV and atheromatous disease has expanded into a complex multifaceted research program, which reflects the complex multifactorial etiology of 135 | P a g e  CAV. Moving forward, research efforts should focus not simply on VEGF alone and its role in CAV; but to begin to elucidate its role, one must consider the balance of numerous factors which may alter the VEGF axis in either a beneficial reparative or deleterious pathological manner, including the role and relativity of splice variants, the variety of primary receptors (and soluble forms), their primary tyrosine phosphorylation-dependent actions and control mechanisms which include homo- and heterodimer configurations of VEGF ligands and also VEGF receptors. Coupled with the function of “decoy” soluble receptors and competitive binding of VEGF family members such as PlGF, there are numerous positive and negative feedback mechanisms, whose ultimate balance may dictate phenotype. The changes in endothelial permeability are intricate and the studies to understand them must be multifaceted. Much remains to be learned about these processes. Foremost are the components of the tight and adherens junctions, especially the sealing elements in tight junctions. Complementing these elements is the function of the different individual junctional components found, and how these differ in cells from different vascular beds. At the other side of the cell lies the extracellular cellular matrix and the role it plays in permeability in vivo. Inside the cell, the role of the cytoskeleton remains to be elucidated. The second messengers are yet another mystery. It is unclear which messenger, subtype or isoenzyme is utilized by each permeability-altering agent. Determining what the targets of the messengers are and how the different pathways interact with each other, as well as how these pathways may be differentially utilized in different vascular beds may offer targets for therapeutic intervention. Certainly, opportunities are wide-open for important discoveries in this area.  136 | P a g e  A revised current concept for the pathogenesis of CAV highlighting the roles of VEGF in CAV is detailed in Figures 39 and 40.  Figure 39 – Revised concept of the early pathogenic mechanisms in cardiac allograft vasculopathy. There are several injurious stimuli, including ischemia/reperfusion injury, hypoxia, reactive oxygen species, hypercholesterolemia and the alloimmune response against the graft, which contribute to the initiation and progression of cardiac allograft vasculopathy (CAV). Early endothelial damage and dysfunction result in the release of not only pro-inflammatory cytokines and chemokines, but also growth factors and other molecules, which enhance the immune response against the graft and induce reparative responses which attempt to maintain endothelial barrier integrity and vascular function. Smooth muscle cells (SMC) in the media produce vascular endothelial growth factor (VEGF) in a physiological attempt to repair the endothelium either through endothelial migration and proliferation along the endothelium or by inducing the homing of endothelia progenitor cells. This expression of VEGF may result in deleterious effects on the vessel wall, resulting in increased endothelial permeability to low-density lipoproteins (LDL) and enhanced chemotaxis of monocytes to the vessel wall. Subsequent to LDL insudation and retention on proteoglycans within the extracellular matrix (ECM), native LDL itself can further induce SMC to produce and secrete VEGF.  137 | P a g e  Monocyte  Stem cell VEGF lumen  ↑VEGF intima  oxLDL LDL  Foam cell  Macrophage  ↑VEGF  media Figure 40 – Revised concept of the later events in the pathogenesis of cardiac allograft vasculopathy. During the progression and propagation of cardiac allograft vasculopathy (CAV), chronic, aberrant expression of vascular endothelial growth factor (VEGF) can continue to alter the vessel microenvironment. It has been well documented that oxidized low-density lipoproteins (oxLDL) can not only induce macrophages to produce and secrete VEGF, but also do so independent of oxLDL uptake in macrophages. As well, VEGF has been shown to have pro-survival effects on macrophages, providing a possible mechanism whereby macrophages which uptake excess low-density lipoproteins (LDL) or oxLDL may be kept alive long enough to form lipid-laden foam cells, which subsequently may contribute to the concentric, lipid-rich lesions characteristic of CAV. Conversely, VEGF may also maintain some beneficial roles within the vessel wall. It has been demonstrated that VEGF can inhibit oxLDL-induced endothelial cell apoptosis. As well, a central role for VEGF in the chemotaxis, signaling, survival and differentiation of stem cells is emerging. It may be possible in the future to augment the VEGF signaling axis to promote the physiological, beneficial effects of VEGF, while minimizing or inhibiting potentially deleterious, pathological effects.  138 | P a g e  References 1. Gardin JM, Siscovick D, Anton-Culver H, Lynch JC, Smith VE, Klopfenstein HS, Bommer WJ, Fried L, O'Leary D, Manolio TA. Sex, age, and disease affect echocardiographic left ventricular mass and systolic function in the free-living elderly. The Cardiovascular Health Study. Circulation. 1995;91:1739-48. 2.  McMurray JJ, Pfeffer MA. Heart failure. Lancet. 2005;365:1877-89.  3. Lloyd-Jones DM, Larson MG, Leip EP, Beiser A, D'Agostino RB, Kannel WB, Murabito JM, Vasan RS, Benjamin EJ, Levy D. Lifetime risk for developing congestive heart failure: the Framingham Heart Study. Circulation. 2002;106:3068-72. 4. Bleumink GS, Knetsch AM, Sturkenboom MC, Straus SM, Hofman A, Deckers JW, Witteman JC, Stricker BH. Quantifying the heart failure epidemic: prevalence, incidence rate, lifetime risk and prognosis of heart failure The Rotterdam Study. Eur Heart J. 2004;25:1614-9. 5. Dickstein K, Cohen-Solal A, Filippatos G, McMurray JJ, Ponikowski P, Poole-Wilson PA, Stromberg A, van Veldhuisen DJ, Atar D, Hoes AW, Keren A, Mebazaa A, Nieminen M, Priori SG, Swedberg K, Vahanian A, Camm J, De Caterina R, Dean V, Funck-Brentano C, Hellemans I, Kristensen SD, McGregor K, Sechtem U, Silber S, Tendera M, Widimsky P, Zamorano JL. ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2008: the Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2008 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC (HFA) and endorsed by the European Society of Intensive Care Medicine (ESICM). Eur Heart J. 2008;29:2388-442. 6.  Neubauer S. The failing heart--an engine out of fuel. N Engl J Med. 2007;356:1140-51.  7. Ross R, Glomset JA. The pathogenesis of atherosclerosis (first of two parts). N Engl J Med. 1976;295:369-77. 8.  Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med. 1999;340:115-26.  9. Abete P, Napoli C, Santoro G, Ferrara N, Tritto I, Chiariello M, Rengo F, Ambrosio G. Agerelated decrease in cardiac tolerance to oxidative stress. J Mol Cell Cardiol. 1999;31:227-36. 10. Ruef J, Peter K, Nordt TK, Runge MS, Kubler W, Bode C. Oxidative stress and atherosclerosis: its relationship to growth factors, thrombus formation and therapeutic approaches. Thromb Haemost. 1999;82:32-7. 11. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005;352:1685-95. 139 | P a g e  12. Perrault LP, Mahlberg F, Breugnot C, Bidouard JP, Villeneuve N, Vilaine JP, Vanhoutte PM. Hypercholesterolemia increases coronary endothelial dysfunction, lipid content, and accelerated atherosclerosis after heart transplantation. Arterioscler Thromb Vasc Biol. 2000;20:728-36. 13. Calkin AC, Allen TJ. Diabetes mellitus-associated atherosclerosis: mechanisms involved and potential for pharmacological invention. Am J Cardiovasc Drugs. 2006;6:15-40. 14. Williams KJ, Tabas I. The response-to-retention hypothesis of early atherogenesis. Arterioscler Thromb Vasc Biol. 1995;15:551-61. 15. A randomized trial of beta-blockade in heart failure. The Cardiac Insufficiency Bisoprolol Study (CIBIS). CIBIS Investigators and Committees. Circulation. 1994;90:1765-73. 16. Effects of enalapril on mortality in severe congestive heart failure. Results of the Cooperative North Scandinavian Enalapril Survival Study (CONSENSUS). The CONSENSUS Trial Study Group. N Engl J Med. 1987;316:1429-35. 17. Pitt B, Waters D, Brown WV, van Boven AJ, Schwartz L, Title LM, Eisenberg D, Shurzinske L, McCormick LS. Aggressive lipid-lowering therapy compared with angioplasty in stable coronary artery disease. Atorvastatin versus Revascularization Treatment Investigators. N Engl J Med. 1999;341:70-6. 18. Cohn JN, Tognoni G. A randomized trial of the angiotensin-receptor blocker valsartan in chronic heart failure. N Engl J Med. 2001;345:1667-75. 19. Pfeffer MA, Swedberg K, Granger CB, Held P, McMurray JJ, Michelson EL, Olofsson B, Ostergren J, Yusuf S, Pocock S. Effects of candesartan on mortality and morbidity in patients with chronic heart failure: the CHARM-Overall programme. Lancet. 2003;362:759-66. 20. Cleland JG, Daubert JC, Erdmann E, Freemantle N, Gras D, Kappenberger L, Tavazzi L. The effect of cardiac resynchronization on morbidity and mortality in heart failure. N Engl J Med. 2005;352:1539-49. 21.  Landsteiner K. On agglutination of normal human blood. Transfusion. 1961;1:5-8.  22. Billingham RE, Brent L, Medawar PB. Acquired tolerance of skin homografts. Ann N Y Acad Sci. 1955;59:409-16. 23. Billingham RE, Medawar PB. The freezing, drying and storage of mammalian skin. J Exp Biol. 1952;29:454-68.  140 | P a g e  24. Mackay IR, Larkin L, Burnet FM. Failure of autoimmune antibody to react with antigen prepared from the individual's own tissues. Lancet. 1957;270:122-3. 25. Dausset J, Colin M. [Research technic for immunologic thrombo-agglutinins; influence of previous heating of platelet suspensions.]. Rev Fr Etud Clin Biol. 1958;3:60-1. 26.  Dausset J. The birth of MAC. Vox Sang. 1984;46:235-7.  27. Carrel A. La technique opératoire des anastomoses vascularies et la trasportation des viscères. Lyon Med. 1902;98:859-64. 28. Carrel A, editor. Les anastomoses vasculaires, leur technique operatoire et leurs indications. Deuxième Congrès de Médecine de Langue Française de l'Amérique du Nord; 1904; Montreal, QC. 29.  Carrel A, Guthrie CC. The transplantation of veins and organs. Am Med. 1905;10:1101-2.  30. Carrel A, Guthrie CC. Uniterminal and biterminal venous transplantation. Surg Gynecol Obstet. 1906;2:266-86. 31. Carrel A. The preservation of tissues and its applications in surgery. J Am Med Assoc. 1912;59:523-7. 32. Sterioff S, Rucker-Johnson N. Frank C. Mann and transplantation at the Mayo Clinic. Mayo Clin Proc. 1987;62:1051-5. 33. Hardy JD, Chavez CM, Kurrus FD, Neely WA, Eraslan S, Turner D, Fabian LW, Labecki TD. Heart transplantation in man: Developmental studies and report of a case. J Am Med Assoc. 1964;88:1132-40. 34. Barnard CN. A human cardiac transplant: An interim report of a successful operation performed at Groote Schuur Hospital, Capetown. S Afr Med J. 1967;41:1271. 35. Haller JD, Cerruti MM. Heart transplantion in man: compilation of cases. January 1, 1964 to October 23, 1968. Am J Cardiol. 1968;22:840-3. 36. Cooley DA, Liotta D, Hallman GL, Bloodwell RD, Leachman RD, Milam JD. Orthotopic cardiac prosthesis for two-staged cardiac replacement. Am J Cardiol. 1969;24:723-30. 37.  Sakakibara S, Konno S. Endomyocardial biopsy. Jpn Heart J. 1962;3:537-43.  141 | P a g e  38. Caves PK, Stinson EB, Billingham M, Shumway NE. Percutaneous transvenous endomyocardial biopsy in human heart recipients. Experience with a new technique. Ann Thorac Surg. 1973;16:325-36. 39. Billingham ME, Cary NR, Hammond ME, Kemnitz J, Marboe C, McCallister HA, Snovar DC, Winters GL, Zerbe A. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: Heart Rejection Study Group. The International Society for Heart Transplantation. J Heart Transplant. 1990;9:587-93. 40. Stewart S, Winters GL, Fishbein MC, Tazelaar HD, Kobashigawa J, Abrams J, Andersen CB, Angelini A, Berry GJ, Burke MM, Demetris AJ, Hammond E, Itescu S, Marboe CC, McManus B, Reed EF, Reinsmoen NL, Rodriguez ER, Rose AG, Rose M, Suciu-Focia N, Zeevi A, Billingham ME. Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection. J Heart Lung Transplant. 2005;24:1710-20. 41. Levi DS, DeConde AS, Fishbein MC, Burch C, Alejos JC, Wetzel GT. The yield of surveillance endomyocardial biopsies as a screen for cellular rejection in pediatric heart transplant patients. Pediatr Transplant. 2004;8:22-8. 42. Hammond EH, Yowell RL, Nunoda S, Menlove RL, Renlund DG, Bristow MR, Gay WA, Jr., Jones KW, O'Connell JB. Vascular (humoral) rejection in heart transplantation: pathologic observations and clinical implications. J Heart Transplant. 1989;8:430-43. 43. Michaels PJ, Fishbein MC, Colvin RB. Humoral rejection of human organ transplants. Springer Semin Immunopathol. 2003;25:119-40. 44. Wahlers T, Mugge A, Oppelt P, Heublein B, Fieguth HG, Jurmann M, Haverich A. Coronary vasculopathy following cardiac transplantation and cyclosporine immunosuppression: preventive treatment with angiopeptin, a somatostatin analog. Transplant Proc. 1994;26:27412. 45. Yeung AC, Davis SF, Hauptman PJ, Kobashigawa JA, Miller LW, Valantine HA, Ventura HO, Wiedermann J, Wilensky R. Incidence and progression of transplant coronary artery disease over 1 year: results of a multicenter trial with use of intravascular ultrasound. Multicenter Intravascular Ultrasound Transplant Study Group. J Heart Lung Transplant. 1995;14:S215-20. 46. Kobashigawa JA, Patel JK. Immunosuppression for heart transplantation: where are we now? Nat Clin Pract Cardiovasc Med. 2006;3:203-12. 47. Schmauss D, Weis M. Cardiac allograft vasculopathy: recent developments. Circulation. 2008;117:2131-41.  142 | P a g e  48. Carrier M, White M, Pelletier G, Perrault LP, Pellerin M, Pelletier LC. Ten-year follow-up of critically ill patients undergoing heart transplantation. J Heart Lung Transplant. 2000;19:43943. 49. Reed EF, Demetris AJ, Hammond E, Itescu S, Kobashigawa JA, Reinsmoen NL, Rodriguez ER, Rose M, Stewart S, Suciu-Foca N, Zeevi A, Fishbein MC. Acute antibody-mediated rejection of cardiac transplants. J Heart Lung Transplant. 2006;25:153-9. 50. Wahlers T, Kotzerke B, Wagenbreth I, Hausen B, Haverich A. Preventive treatment of renal impairment and graft rejection in cardiac transplantation using cyclosporine with an oral prostaglandin analog. Transplant Proc. 1994;26:2743-4. 51. Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med. 1997;336:973-9. 52. Bilchick KC, Henrikson CA, Skojec D, Kasper EK, Blumenthal RS. Treatment of hyperlipidemia in cardiac transplant recipients. Am Heart J. 2004;148:200-10. 53. Choy JC, Granville DJ, Hunt DW, McManus BM. Endothelial cell apoptosis: biochemical characteristics and potential implications for atherosclerosis. J Mol Cell Cardiol. 2001;33:167390. 54. Gonzalez-Pacheco FR, Deudero JJ, Castellanos MC, Castilla MA, Alvarez-Arroyo MV, Yague S, Caramelo C. Mechanisms of endothelial response to oxidative aggression: protective role of autologous VEGF and induction of VEGFR2 by H2O2. Am J Physiol Heart Circ Physiol. 2006;291:H1395-401. 55. Huot J, Houle F, Marceau F, Landry J. Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ Res. 1997;80:383-92. 56. Gutstein WH, Farrell GA. Endothelial defects and blood flow disturbance in atherogenesis. Experientia. 1972;28:1299-300. 57. Tricot O, Mallat Z, Heymes C, Belmin J, Leseche G, Tedgui A. Relation between endothelial cell apoptosis and blood flow direction in human atherosclerotic plaques. Circulation. 2000;101:2450-3. 58. Tsai YC, Hsieh HJ, Liao F, Ni C-W, Chao YJ, Hsieh CY, Wang DL. Laminar flow attenuates interferon-induced inflammatory responses in endothelial cells. Cardiovasc Res. 2007;74:497505.  143 | P a g e  59. Carmeliet P, Moons L, Lijnen R, Janssens S, Lupu F, Collen D, Gerard RD. Inhibitory role of plasminogen activator inhibitor-1 in arterial wound healing and neointima formation: a gene targeting and gene transfer study in mice. Circulation. 1997;96:3180-91. 60. Valantine HA. Cardiac allograft vasculopathy: central role of endothelial injury leading to transplant "atheroma". Transplantation. 2003;76:891-9. 61. Wong D, Dorovini-Zis K. Regualtion by cytokines and lipopolysaccharide of E-selectin expression by human brain microvessel endothelial cells in primary culture. J Neuropathol Exp Neurol. 1996;55:225-35. 62. Xin X, Yang S, Ingle G, Zlot C, Rangell L, Kowalski J, Schwall R, Ferrara N, Gerritsen ME. Hepatocyte growth factor enhances vascular endothelial growth factor-induced angiogenesis in vitro and in vivo. Am J Pathol. 2001;158:1111-20. 63. Zania P, Papaconstantinou M, Flordellis CS, Maragoudakis ME, Tsopanoglou NE. Thrombin mediates mitogenesis and survival of human endothelial cells through distinct mechanisms. Am J Physiol Cell Physiol. 2008;294:C1215-26. 64. Combadiere C, Potteaux S, Gao J-L, Esposito B, Casanova S, Lee EJ, Debre P, Tedgui A, Murphy PM, Mallat Z. Decreased atherosclerotic lesion formation in CX3CR1/apolipoprotein E double knockout mice. Circulation. 2003;107:1009-16. 65. Geng YJ, Henderson LE, Levesque EB, Muszynski M, Libby P. Fas is expressed in human atherosclerotic intima and promotes apoptosis of cytokine-primed human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1997;17:2200-8. 66. Hofnagel O, Luechtenborg B, Stolle K, Lorkowski S, Eschert H, Plenz G, Robenek H. Proinflammatory cytokines regulate LOX-1 expression in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2004;24:1789-95. 67. Winters GL, Kendall TJ, Radio SJ, Wilson JE, Costanzo-Nordin MR, Switzer BL, Remmenga JA, McManus BM. Posttransplant obesity and hyperlipidemia: major predictors of severity of coronary arteriopathy in failed human heart allografts. J Heart Transplant. 1990;9:364-71. 68. McManus BM, Malcom G, Kendall TJ, Gulizia JM, Wilson JE, Winters G, Costanzo MR, Thieszen S, Radio SJ. Lipid overload and proteoglycan expression in chronic rejection of the human transplanted heart. Clin Transplant. 1994;8:336-40. 69. McManus BM, Horley KJ, Wilson JE, Malcom GT, Kendall TJ, Miles RR, Winters GL, Costanzo MR, Miller LL, Radio SJ. Prominence of coronary arterial wall lipids in human heart allografts. Implications for pathogenesis of allograft arteriopathy. Am J Pathol. 1995;147:293308. 144 | P a g e  70. Devlin CM, Leventhal AR, Kuriakose G, Schuchman EH, Williams KJ, Tabas I. Acid sphingomyelinase promotes lipoprotein retention within early atheromata and accelerates lesion progression. Arterioscler Thromb Vasc Biol. 2008;28:1723-30. 71. Holvoet P, Collen D. Oxidized lipoproteins in atherosclerosis and thrombosis. FASEB J. 1994;8:1279-84. 72. Quinn MT, Parthasarathy S, Fong LG, Steinberg D. Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogenesis. Proc Natl Acad Sci U S A. 1987;84:2995-8. 73. Brown MS, Basu SK, Falck JR, Ho YK, Goldstein JL. The scavenger cell pathway for lipoprotein degradation: specificity of the binding site that mediates the uptake of negativelycharged LDL by macrophages. J Supramol Struct. 1980;13:67-81. 74. Ross R, Glomset JA. Atherosclerosis and the arterial smooth muscle cell: Proliferation of smooth muscle is a key event in the genesis of the lesions of atherosclerosis. Science. 1973;180:1332-9. 75. Lougheed M, Moore ED, Scriven DR, Steinbrecher UP. Uptake of oxidized LDL by macrophages differs from that of acetyl LDL and leads to expansion of an acidic endolysosomal compartment. Arterioscler Thromb Vasc Biol. 1999;19:1881-90. 76. Matsuura E, Kobayashi K, Tabuchi M, Lopez LR. Oxidative modification of low-density lipoprotein and immune regulation of atherosclerosis. Prog Lipid Res. 2006;45:466-86. 77. Ramos MA, Kuzuya M, Esaki T, Miura S, Satake S, Asai T, Kanda S, Hayashi T, Iguchi A. Induction of macrophage VEGF in response to oxidized LDL and VEGF accumulation in human atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 1998;18:1188-96. 78. Casscells W, Hathorn B, David M, Krabach T, Vaughn WK, McAllister HA, Bearman G, Willerson JT. Thermal detection of cellular infiltrates in living atherosclerotic plaques: possible implications for plaque rupture and thrombosis. Lancet. 1996;347:1447-51. 79. Desjardins C, Duling BR. Heparinase treatment suggests a role for the endothelial cell glycocalyx in regulation of capillary hematocrit. Am J Physiol. 1990;258:H647-54. 80. Kolodgie FD, Gold HK, Burke AP, Fowler DR, Kruth HS, Weber DK, Farb A, Guerrero LJ, Hayase M, Kutys R, Narula J, Finn AV, Virmani R. Intraplaque hemorrhage and progression of coronary atheroma. N Engl J Med. 2003;349:2316-25. 81. Majno G, Gilmore V, Leventhal M. On the mechanism of vascular leakage caused by histaminetype mediators. A microscopic study in vivo. Circ Res. 1967;21:833-47. 145 | P a g e  82. Majno G, Palade GE. Studies on inflammation. I. The effect of histamine and serotonin on vascular permeability: an electron microscopic study. J Biophys Biochem Cytol. 1961;11:571605. 83. Majno G, Palade GE, Schoefl GI. Studies on inflammation. II. The site of action of histamine and serotonin along the vascular tree. A topographic study. J Biophys Biochem Cytol. 1961;11:607-26. 84. Majno G, Shea SM, Leventhal M. Endothelial contraction induced by histamine-type mediators: an electron microscopic study. J Cell Biol. 1969;42:647-72. 85. Grega GJ, Adamski SW. The role of venular endothelial cells in the regulation of macromolecular permeability. Microcirc Endothelium Lymphatics. 1988;4:143-67. 86. Hansson GK, Holm J, Jonasson L. Detection of activated T lymphocytes in the human atherosclerotic plaque. Am J Pathol. 1989;135:169-75. 87. Libby P, Hansson GK. Involvement of the immune system in human atherogenesis: current knowledge and unanswered questions. Lab Invest. 1991;64:5-15. 88. Frost H, Hess H. [Pathogenesis of arterial occlusive diseases. II. Observations with stereoscan electron microscope on the reparations of endothelial defects in arteries]. Klin Wochenschr. 1969;47:245-9. 89. Constantinides P. The morphological basis for altered endothelial permeability in artherosclerosis. Adv Exp Med Biol. 1977;82:969-74. 90. Frost H. [Endothelial lesions and deposits of blood elements as the initial event in the pathogenesis of arteriosclerosis]. Verh Dtsch Ges Inn Med. 1972;78:1139-45. 91. Shimamoto T, Sunaga T, Yamashita Y, Numano F. [Progress in the study of atherosclerosis by the appearance of scanning electron microscopy--new structure: form and pathological physiology of vascular endothelial folds and intracellular bridge]. Nippon Rinsho. 1970;28:1529-43. 92. Irie S, Tavassoli M. Transendothelial transport of macromolecules: the concept of tissueblood barriers. Cell Biol Rev. 1991;25:317-33, 40-1. 93. Friedman MH, Ehrlich LW. Effect of spatial variations in shear on diffusion at the wall of an arterial branch. Circ Res. 1975;37:446-54. 94. Carew TE, Patel DJ. Effect of tensile and shear stress on intimal permeability of the left coronary artery in dogs. Atherosclerosis. 1973;18:179-89. 146 | P a g e  95. Barath P, Fishbein MC, Cao J, Berenson J, Helfant RH, Forrester JS. Detection and localization of tumor necrosis factor in human atheroma. Am J Cardiol. 1990;65:297-302. 96. Stemme S, Jonasson L, Holm J, Hansson GK. Immunologic control of vascular cell growth in arterial response to injury and atherosclerosis. Transplant Proc. 1989;21:3697-9. 97. Arbustini E, Grasso M, Diegoli M, Pucci A, Bramerio M, Ardissino D, Angoli L, de Servi S, Bramucci E, Mussini A, et al. Coronary atherosclerotic plaques with and without thrombus in ischemic heart syndromes: a morphologic, immunohistochemical, and biochemical study. Am J Cardiol. 1991;68:36B-50B. 98. Rus HG, Niculescu F, Vlaicu R. Tumor necrosis factor-alpha in human arterial wall with atherosclerosis. Atherosclerosis. 1991;89:247-54. 99. Clejan S, Japa S, Clemetson C, Hasabnis SS, David O, Talano JV. Blood histamine is associated with coronary artery disease, cardiac events and severity of inflammation and atherosclerosis. J Cell Mol Med. 2002;6:583-92. 100. Owens GK, Hollis TM. Relationship between inhibition of aortic histamine formation, aortic albumin permeability and atherosclerosis. Atherosclerosis. 1979;34:365-73. 101. Hollis TM, Furniss JV. Relationship between aortic histamine formation and aortic albumin permeability in atherogenesis. Proc Soc Exp Biol Med. 1980;165:271-4. 102. Couffinhal T, Kearney M, Witzenbichler B, Chen D, Murohara T, Losordo DW, Symes J, Isner JM. Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in normal and atherosclerotic human arteries. Am J Pathol. 1997;150:1673-85. 103. Rahmani M, Cruz RP, Granville DJ, McManus BM. Allograft vasculopathy versus atherosclerosis. Circ Res. 2006;99:801-15. 104. Treasure CB, Alexander RW. Relevance of vascular biology to the ischemic syndromes of coronary atherosclerosis. Cardiovasc Drugs Ther. 1995;9:13-9. 105. Adams DH, Tilney NL, Collins JJ, Jr., Karnovsky MJ. Experimental graft arteriosclerosis. I. The Lewis-to-F-344 allograft model. Transplantation. 1992;53:1115-9. 106. Lai JC, Tranfield EM, Walker DC, Dyck J, Kerjner A, Loo S, English D, Wong D, McDonald PC, Moghadasian MH, Wilson JE, McManus BM. Ultrastructural evidence of early endothelial damage in coronary arteries of rat cardiac allografts. J Heart Lung Transplant. 2003;22:9931004.  147 | P a g e  107. Skarsgard PL, Wang X, McDonald P, Lui AH, Lam EK, McManus BM, van Breemen C, Laher I. Profound inhibition of myogenic tone in rat cardiac allografts is due to eNOS- and iNOSbased nitric oxide and an intrinsic defect in vascular smooth muscle contraction. Circulation. 2000;101:1303-10. 108. Moien-Afshari F, Choy JC, McManus BM, Laher I. Cyclosporine treatment preserves coronary resistance artery function in rat cardiac allografts. J Heart Lung Transplant. 2004;23:193-203. 109. Lui AH, McManus BM, Laher I. Endothelial and myogenic regulation of coronary artery tone in the mouse. Eur J Pharmacol. 2000;410:25-31. 110. Walker DC, MacKenzie A, Hosford S. The structure of the tricellular region of endothelial tight junctions of pulmonary capillaries analyzed by freeze-fracture. Microvasc Res. 1994;48:259-81. 111. Dong C, Granville DJ, Tuffnel CE, Kenyon J, English D, Wilson JE, McManus BM. Bax and apoptosis in acute and chronic rejection of rat cardiac allografts. Lab Invest. 1999;79:1643-53. 112. Lin H, Ignatescu M, Wilson JE, Roberts CR, Horley KJ, Winters GL, Costanzo MR, McManus BM. Prominence of apolipoproteins B, (a), and E in the intimae of coronary arteries in transplanted human hearts: geographic relationship to vessel wall proteoglycans. J Heart Lung Transplant. 1996;15:1223-32. 113. Gough PJ, Gordon S. The role of scavenger receptors in the innate immune system. Microbes Infect. 2000;2:305-11. 114. de Lorgeril M, Richard MJ, Arnaud J, Boissonnat P, Guidollet J, Dureau G, Renaud S, Favier A. Lipid peroxides and antioxidant defenses in accelerated transplantation-associated coronary arteriosclerosis. Am Heart J. 1993;125:974-80. 115. de Lorgeril M, Richard MJ, Arnaud J, Boissonnat P, Guidollet J, Dureau G, Renaud S, Favier A. Increased production of reactive oxygen species in pharmacologicallyimmunosuppressed patients. Chem Biol Interact. 1994;91:159-64. 116. Gullestad L, Nordal KP, Forfang K, Ihlen H, Hostmark A, Berg KJ, Cheng H, Schwartz MS, Geiran O, Simonsen S. Post-transplant hyperlipidaemia: low-dose lovastatin lowers atherogenic lipids without plasma accumulation of lovastatin. J Intern Med. 1997;242:483-90. 117. Clough G, Michel CC. Quantitative comparisons of hydraulic permeability and endothelial intercellular cleft dimensions in single frog capillaries. J Physiol (Lond). 1988;405:563-76. 148 | P a g e  118. Thurston G, Baluk P, Hirata A, McDonald DM. Permeability-related changes revealed at endothelial cell borders in inflamed venules by lectin binding. Am J Physiol. 1996;271:H2547-62. 119. Gerrity RG, Richardson M, Somer JB, Bell FP, Schwartz CJ. Endothelial cell morphology in areas of in vivo Evans blue uptake in the aorta of young pigs. II. Ultrastructure of the intima in areas of differing permeability to proteins. Am J Pathol. 1977;89:313-34. 120. Adamson RH. Permeability of frog mesenteric capillaries after partial pronase digestion of the endothelial glycocalyx. J Physiol (Lond). 1990;428:1-13. 121. Henry CB, Duling BR. Permeation of the luminal capillary glycocalyx is determined by hyaluronan. Am J Physiol. 1999;277:H508-14. 122. Huxley VH, Williams DA. Role of a glycocalyx on coronary arteriole permeability to proteins: evidence from enzyme treatments. Am J Physiol Heart Circ Physiol. 2000;278:H1177H85. 123. Ghitescu L, Fixman A, Simionescu M, Simionescu N. Specific binding sites for albumin restricted to plasmalemmal vesicles of continuous capillary endothelium: receptor-mediated transcytosis. J Cell Biol. 1986;102:1304-11. 124. Ghitescu L, Galis Z, Simionescu M, Simionescu N. Differentiated uptake and transcytosis of albumin in successive vascular segments. J Submicrosc Cytol Pathol. 1988;20:657-69. 125. Wagner RC, Robinson CS, Cross PJ, Devenny JJ. Endocytosis and exocytosis of transferrin by isolated capillary endothelium. Microvasc Res. 1983;25:387-96. 126. Omoto E, Minguell JJ, Tavassoli M. Endothelial transcytosis of iron-transferrin in the liver does not involve endosomal traffic. Pathobiology. 1992;60:284-8. 127. King GL, Johnson SM. Receptor-mediated transport of insulin across endothelial cells. Science. 1985;227:1583-6. 128. Solenski NJ, Williams SK. Insulin binding and vesicular ingestion in capillary endothelium. J Cell Physiol. 1985;124:87-95. 129. Poumay Y, Ronveaux-Dupal MF. Endocytosis of low density lipoproteins in human endothelial cells: typical morphological aspects of the high affinity receptor-mediated pathway as revealed by serial sections and acid phosphatase cytochemistry. J Submicrosc Cytol Pathol. 1989;21:627-39. 130. Simionescu N, Simionescu M. Cellular interactions of lipoproteins with the vascular endothelium: endocytosis and transcytosis. Targeted Diagn Ther. 1991;5:45-95. 149 | P a g e  131. Lamaze C, Fujimoto LM, Yin HL, Schmid SL. The actin cytoskeleton is required for receptor-mediated endocytosis in mammalian cells. J Biol Chem. 1997;272:20332-5. 132. Aniento F, Roche E, Cuervo AM, Knecht E. Uptake and degradation of glyceraldehyde-3phosphate dehydrogenase by rat liver lysosomes. J Biol Chem. 1993;268:10463-70. 133. Lamaze C, Schmid SL. Recruitment of epidermal growth factor receptors into coated pits requires their activated tyrosine kinase. J Cell Biol. 1995;129:47-54. 134. Nesterov A, Kurten RC, Gill GN. Association of epidermal growth factor receptors with coated pit adaptins via a tyrosine phosphorylation-regulated mechanism. J Biol Chem. 1995;270:6320-7. 135. Boll W, Gallusser A, Kirchhausen T. Role of the regulatory domain of the EGF-receptor cytoplasmic tail in selective binding of the clathrin-associated complex AP-2. Curr Biol. 1995;5:1168-78. 136. Joly M, Kazlauskas A, Fay FS, Corvera S. Disruption of PDGF receptor trafficking by mutation of its PI-3 kinase binding sites. Science. 1994;263:684-7. 137. Joly M, Kazlauskas A, Corvera S. Phosphatidylinositol 3-kinase activity is required at a postendocytic step in platelet-derived growth factor receptor trafficking. J Biol Chem. 1995;270:13225-30. 138. Sato SB, Taguchi T, Yamashina S, Toyama S. Wortmannin and Li+ specifically inhibit clathrin-independent endocytic internalization of bulk fluid. J Biochem (Tokyo). 1996;119:88797. 139. Holm PK, Eker P, Sandvig K, van Deurs B. Phorbol myristate acetate selectively stimulates apical endocytosis via protein kinase C in polarized MDCK cells. Exp Cell Res. 1995;217:157-68. 140. Bradbury NA, Bridges RJ. Endocytosis is regulated by protein kinase A, but not protein kinase C in a secretory epithelial cell line. Biochem Biophys Res Commun. 1992;184:1173-80. 141. Rothberg KG, Heuser JE, Donzell WC, Ying YS, Glenney JR, Anderson RG. Caveolin, a protein component of caveolae membrane coats. Cell. 1992;68:673-82. 142.  Anderson RG. The caveolae membrane system. Annu Rev Biochem. 1998;67:199-225.  143. Takaoka A, Mitani Y, Suemori H, Sato M, Yokochi T, Noguchi S, Tanaka N, Taniguchi T. Cross talk between interferon-gamma and -alpha/beta signaling components in caveolar membrane domains. Science. 2000;288:2357-60. 150 | P a g e  144. Schmid SL. Clathrin-coated vesicle formation and protein sorting: an integrated process. Annu Rev Biochem. 1997;66:511-48. 145. Smart EJ, Foster DC, Ying YS, Kamen BA, Anderson RG. Protein kinase C activators inhibit receptor-mediated potocytosis by preventing internalization of caveolae. J Cell Biol. 1994;124:307-13. 146. Anderson HA, Chen Y, Norkin LC. Bound simian virus 40 translocates to caveolinenriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae. Mol Biol Cell. 1996;7:1825-34. 147. Feng D, Nagy JA, Hipp J, Dvorak HF, Dvorak AM. Vesiculo-vacuolar organelles and the regulation of venule permeability to macromolecules by vascular permeability factor, histamine, and serotonin. J Exp Med. 1996;183:1981-6. 148. Feng D, Nagy JA, Hipp J, Pyne K, Dvorak HF, Dvorak AM. Reinterpretation of endothelial cell gaps induced by vasoactive mediators in guinea-pig, mouse and rat: many are transcellular pores. J Physiol (Lond). 1997;504:747-61. 149. Wong MK, Gotlieb AI. Endothelial cell monolayer integrity. I. Characterization of dense peripheral band of microfilaments. Arteriosclerosis. 1986;6:212-9. 150. Phillips PG, Lum H, Malik AB, Tsan MF. Phallacidin prevents thrombin-induced increases in endothelial permeability to albumin. Am J Physiol. 1989;257:C562-7. 151. Schnittler HJ, Wilke A, Gress T, Suttorp N, Drenckhahn D. Role of actin and myosin in the control of paracellular permeability in pig, rat and human vascular endothelium. J Physiol (Lond). 1990;431:379-401. 152. Madara JL. Intestinal absorptive cell tight junctions are linked to cytoskeleton. Am J Physiol. 1987;253:C171-5. 153. Stevenson BR, Begg DA. Concentration-dependent effects of cytochalasin D on tight junctions and actin filaments in MDCK epithelial cells. J Cell Sci. 1994;107:367-75. 154. Fasano A, Fiorentini C, Donelli G, Uzzau S, Kaper JB, Margaretten K, Ding X, Guandalini S, Comstock L, Goldblum SE. Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro. J Clin Invest. 1995;96:710-20. 155. Peterson JA, Tian B, McLaren JW, Hubbard WC, Geiger B, Kaufman PL. Latrunculins' effects on intraocular pressure, aqueous humor flow, and corneal endothelium. Invest Ophthalmol Vis Sci. 2000;41:1749-58. 151 | P a g e  156.  Citi S, Cordenonsi M. Tight junction proteins. Biochim Biophys Acta. 1998;1448:1-11.  157. Garcia JG, Davis HW, Patterson CE. Regulation of endothelial cell gap formation and barrier dysfunction: role of myosin light chain phosphorylation. J Cell Physiol. 1995;163:510-22. 158. Moy AB, Shasby SS, Scott BD, Shasby DM. The effect of histamine and cyclic adenosine monophosphate on myosin light chain phosphorylation in human umbilical vein endothelial cells. J Clin Invest. 1993;92:1198-206. 159. Wysolmerski RB, Lagunoff D. Regulation of permeabilized endothelial cell retraction by myosin phosphorylation. Am J Physiol. 1991;261:C32-40. 160. Sheldon R, Moy A, Lindsley K, Shasby S, Shasby DM. Role of myosin light-chain phosphorylation in endothelial cell retraction. Am J Physiol. 1993;265:L606-12. 161. Gilbert-McClain LI, Verin AD, Shi S, Irwin RP, Garcia JG. Regulation of endothelial cell myosin light chain phosphorylation and permeability by vanadate. J Cell Biochem. 1998;70:14155. 162. Hirase T, Staddon JM, Saitou M, Ando-Akatsuka Y, Itoh M, Furuse M, Fujimoto K, Tsukita S, Rubin LL. Occludin as a possible determinant of tight junction permeability in endothelial cells. J Cell Sci. 1997;110:1603-13. 163. Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S. Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol. 1993;123:1777-88. 164. Furuse M, Fujimoto K, Sato N, Hirase T, Tsukita S, Tsukita S. Overexpression of occludin, a tight junction-associated integral membrane protein, induces the formation of intracellular multilamellar bodies bearing tight junction-like structures. J Cell Sci. 1996;109:429-35. 165. Savettieri G, Di Liegro I, Catania C, Licata L, Pitarresi GL, S DA, Schiera G, De Caro V, Giandalia G, Giannola LI, Cestelli A. Neurons and ECM regulate occludin localization in brain endothelial cells. Neuroreport. 2000;11:1081-4. 166. McCarthy KM, Skare IB, Stankewich MC, Furuse M, Tsukita S, Rogers RA, Lynch RD, Schneeberger EE. Occludin is a functional component of the tight junction. J Cell Sci. 1996;109:2287-98. 167. Jiang WG, Martin TA, Matsumoto K, Nakamura T, Mansel RE. Hepatocyte growth factor/scatter factor decreases the expression of occludin and transendothelial resistance (TER) and increases paracellular permeability in human vascular endothelial cells. J Cell Physiol. 1999;181:319-29. 152 | P a g e  168. Li CX, Poznansky MJ. Characterization of the ZO-1 protein in endothelial and other cell lines. J Cell Sci. 1990;97:231-7. 169. Furuse M, Itoh M, Hirase T, Nagafuchi A, Yonemura S, Tsukita S. Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol. 1994;127:1617-26. 170. Itoh M, Furuse M, Morita K, Kubota K, Saitou M, Tsukita S. Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins. J Cell Biol. 1999;147:1351-63. 171. Itoh M, Nagafuchi A, Moroi S, Tsukita S. Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to alpha catenin and actin filaments. J Cell Biol. 1997;138:181-92. 172. Wittchen ES, Haskins J, Stevenson BR. Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3. J Biol Chem. 1999;274:35179-85. 173. Itoh M, Morita K, Tsukita S. Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin. J Biol Chem. 1999;274:5981-6. 174. Haskins J, Gu L, Wittchen ES, Hibbard J, Stevenson BR. ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin. J Cell Biol. 1998;141:199-208. 175. Fanning AS, Jameson BJ, Jesaitis LA, Anderson JM. The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J Biol Chem. 1998;273:29745-53. 176. Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol. 1998;141:1539-50. 177. Furuse M, Sasaki H, Fujimoto K, Tsukita S. A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts. J Cell Biol. 1998;143:391-401. 178. Tsukita S, Furuse M. Overcoming barriers in the study of tight junction functions: from occludin to claudin. Genes Cells. 1998;3:569-73.  153 | P a g e  179. Muresan Z, Paul DL, Goodenough DA. Occludin 1B, a variant of the tight junction protein occludin. Mol Biol Cell. 2000;11:627-34. 180. Morita K, Furuse M, Fujimoto K, Tsukita S. Claudin multigene family encoding fourtransmembrane domain protein components of tight junction strands. Proc Natl Acad Sci U S A. 1999;96:511-6. 181. Tsukita S, Furuse M. Pores in the wall: Claudins constitute tight junction strands containing aqueous pores. J Cell Biol. 2000;149:13-6. 182. Navarro P, Ruco L, Dejana E. Differential localization of VE- and N-cadherins in human endothelial cells: VE-cadherin competes with N-cadherin for junctional localization. J Cell Biol. 1998;140:1475-84. 183. Lampugnani MG, Corada M, Caveda L, Breviario F, Ayalon O, Geiger B, Dejana E. The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, beta-catenin, and alpha-catenin with vascular endothelial cadherin (VE-cadherin). J Cell Biol. 1995;129:203-17. 184. Navarro P, Caveda L, Breviario F, Mandoteanu I, Lampugnani MG, Dejana E. Catenindependent and -independent functions of vascular endothelial cadherin. J Biol Chem. 1995;270:30965-72. 185. Caveda L, Martin-Padura I, Navarro P, Breviario F, Corada M, Gulino D, Lampugnani MG, Dejana E. Inhibition of cultured cell growth by vascular endothelial cadherin (cadherin-5/VEcadherin). J Clin Invest. 1996;98:886-93. 186. Lampugnani MG, Corada M, Andriopoulou P, Esser S, Risau W, Dejana E. Cell confluence regulates tyrosine phosphorylation of adherens junction components in endothelial cells. J Cell Sci. 1997;110:2065-77. 187. Esser S, Lampugnani MG, Corada M, Dejana E, Risau W. Vascular endothelial growth factor induces VE-cadherin tyrosine phosphorylation in endothelial cells. J Cell Sci. 1998;111:1853-65. 188. Wong RK, Baldwin AL, Heimark RL. Cadherin-5 redistribution at sites of TNF-alpha and IFN-gamma-induced permeability in mesenteric venules. Am J Physiol. 1999;276:H736-48. 189. Del Maschio A, Zanetti A, Corada M, Rival Y, Ruco L, Lampugnani MG, Dejana E. Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions. J Cell Biol. 1996;135:497-510.  154 | P a g e  190. Rabiet MJ, Plantier JL, Dejana E. Thrombin-induced endothelial cell dysfunction. Br Med Bull. 1994;50:936-45. 191. Corada M, Mariotti M, Thurston G, Smith K, Kunkel R, Brockhaus M, Lampugnani MG, Martin-Padura I, Stoppacciaro A, Ruco L, McDonald DM, Ward PA, Dejana E. Vascular endothelial-cadherin is an important determinant of microvascular integrity in vivo. Proc Natl Acad Sci U S A. 1999;96:9815-20. 192. Hordijk PL, Anthony E, Mul FP, Rientsma R, Oomen LC, Roos D. Vascular-endothelialcadherin modulates endothelial monolayer permeability. J Cell Sci. 1999;112:1915-23. 193. Castilla MA, Arroyo MV, Aceituno E, Aragoncillo P, Gonzalez-Pacheco FR, Texeiro E, Bragado R, Caramelo C. Disruption of cadherin-related junctions triggers autocrine expression of vascular endothelial growth factor in bovine aortic endothelial cells : effects on cell proliferation and death resistance. Circ Res. 1999;85:1132-8. 194. Gulino D, Delachanal E, Concord E, Genoux Y, Morand B, Valiron MO, Sulpice E, Scaife R, Alemany M, Vernet T. Alteration of endothelial cell monolayer integrity triggers resynthesis of vascular endothelium cadherin. J Biol Chem. 1998;273:29786-93. 195. Schulze C, Firth JA. Immunohistochemical localization of adherens junction components in blood-brain barrier microvessels of the rat. J Cell Sci. 1993;104:773-82. 196. Telo P, Lostaglio S, Dejana E. Structure of intercellular junctions in the endothelium. Therapie. 1997;52:395-8. 197. Aberle H, Schwartz H, Kemler R. Cadherin-catenin complex: protein interactions and their implications for cadherin function. J Cell Biochem. 1996;61:514-23. 198. Schnittler HJ, Puschel B, Drenckhahn D. Role of cadherins and plakoglobin in interendothelial adhesion under resting conditions and shear stress. Am J Physiol. 1997;273:H2396-405. 199. Coll JL, Ben-Ze'ev A, Ezzell RM, Rodriguez Fernandez JL, Baribault H, Oshima RG, Adamson ED. Targeted disruption of vinculin genes in F9 and embryonic stem cells changes cell morphology, adhesion, and locomotion. Proc Natl Acad Sci U S A. 1995;92:9161-5. 200. Rodriguez Fernandez JL, Geiger B, Salomon D, Ben-Ze'ev A. Overexpression of vinculin suppresses cell motility in BALB/c 3T3 cells. Cell Motil Cytoskeleton. 1992;22:127-34. 201. Johnson RP, Craig SW. Actin activates a cryptic dimerization potential of the vinculin tail domain. J Biol Chem. 2000;275:95-105. 155 | P a g e  202. Muhs A, Noll T, Piper HM. Vinculin phosphorylation and barrier failure of coronary endothelial monolayers under energy depletion. Am J Physiol. 1997;273:H608-17. 203. Cruz A, DeFouw LM, DeFouw DO. Restrictive endothelial barrier function during normal angiogenesis in vivo: Partial dependence on tyrosine dephosphorylation of beta-catenin. Microvasc Res. 2000;59:195-203. 204. Brady-Kalnay SM, Mourton T, Nixon JP, Pietz GE, Kinch M, Chen H, Brackenbury R, Rimm DL, Del Vecchio RL, Tonks NK. Dynamic interaction of PTPmu with multiple cadherins in vivo. J Cell Biol. 1998;141:287-96. 205. Bazzoni G, Dejana E, Lampugnani MG. Endothelial adhesion molecules in the development of the vascular tree: the garden of forking paths. Curr Opin Cell Biol. 1999;11:57381. 206. Hynes RO, Bader BL, Hodivala-Dilke K. Integrins in vascular development. Braz J Med Biol Res. 1999;32:501-10. 207.  Zachary I. Focal adhesion kinase. Int J Biochem Cell Biol. 1997;29:929-34.  208. Ilic D, Damsky CH, Yamamoto T. Focal adhesion kinase: at the crossroads of signal transduction. J Cell Sci. 1997;110:401-7. 209. Vepa S, Scribner WM, Parinandi NL, English D, Garcia JG, Natarajan V. Hydrogen peroxide stimulates tyrosine phosphorylation of focal adhesion kinase in vascular endothelial cells. Am J Physiol. 1999;277:L150-8. 210. Yuan Y, Meng FY, Huang Q, Hawker J, Wu HM. Tyrosine phosphorylation of paxillin/pp125FAK and microvascular endothelial barrier function. Am J Physiol. 1998;275:H8493. 211. Schaphorst KL, Pavalko FM, Patterson CE, Garcia JG. Thrombin-mediated focal adhesion plaque reorganization in endothelium: role of protein phosphorylation. Am J Respir Cell Mol Biol. 1997;17:443-55. 212.  Turner CE. Paxillin. Int J Biochem Cell Biol. 1998;30:955-9.  213. Thomas JW, Cooley MA, Broome JM, Salgia R, Griffin JD, Lombardo CR, Schaller MD. The role of focal adhesion kinase binding in the regulation of tyrosine phosphorylation of paxillin. J Biol Chem. 1999;274:36684-92. 214.  Hanks SK, Polte TR. Signaling through focal adhesion kinase. Bioessays. 1997;19:137-45. 156 | P a g e  215. Goldmann WH, Bremer A, Haner M, Aebi U, Isenberg G. Native talin is a dumbbellshaped homodimer when it interacts with actin. J Struct Biol. 1994;112:3-10. 216. Kaufmann S, Piekenbrock T, Goldmann WH, Barmann M, Isenberg G. Talin binds to actin and promotes filament nucleation. FEBS Lett. 1991;284:187-91. 217. Muguruma M, Matsumura S, Fukazawa T. Augmentation of alpha-actinin-induced gelation of actin by talin. J Biol Chem. 1992;267:5621-4. 218. Hildebrand JD, Taylor JM, Parsons JT. An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase. Mol Cell Biol. 1996;16:316978. 219. Schaller MD. Signaling through the focal adhesion kinase. Soc Gen Physiol Ser. 1997;52:241-55. 220. Carbajal JM, Schaeffer RC, Jr. RhoA inactivation enhances endothelial barrier function. Am J Physiol. 1999;277:C955-64. 221. Albelda SM, Oliver PD, Romer LH, Buck CA. EndoCAM: a novel endothelial cell-cell adhesion molecule. J Cell Biol. 1990;110:1227-37. 222. Albelda SM, Daise M, Levine EM, Buck CA. Identification and characterization of cellsubstratum adhesion receptors on cultured human endothelial cells. J Clin Invest. 1989;83:1992-2002. 223. Lampugnani MG, Resnati M, Dejana E, Marchisio PC. The role of integrins in the maintenance of endothelial monolayer integrity. J Cell Biol. 1991;112:479-90. 224. Gao B, Curtis TM, Blumenstock FA, Minnear FL, Saba TM. Increased recycling of (alpha)5(beta)1 integrins by lung endothelial cells in response to tumor necrosis factor. J Cell Sci. 2000;113:247-57. 225. Curtis TM, Rotundo RF, Vincent PA, McKeown-Longo PJ, Saba TM. TNF-alpha-induced matrix Fn disruption and decreased endothelial integrity are independent of Fn proteolysis. Am J Physiol. 1998;275:L126-38. 226. Parker KH, Winlove CP. The macromolecular and ultrastructural basis of the permeability properties of the vascular wall. Eng Med. 1988;17:175-80. 227. Lum H, Malik AB. Regulation of vascular endothelial barrier function. Am J Physiol. 1994;267:L223-41. 157 | P a g e  228. Clough G. Relationship between microvascular permeability and ultrastructure. Prog Biophys Mol Biol. 1991;55:47-69. 229. Svensjo E, Arfors KE, Arturson G, Rutili G. The hamster cheek pouch preparation as a model for studies of macromolecular permeability of the microvasculature. Ups J Med Sci. 1978;83:71-9. 230. Arfors KE, Rutili G, Svensjo E. Microvascular transport of macromolecules in normal and inflammatory conditions. Acta Physiol Scand Suppl. 1979;463:93-103. 231. Lampugnani MG, Caveda L, Breviario F, Del Maschio A, Dejana E. Endothelial cell-to-cell junctions. Structural characteristics and functional role in the regulation of vascular permeability and leukocyte extravasation. Baillieres Clin Haematol. 1993;6:539-58. 232. Steele RH, Wilhelm DL. The inflammatry reaction in chemical injury. II. Vascular permeability changes and necrosis induced by intracutaneous injection of various chemicals. Br J Exp Pathol. 1967;48:592-607. 233. van Hinsbergh WM. Endothelial permeability for macromolecules. Mechanistic aspects of pathophysiological modulation. Arterioscler Thromb Vasc Biol. 1997;17:1018-23. 234. Morel NM, Dodge AB, Patton WF, Herman IM, Hechtman HB, Shepro D. Pulmonary microvascular endothelial cell contractility on silicone rubber substrate. J Cell Physiol. 1989;141:653-9. 235. Lampugnani MG, Resnati M, Raiteri M, Pigott R, Pisacane A, Houen G, Ruco LP, Dejana E. A novel endothelial-specific membrane protein is a marker of cell-cell contacts. J Cell Biol. 1992;118:1511-22. 236. Bolton SJ, Anthony DC, Perry VH. Loss of the tight junction proteins occludin and zonula occludens-1 from cerebral vascular endothelium during neutrophil-induced blood-brain barrier breakdown in vivo. Neuroscience. 1998;86:1245-57. 237. Curry FE. Modulation of venular microvessel permeability by calcium influx into endothelial cells. FASEB J. 1992;6:2456-66. 238. Siflinger-Birnboim A, Malik AB. Regulation of endothelial permeability by second messengers. New Horiz. 1996;4:87-98. 239. Kelly JJ, Moore TM, Babal P, Diwan AH, Stevens T, Thompson WJ. Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+ and permeability. Am J Physiol. 1998;274:L810-9. 158 | P a g e  240. Lum H, Aschner JL, Phillips PG, Fletcher PW, Malik AB. Time course of thrombin-induced increase in endothelial permeability: relationship to Ca2+i and inositol polyphosphates. Am J Physiol. 1992;263:L219-25. 241. Lynch JJ, Ferro TJ, Blumenstock FA, Brockenauer AM, Malik AB. Increased endothelial albumin permeability mediated by protein kinase C activation. J Clin Invest. 1990;85:1991-8. 242. Nagpala PG, Malik AB, Vuong PT, Lum H. Protein kinase C beta 1 overexpression augments phorbol ester-induced increase in endothelial permeability. J Cell Physiol. 1996;166:249-55. 243. Johnson A, Phelps DT, Ferro TJ. Tumor necrosis factor-alpha decreases pulmonary artery endothelial nitrovasodilator via protein kinase C. Am J Physiol. 1994;267:L318-25. 244. Siflinger-Birnboim A, Goligorsky MS, Del Vecchio PJ, Malik AB. Activation of protein kinase C pathway contributes to hydrogen peroxide-induced increase in endothelial permeability. Lab Invest. 1992;67:24-30. 245. Rabiet MJ, Plantier JL, Rival Y, Genoux Y, Lampugnani MG, Dejana E. Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization. Arterioscler Thromb Vasc Biol. 1996;16:488-96. 246. Hoek JB. Intracellular signal transduction and the control of endothelial permeability [editorial; comment]. Lab Invest. 1992;67:1-4. 247. Stanimirovic D, Morley P, Ball R, Hamel E, Mealing G, Durkin JP. Angiotensin II-induced fluid phase endocytosis in human cerebromicrovascular endothelial cells is regulated by the inositol-phosphate signaling pathway. J Cell Physiol. 1996;169:455-67. 248. Tsukita S, Oishi K, Akiyama T, Yamanashi Y, Yamamoto T, Tsukita S. Specific protooncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated. J Cell Biol. 1991;113:867-79. 249. Staddon JM, Herrenknecht K, Smales C, Rubin LL. Evidence that tyrosine phosphorylation may increase tight junction permeability. J Cell Sci. 1995;108:609-19. 250. Tiruppathi C, Song W, Bergenfeldt M, Sass P, Malik AB. Gp60 activation mediates albumin transcytosis in endothelial cells by tyrosine kinase-dependent pathway. J Biol Chem. 1997;272:25968-75. 251. Brass LF. Homologous desensitization of HEL cell thrombin receptors. Distinguishable roles for proteolysis and phosphorylation. J Biol Chem. 1992;267:6044-50. 159 | P a g e  252. Weintraub WH, Negulescu PA, Machen TE. Calcium signaling in endothelia: cellular heterogeneity and receptor internalization. Am J Physiol. 1992;263:C1029-39. 253. Aschner JL, Lum H, Fletcher PW, Malik AB. The differential effects of protein kinase C (PKC) on bradykinin (BK)- and thrombin (T)-mediated phospholipase C (PLC) activation. FASEB J. 1993;7:A719. 254. Pachter JA, Pai JK, Mayer-Ezell R, Petrin JM, Dobek E, Bishop WR. Differential regulation of phosphoinositide and phosphatidylcholine hydrolysis by protein kinase C-beta 1 overexpression. Effects on stimulation by alpha-thrombin, guanosine 5'-O-(thiotriphosphate), and calcium. J Biol Chem. 1992;267:9826-30. 255. Verin AD, Patterson CE, Day MA, Garcia JG. Regulation of endothelial cell gap formation and barrier function by myosin-associated phosphatase activities. Am J Physiol. 1995;269:L99108. 256. Draijer R, Atsma DE, van der Laarse A, van Hinsbergh VW. cGmp and nitric oxide modulate thrombin-induced endothelial permeability. Regulation via different pathways in human aortic and umbilical vein endothelial cells. Circ Res. 1995;76:199-208. 257. Baron DA, Lofton CE, Newman WH, Currie MG. Atriopeptin inhibition of thrombinmediated changes in the morphology and permeability of endothelial monolayers. Proc Natl Acad Sci U S A. 1989;86:3394-8. 258. Joo F. Minireview: regulation by second messengers of permeability in the cerebral microvessels. Neurobiology (Bp). 1993;1:3-10. 259. Kempski O, Villacara A, Spatz M, Dodson RF, Corn C, Merkel N, Bembry J. Cerebromicrovascular endothelial permeability. In-vitro studies. Acta Neuropathol (Berl). 1987;74:329-34. 260. Ide AG, Baker NH, Warren SL. Vascularization of the Brown Pearce rabbit epithelioma transplant as seen in the transparent ear chamber. AJR Am J Roentgenol. 1939;42:891-9. 261. Algire GH, Chalkley HW, Legallais FY, Park HD. Vascular reactions of normal and malignant tissues in vivo. I. Vascular reactions of mice to wounds and to normal and neoplastic transplants. J Natl Cancer Inst. 1945;6:73-85. 262. Greenblatt M, Shubick P. Tumor angiogenesis: transfilter diffusion studies in the hamster by the transparent chamber technique. J Natl Cancer Inst. 1968;41:111-24. 263. 6.  Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971;285:1182160 | P a g e  264. Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science. 1983;219:983-5. 265. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989;246:1306-9. 266. Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, Connolly DT. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science. 1989;246:1309-12. 267. Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O'Shea KS, Powell-Braxton L, Hillan KJ, Moore MW. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996;380:439-42. 268. Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature. 1996;380:435-9. 269. Kowanetz M, Ferrara N. Vascular endothelial growth factor signaling pathways: therapeutic perspective. Clin Cancer Res. 2006;12:5018-22. 270. Red-Horse K, Crawford Y, Shojaei F, Ferrara N. Endothelium-microenvironment interactions in the developing embryo and in the adult. Dev Cell. 2007;12:181-94. 271. Houck KA, Leung DW, Rowland AM, Winer J, Ferrara N. Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem. 1992;267:26031-7. 272. Keyt BA, Nguyen HV, Berleau LT, Duarte CM, Park J, Chen H, Ferrara N. Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis. J Biol Chem. 1996;271:5638-46. 273. Lee S, Jilani SM, Nikolova GV, Carpizo D, Iruela-Arispe ML. Processing of VEGF-A by matrix metalloproteinases regulates bioavailability and vascular patterning in tumors. J Cell Biol. 2005;169:681-91. 274. Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling - in control of vascular function. Nat Rev Mol Cell Biol. 2006;7:359-71. 275. Breier G, Damert A, Plate KH, Risau W. Angiogenesis in embryos and ischemic diseases. Thromb Haemost. 1997;78:678-83. 161 | P a g e  276. Gerber HP, Hillan KJ, Ryan AM, Kowalski J, Keller GA, Rangell L, Wright BD, Radtke F, Aguet M, Ferrara N. VEGF is required for growth and survival in neonatal mice. Development. 1999;126:1149-59. 277. Mattot V, Moons L, Lupu F, Chernavvsky D, Gomez RA, Collen D, Carmeliet P. Loss of the VEGF(164) and VEGF(188) isoforms impairs postnatal glomerular angiogenesis and renal arteriogenesis in mice. J Am Soc Nephrol. 2002;13:1548-60. 278. Ferrara N. Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications. Semin Oncol. 2002;29:10-4. 279. Tjwa M, Luttun A, Autiero M, Carmeliet P. VEGF and PlGF: two pleiotropic growth factors with distinct roles in development and homeostasis. Cell Tissue Res. 2003;314:5-14. 280. Ferrara N. The role of vascular endothelial growth factor in pathological angiogenesis. Breast Cancer Res Treat. 1995;36:127-37. 281. Ferrara N. Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney Int. 1999;56:794-814. 282. Gerber HP, Wu X, Yu L, Wiesmann C, Liang XH, Lee CV, Fuh G, Olsson C, Damico L, Xie D, Meng YG, Gutierrez J, Corpuz R, Li B, Hall L, Rangell L, Ferrando R, Lowman H, Peale F, Ferrara N. Mice expressing a humanized form of VEGF-A may provide insights into the safety and efficacy of anti-VEGF antibodies. Proc Natl Acad Sci U S A. 2007;104:3478-83. 283. Im SA, Gomez-Manzano C, Fueyo J, Liu TJ, Ke LD, Kim JS, Lee HY, Steck PA, Kyritsis AP, Yung WK. Antiangiogenesis treatment for gliomas: transfer of antisense-vascular endothelial growth factor inhibits tumor growth in vivo. Cancer Res. 1999;59:895-900. 284. Khurana R, Martin JF, Zachary I. Gene therapy for cardiovascular disease: a case for cautious optimism. Hypertension. 2001;38:1210-6. 285. Ng EW, Adamis AP. Anti-VEGF aptamer (pegaptanib) therapy for ocular vascular diseases. Ann N Y Acad Sci. 2006;1082:151-71. 286. Shibuya M. VEGF-receptor inhibitors for anti-angiogenesis. Nippon Yakurigaku Zasshi. 2003;122:498-503. 287. Glass CA, Harper SJ, Bates DO. The anti-angiogenic VEGF isoform VEGF165b transiently increases hydraulic conductivity, probably through VEGF receptor 1 in vivo. J Physiol. 2006;572:243-57.  162 | P a g e  288. Ku DD, Zaleski JK, Liu S, Brock TA. Vascular endothelial growth factor induces EDRFdependent relaxation in coronary arteries. Am J Physiol. 1993;265:H586-92. 289. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev. 1997;18:4-25. 290. Greenberg JI, Shields DJ, Barillas SG, Acevedo LM, Murphy E, Huang J, Scheppke L, Stockmann C, Johnson RS, Angle N, Cheresh DA. A role for VEGF as a negative regulator of pericyte function and vessel maturation. Nature. 2008;456:809-13. 291. Unemori EN, Ferrara N, Bauer EA, Amento EP. Vascular endothelial growth factor induces interstitial collagenase expression in human endothelial cells. J Cell Physiol. 1992;153:557-62. 292. Abu-Jawdeh GM, Faix JD, Niloff J, Tognazzi K, Manseau E, Dvorak HF, Brown LF. Strong expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in ovarian borderline and malignant neoplasms. Lab Invest. 1996;74:1105-15. 293. Borgstrom P, Gold DP, Hillan KJ, Ferrara N. Importance of VEGF for breast cancer angiogenesis in vivo: implications from intravital microscopy of combination treatments with an anti-VEGF neutralizing monoclonal antibody and doxorubicin. Anticancer Res. 1999;19:4203-14. 294. Borgstrom P, Hillan KJ, Sriramarao P, Ferrara N. Complete inhibition of angiogenesis and growth of microtumors by anti-vascular endothelial growth factor neutralizing antibody: novel concepts of angiostatic therapy from intravital videomicroscopy. Cancer Res. 1996;56:4032-9. 295. Brown LF, Berse B, Jackman RW, Tognazzi K, Guidi AJ, Dvorak HF, Senger DR, Connolly JL, Schnitt SJ. Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer. Hum Pathol. 1995;26:86-91. 296. Brown LF, Guidi AJ, Schnitt SJ, Van De Water L, Iruela-Arispe ML, Yeo TK, Tognazzi K, Dvorak HF. Vascular stroma formation in carcinoma in situ, invasive carcinoma, and metastatic carcinoma of the breast. Clin Cancer Res. 1999;5:1041-56. 297. Claffey KP, Brown LF, del Aguila LF, Tognazzi K, Yeo KT, Manseau EJ, Dvorak HF. Expression of vascular permeability factor/vascular endothelial growth factor by melanoma cells increases tumor growth, angiogenesis, and experimental metastasis. Cancer Res. 1996;56:172-81. 298. Dvorak HF, Detmar M, Claffey KP, Nagy JA, van de Water L, Senger DR. Vascular permeability factor/vascular endothelial growth factor: an important mediator of angiogenesis in malignancy and inflammation. Int Arch Allergy Immunol. 1995;107:233-5. 163 | P a g e  299. Guidi AJ, Abu-Jawdeh G, Tognazzi K, Dvorak HF, Brown LF. Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in endometrial carcinoma. Cancer. 1996;78:454-60. 300. Li QM, Kan FJ, Min CY. Effect of Weikangning on gastric cancer cell growth and expression of vascular endothelial growth factor and its receptors KDR and Flt-1. World J Gastroenterol. 2005;11:938-42. 301. Mesiano S, Ferrara N, Jaffe RB. Role of vascular endothelial growth factor in ovarian cancer: inhibition of ascites formation by immunoneutralization. Am J Pathol. 1998;153:124956. 302. Slongo ML, Molena B, Brunati AM, Frasson M, Gardiman M, Carli M, Perilongo G, Rosolen A, Onisto M. Functional VEGF and VEGF receptors are expressed in human medulloblastomas. Neuro Oncol. 2007;9:384-92. 303. Verheul HM, Pinedo HM. The role of vascular endothelial growth factor (VEGF) in tumor angiogenesis and early clinical development of VEGF-receptor kinase inhibitors. Clin Breast Cancer. 2000;1 Suppl 1:S80-4. 304. Aiello LP, Bursell SE, Clermont A, Duh E, Ishii H, Takagi C, Mori F, Ciulla TA, Ways K, Jirousek M, Smith LE, King GL. Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta-isoformselective inhibitor. Diabetes. 1997;46:1473-80. 305. Aiello LP, Pierce EA, Foley ED, Takagi H, Chen H, Riddle L, Ferrara N, King GL, Smith LE. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci U S A. 1995;92:10457-61. 306. Nicoletti VG, Nicoletti R, Ferrara N, Meli G, Reibaldi M, Reibaldi A. Diabetic patients and retinal proliferation: an evaluation of the role of vascular endothelial growth factor (VEGF). Exp Clin Endocrinol Diabetes. 2003;111:209-14. 307. Thieme H, Aiello LP, Takagi H, Ferrara N, King GL. Comparative analysis of vascular endothelial growth factor receptors on retinal and aortic vascular endothelial cells. Diabetes. 1995;44:98-103. 308. Ferrara N, Mass RD, Campa C, Kim R. Targeting VEGF-A to treat cancer and age-related macular degeneration. Annu Rev Med. 2007;58:491-504.  164 | P a g e  309. Eremina V, Sood M, Haigh J, Nagy A, Lajoie G, Ferrara N, Gerber HP, Kikkawa Y, Miner JH, Quaggin SE. Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. J Clin Invest. 2003;111:707-16. 310. Malyszko J, Malyszko JS, Mysliwiec M. Endothelial cell injury markers in chronic renal failure on conservative treatment and continuous ambulatory peritoneal dialysis. Kidney Blood Press Res. 2004;27:71-7. 311. Inoue M, Itoh H, Ueda M, Naruko T, Kojima A, Komatsu R, Doi K, Ogawa Y, Tamura N, Takaya K, Igaki T, Yamashita J, Chun TH, Masatsugu K, Becker AE, Nakao K. Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions: possible pathophysiological significance of VEGF in progression of atherosclerosis. Circulation. 1998;98:2108-16. 312. Jaumdally RJ, Varma C, Blann AD, Macfadyen RJ, Lip GY. Indices of angiogenesis, platelet activation, and endothelial damage/dysfunction in relation to ethnicity and coronary artery disease: differences in central versus peripheral levels. Ann Med. 2007;39:628-33. 313. Sasso FC, Torella D, Carbonara O, Ellison GM, Torella M, Scardone M, Marra C, Nasti R, Marfella R, Cozzolino D, Indolfi C, Cotrufo M, Torella R, Salvatore T. Increased vascular endothelial growth factor expression but impaired vascular endothelial growth factor receptor signaling in the myocardium of type 2 diabetic patients with chronic coronary heart disease. J Am Coll Cardiol. 2005;46:827-34. 314. Numnum TM, Rocconi RP, Whitworth J, Barnes MN. The use of bevacizumab to palliate symptomatic ascites in patients with refractory ovarian carcinoma. Gynecol Oncol. 2006;102:425-8. 315. Ananthnarayan S, Bahng J, Roring J, Nghiemphu P, Lai A, Cloughesy T, Pope WB. Time course of imaging changes of GBM during extended bevacizumab treatment. J Neurooncol. 2008;88:339-47. 316. Plotkin SR, Stemmer-Rachamimov AO, Barker FG, 2nd, Halpin C, Padera TP, Tyrrell A, Sorensen AG, Jain RK, di Tomaso E. Hearing improvement after bevacizumab in patients with neurofibromatosis type 2. N Engl J Med. 2009;361:358-67. 317. Rosenfeld PJ, Brown DM, Heier JS, Boyer DS, Kaiser PK, Chung CY, Kim RY. Ranibizumab for neovascular age-related macular degeneration. N Engl J Med. 2006;355:1419-31. 318. Stitt AW, Gardiner TA, Archer DB. Histological and ultrastructural investigation of retinal microaneurysm development in diabetic patients. Br J Ophthalmol. 1995;79:362-7.  165 | P a g e  319. Knudsen ST, Bek T, Poulsen PL, Hove MN, Rehling M, Mogensen CE. Macular edema reflects generalized vascular hyperpermeability in type 2 diabetic patients with retinopathy. Diabetes Care. 2002;25:2328-34. 320. Shen H, Clauss M, Ryan J, Schmidt AM, Tijburg P, Borden L, Connolly D, Stern D, Kao J. Characterization of vascular permeability factor/vascular endothelial growth factor receptors on mononuclear phagocytes. Blood. 1993;81:2767-73. 321. Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marme D. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood. 1996;87:3336-43. 322. Ankoma-Sey V, Wang Y, Dai Z. Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cells. Hepatology. 2000;31:141-8. 323. Blouw B, Song H, Tihan T, Bosze J, Ferrara N, Gerber HP, Johnson RS, Bergers G. The hypoxic response of tumors is dependent on their microenvironment. Cancer Cell. 2003;4:13346. 324. Detmar M, Brown LF, Berse B, Jackman RW, Elicker BM, Dvorak HF, Claffey KP. Hypoxia regulates the expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and its receptors in human skin. J Invest Dermatol. 1997;108:263-8. 325. Jeon SH, Chae BC, Kim HA, Seo GY, Seo DW, Chun GT, Kim NS, Yie SW, Byeon WH, Eom SH, Ha KS, Kim YM, Kim PH. Mechanisms underlying TGF-beta1-induced expression of VEGF and Flk-1 in mouse macrophages and their implications for angiogenesis. J Leukoc Biol. 2007;81:55766. 326. Chua CC, Hamdy RC, Chua BH. Upregulation of vascular endothelial growth factor by angiotensin II in rat heart endothelial cells. Biochim Biophys Acta. 1998;1401:187-94. 327. Stavri GT, Zachary IC, Baskerville PA, Martin JF, Erusalimsky JD. Basic fibroblast growth factor upregulates the expression of vascular endothelial growth factor in vascular smooth muscle cells. Synergistic interaction with hypoxia. Circulation. 1995;92:11-4. 328. Li J, Perrella MA, Tsai JC, Yet SF, Hsieh CM, Yoshizumi M, Patterson C, Endege WO, Zhou F, Lee ME. Induction of vascular endothelial growth factor gene expression by interleukin-1 beta in rat aortic smooth muscle cells. J Biol Chem. 1995;270:308-12. 329. Mountain DJ, Singh M, Singh K. Interleukin-1beta-mediated inhibition of the processes of angiogenesis in cardiac microvascular endothelial cells. Life Sci. 2008;82:1224-30.  166 | P a g e  330. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-9. 331.  Ross R. Atherosclerosis is an inflammatory disease. Am Heart J. 1999;138:S419-20.  332. van der Wal AC, Becker AE, van der Loos CM, Tigges AJ, Das PK. Fibrous and lipid-rich atherosclerotic plaques are part of interchangeable morphologies related to inflammation: a concept. Coron Artery Dis. 1994;5:463-9. 333. St Goar FG, Pinto FJ, Alderman EL, Valantine HA, Schroeder JS, Gao SZ, Stinson EB, Popp RL. Intracoronary ultrasound in cardiac transplant recipients. In vivo evidence of "angiographically silent" intimal thickening. Circulation. 1992;85:979-87. 334. Bourge RC, Naftel DC, Costanzo-Nordin MR, Kirklin JK, Young JB, Kubo SH, Olivari MT, Kasper EK. Pretransplantation risk factors for death after heart transplantation: a multiinstitutional study. The Transplant Cardiologists Research Database Group. J Heart Lung Transplant. 1993;12:549-62. 335. Johnson DE, Gao SZ, Schroeder JS, DeCampli WM, Billingham ME. The spectrum of coronary artery pathologic findings in human cardiac allografts. J Heart Transplant. 1989;8:34959. 336. Senger DR, Asch BB, Smith BD, Perruzzi CA, Dvorak HF. A secreted phosphoprotein marker for neoplastic transformation of both epithelial and fibroblastic cells. Nature. 1983;302:714-5. 337. Senger DR, Perruzzi CA, Feder J, Dvorak HF. A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines. Cancer Res. 1986;46:5629-32. 338. Breslin JW, Pappas PJ, Cerveira JJ, Hobson RW, 2nd, Duran WN. VEGF increases endothelial permeability by separate signaling pathways involving ERK-1/2 and nitric oxide. Am J Physiol Heart Circ Physiol. 2003;284:H92-H100. 339. Nagy JA, Vasile E, Feng D, Sundberg C, Brown LF, Detmar MJ, Lawitts JA, Benjamin L, Tan X, Manseau EJ, Dvorak AM, Dvorak HF. Vascular permeability factor/vascular endothelial growth factor induces lymphangiogenesis as well as angiogenesis. J Exp Med. 2002;196:1497506. 340. Feng Y, Venema VJ, Venema RC, Tsai N, Behzadian MA, Caldwell RB. VEGF-induced permeability increase is mediated by caveolae. Invest Ophthalmol Vis Sci. 1999;40:157-67.  167 | P a g e  341. Clauss M, Gerlach M, Gerlach H, Brett J, Wang F, Familletti PC, Pan YC, Olander JV, Connolly DT, Stern D. Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration. J Exp Med. 1990;172:1535-45. 342. Van Belle E, Rivard A, Chen D, Silver M, Bunting S, Ferrara N, Symes JF, Bauters C, Isner JM. Hypercholesterolemia attenuates angiogenesis but does not preclude augmentation by angiogenic cytokines. Circulation. 1997;96:2667-74. 343. Okuda Y, Tsurumaru K, Suzuki S, Miyauchi T, Asano M, Hong Y, Sone H, Fujita R, Mizutani M, Kawakami Y, Nakajima T, Soma M, Matsuo K, Suzuki H, Yamashita K. Hypoxia and endothelin-1 induce VEGF production in human vascular smooth muscle cells. Life Sci. 1998;63:477-84. 344. Shima DT, Adamis AP, Ferrara N, Yeo KT, Yeo TK, Allende R, Folkman J, D'Amore PA. Hypoxic induction of endothelial cell growth factors in retinal cells: identification and characterization of vascular endothelial growth factor (VEGF) as the mitogen. Mol Med. 1995;1:182-93. 345. Qu H, Nagy JA, Senger DR, Dvorak HF, Dvorak AM. Ultrastructural localization of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) to the abluminal plasma membrane and vesiculovacuolar organelles of tumor microvascular endothelium. J Histochem Cytochem. 1995;43:381-9. 346. Park JE, Keller GA, Ferrara N. The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF. Mol Biol Cell. 1993;4:1317-26. 347. Torry RJ, Labarrere CA, Torry DS, Holt VJ, Faulk WP. Vascular endothelial growth factor expression in transplanted human hearts. Transplantation. 1995;60:1451-7. 348. Lemstrom KB, Krebs R, Nykanen AI, Tikkanen JM, Sihvola RK, Aaltola EM, Hayry PJ, Wood J, Alitalo K, Yla-Herttuala S, Koskinen PK. Vascular endothelial growth factor enhances cardiac allograft arteriosclerosis. Circulation. 2002;105:2524-30. 349. Wong D, Luckhurst J, Toma H, Kuipers N, Loo S, Suarez A, Wilson JE, McManus BM. Vascular endothelial growth factor-D (VEGF-D) is an endothelial hyperpermeability inducing growth factor differentially expressed in human cardiac allografts. J Heart Lung Transplant. 2001;20:156.  168 | P a g e  350. Girnita DM, Brooks MM, Webber SA, Burckart GJ, Ferrell R, Zdanowicz G, DeCroo S, Smith L, Chinnock R, Canter C, Addonizio L, Bernstein D, Kirklin JK, Ranganathan S, Naftel D, Girnita AL, Zeevi A. Genetic polymorphisms impact the risk of acute rejection in pediatric heart transplantation: a multi-institutional study. Transplantation. 2008;85:1632-9. 351. Biselli PM, Guerzoni AR, de Godoy MF, Pavarino-Bertelli EC, Goloni-Bertollo EM. Vascular endothelial growth factor genetic variability and coronary artery disease in Brazilian population. Heart Vessels. 2008;23:371-5. 352. Tambur AR, Pamboukian S, Costanzo MR, Heroux A. Genetic polymorphism in plateletderived growth factor and vascular endothelial growth factor are significantly associated with cardiac allograft vasculopathy. J Heart Lung Transplant. 2006;25:690-8. 353. Kim DH, Lee NY, Lee MH, Sohn SK. Vascular endothelial growth factor gene polymorphisms may predict the risk of acute graft-versus-host disease following allogeneic transplantation: preventive effect of vascular endothelial growth factor gene on acute graftversus-host disease. Biol Blood Marrow Transplant. 2008;14:1408-16. 354. Gunesacar R, Opelz G, Erken E, Pelzl S, Dohler B, Ruhenstroth A, Susal C. VEGF 936 C/T gene polymorphism in renal transplant recipients: association of the T allele with good graft outcome. Hum Immunol. 2007;68:599-602. 355. Lemos FB, Mol WM, Roodnat JI, Uitterlinden A, Ijzermans JN, Weimar W, Baan CC. The beneficial effects of recipient-derived vascular endothelial growth factor on graft survival after kidney transplantation. Transplantation. 2005;79:1221-5. 356. Shahbazi M, Fryer AA, Pravica V, Brogan IJ, Ramsay HM, Hutchinson IV, Harden PN. Vascular endothelial growth factor gene polymorphisms are associated with acute renal allograft rejection. J Am Soc Nephrol. 2002;13:260-4. 357. Torry RJ, Bai L, Miller SJ, Labarrere CA, Nelson D, Torry DS. Increased vascular endothelial growth factor expression in human hearts with microvascular fibrin. J Mol Cell Cardiol. 2001;33:175-84. 358. Reinders ME, Fang JC, Wong W, Ganz P, Briscoe DM. Expression patterns of vascular endothelial growth factor in human cardiac allografts: association with rejection. Transplantation. 2003;76:224-30. 359. Rezai N, Corbel SY, Dabiri D, Kerjner A, Rossi FM, McManus BM, Podor TJ. Bone marrowderived recipient cells in murine transplanted hearts: potential roles and the effect of immunosuppression. Lab Invest. 2005;85:982-91.  169 | P a g e  360. Rezai N, Deisher TA, Heine HL, Wang X, Corbel SY, Leung J, Kerjner A, Rossi FM, Podor TJ, McManus BM. Effects of granulocyte-colony stimulating factor on bone marrow-derived progenitor cells in murine cardiac transplantation. Cardiovasc Pathol. 2010;19:36-47. 361. Rutanen J, Leppanen P, Tuomisto TT, Rissanen TT, Hiltunen MO, Vajanto I, Niemi M, Hakkinen T, Karkola K, Stacker SA, Achen MG, Alitalo K, Yla-Herttuala S. Vascular endothelial growth factor-D expression in human atherosclerotic lesions. Cardiovasc Res. 2003;59:971-9. 362. Lal BK, Varma S, Pappas PJ, Hobson RW, 2nd, Duran WN. VEGF increases permeability of the endothelial cell monolayer by activation of PKB/akt, endothelial nitric-oxide synthase, and MAP kinase pathways. Microvasc Res. 2001;62:252-62. 363. Six I, Kureishi Y, Luo Z, Walsh K. Akt signaling mediates VEGF/VPF vascular permeability in vivo. FEBS Lett. 2002;532:67-9. 364. Feng Y, Venema VJ, Venema RC, Tsai N, Caldwell RB. VEGF induces nuclear translocation of Flk-1/KDR, endothelial nitric oxide synthase, and caveolin-1 in vascular endothelial cells. Biochem Biophys Res Commun. 1999;256:192-7. 365. Fukumura D, Gohongi T, Kadambi A, Izumi Y, Ang J, Yun CO, Buerk DG, Huang PL, Jain RK. Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factorinduced angiogenesis and vascular permeability. Proc Natl Acad Sci U S A. 2001;98:2604-9. 366. Weis S, Shintani S, Weber A, Kirchmair R, Wood M, Cravens A, McSharry H, Iwakura A, Yoon YS, Himes N, Burstein D, Doukas J, Soll R, Losordo D, Cheresh D. Src blockade stabilizes a Flk/cadherin complex, reducing edema and tissue injury following myocardial infarction. J Clin Invest. 2004;113:885-94. 367. Chou MT, Wang J, Fujita DJ. Src kinase becomes preferentially associated with the VEGFR, KDR/Flk-1, following VEGF stimulation of vascular endothelial cells. BMC Biochem. 2002;3:32. 368. Pedram A, Razandi M, Levin ER. Deciphering vascular endothelial cell growth factor/vascular permeability factor signaling to vascular permeability. Inhibition by atrial natriuretic peptide. J Biol Chem. 2002;277:44385-98. 369. Eliceiri BP, Paul R, Schwartzberg PL, Hood JD, Leng J, Cheresh DA. Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability. Mol Cell. 1999;4:915-24. 370. Paul R, Zhang ZG, Eliceiri BP, Jiang Q, Boccia AD, Zhang RL, Chopp M, Cheresh DA. Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke. Nat Med. 2001;7:222-7. 170 | P a g e  371. Seymour LW, Shoaibi MA, Martin A, Ahmed A, Elvin P, Kerr DJ, Wakelam MJ. Vascular endothelial growth factor stimulates protein kinase C-dependent phospholipase D activity in endothelial cells. Lab Invest. 1996;75:427-37. 372. Belgore F, Blann A, Neil D, Ahmed AS, Lip GY. Localisation of members of the vascular endothelial growth factor (VEGF) family and their receptors in human atherosclerotic arteries. J Clin Pathol. 2004;57:266-72. 373. Crews CM, Alessandrini A, Erikson RL. The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science. 1992;258:478-80. 374. Alessi DR, Saito Y, Campbell DG, Cohen P, Sithanandam G, Rapp U, Ashworth A, Marshall CJ, Cowley S. Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. Embo J. 1994;13:1610-9. 375. Rosen LB, Ginty DD, Weber MJ, Greenberg ME. Membrane depolarization and calcium influx stimulate MEK and MAP kinase via activation of Ras. Neuron. 1994;12:1207-21. 376. Cowley S, Paterson H, Kemp P, Marshall CJ. Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell. 1994;77:841-52. 377. Claude P. Morphological factors influencing transepithelial permeability: a model for the resistance of the zonula occludens. J Membr Biol. 1978;39:219-32. 378. McCarthy KM, Francis SA, McCormack JM, Lai J, Rogers RA, Skare IB, Lynch RD, Schneeberger EE. Inducible expression of claudin-1-myc but not occludin-VSV-G results in aberrant tight junction strand formation in MDCK cells. J Cell Sci. 2000;113 Pt 19:3387-98. 379. Ando-Akatsuka Y, Saitou M, Hirase T, Kishi M, Sakakibara A, Itoh M, Yonemura S, Furuse M, Tsukita S. Interspecies diversity of the occludin sequence: cDNA cloning of human, mouse, dog, and rat-kangaroo homologues. J Cell Biol. 1996;133:43-7. 380. Saitou M, Ando-Akatsuka Y, Itoh M, Furuse M, Inazawa J, Fujimoto K, Tsukita S. Mammalian occludin in epithelial cells: its expression and subcellular distribution. Eur J Cell Biol. 1997;73:222-31. 381. Gitay-Goren H, Cohen T, Tessler S, Soker S, Gengrinovitch S, Rockwell P, Klagsbrun M, Levi BZ, Neufeld G. Selective binding of VEGF121 to one of the three vascular endothelial growth factor receptors of vascular endothelial cells. J Biol Chem. 1996;271:5519-23.  171 | P a g e  382. Tessler S, Rockwell P, Hicklin D, Cohen T, Levi BZ, Witte L, Lemischka IR, Neufeld G. Heparin modulates the interaction of VEGF165 with soluble and cell associated flk-1 receptors. J Biol Chem. 1994;269:12456-61. 383. Achen MG, Jeltsch M, Kukk E, Makinen T, Vitali A, Wilks AF, Alitalo K, Stacker SA. Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4). Proc Natl Acad Sci U S A. 1998;95:548-53. 384. Ment LR, Stewart WB, Fronc R, Seashore C, Mahooti S, Scaramuzzino D, Madri JA. Vascular endothelial growth factor mediates reactive angiogenesis in the postnatal developing brain. Brain Res Dev Brain Res. 1997;100:52-61. 385. Yokomori H, Oda M, Yoshimura K, Nagai T, Ogi M, Nomura M, Ishii H. Vascular endothelial growth factor increases fenestral permeability in hepatic sinusoidal endothelial cells. Liver Int. 2003;23:467-75. 386. Roberts WG, Hasan T. Tumor-secreted vascular permeability factor/vascular endothelial growth factor influences photosensitizer uptake. Cancer Res. 1993;53:153-7. 387. Nagy JA, Masse EM, Herzberg KT, Meyers MS, Yeo KT, Yeo TK, Sioussat TM, Dvorak HF. Pathogenesis of ascites tumor growth: vascular permeability factor, vascular hyperpermeability, and ascites fluid accumulation. Cancer Res. 1995;55:360-8. 388. Antonetti DA, Barber AJ, Khin S, Lieth E, Tarbell JM, Gardner TW. Vascular permeability in experimental diabetes is associated with reduced endothelial occludin content: vascular endothelial growth factor decreases occludin in retinal endothelial cells. Penn State Retina Research Group. Diabetes. 1998;47:1953-9. 389. Watanabe H, Sumi S, Urushihata T, Kitamura Y, Iwasaki S, Xu G, Yano S, Nio Y, Tamura K. Immunohistochemical studies on vascular endothelial growth factor and platelet endothelial cell adhesion molecule-1/CD-31 in islet transplantation. Pancreas. 2000;21:165-73. 390. Aramoto H, Breslin JW, Pappas PJ, Hobson RW, 2nd, Duran WN. Vascular endothelial growth factor stimulates differential signaling pathways in in vivo microcirculation. Am J Physiol Heart Circ Physiol. 2004;287:H1590-8. 391. Suarez S, Ballmer-Hofer K. VEGF transiently disrupts gap junctional communication in endothelial cells. J Cell Sci. 2001;114:1229-35. 392. Feng D, Nagy JA, Dvorak AM, Dvorak HF. Different pathways of macromolecule extravasation from hyperpermeable tumor vessels. Microvasc Res. 2000;59:24-37.  172 | P a g e  393. Feng D, Nagy JA, Pyne K, Dvorak HF, Dvorak AM. Ultrastructural localization of platelet endothelial cell adhesion molecule (PECAM-1, CD31) in vascular endothelium. J Histochem Cytochem. 2004;52:87-101. 394. Dvorak AM, Feng D. The vesiculo-vacuolar organelle (VVO). A new endothelial cell permeability organelle. J Histochem Cytochem. 2001;49:419-32. 395. Clark ER, Clark EL. Observations on changes in blood vascular endothelium in the living animal. Am J Anat. 1935;57:385-438. 396. Dejana E, Spagnuolo R, Bazzoni G. Interendothelial junctions and their role in the control of angiogenesis, vascular permeability and leukocyte transmigration. Thromb Haemost. 2001;86:308-15. 397. Spanel-Borowski K, Mayerhofer A. Formation and regression of capillary sprouts in corpora lutea of immature superstimulated golden hamsters. Acta Anat (Basel). 1987;128:22735. 398. Ferrero E, Villa A, Ferrero ME, Toninelli E, Bender JR, Pardi R, Zocchi MR. Tumor necrosis factor alpha-induced vascular leakage involves PECAM1 phosphorylation. Cancer Res. 1996;56:3211-5. 399. Kevil CG, Payne DK, Mire E, Alexander JS. Vascular permeability factor/vascular endothelial cell growth factor-mediated permeability occurs through disorganization of endothelial junctional proteins. J Biol Chem. 1998;273:15099-103. 400. Taylor DO, Edwards LB, Boucek MM, Trulock EP, Keck BM, Hertz MI. The Registry of the International Society for Heart and Lung Transplantation: twenty-first official adult heart transplant report--2004. J Heart Lung Transplant. 2004;23:796-803. 401. Moien-Afshari F, McManus BM, Laher I. Immunosuppression and transplant vascular disease: benefits and adverse effects. Pharmacol Ther. 2003;100:141-56. 402. Choy JC, McDonald PC, Suarez AC, Hung VH, Wilson JE, McManus BM, Granville DJ. Granzyme B in atherosclerosis and transplant vascular disease: association with cell death and atherosclerotic disease severity. Mod Pathol. 2003;16:460-70. 403. Choy JC, Podor TJ, Yanagawa B, Lai JC, Granville DJ, Walker DC, McManus BM. The regulation and consequences of immune-mediated cell death in atheromatous diseases. Cardiovasc Toxicol. 2003;3:269-82. 404. Rahmani M, McDonald PC, Wong BW, McManus BM. Transplant vascular disease: Role of lipids and proteoglycans. Can J Cardiol. 2004;20:58B-65B. 173 | P a g e  405. Rahmani M, Read JT, Carthy JM, McDonald PC, Wong BW, Esfandiarei M, Si X, Luo Z, Luo H, Rennie PS, McManus BM. Regulation of the versican promoter by the beta-catenin-T-cell factor complex in vascular smooth muscle cells. J Biol Chem. 2005;280:13019-28. 406. Wong BW, Wong D, McManus BM. Characterization of fractalkine (CX3CL1) and CX3CR1 in human coronary arteries with native atherosclerosis, diabetes mellitus, and transplant vascular disease. Cardiovasc Pathol. 2002;11:332-8. 407. Dong C, Wilson JE, Winters GL, McManus BM. Human transplant coronary artery disease: pathological evidence for Fas-mediated apoptotic cytotoxicity in allograft arteriopathy. Lab Invest. 1996;74:921-31. 408. Dong C, Winters GL, Wilson JE, McManus BM. Enhanced lymphocyte longevity and absence of proliferation and lymphocyte apoptosis in Quilty effects of human heart allografts. Am J Pathol. 1997;151:121-30. 409. Lin H, Wilson JE, Roberts CR, Horley KJ, Winters GL, Costanzo MR, McManus BM. Biglycan, decorin, and versican protein expression patterns in coronary arteriopathy of human cardiac allograft: distinctness as compared to native atherosclerosis. J Heart Lung Transplant. 1996;15:1233-47. 410. McDonald PC, Huang Y, Steinbrecher U, Lougheed M, Rahimian R, Wong D, Zacher N, Wilson J, McManus B. Immunohistochemical localization of oxidized low-density lipoprotein (OXLDL) and macrophages in coronary arteries: human heart allografts versus human native hearts. J Heart Lung Transplant. 2001;20:187. 411. McDonald PC, Kenyon JA, McManus BM. The role of lipids in transplant vascular disease. Lab Invest. 1998;78:1187-201. 412. Wong BW, Rahmani M, Luo Z, Yanagawa B, Wong D, Luo H, McManus BM. Vascular endothelial growth factor increases human cardiac microvascular endothelial cell permeability to low-density lipoproteins. J Heart Lung Transplant. 2009;28:950-7. 413. Kubrich M, Petrakopoulou P, Kofler S, Nickel T, Kaczmarek I, Meiser BM, Reichart B, von Scheidt W, Weis M. Impact of coronary endothelial dysfunction on adverse long-term outcome after heart transplantation. Transplantation. 2008;85:1580-7. 414. Kobashigawa JA, Starling RC, Mehra MR, Kormos RL, Bhat G, Barr ML, Sigouin CS, Kolesar J, Fitzsimmons W. Multicenter retrospective analysis of cardiovascular risk factors affecting long-term outcome of de novo cardiac transplant recipients. J Heart Lung Transplant. 2006;25:1063-9.  174 | P a g e  415. Rahmani M, Wong BW, Ang L, Cheung CC, Carthy JM, Walinski H, McManus BM. Versican: signaling to transcriptional control pathways. Can J Physiol Pharmacol. 2006;84:77-92. 416. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma S, Edwards NM, Itescu S. Neovascularization of ischemic myocardium by human bone-marrowderived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med. 2001;7:430-6. 417. Bhattacharya V, Shi Q, Ishida A, Sauvage LR, Hammond WP, Wu MH. Administration of granulocyte colony-stimulating factor enhances endothelialization and microvessel formation in small-caliber synthetic vascular grafts. J Vasc Surg. 2000;32:116-23. 418. Kong D, Melo LG, Gnecchi M, Zhang L, Mostoslavsky G, Liew CC, Pratt RE, Dzau VJ. Cytokine-induced mobilization of circulating endothelial progenitor cells enhances repair of injured arteries. Circulation. 2004;110:2039-46. 419. Kong D, Melo LG, Mangi AA, Zhang L, Lopez-Ilasaca M, Perrella MA, Liew CC, Pratt RE, Dzau VJ. Enhanced inhibition of neointimal hyperplasia by genetically engineered endothelial progenitor cells. Circulation. 2004;109:1769-75. 420. Shi Q, Bhattacharya V, Hong-De Wu M, Sauvage LR. Utilizing granulocyte colonystimulating factor to enhance vascular graft endothelialization from circulating blood cells. Ann Vasc Surg. 2002;16:314-20. 421. Kendall RL, Thomas KA. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor. Proc Natl Acad Sci U S A. 1993;90:10705-9. 422. Sugimoto H, Hamano Y, Charytan D, Cosgrove D, Kieran M, Sudhakar A, Kalluri R. Neutralization of circulating vascular endothelial growth factor (VEGF) by anti-VEGF antibodies and soluble VEGF receptor 1 (sFlt-1) induces proteinuria. J Biol Chem. 2003;278:12605-8. 423. Wang Y, Zhou Y, He L, Hong K, Su H, Wu Y, Wu Q, Han M, Cheng X. Gene delivery of soluble vascular endothelial growth factor receptor-1 (sFlt-1) inhibits intra-plaque angiogenesis and suppresses development of atherosclerotic plaque. Clin Exp Med. 2011;11:113-21. 424. Onoue K, Uemura S, Takeda Y, Somekawa S, Iwama H, Imagawa K, Nishida T, Morikawa Y, Takemoto Y, Asai O, Soeda T, Okayama S, Ishigami K, Nakatani K, Kawata H, Horii M, Nakajima T, Akai Y, Iwano M, Saito Y. Reduction of circulating soluble fms-like tyrosine kinase-1 plays a significant role in renal dysfunction-associated aggravation of atherosclerosis. Circulation. 2009;120:2470-7.  175 | P a g e  425. Choy JC, Cruz RP, Kerjner A, Geisbrecht J, Sawchuk T, Fraser SA, Hudig D, Bleackley RC, Jirik FR, McManus BM, Granville DJ. Granzyme B induces endothelial cell apoptosis and contributes to the development of transplant vascular disease. Am J Transplant. 2005;5:494-9. 426. Choy JC, Kerjner A, Wong BW, McManus BM, Granville DJ. Perforin mediates endothelial cell death and resultant transplant vascular disease in cardiac allografts. Am J Pathol. 2004;165:127-33. 427. Wenke K, Meiser B, Thiery J, Nagel D, von Scheidt W, Steinbeck G, Seidel D, Reichart B. Simvastatin reduces graft vessel disease and mortality after heart transplantation: a four-year randomized trial. Circulation. 1997;96:1398-402. 428. Stapleton DD, Mehra MR, Dumas D, Smart FW, Milani RV, Lavie CJ, Ventura HO. Lipidlowering therapy and long-term survival in heart transplantation. Am J Cardiol. 1997;80:802-5. 429. Kobashigawa JA, Kasiske BL. Hyperlipidemia in solid organ transplantation. Transplantation. 1997;63:331-8. 430. Ballantyne CM, Bourge RC, Domalik LJ, Eisen HJ, Fishbein DP, Kubo SH, Lake KD, Radovancevic B, Taylor DO, Ventura HO, Yancy CW, Jr., Young JB. Treatment of hyperlipidemia after heart transplantation and rationale for the Heart Transplant Lipid Registry. Am J Cardiol. 1996;78:532-5. 431. Kapadia SR, Nissen SE, Ziada KM, Rincon G, Crowe TD, Boparai N, Young JB, Tuzcu EM. Impact of lipid abnormalities in development and progression of transplant coronary disease: a serial intravascular ultrasound study. J Am Coll Cardiol. 2001;38:206-13. 432. Sosland RP, Gollub SB, Wilson DB, Moriarty PM. The first case report of the treatment of transplant coronary artery disease with dextran sulfate adsorption lipid apheresis. Ther Apher Dial. 2010;14:218-21. 433. Choy JC, Hung VH, Hunter AL, Cheung PK, Motyka B, Goping IS, Sawchuk T, Bleackley RC, Podor TJ, McManus BM, Granville DJ. Granzyme B induces smooth muscle cell apoptosis in the absence of perforin: involvement of extracellular matrix degradation. Arterioscler Thromb Vasc Biol. 2004;24:2245-50.  176 | P a g e  Appendix – Supplemental information for case materials from Chapter 3  Case no.  Condition  Age (yr)  Sex  Cause of Death  1  Normal  28  M  Self-inflicted gunshot wound  2  Normal  18  F  Blunt trauma to the head and chest  3  Normal  17  M  Blunt trauma to the head and chest  4  Normal  34  F  Drug toxicity  5  Normal  20  M  Carbon monoxide poisoning  6  Normal  17  F  Suffocation  7  Normal  19  M  Shotgun wound to the head  8  Normal  29  F  Gunshot to the abdomen  9  Normal  18  M  Shotgun wound to the head  10  Normal  17  M  Multiple stab wounds to the chest and abdomen  11  Normal  19  F  Blunt trauma to the head, chest and abdomen  12  Normal  22  M  Blunt trauma to the head, chest and abdomen  13  Normal  26  F  Blunt trauma to the head, chest and abdomen  14  Normal  15  F  Blunt trauma to the head and chest  15  Normal  21  F  Blunt trauma to the head, chest and abdomen  16  Normal  32  M  N/A  Table 3 – Case listing for Normal group (Pathobiological Determinants of Atherosclerosis in Youth study). M – male; F – female; N/A – not available.  177 | P a g e  Case no.  Condition  % occlusion  Primary diagnosis  Age (yr)  Sex  Cause of Death  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17  Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis Atherosclerosis  >50% 100% >25% 60-70% 75-90% 50% 65% 50-60% 80% 25% 40-50% 30-40% 65-70% 75-100% 35% 65% 25-30%  N/A CAD ARDS CAD CLL CAD CAD CAD CAD N/A CAD CAD CAD CAD CAD CAD CAD  78 76 51 69 53 41 52 77 84 24 74 69 76 52 78 87 68  M M M F F F M F M M M M M M M F F  Respiratory, cardiac, renal failure Acute myocardial infarction Ruptured aortic aneurysm Cardiac arrest Acute myocardial infarction AIDS, mycobacterium, HepC, HepB Massive pulmonary embolism Hemopericardium, aortic dissection Subendocardial necrosis Arrhythogenic right ventricular dysplasia Focal myocyte necrosis, ischemia Cerebral infarction, thrombus from heart Aortic dissection, sudden death Cardiac arrest Chronic obstructive lung disease Cardiac arrest Acute myocardial infarction  Table 4 – Case listing for Native Atherosclerosis (NA) group. M – male; F – female; N/A – not available; CAD – coronary artery disease; ARDS – acute respiratory distress syndrome; CLL – chronic lymphocytic leukemia; AIDS – acquired immune deficiency syndrome; HepC – hepatitis C; HepB – hepatitis B.  Case no.  Condition  % occlusion  Primary diagnosis  Age (yr)  Sex  Cause of Death  1  Diabetic  20-40%  CAD  63  M  Heart transplant  2  Diabetic  >75%  CAD  49  M  Heart transplant  3  Diabetic  50-75%  CAD  55  M  Heart transplant  4  Diabetic  50-75%  CAD  54  M  Heart transplant  5  Diabetic  <25%  CAD  57  M  Heart transplant  6  Diabetic  25-50%  CAD  67  F  Congestive heart failure  7  Diabetic  100%  CAD  N/A  N/A  N/A  8  Diabetic  N/A  CAD  69  F  Arrhythmia, severe atherosclerotic heart disease Rhythm disturbance, myocardial necrosis  9  Diabetic  40%  CAD  77  F  10  Diabetic  50%  CAD  65  M  Myocardial ischemia  11  Diabetic  75%  CAD  69  M  Respiratory failure, severe acute lung disease  12  Diabetic  N/A  CAD  85  F  Myocardial infarction  13  Diabetic  50%  CAD  59  F  14  Diabetic  70%  CAD  64  M  15  Diabetic  75%  CAD  71  F  Acute myocardial infarction Acute myocardial infarction, non-insulin dependent diabetes mellitus Cardiac arrest, non-insulin dependent diabetes mellitus  Table 5 – Case listing for Diabetes Mellitus (DM) group. M – male; F – female; N/A – not available.  178 | P a g e  Case no. 1 2  3 4 5 6 7 8 9 10  11 12 13 14 15 16 17 18  19 20 21  Condition heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant heart transplant  % occlusion  Primary diagnosis  Implant Duration (days)  Age (yr)  Sex  Cause of Death  Donor age (yr)  Donor sex  >25%  Heart failure  540  48  F  Myocardial infarction  N/A  N/A  100%  CAD  611  16  M  Heart rejection  47  M  <25%  N/A  102  60  M  Unknown  N/A  N/A  >25%  CHF  360  67  F  Heart failure  N/A  N/A  ~50%  N/A  N/A  N/A  N/A  N/A  N/A  N/A  0-25%  CMY  21  15  F  Rejection  15  M  0-25%  CMY  208  21  F  Rejection  15  M  51-75%  IDC  55  58  M  Pneumonia, Aspergillosis, CMV  30  M  0-25%  IDC  238  51  M  Rejection  17  M  26-50%  CMY  311  21  F  Rejection  21  M  0-25%  VHD  153  39  F  Allograft failure  N/A  M  N/A  CMY  36  43  M  Rejection  35  M  0-25%, 26-50%  IDC  639  37  M  Heart failure, rejection  17  M  0-25%  IHD  13  38  M  Rejection  17  M  51-75%  IHD  45  55  M  Acute myocardial infarction, rejection  21  M  0-25%  CMY  294  47  M  Acute myocardial infarction  31  M  >75%  IHD  638  39  M  Liver failure, cirrhosis  37  M  26-50%, 51-75%  IHD  426  26  M  Rejection  25  M  N/A  IDC-post partum  1432  23  F  Heart failure  27  M  N/A  DCM  46  60  F  Sepsis, islet tumor pancreas, APE, LD  44  F  N/A  CMY  718  53  M  PTCD  27  M  Table 6 – Case listing for Cardiac Allograft Vasculopathy (CAV) group. M – male; F – female; N/A – not available; CAD – coronary artery disease; CHF – congestive heart failure; CMY – cardiomyopathy; IDC – intravascular disseminated coagulopathy; VHD – vascular heart disease; DCM – dilated cardiomyopathy; CMV – cytomegalovirus; APE – acute pulmonary edema; LD – lymphoproliferative disorder; PTCD – percutaneous transhepatic cholangiographic drainage.  179 | P a g e  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.24.1-0105116/manifest

Comment

Related Items