Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Quantitative and qualitative assessment of aortic structure and function, and elastic fiber ultrastructure… Cui, Zhe (Jason) 2017

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

Item Metadata

Download

Media
24-ubc_2017_november_cui_zhe.pdf [ 3.86MB ]
Metadata
JSON: 24-1.0357258.json
JSON-LD: 24-1.0357258-ld.json
RDF/XML (Pretty): 24-1.0357258-rdf.xml
RDF/JSON: 24-1.0357258-rdf.json
Turtle: 24-1.0357258-turtle.txt
N-Triples: 24-1.0357258-rdf-ntriples.txt
Original Record: 24-1.0357258-source.json
Full Text
24-1.0357258-fulltext.txt
Citation
24-1.0357258.ris

Full Text

         Quantitative and qualitative assessment of aortic structure and function, and elastic fiber ultrastructure in the mouse model of Marfan syndrome   by   Zhe (Jason) Cui    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate and Postdoctoral Studies  (Pharmacology and Therapeutics)    THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2017     © Zhe (Jason) Cui, 2017 ii  Abstract  Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the fibrillin-1 gene, with aortic aneurysm considered as the most life-threatening complication. Previous studies have shown that doxycycline, a general inhibitor of matrix metalloproteinases (MMPs), can improve aortic contractility and elastin structure in the mouse model of MFS. However, the longitudinal effects of MMPs inhibition on the gradual progression of aneurysm and aortic wall biophysical properties in a live animal have not yet been investigated. Therefore, we assessed the hypothesis that a long-term treatment with doxycycline would delay the progression of aortic aneurysm, improve aortic wall elasticity, and protect the ultrastructure of elastin in MFS mice.  In this study, non-invasive and label-free multiphoton microscopy imaging was used to quantify fiber morphology and volumetric density of aortic and cutaneous elastin and collagen in MFS mice. We also utilized non-invasive high-resolution echocardiography to conduct a longitudinal in vivo study of the structural, functional, and biophysical properties of the aortic wall in control and MFS mice in the absence and presence of doxycycline treatment. The ultrastructure of aortic elastic fibers was also assessed by electron microscopy.  Multiphoton imaging revealed significant elastin fragmentation and disorganization within the aortic wall of MFS mice, which was also associated with reduction in cutaneous volumetric density of elastin and collagen. Ultrasound imaging showed that aortic pulse wave velocity (PWV) was significantly elevated in MFS mice starting at the age of 6-month-old, which was associated with a distinct increase in aortic root dimeter in the regions of aortic annulus and sinus of Valsalva. Long-term treatment with doxycycline resulted in a significant improvement in elastin organization, reduction of aortic root growth and aortic iii  PWV in MFS mice. These findings underscore the key role of MMPs in the pathogenesis, and provide new insights into the potential therapeutic value of doxycycline in blocking MFS-associated aneurysm.  Furthermore, the use of multiphoton imaging to detect the signs of elastin degradation in the skin dermis may be considered as the first step towards the potential development of a non-invasive approach for monitoring the aneurysm progression in MFS patients.                                    iv  Lay Summary  Marfan syndrome (MFS) is an inherited systemic connective tissue, with characteristic cardiovascular malformations. The most common abnormality is dilatation of the aorta, an enlargement of the main artery in the human body. MFS is caused by mutations in gene encoding for fibrillin 1, a structural protein essential for the formation of elastic fibers. Severe elastic fiber fragmentation and disorganization were quantified in the MFS mice by the non-invasive multiphoton imaging. The aortic structure and function were determined in the living MFS mice by an ultrasound, and significant aortic dilatation and stiffness were found in these animals. The therapeutic effects of doxycycline, an antibiotic found to prevent the elastic fiber fragmentation and slow down the progression of aortic dilatation and stiffness, were investigated by performing a longitudinal study with the use of ultrasound imaging and electron microscopy. These findings underscore the potential use of doxycycline in blocking MFS-associated aneurysm.                      v  Preface  This thesis is devoted to the investigation of mechanisms underlying the progression of aortic dilatation in a well-established mouse model of Marfan syndrome, and includes characterization and assessment of the integrity of extracellular matrix of the aortic wall by multiphoton microscopy, determination of the elastic fiber ultrastructure by electron microscopy, and in vivo investigation of aortic wall elasticity and stiffness by high frequency, high-resolution ultrasound imaging. In addition, this study evaluates the potential therapeutic value of long-term treatment with a sub-antibiotic dose of doxycycline in the Marfan mouse.  All of the work presented henceforth was conducted in the Child & Family Research Institute and James Hogg Research Centre at the University of British Columbia. All animal procedures were approved by the University of British Columbia’s Animal Ethics Board [Reference #A11-0018], and all animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals (www.nap.edu/catalog/5140.html).  Portions of the introductory text are modified from previously written introductory material from my Ph.D. comprehensive exam research proposal entitled “Investigation of effects of losartan, mild aerobic exercise and their combination on Loeys-Dietz syndrome-associated aortic aneurysm in mice” (2016) completed at the University of British Columbia. I created Table 1, by adapting and modifying Table 1 in (Stechmiller et al., 2010). I also created Figure 1, by adapting and modifying Figure 4 in (Fedak et al., 2002).  Project 1 of this thesis, ex vivo study of elastic fiber morphology in the mouse model of Marfan syndrome by multiphoton microscopy, was based on my previous publication in Journal of Structural Biology as the first author (Cui et al., 2014). Figures 2 and 5–10, along vi  with portions of the text in the results and discussion sections are used with permission from licensed content publisher Elsevier, with a license #4146090966326 to reuse in a thesis/dissertation. I was responsible for performing all the experiments, samples collection and preparation, data acquisition and analysis, and writing the paper.  Project 2 of this thesis, in vivo study of aortic structure and function in the mouse model of MFS by ultrasound imaging, was based on part of my previous publication in PLOS ONE as the second author (Lee et al., 2016). Figures 11–14, along with portions of the text in the results and discussion sections are used with a written permission from the journal’s editorial manager, indicating that “all manuscripts are CC-BY which means that they can be used, reused, and copied for free as long as they are properly cited. You may use your publication in your upcoming thesis.” I created Table 2, which is adapted and modified from Table 1 in this publication. I performed parts of the experiments, samples collection and preparation, data acquisition and analysis, with a significant contribution from Dr. Glen Tibbits’ laboratory.  Project 3 of this thesis, in vivo and ex vivo study of doxycycline effects on biophysical and ultrastructural properties of the aorta in a mouse model of Marfan syndrome by echocardiography and transmission electron microscopy, was based on the manuscript ready for submission, entitled “Evaluation of the effects of long-term doxycycline intervention on the progression of aortic dilatation in Marfan mice by high-resolution ultrasound imaging and electron microscopy”. The portion using the echocardiography is a refinement and extension of the method developed by Lee L. and Tibbits G.F., while I jointly developed the method for quantitative analysis of the ultrastructure on electron microscopy, and was responsible for designing and performing parts of the experiments, conducting all the long-term (12-14 vii  months) of animal care and drug treatments, data acquisition and analysis, and drafting the manuscript, with collaboration with Dr. Glen Tibbits’ laboratory.                         viii  Table of Contents  Abstract··································································································· ii Lay Summary ··························································································· iv Preface ···································································································· v Table of Contents ····················································································· viii  List of Tables ···························································································· xi  List of Figures ·························································································· xii  List of Abbreviations ················································································ xiv  Acknowledgements ··················································································· xvi Chapter 1. Introduction ··············································································· 1  1.1 Genetics and classification of Marfan syndrome and its clinical manifestations ······ 1 1.1.1 Genetic causes of Marfan syndrome and its classification ··························· 1 1.1.2 Clinical manifestations of Marfan syndrome ·········································· 2  1.2 Transferring growth factor-beta (TGF-β) and Marfan syndrome ························ 4 1.2.1 TGF-β superfamily and its maturation process and related precursors ············· 4 1.2.2 TGF-β downstream signaling pathways in Marfan syndrome ······················· 7  1.3 Angiotensin II signaling in Marfan syndrome ·············································· 7 1.3.1 Angiotensin II and its receptors ·························································· 7 1.3.2 Ang II receptor blockers (ARBs) in Marfan syndrome ······························· 9  1.4 Matrix metalloproteinases (MMPs) and their inhibitors in MFS aortic aneurysms ·· 10 1.4.1 MMP sub-types and their roles in diseases ············································ 10 1.4.2 Inhibition of MMPs by their intrinsic and extrinsic inhibitors ····················· 14  1.5 Major transgenetic animal models of Marfan syndrome and their main features ···· 15  1.6 Emerging novel techniques for in vivo and ex vivo studies of biophysical   and structural properties of the aorta ························································ 17 1.6.1 Multiphoton microscopy and its applications ········································· 17 1.6.2 High frequency high-resolution ultrasound imaging and its applications ········ 19 1.6.3 Electron microscopy and its applications in characterization of elastic   fiber ultrastructure········································································· 22  1.7 Three objectives of the present study ······················································· 24 1.7.1 Ex vivo study of elastic fiber morphology in the mouse model of MFS by   MPM ························································································ 24 1.7.2 In vivo study of aortic structure and function in the mouse model of MFS  ix   by ultrasound imaging ···································································· 25 1.7.3 In vivo and ex vivo study of doxycycline effects on biophysical and   ultrastructural properties of the aorta in a mouse model of MFS by echo and   TEM ························································································· 26  Chapter 2. Materials & Methods ·································································· 29  2.1 Experimental animals for ex vivo and in vivo studies ···································· 29   2.2 Preparation of aortic and skin tissue samples for MPM imaging ······················· 29  2.3 Features of the MPM system and experimental set-up ··································· 30  2.4 Preparation of aortic and skin tissue samples for histological staining ················ 31  2.5 Quantitative and statistical analysis on MPM imaging ··································· 32  2.6 Timeline for doxycycline treatment in mice ··············································· 33  2.7 Features of the echocardiography ultrasound imaging system and experimental   set-up ···························································································· 35  2.8 Preparation of aorta tissue samples for TEM imaging ··································· 37  2.9 TEM image acquisition and quantitative statistical analysis ···························· 38  Chapter 3. Results ····················································································· 40  3.1 Comparison of aortic elastic fiber morphology and volumetric density of elastin   and collagen between control and MFS mice ············································ 40  3.2 Quantitative determination of aortic elastic fiber fragmentation and organization   in control and MFS mice ···································································· 44  3.3 Comparison of cutaneous elastin and collagen morphology between control and   MFS mice ······················································································ 48  3.4 Total volumetric density of cutaneous elastin and collagen, and the thickness   of dermal layer in control and MFS mice ················································· 50  3.5 Longitudinal comparison of aortic structure between control and MFS mice   by ultrasound imaging ········································································ 52  3.6 Longitudinal comparison of aortic function between control and MFS mice   by ultrasound imaging ········································································ 53  3.7 Quantitative assessment of the progression of aortic elastic fiber   fragmentation between control and MFS mice ··········································· 56  x  3.8 Longitudinal comparison of pulse wave velocity between control and MFS mice   with and without long-term doxycycline intervention ··································· 58  3.9 Longitudinal comparison of regional aortic diameters between control and MFS   mice with and without long-term doxycycline intervention ····························· 60  3.10 Correlation between aortic structure and function over time in control and MFS   mice with and without long-term doxycycline treatment ······························ 65  3.11 Comparison of cardiovascular gross structure and elastic fibers morphology   between control and MFS mice with and without long-term   doxycycline treatment ······································································ 69  3.12 Ultrastructural changes at break points of elastic fibers associated with MFS   and MMPs inhibition ······································································· 72  Chapter 4. Discussion, Conclusions and Future Direction ··································· 76  Chapter 5. General Conclusions and Significance ············································· 88  Bibliography ···························································································· 90  Appendices ····························································································· 113 Appendix A: Publications and Abstracts ····················································· 107 Appendix B: Conference Presentations ······················································ 108                         xi  List of Tables  Table 1. Matrix metalloproteinases (MMPs) sub-types and actions ···························· 12  Table 2. Echocardiographic functional analysis for control and MFS mice ··················· 52                                            xii  List of Figures   Figure 1. The signaling mechanisms for MFS-associated aortic aneurysm. ···················· 6  Figure 2. A simplified schematic representation of multiphoton microscopy system. ······· 31  Figure 3. Animals were divided into doxycycline treatment and no treatment groups. ······ 34  Figure 4. Echocardiography B-mode and pulse wave Doppler-mode image views of  control and MFS mouse aorta. ········································································ 37  Figure 5. Morphology of elastin fibers and collagen in the aortic wall. ························ 41  Figure 6. Assessment of volumetric density of elastin and collagen in aorta. ················· 43  Figure 7. Quantitative assessment of aortic elastic fiber fragmentation. ······················· 44  Figure 8. Analysis of aortic elastic fiber organization by FFT algorithm. ····················· 47  Figure 9. Visualization of cutaneous elastin and collagen morphology in control and  MFS skin samples. ······················································································ 49  Figure 10. Total volumetric density of cutaneous elastin and collagen, and dermal  layer thickness in control and MFS mice skin. ····················································· 51  Figure 11. Aortic root diameters of control and MFS mice. ····································· 53  Figure 12. Pulse wave velocity (PWV) of aortic arch. ············································ 54  Figure 13. Peak velocity was calculated from pulsed-wave Doppler mode from an  aortic arch view. ························································································· 56  Figure 14. Histological analysis of control and MFS mice aorta. ······························· 57  Figure 15. Longitudinal measurements of PWV in control and MFS mice groups with and without doxycycline treatment. ···························································· 59  Figure 16A. Measurements of diameters at the aortic annulus. ································· 61  Figure 16B. Measurements of diameters at the aortic sinus of Valsalva. ······················ 63  Figure 16C. Measurements of diameters at the aortic sinotubular junctions. ················· 64  Figure 17A. Correlations between PWV and aortic diameters in three aortic regions,  aortic annulus, sinus of Valsalva, and sinotubular junction. ······································ 66  Figure 17B. Correlations between PWV and aortic diameters in sinus of Valsalva, in  four groups of experimental animals. ································································ 68 xiii   Figure 18. Representative images of dissected heart and aorta samples. ······················ 70  Figure 19. Representative images of van Gieson’s staining of the aortic root. ················ 71  Figure 20.  Representative images of ultrastructure of the aortic elastic fibers. ·············· 73  Figure 21. Bar graphs presenting measurements of elastin irregularity  index (circumference/width) in control, MFS (with doxycycline) and MFS  (without doxycycline). ················································································· 74                                        xiv  List of Abbreviations   2-D   two-dimensional 3-D   three-dimensional AAA   abdominal aortic aneurysms ACEIs   angiotensin-converting-enzyme inhibitors Ang II   angiotensin II ARB   angiotensin II type 1 receptor blocker AT1R   angiotensin II type 1 receptor AT2R   angiotensin II type 2 receptor BW   body weight ECG   electrocardiogram ECM   extracellular matrix EF    ejection fraction EM   electron microscope ESMA   elastin-specific magnetic resonance contrast agent FBN1   fibrillin-1 FFT   fast Fourier transform FS    fractional shortening HR    heart rate LAP    latency-associated peptide LDS   Loeys–Dietz syndrome LLC   large latent complex LTBP   latent TGF-β binding proteins MFS    Marfan syndrome xv  MLC    myosin light chain MMPs   matrix metalloproteinases MPM   multiphoton microscopy MRI   magnetic resonance imaging NO    nitric oxide PCM   panniculus carnosus muscle PW   pulse wave PWV   pulse wave velocity RAS   renin-angiotensin system ROS    reactive oxygen species RSMAD   receptor-regulated SMAD SEM   scanning electron microscope SHG   second harmonic generation TAA    thoracic aortic aneurysm TEM   transmission electron microscope TGF-β   transforming growth factor-beta TGFBR1  TGF-βreceptor subtypes type I  TGFBR2  TGF-βreceptor subtypes type II  TIMP   tissue inhibitor of MMP TPF   two-photon excited fluorescence Tsp-1   thrombospondin-1 VSMCs  vascular smooth muscle cells WT   wild type   xvi   Acknowledgements  First of foremost, I wish to express my sincere thanks to my supervisor Dr. Cornelis van Breemen for his immense knowledge, patient guidance, and professionalism throughout my graduate training. He has been an influential role model for me and I greatly appreciate his continuous encouragement, support and instruction.  I am very grateful to my mentor, Dr. Mitra Esfandiarei for her constant advice, support and encouragement on all aspects of these projects during the past several years. Her intelligence is inspiring and she has taught me a lot that were crucial to my understanding of many topics. I am more than grateful to both Drs. van Breemen and Esfandiarei for providing me with such a wonderful learning experience and great opportunity for my intellectual growth.  I am also very grateful to Dr. Glen Tibbits and his laboratory, especially Ms. Lilian Lee and Dr. Helen Sheng, as well as Dr. George Sandor, Dr. David Walker and Dr. Fanny Chu, for their tremendous collaborative efforts on these Marfan projects, and continuous support and technical assistance throughout the years. Their generosity and creativity are inspirational and I truly treasure our friendships.  I would like to extend my gratitude to my committee members Dr. David Fedida, Dr. Pascal Bernatchez and Dr. James Wright for their professional counselling, invaluable advice, and sharing of knowledge and contribution during these years.  I am thankful to Mr. Arash Tehrani, Ms. Gabriela Ziomek and the rest of the members of Dr. van Breemen and Tibbits laboratories for their encouragements and assistance. I appreciate everything they have taught me and the friendships we have formed.  I would like to extend my thanks to Dr. Tim Bradley for his contribution to my thesis as an External Examiner, and to Drs. Brian Rodrigues and Chun Y. Seow for their participation in my oral defense as University Examiners.  Lastly, special thanks to my parents and my significant other, Ms. Shelly Zhu, as well as her parents, for their unconditional, unending love and support. Many thanks to my best friend Allen and family, Mrs. Lv and family, Uncle Andrew and Aunt Rosemary, Uncle Chin and family, their compliments are much appreciated.     1  Chapter 1. Introduction: 1.1 Genetics and classification of Marfan syndrome and its clinical manifestations 1.1.1 Genetic causes of Marfan syndrome and its classification  Marfan syndrome (MFS) is an inherited, autosomal dominant disorder of connective tissue caused by mutations in the FBN1 gene located on chromosome 15 (15q21.1), which encodes for fibrillin-1 (FBN1) (Dietz et al., 1991b). MFS was first described by Dr. Antoine Bernard-Jean Marfan (1858-1942) in 1896, a pediatrician in France (Marfan, 1896), and since then it has been characterized by defects in multiple systems, including cardiovascular, pulmonary, skeletal, and ocular systems (Dietz et al., 1991a; Pyeritz, 2000), with a frequency of approximately 1 in 5,000 people (Judge and Dietz, 2005).   Fibrillin-1 is a large extracellular matrix (ECM) glycoprotein broadly distributed in elastic and non-elastic tissues. FBN1 monomers self-associate to form a major component of the ECM macro-aggregates, known as microfibrils, which provide a scaffold that plays an essential role in construction and maturation of elastic fibers (Kielty et al., 2005). Over a thousand of mutations have been identified distributing throughout the sequence of FBN1 so far (Ammash et al., 2008; Keane and Pyeritz, 2008), of which missense mutations are responsible for the vast majority. It is believed that each of these mutations alter a single one of the amino acids that constitute the protein, and thus numerous effects at the protein level have been reported, such as affecting cysteine residues that are involved in calcium binding, altering the secondary structure, delaying secretion or enhancing protease susceptibility (Mizuguchi and Matsumoto, 2007). Furthermore, most mutations that cause neonatal MFS are usually located in exons 24-32, and they are responsible for the most severe phenotype of the disease (Faivre et al., 2007). Various degrees of genetic deficiency in FBN1 have been reported previously, including a decrease in microfibril production in fibroblasts obtained from patients with MFS (Godfrey et al., 1990b), and 2  pathological aging of arteries in a mouse model of MFS (Mariko et al., 2011).   Previously, mutations of the FBN1 gene were believed to be the main cause of the majority of cases of MFS, however, mutations in the genes located on chromosome 3 that primarily encode for two transforming growth factor-beta (TGF-β) receptor subtypes, type I (TGFBR1) and II (TGFBR2), have been also implicated collectively as the causes of MFS type 2. These mutations on TGFBR1 and TGFBR2 are currently referred to as Loeys–Dietz syndrome (LDS), which is further categorized as LDS type-I and LDS type-II, respectively (Loeys et al., 2005; Loeys et al., 2006; Mizuguchi et al., 2004). This syndrome is characterized by dysmorphic symptoms similar to those more generally recognized as in the patients with MFS, with the exception of the finding of lenticular dislocation in ocular system, which are only present in patients with MFS (Zangwill et al., 2006).  1.1.2 Clinical manifestations of Marfan syndrome The pleiotropic manifestations of MFS in humans include aortic root dilatation, mitral valve prolapse, pulmonary disease, lenticular dislocation, skeletal and neurologic abnormalities. The cardiovascular complications are the leading cause of morbidity and mortality, with mitral valve prolapse being the most common of these features in MFS patients. However, aortic root dilatation and dissection are the most lethal complications, and has the biggest influence on prognosis. (Citing quotations from (Cui, 2016) “Patients with MFS are typically characterized by an early and rapidly progressive enlargement of the aorta. Weakening and stretching of the aorta will result in root dilatation and aneurysms that can eventually lead to aortic dissection, a sudden aortic wall medial layers separation, which is a life-threatening complication that can occur without symptoms (Loeys et al., 2005; Loeys et al., 2006). Besides the aorta, MFS patients also present with skeletal features, for instance, thin fingers and long limbs, as well as joint laxity and 3  scoliosis (Boileau et al., 2012; Lindsay et al., 2012).”   Aortic aneurysm is characterized by degeneration of elastin and collagen in the tunica media and adventitia, transmural infiltration of lymphocytes and macrophages, loss of medial vascular smooth muscle cells (VSMCs) with thinning of the vessel wall (resulting in impairment of vascular cell function), and deterioration of aortic mechanical properties (Ailawadi et al., 2003). MFS-associated aneurysms and dissections can also occur in other arteries. In comparison to thoracic and abdominal aortic aneurysms (TAA and AAA) that usually affect the ascending aorta in the tubular area or the descending aorta, in majority of adult MFS patients, aortic dilatation typically locates in the aortic root area (Milewicz et al., 2005). To be more specific, due to more cyclic torsion and increasing wall stress during ventricular ejection as well as its higher elastic fiber content, aortic dilatation usually occurs in the sinus of Valsalva, while the dilatation may extend to other areas of the aorta.  Destruction of ECM reduces the load-bearing capacity of the aorta, contributing to micro-dissection and rupture of the aorta. The primary load-bearing components of the aortic wall are elastic fibers (its assembly involves fibrillin-1), collagen fibrils, and VSMCs. In patients with MFS, the medial layer of the aorta displays extensive irregularities, such as elastic fiber disorganization, fragmentation, and loss of the fiber integrity and its replacement by a basophilic material primarily composed of glucosaminoglycans (Chung et al., 2007a; Schlatmann and Becker, 1977). Degeneration of elastin and alterations in collagen reduce the load bearing capacity of the aorta in MFS, making it more susceptible to micro-dissection. Consequently, progressive thoracic aortic aneurysm, dissection, and rupture are the leading causes of death in MFS (Judge and Dietz, 2005). This medial degeneration is sometimes thought incorrectly to be pathognomonic of MFS, however, it is non-specific and can be found in other kinds of TAA (El-4  Hamamsy and Yacoub, 2009). Therefore, investigations in the progression of elastic fiber deterioration in Marfan-associated aortic aneurysm are crucial for understanding the underlying mechanisms and improving the clinical management of patients with MFS.  The clinical presentation and diagnosis of aortic dilatation in patient is age-dependent, however, it can begin during intrauterine life in the most severe form of the disease (Lalchandani and Wingfield, 2003). “Patients with MFS, who reportedly tend to have aortic dilatation or rupture at a young age and at small dimensions, are at a relatively high risk compared to those with other aneurysm syndromes (Loeys et al., 2005). Therefore, intervention through operation is recommended promptly at ascending aortic diameters of larger than 4.2 centimeter by transesophageal echocardiography or larger than 4.4~4.6 centimeter by computed tomography (Booher and Eagle, 2011). Previous studies reported that patients with aortic aneurysm could ultimately develop aortic dissection at as early as 3 months of age, and cerebral hemorrhage as the most severe form and the leading cause of mortality, with a mean age of death less than 27 years old (Malhotra and Westesson, 2009; Williams et al., 2007a). Therefore, this condition is of medical urgency making early and correct diagnosis, better surveillance and treatment necessary in order to extend the life span of affected patients.”   1.2 Transferring growth factor-beta and Marfan syndrome 1.2.1 TGF-β superfamily and its maturation process and related precursors “Transferring growth factor-beta is a superfamily of multi-functional cytokines that regulates a number of cellular activities (Massague, 1990), including cell growth and differentiation, apoptosis, cellular homeostasis, as well as ECM deposition (Bierie and Moses, 2010; Pelton et al., 1991; Pelton and Moses, 1990). TGF-β superfamily exists in five isoforms, with three of them (TGF-β1, -β2, and -β3) mainly expressing in mammals (Lawrence, 1996). The TGF-β ligands 5  along with TGF-β receptors are ubiquitously expressed in a majority of cell lines and normal tissue (Hayashi and Sakai, 2012; Lawrence, 1996; Segarini, 1993; Wrana, 1998). TGF-β is encoded as a large precursor protein, and each individual gene is responsible for encoding for a separate isoform of TGF-β (Derynck et al., 1985; Gentry and Nash, 1990; Schlunegger and Grutter, 1992; ten Dijke et al., 1990). Prior to their secretion from the cell as a large latent complex (LLC), TGF-β proteins undergo a variety of intracellular processing, including proteolysis of the precursor proteins by the endopeptidase furin and assembling into dimers, which form the 65–75-kDa latency-associated peptide (LAP), and the 25-kDa dimer mature TGF-β (Blanchette et al., 1997; Dubois et al., 1995). It has been reported that the relatively larger molecular weight LAP protein facilitates transportation of mature TGF-β from the cell and constrains the bioactivities of TGF-β (Lopez et al., 1992). The LLC secreted from the cell consists of the active cytokine and one of three latent TGF-β binding proteins (LTBP), LTBP-1, LTBP-3, or LTBP-4 (Saharinen et al., 1996). Association of any LTBP proteins with latent TGF-β (also referred to as small latent TGF-β) leads to the formation of the large latent TGF-β (Hayashi and Sakai, 2012; Munger et al., 1997), which is bound and kept inactive to the ECM through interactions between LTBP proteins and fibrillin and fibronectin complexes (Goumans et al., 2009). It is suggested that defective fibrillin-1 protein (due to genetic defects in FBN1 genes) fails to sequester the latent form of TGF-β in the ECM, leading to excessive activation of TGF-β signaling pathway (Neptune et al., 2003). The active TGF-β ligands bind to TGFBR2, subsequently recruiting and phosphorylating TGFBR1. The receptor-regulated SMADs (R-SMADs) is then phosphorylated by TGFBR1, and translocate to the nucleus (Wrana et al., 1992). Once in the nucleus, SMADs act as transcription factors, targeting a variety of DNA binding proteins, and regulating transcriptional responses and downstream signaling, such as increased transcription of TGF-β, TGFBR1, TGFBR2, and matrix metalloproteinase (MMP)-2 and -9 (Hijova, 2005; Lin and Pan, 2008; Wang et al., 2006)” (Fig. 1). 6   Figure 1. The signaling mechanisms for MFS-associated aortic aneurysm. Fibrillin-1 contains calcium-binding sites that are crucial in stabilizing the microfibrils against degradation within the ECM. Fibrillin-1 also binds to LTBP, sequestering TGF-β activity within the ECM. A mutated fibrillin-1 leads to failed sequestration of LLC, and releasing its active form from the ECM, subsequently, allowing its interaction with TGFBRs, initiating the downstream signaling pathway, such as phosphorylation of SMAD2/3. The phospho-SMAD2/3 will be translocated to the nucleus, and they would target different DNA binding proteins to regulate transcriptional responses, including activation of MMP-2 and -9. Increased MMPs activity will result in breakage of elastin and collagen fibers, and therefore, the loss of vessel wall integrity. Eventually, aortic dilatation or aneurysms may be the ultimate outcome. (Adapting and modifying the figure from (Fedak et al., 7  2002).  1.2.2 TGF-β and its downstream signaling pathways in Marfan syndrome The overall abnormalities in the homeostasis of the ECM are believed to be responsible for a variety of manifestations of MFS described before, in which the mutated forms of FBN1 play a critical role in modifications in the mechanical characteristic of the connective tissues, elevated TGF-β activity and downstream signaling, and loss of interactions between cells and the ECM (El-Hamamsy and Yacoub, 2009). The irregular homeostasis results in vascular remodeling, characterized by over expression and activation of MMP-2 and -9, leading to an exaggerated elastolysis and increased hyaluronan content (Nataatmadja et al., 2006). It has been shown in samples of dilated aortas of patients with MFS that apoptosis of VSMCs might be part of the process of vascular remodeling that contributes to cystic medial necrosis (Nagashima et al., 2001), but the role of apoptosis process still remains unclear. A previous study reported that up-regulated TGF-β signaling had an important impact on the development of aortic root aneurysms in a mouse model of MFS (Habashi et al., 2006). Administration of TGF-β-neutralizing antibodies was shown to have a beneficial effect on the phenotype by slowing down the growth of the aortic root and reducing the fragmentation of elastin in the aortic wall of treated mice, when comparing against the placebo group (Habashi et al., 2006). The same research group also showed that aortic root aneurysm in the MFS mouse model was diminished by pharmacological inhibition of the TGF-β signaling pathway (Habashi et al., 2011).  1.3 Angiotensin II signaling in Marfan syndrome 1.3.1 Angiotensin II and its receptors “Angiotensin II (Ang II) is a key effector molecule of cardiovascular homeostasis that exerts multiple functions in various target tissues, such as controlling blood pressure, blood volume and 8  vascular tone in the cardiovascular system, hormone secretion, tissue growth, and neuronal activities (Whitebread et al., 1989). Most importantly, Ang II triggers ECM remodeling and vascular hypertrophy, and stimulates collagen formation, while reducing elastin synthesis, depressing nitric oxide (NO)-dependent signaling, and increasing reactive oxidant stress (ROS) production, which collectively contributes to aortic stiffness (Dzau, 1986). Ang II mainly interacts with two pharmacologically different sub-types of cell surface receptors, Ang II type 1 receptor (AT1R) and Ang II type 2 receptor (AT2R) (Iwai and Inagami, 1992), with the former being the dominant receptor responsible for the majority of the proinflammatory effects of Ang II in adult tissues. AT1R mediates the activation of the phosphoinositide or calcium pathway, as well as the inhibition of adenylate cyclase activity (Murphy et al., 1991; Sasaki et al., 1991; Sasamura et al., 1992); whereas AT2R seems to mediate vasculo-protective effects through the activation of bradykinin / NO-mediated pathway (Mukoyama et al., 1993). In addition, the abundant expression of AT2R in developing fetal tissues and brain and healing skin wounds has brought forward the suggestion that AT2R activation is critical for normal growth and development (Chiu et al., 1989). It has been known that the interaction of Ang II with AT1R results in decreased apoptosis while increased cellular proliferation and fibrosis, as well as increased MMP-2 and -9 activities, contributing to the degradation of elastin and collagen, and the loss of fiber integrity. On the contrary, the AT2R is thought to increase apoptosis, while decreasing proliferation, fibrosis, and MMP-2/-9 activities (Chung et al., 2007a; Habashi et al., 2006). However, the role that AT2R plays in the progression of MFS aortic aneurysm remains controversial. Some studies have shown that AT2R activation can oppose AT1R-mediated enhancement of TGF-β signaling in some cell types and tissues, suggesting a protective role for AT2R in MFS aneurysm (Doyle et al., 2012; Jones et al., 2004; Rodriguez-Vita et al., 2005).”   9  1.3.2 Ang II receptor blockers (ARBs) in Marfan syndrome The renin-angiotensin system (RAS) is a hormone system that is critically involved in vasodilation and has been used as a potential therapeutic target in the treatment of a wide variety of cardiovascular diseases, such as cardiomyopathy and hypertension. “Vasoconstriction induced by Ang II signaling in VSMCs can be blocked by a commonly prescribed angiotensin converting enzyme inhibitor (ACEI), captopril, through prevention of the conversion of angiotensin I (Ang I) to Ang II, limiting signaling through both AT1R and AT2R, and has been used successfully to treat numerous cardiovascular diseases with few side effects. On the other hand, vasoconstriction induced by Ang II signaling can also be disrupted by selective blockers of AT1R such as losartan (Bahk et al., 2008). Losartan is often prescribed for cardiovascular conditions such as hypertension where ACEIs cannot be used due to their bradykinin-mediated side effects. Unlike the ACEIs, losartan does not play a role in inhibition of VSMCs apoptosis, and as such may not diminish cystic medial degeneration, the histological irregularity observed in aortic dissection (Williams et al., 2008). Nonetheless, other AT1R blockers, for instance irbesartan, are found to decrease MMPs activities (Schieffer et al., 2004). Furthermore, the potential role of TGF-β receptor antagonism by ARBs has been reported to reduce blood pressure and attenuate the deterioration of renal function in uraemic rats (Lavoie et al., 2005). A previous study has shown that the expression of TGF-β1 and collagen in a mouse model of hypertrophic cardiomyopathy, as well as the interstitial fibrosis can be reversed by losartan (Lim et al., 2001). Activation of AT1R by Ang II can also increase the generation of thrombospondin-1 (Tsp-1), a powerful regulator of latent TGF-β activation. Elevated levels of Tsp-1 activate latent TGF-β in response to its signaling through AT1R, resulting in increased expression of active TGF-β ligands and downstream signaling (Albo et al., 1999). Losartan blocks TGF-β activation through inhibition of Tsp-1 production. A recent in vivo study of TGF-β signaling in the aortic wall reported that the SMADs phosphorylation and TGF-β target gene output were up-regulated progressively, which 10  paralleled deterioration of aneurysm pathology and coincided with up-regulation of TGF-β protein expression, all of which were reversed by losartan (Gallo et al., 2014).”  1.4 Matrix metalloproteinases (MMPs) and their inhibitors in MFS aortic aneurysms 1.4.1 MMP sub-types and their roles in diseases “Matrix metalloproteinases are a family of over 25 zinc-dependent endopeptidases with proteolytic activity that break down most of the components of ECM including microfibrils, and regulate many normal and pathological processes (Nagase and Woessner, 1999; Yang and Rosenberg, 2011). They play a central role in organ development and subsequent tissue remodeling and degradation, as well as participating in inflammation and injury.” Collagenase, the founding member of the MMPs family, was the first MMP described in 1962 by Gross and Lapiere, who found that this protease is capable of degrading fibrillar collagen during tadpole tail metamorphosis (Brinckerhoff and Matrisian, 2002; Gross and Lapiere, 1962). This protease was later renamed as MMP-1, following the identification of a similar collagenase in human skin, and inspired a huge body of research on the physiological and pathological role of MMPs. Later in 1960s to 1970s, it was reported that MMPs were up-regulated in various human diseases, such as cancer and rheumatoid arthritis. Furthermore, the increased MMPs activity was usually associated with poor prognosis in human patients (Egeblad and Werb, 2002). However, the roles of MMPs in diseases are controversial in previous clinical research; for instance, elevated MMP activity can either augment tumor progression or can prevent it (Coussens et al., 2002). This complicated relationship between MMP expression and cancer has raised the awareness and drawn more interest in understanding the underlying mechanism of MMP function and cancer pathology, but relatively less focus has been paid to the normal and physiological roles of these enzymes.  11  MMPs have been found to play crucial roles in the regulation of a variety of physiological and pathological processes, such as vascular angiogenesis and remodeling, and are also involved in vascular diseases including aortic aneurysm, atherosclerosis and hypertension, as well as varicose veins. Traditionally, MMPs are thought to be the main enzymes responsible for degradation of many structural components of the ECM, including fibrin, microfibrils, tendons, and cartilage, to facilitate normal turnover of cellular cytoskeleton and ECM, cell migration, and tissue regeneration and development. However, because cells have receptors for the ECM components, for example integrins, MMPs proteolysis can also influence cellular functions by modulating the matrix proteins with which the cells interact (Streuli, 1999). The regulatory functions of MMPs include space creation for cell migration, regulation of tissue structure on the ECM, production of substrate-cleavage fragments with independent biological activity, as well as modification of the activity of signaling molecules (Sternlicht and Werb, 2001).   There are as many as 25 different MMP sub-types identified so far (Kim and Hwang, 2011), with 8 different MMP genes being identified on a chromosome 11 (Arakaki et al., 2009), and together, these 25 MMP sub-types possess the enormous and overlapping variety of enzyme substrate (Roy et al., 2009). However, each one of the MMPs is classified based on its substrates, for instance, collagenases, matrilysins, stromelysins, and gelatinases, as well as the membrane-type MMPs (Kim and Hwang, 2011) (Table 1). Furthermore, the activation of signaling pathway molecules including growth factors and death receptors is regulated by MMPs (Rosenberg, 2009). Most of these MMPs are secreted from the cell, and their activation requires calcium ions and proteolytic cleavage at a neutral pH environment (Tezvergil-Mutluay et al., 2010), with some being activated within the cell (Vincenti and Brinckerhoff, 2002). Among a variety of MMP downstream target molecules, TGF-β is an important one. The activation of TGF-β often modifies cell migration, for instance, MMP-9 confines corneal epithelial migration through the stimulation 12  of TGF-β (Mohan et al., 2002). Both MMP-2 and MMP-9 have a close association with TGF-β, leading to the release of TGF-β from an inactive extracellular complex that is composed of TGF-β, LAP (the pro-domain of TGF-β), and LTBP (Yu and Stamenkovic, 2000). Increased MMP-2 and MMP-9 activities negatively impact the normal turnover of elastin and collagen within the ECM, resulting in disruption and disorganization of elastic fibers and loss of ECM structural integrity, as well as reduction of VSMCs contraction (Chung et al., 2007a; Cui et al., 2014).   Table 1. Matrix metalloproteinases (MMPs) sub-types and actions. (Adapting and modifying the table from (Stechmiller et al., 2010).    The denatured forms of collagenous fibrils of both types I and III collagen, previously thought degraded by collagenases, are found to be cleaved partially by MMP-2 and MMP-9, also known as gelatinase A and B, respectively (Stechmiller et al., 2010). Gelatinases cleave other subtypes of non-fibrillar collagens and ECM constituents, such as elastin, type IV and X collagen, and small diameter fibrillary collagen (type V), as well as epithelial anchoring collagen (type VII) 13  (Birkedal-Hansen, 1995). Following the removal of collagen types I, II, and III from the triple helix, these collagens are further degraded by gelatinases (Agren, 1994). MMP-2 is produced by fibroblasts, while MMP-9 is secreted by leukocytes and keratinocytes (Pilcher et al., 1999). Both MMP-2 and MMP-9 are believed to result in cardiac rupture following myocardial infarction, while MMP-2 could also be responsible for cardiac damage through the digestion of poly(ADP-ribose) polymerase and myosin light chain (MLC) (Nagase et al., 2006). Gene expression and activity of MMP-2 are also evident in skeletal muscle from patients with Duchenne muscular dystrophy (Lluri and Jaworski, 2005). In addition, MMP-2 is thought to have an anti-inflammatory role, and is involved in differentiation of mesenchymal cells, generation of vasoconstrictors, as well as the neurite outgrowth (Nagase et al., 2006). MMP-9, itself activated by MMP-3, is mainly involved in degradation of elastin and collagen (type IV). MMP-9 is also believed to cleave and modify chemokine and cytokine, as well as activity of growth factor (Steenport et al., 2009). Furthermore, it is shown that expression of MMP-9 is usually reduced in normal tissues but is noticeably increased during inflammation, neoplasia, as well as wound healing (Colnot et al., 2003; Galis et al., 2002; Page-McCaw et al., 2007). Increased expression of MMPs, both the mRNA and protein, has been observed in human aneurysm tissue (Curci et al., 1998a; Davis et al., 1998; Freestone et al., 1995; Thompson et al., 1995) and MFS mice aorta (Chung et al., 2007a; Chung et al., 2007b; Yang et al., 2010). In a mouse model of MFS associated aneurysm, increased MMP-2 and MMP-9 activities are concurrent with extensive destruction of elastin and collagen, which eventually results in reduction of VMSCs contractility, loss of vascular matrix integrity, and endothelial dysfunction (Chung et al., 2007a; Chung et al., 2008). “The loss of balance between MMP-2/-9 and the tissue inhibitors of MMPs (TIMP)-1/-2, attributed to over-activation of the TGF-β signaling pathway, plays a crucial role in the pathogenesis of aortic aneurysm, which carries the risk of rupture eventually (Allaire et al., 1998; Longo et al., 2002; Manabe et al., 2004; Sakalihasan et al., 1996; Tamarina et al., 1997).”  14   1.4.2 Inhibition of MMPs by their intrinsic and extrinsic inhibitors MMP activity is inhibited by specific and non-specific inhibitors including endogenous TIMPs and pharmacological inhibitors, respectively, for example, zinc chelators, marimastat and doxycycline. Together, MMPs and their intrinsic inhibitors contribute to the tightly regulated turnover of elastic and collagenous fibers within the ECM (Baramova and Foidart, 1995; Matrisian, 1990; Matrisian, 1992; Mauch et al., 1994). Besides endogenous TIMPs, MMP-2 activity has been demonstrated to be suppressed by beta-amyloid precursor protein, a C-terminal fragment of procollagen C-proteinase enhancer protein, and RECK (reversion-inducing-cysteine-rich protein with kazal motifs), an angiogenesis suppressing glycoprotein (Nagase et al., 2006).   Doxycycline, one of the most active antibiotics of the tetracycline family, was found to be a general inhibitor of MMPs activity at sub-antimicrobial doses and the only widely available inhibitor in clinical practice (Liu et al., 2003). Doxycycline has been tested in a wide variety of conditions associated with high levels of MMP activity, and is traditionally administered intravenously or orally. Tetracycline derivatives, especially doxycycline, have been widely investigated in human and animal disease models. In humans, these include rheumatoid arthritis (Lauhio et al., 1994), non-infected corneal ulcers (Perry and Golub, 1985), osteoarthritis (Shlopov et al., 1999; Yu et al., 1991), adult periodontitis (Golub et al., 1995), and chronic wounds (Chin et al., 2003; Siemonsma et al., 2003; Smith et al., 1999). In laboratory animal models, doxycycline and other tetracycline derivatives have been studied in ulcerative disease models, for instance, alkali-burned rabbit corneas (Burns et al., 1989; Seedor et al., 1987), alveolar bone loss in rodents (Chang et al., 1994), and in angiogenic morphogenesis models where MMP activity is required (Tamargo et al., 1991), as well as in model of MFS associated aneurysm (Chung et al., 2008). In a number of animal studies, oral treatment with doxycycline or 15  tetracycline derivatives has improved healing outcomes (Ramamurthy et al., 1998; Seedor et al., 1987; Siemonsma et al., 2003). A previous study in the MFS mouse model showed that progression of aortic aneurysm in MFS is associated with up-regulation of MMP-2 and -9, and that doxycycline could preserve elastin structure and organization, improve aortic contractility, and normalize aortic function (Chung et al., 2008). The same study reported that doxycycline seemed to be more effective than atenolol in preventing aneurysm progression in mice (Chung et al., 2008).  1.5 Major transgenetic animal models of Marfan syndrome and their main features The development of several mouse models of MFS associated aortic aneurysm has contributed greatly to our current knowledge of molecular pathogenesis of aortic aneurysm in MFS and other related connective tissue disorders. The first knockout mouse model was homozygous for hypomorphic alleles of Fbn1 gene: the mgΔ (Fbn1mgΔ/mgΔ) and mgR (Fbn1mgR/mgR) models (Pereira et al., 1997). (Citing quotations from (Lee et al., 2016) “The mouse model for MFS-mgΔ was generated by Pereira and colleagues in 1997, in which exons 19 to 24 were replaced with the neomycin (neo) gene under the control of the PGK promoter, to mimic the dominant-negative effect of FBN1 mutations observed in patients with MFS. A truncated form of FBN1 is expressed in the mgΔ model at relatively low levels, leading to early postnatal lethality. Meanwhile, a MFS-mgR homozygote mouse model was created by inserting a PGK neo-cassette between exons 18 and 19 of the endogenous gene, recapitulating the adult lethal form of MFS (Pereira et al., 1999). This results in a hypomorphic mutation, and generation of Fbn1mgR/mgR mice that exhibit an approximately 5-fold reduction in the expression of the mgR allele, which produces low levels of normal fibrillin-1.” Histologically, the mgΔ and mgR heterozygote mice were indistinguishable from their wild type littermates, which suggest the lack of the dominant negative effects of FBN1 mutations observed in patients with MFS. However, because of the 16  severe cardiovascular failure, the mgΔ and mgR homozygote animals died at the age of 3 to 6 months old. Furthermore, expression analysis showed that the transcript level is 10-fold lower in the mgΔ allele vs. the normal Fbn1 allele. Therefore, it was assumed that expression of the mutant allele had been interfered with by the neoR cassette sequence, and thus confining the dominant-negative effect of FBN1 mutation. Although the MFS-mgΔ mouse model was generated to characterize a crucial function of FBN1, the premature death of these mice hindered the precise identification of the pathological mechanism resulting in an aortic aneurysm. Besides these premature death knockout mice models, a third transgenetic animal model, a heterozygous mutation for the C1039G (Fbn1C1039G/+), was developed by the cysteine for glycine substitution (C-to-G) at residue 1039 in an epidermal growth factor domain of FBN1, resembling to one of the most often seen FBN1 mutations in patients (Judge et al., 2004). The C1039G heterozygote animals exhibit bone deformity, and insufficient FBN1 microfibril deposition in the ECM, as well as progressive weakening of the elastic fiber structure in the aortic wall (Ng et al., 2004). “These features are in agreement with a model that invokes haploinsufficiency for wild type FBN1 protein, rather than production of mutant protein, as the primary determinant of failed microfibrillar assembly. The haploinsufficiency results in approximately half of the normal fibrillin concentration, which directly contributes to the progression of an aortic aneurysm.”  Taken together, these 3 animal models described above have been characterized in the B6 inbred strain (Dietrich et al., 1993; Pereira et al., 1997; Whiteman and Handford, 2003). These mice generally develop advanced aortic aneurysms by the age of 9-month-old, but early signs of elastic fiber fragmentation are observed at around 3-month-old (Cui et al., 2014), and death due to aortic aneurysm usually occurs at 12–18 months of age (Judge et al., 2004). Recently, a novel mouse model of MFS-mgΔloxPneo was established to recapitulate several key manifestations in patients with MFS, presenting the clinical characteristics and individual variability in human 17  disease (Lima et al., 2010). The main difference between this novel model and the original Fbn1mgΔ/mgΔ model is the presence of 2 loxP sites flanking neoR alleles in the new MFS-mgΔloxPneo model. Both mutant alleles are believed to be responsible for the in-frame deletion, encompassing exons 19–24 of Fbn1 gene. However, compared to the mgΔ mutant allele, the relative expression levels of the mgΔloxPneo allele increases by 47%, which can elucidate the features of the disease in mgΔloxPneo heterozygotes based on the dominant-negative model of pathogenesis for MFS (Lima et al., 2010).  1.6 Emerging novel techniques for in vivo and ex vivo studies of biophysical and structural properties of the aorta 1.6.1 Multiphoton microscopy and its applications Two-photon excitation laser scanning microscopy, also referred to as multiphoton microscopy (MPM), was invented in early 1990s by Denk and colleagues, as an advanced fluorescence imaging technique that enables imaging of live samples up to approximately 1 mm in depth (Denk and Webb, 1990). MPM can be a superior alternative to confocal microscopy, where excitation of fluorescent probes occurs through the simultaneous absorption of two or more photons of longer wavelengths, in the near-infrared regions (Denk, 1994; Zipfel et al., 2003). The MPM imaging system provides multiple advantages over one-photon techniques for microscopy in scattering specimens, allowing high-contrast and high-resolution fluorescence imaging deep in the tissue (Denk et al., 1994). Firstly, the excitation wavelengths utilized in MPM, such as deep red and near-infrared, penetrate much deeper into tissue, compared to the visible wavelengths utilized in one-photon microscope. The reduced absorption and scattering by endogenous chromophores contribute to the improved penetration by MPM (Oheim et al., 2001; Svoboda and Block, 1994; Yaroslavsky et al., 2002). Secondly, scattered excitation photons are too dilute to generate noticeable absorption due to the property of nonlinear excitation. 18  Furthermore, in normal circumstances most of the incident photons are scattered, even deep in tissue, and thus, fluorescence excitation is restricted only to a small focal volume. Thirdly, all the emitted photons from multiphoton excitation, including scattered and ballistic, establish valuable signal if they are detected as a result of localization of excitation. On the contrary to MPM, scattered fluorescence photons are either worse or lost, contribute to background in wide-field and confocal microscopy (Centonze and White, 1998). Therefore, these advantages of MPM could be huge in imaging tissue as the majority of fluorescence photons are scattered.  (Citing quotations from (Cui et al., 2014) “Due to its efficient light detection, deeper tissue penetration, and reduced phototoxicity, MPM has been employed to yield three-dimensional (3-D) rendering of the structures in living tissue up to a very high depth. In addition, because MPM is able to non-invasively reveal the ultrastructure of elastin and collagen (Abraham and Hogg, 2010), this technique has gained considerable favor in cardiovascular research and dermatology (Chen et al., 2011; Lin et al., 2007). Its clinical advantage is the capability of observing non-fixed, unstained tissue samples (Abraham et al., 2011), with the potential of performing non-invasive in vivo measurements directly on a patient (Masters et al., 1997). The aorta and skin contain naturally occurring fluorophores that can be imaged using MPM without the need for exogenous contrast agents. These include elastin, collagen, melanin, keratins, porphyrins, nitrate reductase (NAD(P)H) and flavins (Levitt et al., 2011). Collagen in the aorta and dermis of the skin produces a second harmonic generation (SHG) signal, which can be differentiated from two-photon excited fluorescence (TPF) generated by elastin (Tang et al., 2006).” The basic outline of a MPM system capable of both TPF and SHG signal detections is presented in Figure 2 and is described in details elsewhere (Abraham et al., 2012). In this study, SHG signal originating from collagen was obtained from the emission wavelength at 440 nm, which only arises at half of the excitation wavelength at 880 nm. The TPF signal originating from elastin was also obtained from 19  the excitation wavelength at 880 nm, attributing to the measured broadband emission spectrum ranging from 400–650 nm with a peak at 500 nm, as previously described (Abraham and Hogg, 2010).  There are more than a thousand publications, which have developed, employed, or reviewed MPM (Denk and Svoboda, 1997; Helmchen and Denk, 2002; Helmchen and Denk, 2005; So et al., 2000; Zipfel et al., 2003), and MPM has been used in numerous disease diagnosis and medical research areas, for instance, neurobiology, embryology, physiology, and tissue engineering. Virtually transparent tissues, such as thin skin sections, have been visualized at high-resolution because of the 3-D rendering by MPM (Masters et al., 1997). MPM’s high-speed imaging capacity could potentially be used in non-invasive optical biopsy (Bewersdorf et al., 1998). Furthermore, in cell biology, MPM has been utilized for creating localized chemical reactions (Denk et al., 1994).  1.6.2 High frequency high-resolution ultrasound imaging and its applications An echocardiogram, also referred to as a “cardiac ultrasound” (a term was developed in the 1960s and 1970s), is a sonogram of the heart, and a non-invasive diagnostic technique which provides information on cardiac function, morphology and hemodynamics. Echocardiography uses standard 2-D (two-dimensional), 3-D, and Doppler ultrasound to create images of the heart and aorta, which is the most broadly used cardiovascular diagnostic test (Feigenbaum, 1996). With recent advances, echocardiography has been playing a crucial role in cardiovascular medicine, and become regularly used in the diagnosis, management, and follow-up of patients with any suspected or known cardiovascular diseases. Echocardiography can provide a variety of valuable estimates, such as the cardiac and aortic structure (for example, size and shape of the heart and aorta), cardiac and aortic function (for instance, pumping capacity, calculation of the 20  ejection fraction, cardiac output, diastolic function, and pulse wave velocity (PWV), as well as the location and extent of any tissue damage. Furthermore, echocardiography can also generate precise assessment of the blood flow by Doppler echocardiography, using continuous- or pulsed-wave Doppler ultrasound, which enables the determination of blood flow through the vessels and chambers of the heart. In addition, the Doppler technique can also be used for PWV and tissue motion measurement, by tissue Doppler echocardiography.  (Citing quotations from (Cui, 2016) “Aortic wall stiffness is an indicator of aortic wall structure and reflects its components, such as the amounts of calcium, collagen and elastic fibers (Roach and Burton, 1957). It is also a direct indicator of arterial distensibility, a determinant of stress on the vessel wall and a reciprocal of stiffness which has been shown to be predictive of cardiovascular events and all-cause mortality (Laurent et al., 2006; Vlachopoulos, 2012; Vlachopoulos et al., 2010), for instance, in type II diabetes patients (Cruickshank et al., 2002). Arterial stiffness is quantitatively expressed as compliance and distensibility (Safar et al., 2003).” Compliance is defined as “a change in cross-sectional area for a given change in pressure” (Tozzi et al., 2000), and distensibility is “a fractional change in volume or cross-sectional area for a given change in pressure” (Hughes et al., 2004). “These are quantitative parameters with appropriate units of measurement (Gosling and Budge, 2003). Stiffening affects elastic arteries, predominantly the aorta. There have been various indirect methods to quantify aortic stiffness, including 1) measuring PWV by echocardiography; 2) relating variations in aortic diameter to distending pressure; and 3) assessing aortic pressure wave formation. PWV is the most broadly accepted technique of quantification of aortic wall stiffness, and has been a valuable non-invasive method to assess stiffness of cardiothoracic arteries (O'Rourke et al., 2002). PWV, affected by the aortic intrinsic elasticity, is “the velocity of the blood pressure wave as it travels a known distance between two anatomic sites within the arterial system” (Oliver and Webb, 2003). In other words, 21  PWV can be calculated as the velocity of flow wave propagation of pressure through a defined portion of the arterial tree, which provides an indirect measure of an alteration in the mechanical properties of an aortic segment, and positively correlates with aortic distensibility and stiffness. In addition to determining the aortic stiffness by PWV measurement (Bradley et al., 2005), echocardiography and color Doppler has been employed to evaluate the structural and functional properties of the heart and aorta, and to monitor a variety of cardiovascular manifestations in MFS include mitral valve prolapse, mitral annular calcification, ascending and descending aortic dilatation and dissection, aortic regurgitation, and dilated cardiomyopathy (Arnlov et al., 2004; Hirata et al., 1991; Levenson, 2010). These applications and findings highlight the robustness of echocardiography and its value in cardiovascular risk assessment.”  (Citing quotations from (Lee et al., 2016) “Aortic root aneurysm is the most lethal cardiovascular complications in MFS patients (Loeys et al., 2010). Increased aortic stiffness and loss of wall elasticity are considered to be important detrimental factors contributing to aneurysm progression. Early studies in a mouse model of MFS have used ex vivo approaches to indirectly measure aortic wall stiffness or elasticity including the use of isometric wire myography (Chung et al., 2007a). Later, non-invasive high-resolution high frequency ultrasound imaging techniques provided a more powerful tool for in vivo measurements of both cardiac and aortic structure and function in real time (El-Hamamsy and Yacoub, 2009). Even without aortic root dilation, MFS patients have been shown to have increased aortic stiffness (Bradley et al., 2005). PWV has been reported to be a robust marker of aortic stiffness and can be measured non-invasively using echocardiography in patients (Hirata et al., 1991). PWV has been demonstrated in different populations including elderly, hypertensive, diabetic and renal patients as an index of aortic stiffness and the earliest predictor of cardiovascular risk (Cruickshank et al., 2002; Laurent et al., 2006; Laurent et al., 2003; Sutton-Tyrrell et al., 2005). Using echocardiography or magnetic 22  resonance imaging (MRI), previous studies have confirmed increased aortic stiffness in MFS (Groenink et al., 2001; Kiotsekoglou et al., 2011; Kroner et al., 2013; Westenberg et al., 2011), and demonstrated that aortic stiffness was increased with age and aortic diameter (Jeremy et al., 1994). Bradley et al. introduced an echocardiographic Doppler method of assessment of the biophysical properties of the aorta for use in humans, and reported an increased PWV in pediatric patients MFS patients as compared with normal subjects (481 ± 70 vs. 357 ± 61 cm/s, p<0.001) (Bradley et al., 2005).” Although normal systolic function has been demonstrated in patients by conventional echocardiography using M-mode and 2-D parameters (Chatrath et al., 2003; Das et al., 2006; Meijboom et al., 2005; Roman et al., 1989), more sophisticated techniques including cardiac magnetic resonance imaging and Doppler tissue imaging have provided evidence of compromised systolic and diastolic function (De Backer et al., 2006; Kiotsekoglou et al., 2009; Rybczynski et al., 2007; Savolainen et al., 1994). Hence, in this study, we have conducted an in vivo study in the mouse model of MFS, to duplicate studies conducted in patients with MFS showing dilatation of the sinuses of Valsalva and sinotubular junction, and progressive loss of aortic root elasticity using the high-resolution and non-invasive ultrasound technique (El-Hamamsy and Yacoub, 2009; Hirata et al., 1991; Nollen et al., 2004).   1.6.3 Electron microscopy and its applications in characterization of elastic fiber ultrastructure An electron microscope (EM), first developed and constructed in 1920s and 1930s by Hans Busch and Ernst Ruska (Ruska, 1987), is a microscope that utilizes a beam of accelerated electrons to illuminate the specimen. Compared to light microscopes, the resolving capability is far more powerful in electron microscopes, due to the wavelength of an electron which is up to a hundred thousand times shorter than those of visible light photons. Therefore, EM can reveal the ultrastructure of various inorganic and biological samples, such as cells, crystals, microorganisms, 23  large molecules and biopsy samples. A transmission electron microscope (TEM), the original form of EM, uses a high-voltage electron gun as the illumination source to irradiate the thin specimen and generate an image. The image resolution of a TEM is generally higher than that of a scanning electron microscope (SEM). However, because the TEM images both the surface and interior of a specimen, the electrons have to travel through the very thin sample section. The need for extremely thin sections of the specimens (approximately 100 nanometers) is one of the major challenges of the TEM, as processing such extremely thin sections for biological specimens is technically difficult. Typically, biological materials are initially formaldehyde-fixed and methanol-dehydrated, followed by embedding in polymer resin, to harden the blocks sufficiently to allow them to be ultra-thin sectioned. Sections of organic polymers and similar specimens may require further staining with heavy atom labels in order to enhance the image contrast.  Previously, both the TEM and the conventional light microscopy were employed to reveal the elastic laminae in the human bronchial and tracheal mucosa. Three types of fibers were characterized through their staining properties and ultrastructural morphology, including elastic fibers, elaunin fibers, and oxytalan fibers (Bock and Stockinger, 1984). The investigators found that elastic fibers are mainly composed of elastin and few microfibrils. They also reported that the major difference between elastic fibers and elaunin fibers is that the relative amount of elastin over microfibrils is smaller in elaunin fibers. On the contrary, oxytalan fibers are bundles of pure microfibrils. Traditional light microscope showed that the submucosa of the normal mucous membrane and the lamina propria are separated by the elastic laminae, a well-defined lamina composed of coarse strands of elastic fibers that are running longitudinally. In addition, sub-epithelial elastic layer, the epithelial basement membrane, was found to attach to a sophisticated meshwork of elastica-positive fibers. In terms of the ultrastructural morphology, electron microscopy revealed that elastic fibers predominantly form the lamina elastica, while elaunin 24  fibers form the sub-epithelial elastic layer. Furthermore, oxytalan fibers were observed to penetrate the thickened basement membrane layer of the epithelium. Taken together, all 3 types of fibers can be found in the lamina propria and throughout the submucosa.  1.7 Three objectives of the present study 1.7.1 Ex vivo study of elastic fiber morphology in the mouse model of MFS by MPM (Citing quotations from (Cui et al., 2014) “Skin is the largest organ of the body based on surface area. Like the aorta, elastin and collagen are two major ECM components of the skin dermis (Chen et al., 2011). Elastin provides the majority of the resilience and elastic properties of skin, whereas collagen gives the skin strength, texture, durability, mechanical and structural integrity (Mochizuki et al., 2002). Previous studies have shown that various proteases, including MMPs, are responsible for the degradation of elastin and collagen in skin (Brenneisen et al., 1997; Brenneisen et al., 1996). Thus far, quantitative analyses of elastic fiber damage and breakdown of collagen in MFS skin have not been explored. Existing studies are based on histological methods, electron microscopy, and immunohistochemical techniques (Amadeu et al., 2004; Godfrey et al., 1990a). These methods usually involve fixation, staining, and dehydration of biopsied tissue specimen, which alter the native morphology of elastin and collagen.” As described above, the main advantages of multiphoton microscope include the capability of non-invasively revealing the ultrastructure of elastin and collagen, observing non-fixed, unstained tissue samples (Abraham et al., 2011), and potentially performing non-invasive in vivo measurements directly on a patient (Masters et al., 1997). “The aorta and skin contain naturally occurring fluorophores, such as elastin and collagen, that can be imaged using MPM without the need for exogenous contrast agents (Levitt et al., 2011). Collagen in the aorta and dermis of the skin produces a SHG signal, which can be differentiated from TPF generated by elastin (Tang et al., 2006). The aim of this ex vivo study (as part I of the present thesis), is to use MPM for 25  assessing the progress of elastin and collagen damage in the large arteries and skin dermis of a mouse model of MFS. The morphology and total volumes of elastin and collagen measured using MPM in MFS mice aorta and skins are compared with control mice in different age groups. The novel aspect of this study is the non-invasive recording and comparison of defects in elastin and collagen present in aorta and the inner layer of the skin dermis; with the long-term aim of developing an in vivo skin test for early non-invasive diagnosis of MFS and other connective tissue disorders.”  1.7.2 In vivo study of aortic structure and function in the mouse model of MFS by ultrasound imaging (Citing quotations from (Lee et al., 2016) “Echocardiography has been of great importance in the diagnosis and follow-up of patients with various connective tissue diseases to detect and evaluate their cardiovascular structure and function including thoracic aortic aneurysm (Levenson, 2010). In addition to evaluating the structural and functional properties of the aorta, such as measuring the aortic root diameter, PWV assessment has the capacity to estimate the level of aorta stiffness (Bradley et al., 2005; Evangelista et al., 2010).” It has also been demonstrated that the fragmentation and disorganization of elastic fibers are detrimental factors contributing to loss of elasticity and increased stiffness in the aortic wall, ultimately resulting in the progression of aneurysm (Chung et al., 2008). Aortic stiffness is determined by chemical components, such as elastin, collagen and calcium, as well as their structural arrangement (Roach and Burton, 1957). Increased aortic stiffness has been shown to be predictive of cardiovascular events and all-cause mortality in the general population (Laurent et al., 2006; Vlachopoulos, 2012; Vlachopoulos et al., 2010). There have been various indirect methods to quantify aortic stiffness including the carotid-femoral PWV, a broadly accepted measure of arterial stiffness of elastic arteries (Asmar et al., 1995; Yildiz et al., 2006). Measuring thoracic aorta PWV by echocardiography is less 26  accepted, but has been a valuable non-invasive technique to assess stiffness of cardiothoracic arteries (O'Rourke et al., 2002; Sandor et al., 2015). In addition to determining the aortic stiffness, echocardiography and color Doppler have been employed to monitor a variety of cardiovascular manifestations, highlighting the robustness of the technique and its value in cardiovascular risk assessment in MFS patients. “Both the mgR (Fbn1mgR/mgR) and the C1039G (Fbn1C1039G/+) transgenetic mouse models of MFS as described before continue to reveal tremendous insight into the impact and consequences of FBN1 mutations in the etiology of MFS, however, there have not been any in vivo non-invasive echocardiographic studies of the clinical phenotype of these mice to determine the degree to which they recapitulate the human condition (Judge et al., 2004). Due to its more natural progression of the disease, the C1039G (Fbn1C1039G/+) mouse model of MFS allows us to examine the time course of the pathological phenotypes of MFS over a 12-month period.” Therefore, the aim of this in vivo study (as part II of the thesis), was to “investigate the structural and functional properties of the aorta in the Fbn1C1039G/+ mice, particularly PWV and other well characterized indices known to be abnormal in MFS patients, using a non-invasive and high-resolution ultrasound technique, and in order to duplicate previously published studies in MFS patients showing dilatation of the sinuses of Valsalva and sinotubular junction, and progressive loss of aortic root elasticity (El-Hamamsy and Yacoub, 2009; Hirata et al., 1991; Nollen et al., 2004). Moreover, an important advantage of the echo Doppler method in the Fbn1C1039G/+ mouse is that it allows for gradual and longitudinal evaluation of the disease progression in the same animal.”  1.7.3 In vivo and ex vivo study of doxycycline effects on biophysical and ultrastructural properties of the aorta in a mouse model of MFS by echo and TEM Rapidly progressive aortic aneurysm, a distinct feature of the aggressive vascular pathology of MFS, warrants close monitoring with the necessity of subsequent endovascular repair or aortic 27  replacement surgeries. “Previous studies in human and animal models have revealed the involvement of up-regulated TGF-β signaling in MFS-associated aortic aneurysm (Gallo et al., 2014), therefore, the administration of therapeutic agents that are known to antagonize TGF-β signaling through inhibition of TGF-β expression and activation such as losartan may provide certain positive outcomes for patients with MFS (Lavoie et al., 2005; Loeys et al., 2006; Malhotra and Westesson, 2009). However, such treatment has been shown to postpone the onset of the disease, without preventing the ultimate need for the aortic replacement surgery.” In addition, our previous studies in the mouse model showed that progression of aortic aneurysm is accompanied by up-regulation of MMP-2 and MMP-9 in MFS. Elevated MMP activity is associated with extensive destruction of elastin and collagen, which eventually results in endothelial dysfunction, reduction of VMCS contractility, and loss of ECM integrity (Chung et al., 2007a; Chung et al., 2007b; Yang et al., 2010). We have also been able to demonstrate that doxycycline can preserve elastic fiber structure and organization, as well as improving aortic contractility and normalizing aortic function. Besides, doxycycline seems to be more effective than atenolol in preventing thoracic aortic aneurysm in a mouse model of MFS (Chung et al., 2008). However, critical gaps remain in our understanding of the effects of the long-term administration of a sub-antibiotic dose of doxycycline on aortic aneurysm progression, and its effects on ultrastructure of elastic fibers in Marfan mice. As part III of the present thesis, this longitudinal study was designed to determine the possibility of performing a long-term therapy research in vivo in MFS mice, and to assess the outcomes by evaluating the alterations in aortic function and structure, and elastic fiber ultrastructure. To address these objectives, a series of experiments were conducted to measure the thoracic aortic diameters, PWV, and the functional characterization of the aortic wall elasticity in vivo (by echocardiography), and ex vivo (by TEM) in a mouse model of MFS.  The novel aspect of this study is the longitudinal non-invasive monitoring of progression of aortic dilatation and stiffness, and the therapeutic effects of doxycycline on blocking this progression in the same 28  experimental subjects, with the hope of providing new insights into the potential therapeutic value of long-term doxycycline intervention in blocking Marfan-associated aneurysm, and establishing the rationale for a future similar clinical trial in human MFS patients.                        29  2. Chapter 2. Materials & Methods: 2.1  Experimental animals for ex vivo and in vivo studies (Citing quotations from (Cui et al., 2014) “All experiments were performed using a previously described transgenic mouse model, harboring an Fbn1 allele encoding mutation C1039G (a cysteine substitution Cys1039Gly), in an epidermal growth factor–like domain of fibrillin-1 (Fbn1C1039G/+) (Chung et al., 2007a; Chung et al., 2008). Heterozygous mice were bred with wild type mice (C57BL/6) to generate control (Fbn1+/+) and MFS (Fbn1C1039G/+) mice, which were housed in the institutional animal facility. All animal procedures were approved by the institutional animal ethics board [reference number A11-0018], and all animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals (www.nap.edu/catalog/5140.html).”   For ex vivo study of elastic fiber morphology by MPM, “mice were anaesthetized by inhalation of 3% of isoflurane (Baxter Corporation, Mississauga, Canada), and the adequacy of anesthesia was confirmed by pedal reflex. Mice were then sacrificed by cervical dislocation;” for in vivo study of aortic structure and function, six- and twelve-month old MFS mice and their control wild type (WT) littermates were subjected to high-resolution ultrasound imaging.  2.2  Preparation of aortic and skin tissue samples for MPM imaging “The thoracic aorta (~10–14 mm) was dissected from control and MFS mice, at the age of 3-, 6-, and 9-month (3-month: control n=5, MFS n=4; 6-month: control n=5, MFS n=4; 9-month: control n=3, MFS, n=4). Specimens were washed in cold PBS (pH=7.4) and cut into segments for use in MPM and histologic analysis. The ascending aortic root was transected above the level of the aortic valve, and 2–3 mm transverse sections were cut to two or three pieces (length, 1 mm/each). The curving aortic arch (length, ~3 mm) and the descending aorta (length, 3-5 mm) 30  were cut to 3 or 4 pieces, respectively (1 mm/each piece). The aortic tube segments were then mounted vertically on a petri dish, after which they were washed and immersed in PBS for multiphoton imaging.” “The skin from the dorsal surface was dissected from control and MFS mice (3-, 6- and 9-month old) after the hairs were shaved and removed by applying hair removal, followed by washing in cold PBS. The harvested skin specimens were flattened and cut into two halves. One half was embedded in optimum cutting temperature compound (VWR, West Chester, PA), snap frozen in liquid nitrogen, and cut into transverse sections at 50 μm thick for multiphoton imaging. The other half was utilized for histochemistry.”  2.3  Features of the MPM system and experimental set-up “The basic outline of a MPM system capable of both TPF and SHG signal detections is presented in figure 2 and was described in details elsewhere (Abraham et al., 2011). In our present study, SHG signal originating from collagen was obtained from the emission wavelength at 440 nm, which only arises at half of the excitation wavelength 880 nm. The TPF signal originating from elastin was also obtained from the excitation wavelength at 880 nm, attributing to the measured broadband emission spectrum ranging from 400–650 nm with a peak at 500 nm, as previously described (Abraham and Hogg, 2010).”  31    Figure 2. A simplified schematic representation of multiphoton microscopy system. The laser beam is focused on the specimen through a high-resolution water dipping objective. The backscattered emissions of SHG and TPF signals from the thick aorta and skin tissues are collected through the same objective lens and directed to the photomultiplier tube (PMT) detectors in the reflection geometry. TPF signals from elastin are illustrated in green, whereas SHG signals from collagen are present in dark purple. (Citing the figure and legend from (Cui et al., 2014)  2.4  Preparation of aortic and skin tissue samples for histological staining For ex vivo study on elastic fiber morphology, “three parts of aorta and the second half of dorsal skin samples were fixed in 10% buffered formalin for 48 hours, after which they were immersed in 70% ethanol overnight at 4°C, and embedded in paraffin. Specimens were cut into 5 32  μm thick cross-sections. Tissue sections were deparaffinized in xylene and rehydrated in graded ethanol. Elastic fibers of aortas and skins were stained by use of Accustain® Elastic Stain kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s standard procedure. Briefly, rehydrated tissue sections were placed in a Coplin jar containing working elastic stain solution for 10 minutes. Sections were rinsed in deionized water and then differentiated in working ferric chloride solution for 30–60 seconds. Differentiation was stopped with several changes of tap water. Sections were then rinsed in 95% alcohol to remove iodine and then in Van Gieson solution for 3–5 minutes (Lillie, 1965). Following the staining procedure, samples were dehydrated through graded ethanol and xylene, and mounted with mounting medium with coverslip” for light microscope imaging.  At the end of the longitudinal in vivo study (at the age of 12 months), sham or doxycycline treated control and MFS mice (n=4) were euthanized by inhalation of 3% of isoflurane followed by cervical dislocation. “The thoracic aorta (~10–14 mm) was dissected from control and MFS mice, and the tissue specimens were transversely cut into 5 μm thick cross-sections, and followed by deparaffinized in xylene and rehydrated in graded ethanol. The fixation of the samples was described briefly as above, and again, the elastic fibers of aortas were stained by use of Accustain® Elastic Stain kit (Sigma-Aldrich, St. Louis, MO)” as described previously (Cui et al., 2014).  2.5  Quantitative and statistical analysis on MPM imaging “3-D reconstruction of aortic segments and skin, as well as the quantification of volumetric density of elastin and collagen distinguished by TPF and SHG signals, respectively, was performed using Volocity® 6.1.1 image-processing software (PerkinElmer, Waltham, MA) as previously descried (Suzuki et al., 2012). The elastic fiber fragmentation was determined using 33  Image-Pro-Plus 6.0 software package (Media Cybernetics, Bethesda, MA), by tracing elastin and measuring their lengths in pixels followed by unit conversion to micrometer as previously described (Zhou et al., 2012).  In the present study, we have also used the 2-D fast Fourier transform (FFT) algorithm, which convert complex spatial patterns (represented by changes in pixel intensity values) into directionally dependent frequency components (Petroll, 2006), to determine the elastic fiber orientation and anisotropy of the elastin. Briefly, FFT was performed on each image of dimensions 512×512 pixels, converting it from complex spatial signals of intensity and position (x, y) into directionally dependent frequency components (u, v) using MATLAB (Mathworks, Natick, MA). Using FFT analysis, intensity signals can be transformed from the spatial domain to the frequency domain, and the histograms of both the horizontally and the vertically occurring frequencies can be examined. The theory of FFT algorithm has been previously described in details (Abraham et al., 2012). Numerical values were expressed as means ± SEM unless otherwise indicated. Comparisons of parameters among the three groups (harvested at 3-, 6- and 9-month) were made using 1-way ANOVA followed by Tukey multiple comparison test. Comparisons of parameters between two groups were made by two-tailed Student's t-test, where a p value of p < 0.05 was considered significant.”  2.6  Timeline for doxycycline treatment in mice Beginning 6 weeks of age, control (n=13) and MFS (n=12) mice were given a sub-antimicrobial dose of doxycycline hyclate (Alfa Aesar, Ward Hill, MA) in their drinking water at a concentration of 0.24 g/L/day, a therapeutic dose already shown in our laboratory to effectively inhibit MMP activity in MFS mice (Chung et al., 2008); whereas another group of control (n=12) and MFS (n=12) mice were given plain drinking water for comparison (Fig. 3). Because 34  doxycycline hyclate is light-sensitive and only stable in water for 48 hours, the doxycycline water was shielded from light and changed every other day. At 3-, 6-, 9- and 12 months of age, aortic diameters and PWV of the experimental animals were measured by ultrasound imaging.    Figure 3. Animals were divided into doxycycline treatment and no treatment groups. Each groups had 12-13 wild type or Marfan mice. At 6-week of age, treatment groups were given doxycycline in their drinking water at a dose of 0.24 mg/ml, which is a sub-antibiotic dose that was found to be active from the previous studies. Because doxycycline is light-sensitive and only stable in water for 48 hours, the doxycycline water was shielded from all light and changed every other day. At 3, 6, 9, and 12-month, PWV and aortic root diameter were measured by echocardiography, and the blood samples were collected at the same time for future use of biomarkers assay. At the end of study, aortic segments and skin samples were dissected and prepared for MPM/EM to determine fiber damages.  35  2.7  Features of the echocardiography ultrasound imaging system and experimental set-up The high-resolution, high-contrast ultrasound imaging system Vevo® 2100 (Visual Sonics, Toronto, ON, Canada) equipped with a MS550 transducer was employed to conduct longitudinal experiments, to measure aortic diameter (indication of aortic dilatation) and PWV (indication of aortic elasticity/stiffness) in mice. The main characteristics of the transducer include “a central frequency of 40 MHz, a focal length of 7.0 mm, and a frame rate of 557 fps (single zone, 5.08 mm width, B-mode). The maximum field of view of 2-D imaging was 14.1 x 15.0 mm with a spatial resolution of 90 μm (lateral) by 40 μm (axial).”  Aortic diameters and PWV were measured by ultrasound imaging system as previously described in detail (Lee et al., 2016). “Briefly, the experimental animal was anesthetized in an induction chamber using 3% isoflurane (Baxter Corporation, Mississauga, Canada) and 1 L/min 100% oxygen for 1–2 minutes. After testing the anesthesia state by confirming the loss of its righting reflex, the animal was laid supine on a heated platform with its nose enveloped in a nose cone to maintain anesthetized by 1.5–2% isoflurane (Gao et al., 2011; Roth et al., 2002).” Heart rate and respiratory rate were being monitored during the echocardiography procedure, as well as the electrocardiogram (ECG), which was measured by connecting the limbs of the mouse with “ECG electrodes that were imbedded inside the platform. In addition, body temperature was also monitored through a rectal probe, which was maintained at 36–38°C with a heating lamp.”  With the use of echocardiography, the diameters at three different aortic regions (i.e. L1=aortic annulus, L2=sinuses of Valsalva, and L3=sinotubular junctions) can be measured from the B-mode aortic arch view, as shown on a representative pulse wave (PW) Doppler tracing (Fig. 4A, B), and the developing aortic dilatation with age in MFS mice, as well as the potential therapeutic effects of long-term doxycycline intervention on reducing aortic dilatation can be 36  followed and evaluated. “The ascending and descending aortic peak velocities were measured from the PW Doppler-mode aortic arch view. PWV was calculated indirectly from the parameters obtained from the B-mode and Doppler-mode aortic arch view, by the formula: PWV = aortic arch distance / transit time (cm.s-1) (Bradley et al., 2005). The aortic arch distance was measured as d–d0 (mm) between the 2 sample volume positions, ascending and descending aorta labeled as d0 and d, respectively, along the central axis of aortic arch on the B-mode image. The PW Doppler-mode sample volume was placed in the ascending aorta and the time from the onset of the QRS complex to the onset of the ascending aortic Doppler wave form was measured as T1. Meanwhile, when the PW Doppler-mode sample volume was placed as distal as possible in the descending aorta, the time from the onset of the QRS complex to the onset of the ascending aortic Doppler wave form was measured as T2” (Fig. 4C). The means for T1 and T2 were calculated from 10 cardiac cycles, and the transit time was calculated by T2 –T1 (ms). Therefore, indirect measurement of PWV was calculated by the equation of PWV=[d-d0]/[T2-T1].   37    Figure 4. Echocardiography B-mode and pulse wave Doppler-mode image views of control and MFS mouse aorta. (A) Control mouse: Aortic arch length from d0 to d was measured on B-mode view as the distance; (B) Marfan mouse: Measurements of diameters in aortic root, L1=aortic annulus, L2=sinuses of Valsalva, L3=sinotubular junctions; (C) Tracing recordings on pulse wave Doppler-mode view: time interval T1 in the ascending aorta (upper panel) and T2 descending aorta (lower panel). Pulse Wave Velocity formula: PWV = [d-d0] / [T2-T1].  2.8  Preparation of aorta tissue samples for TEM imaging At 12 months of age, mice (control and MFS, with and without doxycycline treatment, n=4) were sacrificed, and the thoracic aorta were dissected. Specimens were washed in zero Ca2+ HEPES buffer, and the ascending aortic root was transected above the level of the aortic valve, and transversely cut into three or four ring segments (length, 0.5 mm/each). The segments were then transferred into the glutaraldehyde fixative solution (contains 1 ml of 25% glutaraldehyde, 4 38  ml of ddH2O + 5 ml of 0.2M sodium cacodylate buffer, pH=7.4) and fixed for 1-1.5 hours. After the primary fixation, the ring segments were washed by 0.1 M sodium cacodylate buffer to get rid of the excessive glutaraldehyde, and then incubated in osmium tetroxide fixative solution (mixes with 5 ml of 0.2 M sodium cacodylate buffer, 2.5 ml of 6% of KFe3(CN)6, and 2.5 ml of 4% osmium) in room temperature for 2 hours. Osmium mix solution is used as a secondary fixative after glutaraldehyde fixative because its rate of penetration is too slow to prevent artifacts if used initially. After osmium binding the components of the aortic tissues, the ring segments became rigid and stable with the color turning brown. Following the fixation procedure, aortic segments were dehydrated through a series of graded acetone, and then infiltrated with epoxy resin and embedded in blocks for further processed for TEM.  2.9  TEM image acquisition and quantitative statistical analysis Twelve blocks of aorta specimens from three groups of experimental animals described as above were randomly picked and sectioned at 60 to 90 nm thickness on a Reichert ultramicrotome. The thin sections were stained and viewed on a Hitachi H7600 TEM. Digital TEM images were captured at x15,000 magnification for a variety of extracellular matrix components, including elastin, collagen, smooth muscle cells, as well as the basal lamina apertures. For each cross-sectioned aorta, TEM images were acquired at four directions, north, south, east and west, and the quantitative analysis was performed. The gaps between the elastic fibers in a horizontal direction were spotted as the breakages, while the circumference or length (T) of each fragmented elastic fiber was traced along the fuzzy border up to the ends at which the thickness (D) was measured. Because the elastic fibers have fuzzy borders with a notched appearance in the MFS aorta, the extended measurement of circumference is terminated at the cross point where the perpendicular line between them is the thickness of the fiber. The irregularity index (IR index) was introduced for morphometric calculation, by dividing the length 39  of the border lines by the width of each fragmented elastic fiber (IR index=T/D), and the average elastic fibers IR indices were compared among the experimental groups. The higher the IR index, the more irregular and fragmented the elastic fibers are.  “Numerical values were expressed as means ± SEM unless otherwise indicated. Comparisons of parameters among treatment groups were made using one-way analysis of variance (1-way ANOVA) followed by Tukey’s multiple-comparison test. Comparisons within the same group among different time points were made using 1-way repeated measures ANOVA. Comparisons of parameters between two groups were made by two-tailed Student's t-test, where a p value of p < 0.05 was considered significant.”                40  3. Chapter 3. Results: 3.1  Comparison of aortic elastic fiber morphology and volumetric density of elastin and collagen between control and MFS mice (Citing quotations from (Cui et al., 2014) “In order to compare the morphological differences of elastic fibers in the aorta between control and MFS mice, we used both conventional Van Gieson’s staining, and MPM imaging of freshly isolated aorta. Elastic fibers in cross sections of ascending aorta were stained in dark blue to purple/black with the Van Gieson’s staining. The zigzag shape of elastin appears intact, without fragmentation or breakage in control mice, while elastic fibers in MFS mice display severe fragmentation and disorganization (Fig. 5A), confirming our earlier observations (Chung et al., 2007a).  MPM imaging provides several advantages over conventional histology including specificity, sensitivity, and high spatial resolution without staining, but most importantly, it allows imaging of biological thick specimens in vivo & ex vivo without invasive fixation. In order to investigate differences in structural organization of elastic fibers and collagen within the aortic walls, we captured TPF (green signal for elastin) and SHG (purple signal for collagen) images of aortic samples from control and MFS mice (Fig. 5B). The MPM imaging of the freshly isolated aortic segments from the MFS mouse (Fig. 5B) presented the same structural disorganization and fragmentation of elastin fibers that was observed in formalin-fixed aortic tissue (Fig. 5A). Since we observed a significant difference in elastin fibers organization and length between control and MFS aortic sections, the independent TPF channel in green was acquired to assess elastic fibers morphology in three aortic regions, root, arch, and descending (Fig. 5C). Elastic fibers appear intact, regardless of ages or segment location in the control aorta. In contrast, MFS mice display fragmented and disorganized elastic fibers at 6-month; minor fiber fragmentation can even be observed as early as at 3-month in the aortic arch. The degree of aortic wall damage appears to 41  progress with age, but is less intense in the distal segments.   Figure 5. Morphology of elastin fibers and collagen in the aortic wall. (A) Representative image showing the structure of elastin and collagen in the cross-section of the ascending aorta from control and MFS (9-month) mice stained with Van Gieson's staining. Elastic fibers are stained dark blue and collagen is stained pink. Elastin fibers disorganization and fragmentation is evident in the MFS aorta as compared with control samples. (B) Representative MPM images of control and MFS aortic sections for both TPF (green for elastin) and SHG (purple for collagen) signals shows elastin disorganization and fragmentation in the MFS aortic wall. (C) Representative MPM images (TPF signal) showing the morphology of elastic fibers within the aortic wall in three aortic segments (root, arch and descending) from control and MFS mice at 42  different ages (3-, 6-, and 9-month). As shown, elastin is disorganized and fragmented in all MFS aortic segments. (Citing the figure and legend from (Cui et al., 2014)  3-D structures of aortic segments from control and MFS mice at different ages were reconstructed by MPM (Fig. 6A). TPF channel in green represents elastin, whereas SHG signals from collagen are present in purple. This 3-D reconstruction allows for assessment of volumetric density of elastin and collagen in aorta, which is not feasible with conventional staining. However, no significant difference was observed in the total volume of elastin and collagen between control and MFS mice in any of the age groups (Fig 6B). Interestingly, a decreasing trend in control and an increasing trend in MFS mice with age were observed in the total volume of collagen. These findings suggest that volumetric density of elastic fiber may not be an ideal parameter to determine the differences in morphology between control and MFS mice, and therefore, other quantitative parameters such as elastin fiber fragmentation and organization may be a more accurate measure of the wall structural integrity in MFS aorta.” 43    Figure 6. Assessment of volumetric density of elastin and collagen in aorta. (A) Representative images of 3-D reconstruction of aortic segments from control and MFS mice (ages of 3-, 6-, and 9-month). Elastin is illustrated in green (TPF signal), whereas collagen is present in purple (SHG signal). (B) Bar graphs represent quantification of volumetric density of aortic elastin and collagen from control and MFS mice. As shown, no significant difference was found among different age groups (means ± SEM, n= as indicated for each column). (Citing the figure and legend from (Cui et al., 2014)  44  3.2  Quantitative determination of aortic elastic fiber fragmentation and organization in control and MFS mice “The tracing of many elastic fibers indicate that they are intact in control mice, but truncated in MFS mice (Fig. 7A). In 3-month old MFS mice, the length of elastic fibers in some aortic segments is slightly decreased compared to controls, but these changes are not significant (Fig. 7B). However, in both 6-month and 9-month old MFS mice, fiber lengths are significantly decreased compared to controls, regardless of the aortic segment location (root, arch, & descending) indicating the elastin fibers fragmentation within the aortic wall in MFS mice (Fig. 7B).   Figure 7. Quantitative assessment of aortic elastic fiber fragmentation. (A) Representative MPM images showing the tracing of aortic elastic fibers in the aortic cross section from control and MFS mice (ages of 3-, 6-, and 9-month). (B) Comparison of aortic elastic fiber lengths in 45  aortic root, arch, and descending aorta from control and MFS mice of different ages (means ± SEM, *p < 0.05, **p < 0.01, n= as indicated on each bar). (Citing the figure and legend from (Cui et al., 2014)  We further analyzed and determined elastin fibers organization using the FFT algorithm. To determine each fiber direction on individual optical section (z-plane), the spatial frequencies (i.e. horizontal and vertical occurring) were plotted in 2-D frequency space as a long and a short axis, assuming an elliptical shape (Fig. 8A). For comparison between groups, the ratio of the short axis to the long axis was calculated to yield a fiber orientation index. The orientation index ranges from 0 to 100, representing the levels of order; the higher the number, the greater the degree of organization. As shown by the representative images in figure 8A, the orientation index in the aorta of a 3-month old control mouse (≈56) is more than twice that in the MFS counterpart (≈24). Furthermore, in 2-D frequency space, the frequency signals appear less symmetric in the MFS mouse, indicating that aortic elastic fibers in MFS are less organized than in the control. Additionally, a representative 2-D analysis of the relationship between fiber angular direction and intensity signals is shown in figure 8B. The x-axis representing angular values from 0 to 360 degrees in the 2-D frequency space was transcribed from the radar grid in figure 8A. Since the multiphoton images of aortic sections are roughly at 45-degree angle, the two ends of the frequency distribution point in the 45- and 225-degree directions, respectively. The y-axis represents the signal frequency magnitude. Higher frequencies represent higher directional order of the fibers. In the 3-month control aortic root, the average frequency magnitude of elastic fibers in two main directions reaches almost 4.5 x 104, but in the MFS aorta, it is barely 2.5 x 104, indicating that almost twice as many elastic fibers are pointing in the same direction in control as compared to MFS. Moreover, the signals and noise of elastin in the MFS mouse reveal jagged and rough parts on the shoulders of the peaks as compared to the control, which appear much 46  smoother (Fig. 8B). The breadth of the signal base is also greater in MFS than in controls (Fig. 8B). Together these results quantify elastic fiber disorganization in MFS mice, which could be of prognostic value.  The directions of elastic fibers vary in different z-places of aortic sections, which will result in differences in the axial/equatorial signals and relative orientation index in each plane. Therefore, the average orientation index for each aortic segment was determined from multiple z-planes using the FFT algorithm (Fig. 8C). In ascending aorta, significantly lower orientation indices are observed in MFS mice, regardless of age, suggesting that elastic fiber organization is lost at as early as 3 months in MFS mice. Interestingly, this difference between control and MFS mice diminishes with age (Fig. 8C), indicating that elastic fiber disorganization naturally occurs during aging in control mice.”  47    Figure 8. Analysis of aortic elastic fiber organization by FFT algorithm. (A) Representative images showing how the intensity signals from 3-month old control and MFS aorta were transformed and plotted in 2-D frequency spaces as long and short axes. The orientation index (N), was calculated from the ratio of the short axis to the long axis, representing the level of order. (B) Following the frequency space transfer, the relationship between fiber angular direction (angular values from 0 to 360 degrees transcribed from radar grid in the 2-D frequency) and intensity signals (average frequency magnitude) was analyzed. Elastin signal appears much smoother in control (single arrow); jagged and rough parts are revealed on the shoulders of the peak (double arrows) in the MFS mouse. (C) Average orientation indices of aortic elastic fibers in 48  ascending and descending aorta (from multiple z-planes), from 3-, 6-, and 9-month old control and MFS mice (means ± SEM, *p < 0.05, **p < 0.01, n= as indicated on each bar). (Citing the figure and legend from (Cui et al., 2014)  3.3  Comparison of cutaneous elastin and collagen morphology between control and MFS mice “The basic structure of skin from control and MFS mice is demonstrated with hematoxylin and eosin (H&E) staining (Fig. 9A). The dermal layer in 3- and 6-month old control mice is thicker than in MFS mice, but this difference disappears at 9 months of age. As expected, the thickness of dermis also decreases in control groups with age, such that samples from 9-month old control mice resemble those from 3-month old MFS mice, except for the thicker appearance of hypodermal fat layer. Meanwhile, this relatively thin layer remains constant in thickness in the MFS groups regardless of age. 49    Figure 9. Visualization of cutaneous elastin and collagen morphology in control and MFS skin samples. (A) Histological H&E staining of skin from control and MFS mice (ages of 3-, 6-, and 9-month) demonstrates the basic layering of mouse skin: epidermis, dermis, hypodermis, and panniculus carnosus muscle (PCM). Note the significant thinning of the dermis layer of skin of MFS mice compared to controls. (B) Representative 2-D MPM images of gross layering structure of skin from control and MFS mice. (Citing the figure and legend from (Cui et al., 2014)  To record changes in cutaneous elastin and collagen in MFS mice, TPF/SHG multiphoton images were acquired from the dermal layer (Fig. 9B). Besides the dermis, also the hypodermis, panniculus carnosus muscle (PCM) and hairs can be imaged as their signal wavelengths are close 50  to the elastin TPF signal range. However, their signal intensities are quite distinct from elastin TPF, with intensities highly saturated in the dermal layer, but only barely observable in other layers. Therefore, in the following MPM image acquisitions, the intensities were optimized and adjusted to focus on elastin and collagen signals in the dermis only. Consistent with previous observations with H&E staining (Fig. 9A), the dermis in control mice appears thicker than those in the MFS counterparts, with the exception of the 9-month groups, where the difference between old age control and MFS mice disappeared.”  3.4  Total volumetric density of cutaneous elastin and collagen, and the thickness of dermal layer in control and MFS mice “In this part of the study, the intensities were optimized and adjusted to focus on elastin and collagen signals in the dermis only (Fig. 10A). The elastin volume was significantly decreased in 3- and 6-month MFS compared to control mice, while no difference was observed in the 9-month old groups (Fig. 10B). Unlike elastin, the volumetric density of collagen increases with age in control mice, with significant higher contents observed in 6- and 9-month groups compared to MFS groups (Fig. 10C). However, similar to elastin, the collagen content remains low in MFS mice regardless of age. Furthermore, the thickness of the dermal layer is significantly less in 3- and 6-month MFS mice compared to controls, but they are similar in the oldest groups (Fig. 10D). These measurements confirm our histological observations in Fig. 9A, that the dermis is relatively thinner in all MFS mice, resembling that of the oldest controls.  As predicted by the above findings, the ratio of elastin-to-collagen volume is significantly decreased in 3- and 6-month MFS compared to control mice (Fig. 10E). We observed a gradual and age dependent decrease in the elastin to collagen ratio in the controls, but not in the MFS mice, suggesting pre-mature aging of the skin in MFS mice. It is noteworthy to mention that the 51  elastin/collagen ratios in 3- and 6-month control mice are higher than 1.0, indicating a relatively higher elastin content than collagen.”    Figure 10. Total volumetric density of cutaneous elastin and collagen, and dermal layer thickness in control and MFS mice skin. (A) Representative MPM images of skin elastin and collagen from control and MFS mice (ages of 3-, 6-, and 9-month). TPF channel in green represents elastin fibers; SHG channel in purple represents collagen. (B) Measurements of total volumetric density of elastin in mice skin dermis. (C) Measurements of total volumetric density of 52  collagen in mice skin dermis. (D) Measurements of thickness of dermal layer in mice skin. (E) Comparison of ratio of elastin to collagen volume in mice skin (means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, n= as indicated on each bar). (Citing the figure and legend from (Cui et al., 2014)  3.5  Longitudinal comparison of aortic structure between control and MFS mice by ultrasound imaging (Citing quotations from (Lee et al., 2016) “There were no significant differences in heart rate (HR), body weight (BW), ejection fraction (EF) and fractional shortening (FS) between control (WT) and MFS mice groups at both 6 and 12 months of age (Table 2).”  Table 2. Echocardiographic functional analysis for control and MFS mice.  All parameter measurements are presented as Means ± SEM (n=8 mice). N, normalized with body weight. EF Ejection fraction; FS Fractional shortening. (Adapting and modifying the table from (Lee et al., 2016).  “The aortic annulus diameter was significantly increased by 18% in MFS versus control (WT) in the 6-month (p=0.046) and by 27% in the 12-month (p=0.001) groups, respectively (Fig 53  11). The diameter of the sinus of Valsalva was also significantly increased in MFS mice versus control (WT) by 19% in 6-month (p=0.01) and by 27% in 12-month (p< 0.001) groups, respectively (Fig 11).”    Figure 11. Aortic root diameters of control and MFS mice. (A) B-mode view of the aortic arch from a 6-month MFS mouse. Diameters of the (B) aortic annulus [L1], (C) sinus of Valsalva [L2] and (D) sinotubular junction [L3] were significantly increased in MFS mice versus controls (WT). The larger aortic root diameter indicates significant aortic dilatation. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001 (n=8). (Citing the figure and legend from (Lee et al., 2016)  3.6  Longitudinal comparison of aortic function between control and MFS mice by ultrasound imaging “PWV was increased by 79% in 6–8 months old MFS mice compared to controls (WT) 54  (p=0.003) and by 124% in 12–16 month MFS mice compared to controls (WT) (p<0.001).  The relationship between age (x-axis) and PWV (y-axis) of MFS and control (WT) mice is shown in Fig 12. PWV in MFS mice increased linearly with age (R-squared=0.356, p=0.02), but not in control (WT) mice (R-squared=0.008, p=0.73). The estimated regression lines are y=0.45x+275.49 in the MFS group and y=0.06x+187.01 in the control (WT) group.”    Figure 12. Pulse wave velocity (PWV) of aortic arch. (A) B-mode view of aortic arch of a 6-month control (WT) mouse. The distance between the ascending and descending aorta pulse wave Doppler recordings is indicated by the blue line (d0 to d). Scale bars, 2 mm. (B) Pulse wave Doppler tracing of the ascending (upper panel) and descending aorta (lower panel). The X-axis represents time (ms) and Y-axis represents blood flow velocity (mm/s). T1 is measured from the beginning of the QRS wave on the ECG to the beginning of the ascending aortic peak velocity and T2 is the beginning of the QRS wave on the ECG to the beginning of the descending aortic peak 55  velocity. Pulse wave velocity was calculated using the distance between d0 and d in the aortic arch divided by the transit time (i.e. [d-d0] / [T2-T1]). (C) Aortic PWV of control (WT) and MFS mice from two age groups (6-8 months and 12-16 months group). PWV was significantly increased in 6-8 months MFS mice compared to controls (WT) (p=0.003) and in 12-16 months MFS mice compared to controls (WT) (p<0.001), respectively. (D) Correlations between age (x-axis) and PWV (y-axis) of control (WT) (●) and MFS (∎) mice. PWV in MFS mice was directly proportional to age (R-squared=0.356, p=0.02), but not in control (WT) mice. ** indicates p<0.01, *** indicates p<0.001 (n=8). (Citing the figure and legend from (Lee et al., 2016)  “Fig 13A shows that the ascending aortic peak velocities were significantly decreased by 25% (p=0.04) in 12-month MFS versus control (WT) mice. Descending aortic peak velocity was decreased by 28% in 12-month MFS versus control (WT) mice (p<0.001) and by 18% in 12-month versus 6-month MFS mice (p=0.01) (Fig 13B).”  56    Figure 13. Peak velocity was calculated from pulsed-wave Doppler mode from an aortic arch view. (A) Ascending aortic peak velocity of control (WT) and MFS mice from two age groups (6- and 12-month). Ascending aortic peak velocity was significantly decreased in 12-month MFS mice versus control (WT) mice (p=0.04). (B) Descending aortic peak velocity was significantly decreased in 12-month MFS mice (p<0.001) versus control (WT) mice. * indicates p<0.05, *** indicates p<0.001 (n=8). (Adapting the figure and legend from (Lee et al., 2016)  3.7 Quantitative assessment of the progression of aortic elastic fiber fragmentation between control and MFS mice “Histological imaging of the aortas from 6-month and 12-month control (WT) and MFS 57  showed the loss of organization and disruption of the elastin fibers as illustrated in Fig 14A. With van Gieson’s staining, elastin is illustrated in dark purple, whereas collagen is present in light pink. Elastin fibers fragmentation is visible in the MFS aorta compare to control (WT). The area of elastin fibers was significantly increased in MFS mice versus controls (WT) in 6-month of age (26%, p=0.02). The number of elastin fibers was significantly increased in 12-month MFS mice versus 12-month controls (WT) (125%, p=0.03) and versus 6-month MFS mice (123%, p=0.03) ” (Fig. 14B).    Figure 14. Histological analysis of control and MFS mice aorta. (A) Representative histological images stained with Van Gieson's staining reveal the arrangement of elastin (dark blue) and collagen (pink) in the cross-section of the aorta from 6-month and 12-month control (WT) and MFS mice. Elastin fibers display severe fragmentation and disorganization in the MFS aorta as compared with control (WT) samples. (B) The area and (C) number of elastin fibers were measured by tracing the elastin fibers on the transverse section of mouse aorta. *indicates p<0.05 (n=3). (Adapting the figure and legend from (Lee et al., 2016) 58   3.8 Longitudinal comparison of pulse wave velocity between control and MFS mice with and without long-term doxycycline intervention In vivo ultrasound imaging technique has allowed us to study structural and functional changes occurred overtime and during the progression of aortic aneurysm in the absence or presence of treatment in the same experimental subject, without jeopardizing the health of the animals. Initially at 3 months of age, we have shown that PWV is significantly increased in MFS mice compared to controls (p<0.01), with doxycycline having no impact on the progression of aneurysm in MFS mice (Figure 15). Likewise, at 6 months of age, PWV is significantly increased in MFS mice compared to controls (p<0.001), but doxycycline treated MFS mice have significantly lower PWV than non-treated MFS mice (p<0.01), and they are not different from either the treated or non-treated control mice. At 9 months of age, treated MFS mice have also significantly lower PWV than non-treated MFS mice (p<0.05), although it stays markedly above the value recorded in age-matched control mice. Taken together, our data shows that PWV in the non-treated MFS mice increases significantly with age and that doxycycline attenuates this age-related increase in PWV, but does not completely block its development (Fig. 15). 59    Figure 15. Longitudinal measurements of PWV in control and MFS mice groups with and without doxycycline treatment. PWV is significantly increased in MFS mice compared to controls without treatment at in 3-month old mice (**p<0.01), but the difference in PWV is not significant in doxycycline-treated MFS mice compared to controls. PWV is significantly increased in MFS mice compared to controls without treatment at 6-month old (***p<0.001); doxycycline-treated MFS mice have significantly lower PWV than non-treated ones (**p<0.01), while they do not display any significant difference in PWV from both treated and non-treated control mice. At 9 and 12 months of age, PWV is significantly increased in MFS mice compared to non-treated controls (***p<0.001); doxycycline-treated MFS mice have significantly lower PWV than non-treated MFS mice (*p<0.05), however, they still show a significantly increased PWV compared to treated control mice (**p<0.01). The findings suggest that long term doxycycline treatment results in a significant reduction in aortic stiffness (as indicated by PWV measurement) in MFS mice starting at the age of 6 months.  60  3.9  Longitudinal comparison of regional aortic diameters between control and MFS mice with and without long-term doxycycline intervention Aortic diameters in three distinct areas, aortic annulus [L1], sinuses of Valsalva [L2], and sinotubular junctions [L3], were measured from the echocardiography B-mode aortic arch view. Diameters of aortic annulus are significantly increased in MFS mice compared to controls with and without doxycycline treatment at 3-month of age (p<0.01, p<0.001, respectively) (Figure 16A). The diameters of the treated Marfan mice are not different from those in treated controls at age of 6 and 9 months, while the aortas are all significantly larger in MFS mice without doxycycline treatment (p<0.001) compared to their control counterparts, demonstrating significant aortic root dilatation with age in MFS mice. After over ten months on intervention, doxycycline suppressed the aortic dilatation in this root area effectively, with significant decreases in aortic diameters in treated MFS mice compared to non-treated MFS mice at the age of 12 months (p<0.05), and without differences from those treated control mice. Meanwhile, the differences in diameters remained significant among MFS and control mice without treatment, the same as they were at earlier ages.   61    Figure 16A. Measurements of diameters at the aortic annulus. Diameters are significantly increased in MFS mice compared to control at 3 months of age (**p<0.01). Aortic annulus diameters of the treated Marfan mice are not different from those in control at the age of 6 and 9 months, while the aortas are all significantly larger in non-treated MFS mice (***p<0.001). Aortic annulus diameters of the treated Marfan mice are significantly decreased compared to non-treated MFS mice at the age of 12 months (*p<0.05), but without difference from the controls.  62  Similar to aortic annulus, diameters of sinuses of Valsalva are significantly increased with age in MFS mice compared to controls without doxycycline treatment, at 3-, 6-, 9- and 12-month old (p<0.001, <0.01, <0.001, <0.01, respectively) (Figure 16B). Furthermore, with long-term doxycycline treatment, the significant difference found in these aortic sinus of Valsalva diameters in MFS than in controls at 3-month old (p<0.001) disappeared starting from 6-month of age, and they are no longer different from those in controls in the treatment group at older ages. In particular, at 6- and 12-month old, treated MFS mice display significantly decreased aortic sinus of Valsalva diameters compared to their age-matched MFS counterparts without treatment (p<0.05), suggesting potential protective effects of doxycycline on reduction of aortic dilatation in this particular aortic root area. At the relatively distant area from the left ventricle, aortic sinotubular junction diameters show no differences between MFS and control mice (Figure 16C), regardless of age or treatment with doxycycline, even though a trend of dilated aortic sinotubular junction diameters in MFS mice can be detected.  63    Figure 16B. Measurements of diameters at the aortic sinus of Valsalva. Aortic sinus of Valsalva diameters are significantly increased in MFS mice compared to controls at 3-month of age (***p<0.001). Aortic sinus of Valsalva diameters of the treated Marfan mice are not different from those in controls at the age of 6 and 9 months, while aortic sinus of Valsalva diameters are significantly larger in non-treated MFS mice (**p<0.01, ***<0.001, respectively). Furthermore, at 6 months of age, treated MFS mice display significantly decreased aortic sinus of Valsalva diameters compared to their age-matched MFS counterparts without treatment (*p<0.05), suggesting potential protective effects of doxycycline on reduction of aortic sinus of Valsalva dilatation. Aortic sinus of Valsalva diameters of the treated Marfan mice are significantly 64  decreased compared to non-treated MFS mice at the age of 12 months (*p<0.05, but without difference from the controls.    Figure 16C. Measurements of diameters at the aortic sinotubular junctions. At this relatively distant area of aorta from the left ventricle, there is no difference in the aortic diameters between MFS and control mice, regardless of sex and doxycycline treatment, even though trends of dilated aortas in MFS mice can be observed.  65  3.10 Correlation between Aortic Structure and Function over time in control and MFS mice with and without long-term doxycycline treatment Correlations between aortic diameters and PWV in three aortic regions reveal positive linear regressions among these experimental animal groups regardless of doxycycline treatment (Figure 17A). The gap of the linear regressions between sinotubular junction and aortic annulus is narrowed in MFS mice compared to controls, while the gap is widening between these two regions and sinus of Valsalva. A particularly high correlation is observed in sinus of Valsalva, suggesting that the aortic function in this proximal part is relatively more susceptible to the changes in structure, which may be more likely to be influenced by suppressing effects of doxycycline on aortic dilatation. The correlations with age and doxycycline treatment warrant further investigation in this particular area of the aorta.     66    Figure 17A. Correlations between PWV and aortic diameters in three aortic regions, aortic annulus, sinus of Valsalva, and sinotubular junction. (A) non-treated control mice, (B) non-treated MFS mice, (C) doxycycline-treated control mice, and (D) doxycycline-treated MFS mice. The gap of the linear regressions between sinotubular junction and aortic annulus is narrowed, while the gap between these two regions and sinus of Valsalva is widened, in MFS mice compared to controls, regardless on doxycycline treatment or not. Out of the three aortic regions, correlation in sinus of Valsalva stands out as the highest level and is worthy further investigation.  The correlations show that at 3 months of age, aortic diameters are not positively correlated 67  with PWV in control mice, the same findings are observed in treated and non-treated MFS mice, even though these correlations in MFS mice are at relatively higher levels (Figure 17B). However, at 6 months of age, a positive correlation is found between aortic diameters and PWV in non-treated control mice, as well as in both treated and non-treated MFS mice. As mice age (6 – 9 months), linear regression level in treated MFS mice starts to falls under the non-treated MFS mice, while the latter ones remain dominant at the higher scale, suggesting that doxycycline can be effective in suppressing aortic dilatation after two months of treatment, while a wider and stiffer aorta is developed in non-treated MFS mice. Eventually, at the end stage of the experiment, linear regression level in treated MFS group falls down to the level that is similar to the control groups, while non-treated MFS group continues to stay at the higher range, suggesting that long-term doxycycline treatment can improve both the aortic structure and function in the area of sinus of Valsalva, by suppressing the dilatation and limiting the progression of aortic wall stiffness.  68    Figure 17B. Correlations between PWV and aortic diameters in sinus of Valsalva, in four groups of experimental animals. (A and B) At 3 months of age, diameters do not display positive correlation with PWV in control mice without treatment, the same findings are observed in both MFS mice with and without doxycycline treatment, even though they are at relatively higher level; while at 6 months of age, positive correlation is found between diameters and PWV in control mice without treatment, as well as in both MFS mice with and without doxycycline treatment. Linear regression level in treated MFS mice falls under the non-treated MFS mice starting at 6-month of age. (C) At 9-month of age, correlation between PWV and aortic diameters continues to fall in treated MFS mice, while non-treated MFS remain dominant at the higher range. (D) At 12-69  month of age, linear regression of the correlation in MFS-DOX group falls down to the level that is similar to the control-NT and control-DOX groups, while MFS-NT group continues to stay at the higher range.  3.11 Comparison of cardiovascular gross structure and elastic fibers morphology between control and MFS mice with and without long-term doxycycline treatment At the end of study, we assessed the gross structure of the aorta of 12-month old MFS and control mice. As shown in figure 18, intact aorta of normal size and a sigmoid shape is observed in the control mouse, with the typical three major branches in the aortic arch, from proximal to distal, the brachiocephalic trunk, the left common carotid artery, and the left subclavian artery (Figure 18A, B). In the absence of doxycycline treatment, a balloon-like bulge with severe dilatation in aortic root proximal to the aortic arch three branches is observed in the MFS mouse. Meanwhile, the size of the left ventricle is also increased (Figure 18C, D). With doxycycline treatment, the cardiovascular gross structure is protected, with both aorta and heart of the MFS mouse being observed in a relatively normal shape and size in the MFS mouse, similar to those in the control mouse (Figure 18E, F), suggesting that doxycycline may not only improves the aortic structure and function, but may also keep the heart from left ventricular dilatation with regard to its gross structure.  70    Figure 18. Representative images of dissected heart and aorta samples from (A, B) control and MFS mice (C, D) without and (E, F) with doxycycline treatment at the age of 12 months of age. (A, B) Intact mouse aorta with the typical three major branches in the aortic arch,  from proximal to distal, the brachiocephalic trunk, the left common carotid artery, and the left subclavian artery, attached to a normally shaped heart in a control mouse. (C, D) Severe aortic dilatation in MFS mouse in the absence of doxycycline treatment, a “balloon-like” dilation is observed close to the aortic arch three branches. The left ventricle of MFS mouse heart seems to be enlarged. (E, F) Relatively normal shape and size aorta and heart can be observed in MFS mouse treated with doxycycline.  Aortic cross sections from 12-month old mice were subjected to the conventional Van Gieson’s staining, to investigate the morphology of elastic fibers in the aortic root, particularly in the sinus of Valsalva. Elastic fibers are stained in dark blue/purple, while collagenous fibers are stained in light pink. The zigzag shape of elastin appears intact, without fragmentation or breakage in control mice (Figure 19A, C). However, elastic fibers in non-treated MFS mice 71  display severe fragmentation and disorganization (Fig. 19B), confirming our previous observations (Chung et al., 2007a). With more than 10 months of doxycycline treatment, there appears to be much less fragmentation of elastic fibers in the aortic wall of MFS mice as compared to non-treated group.    Figure 19. Representative images of van Gieson’s staining of the aortic root from (A) control and (B) Marfan mice without doxycycline treatment, and (C) control and (D) Marfan mice with doxycycline treatment, at 12-month of age, illustrating elastin (dark blue/purple) and collagen (light pink). Long-term doxycycline treatment appears to show protective effects on maintaining elastin fibers organization and aortic wall integrity on Marfan mice.   72  3.12 Ultrastructural changes at break points of elastic fibers associated with MFS and MMPs inhibition Our previous quantitative analysis of elastin in MFS aorta using multiphoton imaging demonstrated that elastic fiber length in all three aortic sections (root, arch and descending) decreased in MFS mice compared to controls. In addition, the elastic fiber orientation indices in MFS ascending aorta were lower than in the controls (Cui et al., 2014). Breakage and loss of elastic fiber integrity are considered as the main causes of aneurysm in patients with MFS. The apparent breakage and irregular border lines of elastic fibers could be the result of elevated levels of MMPs and their proteolytic activity. Therefore, TEM was used to investigate possible changes in the ultrastructure of the elastin fiber ends in sinus of Valsalva which could be associated with the increase in breakage observed in MFS aortic wall. The representative images in figure 20A shows that the borders of the elastic fibers are relatively clean and sharp in control aorta, while irregular and fuzzy borders are observed in aortic sections from MFS mice (Fig. 20B). However, with doxycycline treatment, the elastin appeared to retain its normal shape on the terminal ends of the fibers, including that inhibition of MMPs by doxycycline appeared to reverse the irregularities of the elastic fibers within the aortic wall (Fig. 20C).   73    Figure 20. Representative images of ultrastructure of the aortic elastic fibers from 12-month old (A) control and (B) MFS mice without doxycycline treatment, and (C) MFS mice with doxycycline treatment. The terminal ends of the elastic fibers are relatively clean and sharp in control aorta; while irregular and fuzzy boarder can be observed in MFS mice. However, with long-term doxycycline treatment, the elastic fibers appear to maintain their normal shape on border lines.  74  Apart from the qualitative ultrastructure observations, quantitative analysis was also performed to compare the irregularities of elastic fibers among the control and MFS mice. The average irregularity index of elastic fibers terminals is significantly increased in MFS aortas compared to controls in the absence of treatment (p<0.001); while the treated MFS aortas have significantly lower irregularities index than non-treated ones (p<0.001), which are similar to the values seen in controls (Fig. 21). This is consistent with the expectation that the chemical degradation of elastic fibers by MMPs is inhibited by doxycycline. Taken together, both the qualitative and quantitative evaluations suggest that long-term doxycycline intervention is able to protect the elastic fibers from further degradation at the ultrastructural level.  Figure 21. Bar graphs presenting measurements of elastin irregularity index (circumference/width) in control, MFS (with doxycycline) and MFS (without doxycycline). At 75  12 months of age, the irregularity indices of elastic fibers are significantly increased in MFS mice as compared to control (***p<0.001), whereas doxycycline-treated MFS mice have significantly lower irregularity index than non-treated MFS mice (***p<0.001), normalizing to the level observed in control mice.                      76  4. Chapter 4. Discussion, Conclusion and Future Direction: In the part I of this thesis, (citing quotations from (Cui et al., 2014) “we used a non-invasive MPM imaging technique to quantify morphological changes in elastin and collagen in MFS mouse aorta and skin. Our aim was to establish a reliable new method for studying the structural changes involved in formation and progression of aneurysm in MFS. In addition, the data presented herein provide proof of principle for a potential non-invasive skin test for early diagnosis or monitoring the progression of aortic abnormality in MFS.”  “In aorta, elastic fiber fragmentation has been widely documented in MFS patients, as well as in the mouse model (Chung et al., 2007a; Chung et al., 2008; Marque et al., 2001; Segura et al., 1998). However, prior histologic studies necessarily involved alterations due to tissue fixation and staining, while quantification of fiber fragmentation has not been explored. By measuring the aortic elastic fiber lengths on MPM images, we quantitatively compared the fragmentation in MFS and control mice of different ages, and demonstrated that the lengths of elastic fibers decreased with age from 3- to 9-month in MFS mice, and became significantly shorter than in the controls as mice aged (6- and 9-month old), in all three aortic sections (root, arch and descending) (Fig. 7).”  “Another important characteristic of MFS aorta is the structural disorganization of elastin. This is important as a previous study reported that the orientation of elastic fibers determined their resistance to strain, which would eventually influence the load bearing capacity of the aorta (Lillie et al., 1998). However, the organization of elastin had heretofore not been quantitatively analyzed in MFS aorta. By using the FFT algorithm, our results revealed significantly lower orientation indices in MFS ascending aorta compared to aorta from control mice as early as 3 months of age (Fig. 8). As the mice aged, the elastic fibers became more disorganized in both 77  MFS and control mice in descending aorta, as was evident by a decrease in the orientation index. It is of importance that the elastic fiber disorder preceded fragmentation, suggesting that quantitative determination of elastic fiber disorganization could provide an early indication of abnormalities in the aortic wall. In this respect it is interesting to note that in control mice aging led to significant disorganization of the elastic fibers, whereas at nine months of age fragmentation was not yet observed and elastic content remained constant.”  Our laboratory “demonstrated that in MFS aneurysmal aorta from 3- to 9-month mice, progressive fragmentation of elastic fibers and stiffness is accompanied by increased expression and activity of MMP-2 and -9 (Chung et al., 2008), which have been suggested to be involved in pathological vascular remodeling (Rodriguez-Pla et al., 2005). Quantification of elastin fragmentation and organization in aorta may help in future studies of cause and effect in large artery disease in MFS.”  “We also captured high-resolution MPM images of the skin, with the main focus on the dermal layer, from control and MFS mice at different ages (Fig. 9 & 10). Histology was also obtained as an independent corroboration of the MPM imaging results, even though it is less suited for determining the alignment and packing of elastin and collagen fibers. Previous studies compared the architecture of the dermal elastic fibers among individuals of different ages by scanning electron microscopy (Tsuji and Hamada, 1981), and light microscopy (Jarrett, 1974), but none of them has quantified the fiber contents, due to limitations of the conventional methods. Based on our observation, morphology and volumetric density of elastin and collagen in MFS skin were clearly different from controls and, interestingly, but similar to what we observed in the older control mice, which is suggestive of pre-mature aging of the skin the MFS mouse. Our findings in the dermis provide quantitative evidence to support previous indirect 78  immunofluorescent observations of apparent deficient content of elastin-associated microfibrillar fibers in MFS skin (Godfrey et al., 1990a). Another important new finding in this study is the significantly thinner dermal layer in young MFS mice vs. controls, which indicates that the aging process of skin is accelerated in MFS mice. ”  “Our observations clearly suggest that early thinning of skin in MFS preceded the abnormalities in the aorta and might thus be exploited for the development of a non-invasive diagnostic test for early stages of the disease. Traditionally, the diagnosis of MFS is dependent on the demonstration of the multisystem clinical problems, which present themselves at a more advanced stage of the disease (Beighton et al., 1988; De Paepe et al., 1996), combined with the medical history of the patient’s family (Canadas et al., 2010). Our observations in MFS skin may shed light on other potential uses of the current clinical application of the MPM imaging. A modified version of MPM imaging has already been used to investigate biomechanical properties of human skin in vivo during skin aging (Koehler et al., 2012; Koehler et al., 2009), in skin cancer diagnosis (Dimitrow et al., 2009a; Dimitrow et al., 2009b), and for drug screening and monitoring (Konig et al., 2006). Such non-invasive MPM diagnostic testing could potentially be extended to MFS and other associated diseases such as Loeys–Dietz syndrome (Loeys et al., 2005), and connective tissue disorders caused by a defect in the synthesis of collagen, such as Ehlers–Danlos syndrome (Coe and Silvers, 1940), and the deficiency of fibrillin-4 associated with the FBN4 gene defect, known as cutis laxa syndrome (Hucthagowder et al., 2006).”   (Citing quotations from (Lee et al., 2016) “Aortic root aneurysm is the most prominent and life-threatening feature of MFS cardiovascular complications (Loeys et al., 2010). Increased aortic stiffness and loss of wall elasticity are considered as important detrimental factors contributing to aneurysm progression. Early studies in mouse models of MFS have used ex vivo 79  approaches to indirectly measure aortic wall stiffness/elasticity including the use of isometric wire myography (Chung et al., 2007a). Later, non-invasive high-resolution high frequency ultrasound imaging techniques provided a more powerful tool for in vivo measurements of both cardiac and aortic function and structure in real time. The present study is the first in vivo mouse study to duplicate studies in MFS patients showing dilation of the sinuses of Valsalva, sino-tubular junction and progressive loss of aortic root elasticity (El-Hamamsy and Yacoub, 2009; Hirata et al., 1991; Nollen et al., 2004) using a non-invasive and high-resolution ultrasound technique. As one would predict, as the vessel diameter increased, the peak blood flow velocity decreased (Rhoades R, 2013). Our study also showed a significantly decreased peak velocity in both the ascending and descending aortas. These results were more pronounced in the 12-month group, which is consistent with progressive dilation in the aorta of MFS mice with aging. Furthermore, PWV was significantly increased in 6-8- and 12-16-month old MFS mice compared with control (WT) and increased linearly with age. These data indicate a continuing process of aortic stiffening in MFS mice with aging. This may increase the LV afterload, potentially leading to cardiac remodeling.”  “The etiology for the increased PWV in MFS is likely based on a loss of aortic elastin fibril organization and integrity. In part II of the thesis, this was clearly demonstrated in both the 6-month old and 12-month old groups using histology in the fixed aortic samples (Fig 14). A more detailed examination of this approach is described above in part I of the thesis, the basic results of which are validated. Recently an MRI-based study was performed with LMI1174—a gadolinium-based elastin-specific magnetic resonance contrast agent (ESMA), which accurately measures elastin bound gadolinium within the aortic wall (Okamura et al., 2014). In this study, they found that in the 8-month Fbn1C1039G/+ MFS mouse, there was a significant decrease in aortic wall elastin compared to controls.” 80   “Our data are also consistent with previous human studies using PWV analysis methods on MFS patients (Bradley et al., 2005; Kiotsekoglou et al., 2011), and therefore, confirming that PWV measurement using 2D and Doppler echocardiography as performed in this study can be reliably applied to the mouse for evaluating aortic stiffness. The difficulties of this method in mice include the extreme shortness of the aortic arch (~5.5 mm) and the very rapid transit time due to the high heart rate. However, with repeated measurements over ten cardiac cycles, the accuracy can be excellent making this a robust technique for the evaluation of aortic stiffness in the mouse. Similar observations of increased aortic stiffness were made by our group previously in the ex vivo MFS mouse model using wire myography (Chung et al., 2007a). Different methods of measuring PWV have been reported in mice. They also differ in what they measure, some measure the velocity of the flow wave, others determine the velocity of the pressure wave in the aortic wall and others a combination of both. Hartley and colleagues (Hartley et al., 1997) calculated upper abdominal aortic PWV in mice by dividing the fixed distance from the aortic arch to the abdominal aorta 4 cm downstream by the pulse transit time which they measured using the Doppler waveform synchronized with ECG. PWV has also been measured by using a flow/area (QA) method involving aortic Doppler flow and aortic area change (Di Lascio et al., 2014; Williams et al., 2007b). However, the QA method is complex as it requires angle correction of the Doppler velocity and needs careful imaging of the aorta. Rabben and colleagues compared the QA method with the Bramwell–Hill (BH) method and concluded that the QA method needed refining (Rabben et al., 2004). Another study applied carotid-femoral applanation tonometry, a reliable technique commonly used clinically to assess PWV in mice (Leloup et al., 2014). In comparison, our method is relatively simple and direct, without the need for simultaneous blood pressure measurements or assumptions about Doppler angle to obtain flow volumes.” In addition, the technique presented in this part of the thesis focuses on the localized distance of aortic root, 81  which is the most important area of the aorta for the MFS characterization. It has the potential to be repeated and paves the path for a long-term longitudinal in vivo study such as discussed below, to evaluate the doxycycline therapeutic effects on aortic aneurysm by echocardiographic ultrasound imaging.  Loss of elasticity and increased stiffness in the aortic wall, as a result of the fragmentation and disorganization of elastic fibers, are considered crucial detrimental factors contributing to the progression of aneurysm (Chung et al., 2008). By using ex vivo approaches such as isometric wire myography, previous studies have measured aortic elasticity and stiffness in MFS mice (Chung et al., 2007a) and concluded that the reduced elasticity in MFS aorta may be primarily attributed to the disruption of elastic fibers in the ECM. With the use of high-resolution ultrasound imaging techniques, the progressive dilatation and stiffness in aorta can be studied in real time in both MFS patients and MFS mouse models (El-Hamamsy and Yacoub, 2009; Hirata et al., 1991; Lee et al., 2016; Nollen et al., 2004). Our findings of the increased aortic root diameters with age in MFS mice without doxycycline treatment are in agreement with the previous study comparing only 6 and 12 months groups, with significantly larger diameters of both aortic annulus and sinus of Valsalva in MFS mice. Furthermore, no difference was found in the distant aortic region of sinotubular junction (Fig. 16A, B, C). In addition, by expanding the age groups and filling the gaps between 6 and 12 months age groups, we were able to establish a developmental profile of gradual aortic root growth in the mouse model to allow us to gain a better understanding of the different stages of the aneurysm progression. It is noteworthy that our in vivo observations of aortic dilatation at the aortic annulus and sinus of Valsalva at as early as 3 months of age in MFS mice re-confirms our previous ex vivo quantitative analysis of early detections of loss of elastic fiber organization using MPM and conventional histology (Cui et al., 2014). An increase in aortic diameter with age in humans has been widely reported in various clinical studies. A clear-cut 82  increase in aortic surface area with age has been shown in autopsy studies (Mitchell and Schwartz, 1965), whereas to a lesser degree, cross-sectional studies of aortic diameter by both ultrasound (Agmon et al., 2003; Gerstenblith et al., 1977; Roman et al., 1993; Vasan et al., 1995) and angiography (Nichols et al., 1985) still show definite increase with age. Meanwhile, blood pressure increase associated with aging may also play a role in aortic dilatation, adding some controversy to the competing effects of age (Kim et al., 1996). Progressive aortic dilatation with age has also been reported in patients with MFS and cystic aortic necrosis in longitudinal studies, but the rate of dilatation is slowed down by antihypertensive agents such as beta-adrenergic blockers (Shores et al., 1994).  “Aortic wall stiffness is a functional indicator of aortic structure that reflects its major ECM components such as elastic and collagenous fibers. Possible beneficial changes in aortic stiffness could affect the cardiac structure and function, such as left ventricular size and mass, and interventricular end diastolic septal thickness, as well as the echocardiography-based diastolic assessment of early filling velocity and early (E) to late (A) ventricular filling velocities ratio (E/A).” However, the primary focus of this present study was to gain insight into the potential protective effects of doxycycline on aortic structure and function in MFS mice. PWV is believed to be a powerful index of arterial stiffness, which can be measured by ultrasound imaging in humans non-invasively (Hirata et al., 1991). As the pioneer to predict cardiovascular risk, increased PWV has been shown in various populations, such as diabetic, hypertensive, elderly and renal patients (Cruickshank et al., 2002; Laurent et al., 2006; Laurent et al., 2003; Sutton-Tyrrell et al., 2005). Bradley and colleagues previously demonstrated an elevated PWV in pediatric patients with MFS vs. control subjects. The same group also established a reliable echocardiographic Doppler method, to determine the aortic stiffness in humans indirectly (Bradley et al., 2005). The 2-D and Doppler echocardiography method were also shown to be 83  feasible and reliable to assess a variety of biophysical properties in a mouse model of MFS (Lee et al., 2016). Therefore, in the present in vivo study, the alterations of aortic elasticity or stiffness in both control and MFS mice were followed and evaluated longitudinally through indirect measurements of PWV by the ultrasound imaging. Our findings show that PWV is significantly increased in MFS mice compared to their control counterparts in all age groups, with the increases ranging from 1.5-fold to over 2-fold at different ages, beginning at 3 months of age (Fig. 15).   Data presented in this study are not only in agreement with previous studies utilizing echocardiography on MFS patients and mice (Bradley et al., 2005; Groenink et al., 2001; Kiotsekoglou et al., 2011; Kroner et al., 2013; Westenberg et al., 2011), but also expand the age groups and display an extended upward trend in PWV. The observed increase in aortic PWV is generally attributed to the corresponding increased stiffness of the aortic wall with age. When arterial stiffening is measured as PWV, it has been reported that there is an approximately 100% increase in aortic PWV between ages 20 and 80 years in humans (Avolio et al., 1983). However, in contrast to big arteries like aorta, there is little increase with age in PWV within muscular conduit arteries of the limbs (Lakatta and Levy, 2003; Mitchell et al., 2004), which are the medium-sized vessels known as distributing arteries. Furthermore, our data also show that PWV is significantly higher in MFS aorta vs. controls at as early as 3 months of age, which has not been reported previously, indicating that stiff aorta has already developed at an early stage in MFS mice. Whether the pathologic process of aortic stiffening in MFS starts at a younger age (<3 months) or even during the pre-weaning period in mice, is still unclear and worth further investigations.   Similar findings of increased aortic stiffness were previously demonstrated in our laboratory 84  in mouse model of MFS ex vivo by the use of wire myography (Chung et al., 2007a). Other studies also reported that aortic stiffness increased with aortic diameter (Jeremy et al., 1994), or even in the absence of aortic root dilatation (Bradley et al., 2005). In the current study, we have shown that both aortic diameters and PWV increase with age in MFS mice, and that these structural and functional changes can be the direct result of age-dependent degradation of elastic fibers, a process that was significantly blocked, especially in the sinus of Valsalva, by long-term administration of doxycycline (Fig. 17A-D).   In the present in vivo study, one of the goals was to evaluate the effects of long-term doxycycline intervention on delaying the progression of MFS-associated aortic dilatation and mitigating the aortic stiffness in the mouse model at various ages. Our data show that beginning at 6 months of age, PWV measurements dropped significantly in treated MFS mice compared to those without intervention, and such significant decrease was observed in the later age groups as well (Fig. 15). Such results were not evident in non-treated MFS mice at different age groups, suggesting that doxycycline can effectively delay, although not completely block, the progression of aortic stiffness. With respect to aortic root diameter, doxycycline markedly reduced the progression of aortic root growth at the aortic annulus and sinus of Valsalva to levels that was comparable to those observed in age-matched control mice, starting at 6 months of age (Fig. 16A, B). Interestingly, when the correlations between PWV and aortic diameters with age were studied in particular in sinus of Valsalva (Fig. 17B), it was prominent as the highest level among all three regions of the aortic root evaluated in this study, suggesting that the aortic function in this proximal part of aortic root is relatively more sensitive to the therapeutic and beneficial effects of doxycycline treatment.  Conventional van Gieson’s staining of the aortic root confirms our previous findings of loss 85  of organization and severe fragmentation of the elastic fibers in non-treated MFS mice compared to controls (Cui et al., 2014) (Fig. 7). It has been demonstrated that doxycycline intervention appears to rescue elastin fiber organization, and preserve aortic wall integrity in MFS mice through inhibiting proteolytic activities of MMPs (Chung et al., 2008).   In order to present a clear picture of the ultrastructural properties of the elastic fibers within the aortic wall in both treated and not treated MFS and control mice, we utilized TEM imaging and quantitative analysis of elastic fiber irregularities presented as the “irregularity index”. TEM imaging illustrates that the borders of the elastic fibers are relatively clean and sharp in controls, while irregular and fuzzy borders are visible in the MFS aorta. However, it appears that the elastic fibers maintained a normal shape on border lines, the irregularity in MFS aorta was reversed after treatment with doxycycline (Fig. 8). Furthermore, quantitative analysis reveals a significantly lower elastin irregularities index in treated MFS aorta compared to non-treated counterparts, with a similar average index as in controls (Fig. 9). Taken together, this suggests that the thinning and fraying of the elastic fibers with irregular boarders in MFS, particularly in the sinus of Valsalva, can be prevented by doxycycline treatment.   Data presented in this report highlights the beneficial and protective effects of doxycycline on both structure (elastic fiber organization and integrity, aortic root diameter) and function (PWV, aortic wall stiffness). Interestingly, the protective effects are not limited to MFS associated aneurysm, since a previous study comparing the effects of doxycycline and numerous non-antibiotic chemically modified tetracyclines in a rat model of elastase-induced abdominal aortic aneurysms (AAA), and showed that tetracycline derivative medications could inhibit the progression of AAAs in the rat (Curci et al., 1998b). The inhibitory effects of doxycycline on aneurysm seem to depend on dose and different from its antibiotic properties, and they are 86  concurrent with the structural preservation of medial elastic fibers (Curci et al., 1998b). Clinical trials in human AAA patients also reported that a short-term treatment of doxycycline has a prominent but selective effect on vascular inflammation, suggesting that doxycycline may favorably alter the outcome of patients with small AAA (Lindeman et al., 2009; Mosorin et al., 2001). Another study employing a rodent model of connective tissue disruption also demonstrated the effects of non-antimicrobial property of tetracyclines on inhibiting MMPs and other tissue degradation pathways (Golub et al., 1998). It was shown that tetracyclines are capable of binding Ca2+/calmodulin, an intracellular Ca2+-binding protein that stimulates phosphodiesterase, allowing this enzyme to hydrolyze cyclic AMP, which subsequently could affect the expression of MMPs in a variety of cells such as monocytes (Zhang et al., 1997) and cartilage cells (Richard et al., 1991). Therefore, preventing the activation of cAMP-dependent phosphodiesterase by tetracyclines could serves as another cell-regulatory mechanism for suppressing MMP-mediated connective tissue disruption (Schlondorff and Satriano, 1985).   In the aging human aorta, the thickness of the load bearing aortic media layer was found to remain relatively constant throughout life, and the increased width of the intimal layer is believed to be the predominant result of the aortic wall thickening (O'Rourke et al., 1987; Virmani et al., 1991). While in patients with MFS, there are fragmentation and thinning of the elastic fibers, along with progressive disorganization of the aortic media layer, as well as collagenous remodeling, which together ultimately develop aortic dilatation, dissection and rupture (Rubin, 1999; Underwood, 2000). Fragmentation of the elastic fibers is believed to be the main contributor to the progression of aortic dilatation and stiffening associated with aging and in individuals with genetic predisposition such as MFS patients (Nichols and O’Rourke, 2005). The majority of the aorta's stress load is normally absorbed by elastic fibers in the media layer. However, when the fibers are destroyed due to increased proteolytic activities by endopeptidases 87  like MMPs, the artery dilates following the stretches of aortic wall. Stress is then transferred to the less extensible collagen constituents in the normal intima, which usually bear only a fraction of the stress load. The latter event is the result of the well-known nonlinear pressure-diameter relationship in arteries (Rahbar et al., 2012; Valdez-Jasso et al., 2011).  It is still not established whether there are any effects of doxycycline on the quantitative and volumetric analysis of elastic and collagenous fibers in both MFS aorta and dermis, therefore it is warranted to further investigate this issue by the use of MPM imaging, to quantify the abnormal features, including fragmentation and disorganization. Since blood samples have already been collected from the doxycycline-treated and non-treated mice in the longitudinal study, their plasma levels of TGF-β, MMP-2, and MMP-9 will be determined by enzyme-linked immunosorbent assay. In addition, we plan to further establish a potential correlation between the plasma biomarkers and aortic pathology. Apart from aortic pathology, we will also measure aberrations in cardiac function (for example, pumping capacity, cardiac output, stroke volume, calculation of the ejection fraction, and mitral valve early and atrial velocities ratio) and structure (such as left ventricular mass and wall thickness) in the MFS mice. Furthermore, the blood flow through the vessels and chambers of the heart will be assessed by Doppler echocardiography to determine severity of aortic valve regurgitation. Eventually, our ultimate goal is to correlate these changes in the heart with those as seen in the aorta.       88  5. Chapter 5. General Conclusions and Significance: To our best knowledge, the study presented in part I of this thesis is the first quantitative study of aortic and cutaneous elastin and collagen abnormalities in MFS determined by MPM imaging. “Our results demonstrate that MPM imaging can be effectively used for the quantitative analysis of abnormal elastin and collagen in MFS aorta and skin. Our important findings of early signs of fiber degradation and thinning of skin dermis support the potential development of a novel non-invasive approach for early diagnosis and potentially risk stratification and a clinical monitoring tool of MFS and other connective tissue disorders. Such a new non-invasive diagnostic procedure would offer considerable benefits in terms of early management of patients suffering from MFS.”  The study presented in part II of this thesis is the first longitudinal in vivo study in MFS mice that uses high-resolution ultrasound imaging to measure parameters very similar to those collected in human MFS studies. The larger aortic root diameter and higher PWV in MFS mice are clear indications of significant aortic dilatation and central aortic stiffness. Data collected in this study shows a pattern of aortic dilatation and increased PWV similar to those reported in human MFS patients, highlighting the value of in vivo ultrasound imaging as a reliable tool to study the gradual pathological changes in the aortic function and structure of the aortic wall, from the initial phase towards the end stage aneurysm, in the mouse model of MFS-associated aortic aneurysm.  In this longitudinal study, the protective effects of long-term doxycycline intervention on aortic structure and function were determined in MFS mice. We confirmed that doxycycline prevented aortic dilatation seen in MFS mice, and also attenuated the increase in PWV, implying that aortic stiffness was less in the treated MFS mice. In addition, the ultrastructural changes of 89  elastin contributing to the formation of aneurysm in MFS aorta were also investigated by TEM imaging, and quantitatively analyzed. The morphometric modifications of elastic fibers were significantly improved after doxycycline intervention. In summary, data presented herein may provide new insights into the potential therapeutic value of sub-antibiotic dose of doxycycline in blocking MFS-associated aneurysm, and may strengthen the rationale for a similar clinical trial in human MFS patients in the future.                    90  Bibliography:  Abraham, T., and J. Hogg. 2010. Extracellular matrix remodeling of lung alveolar walls in three dimensional space identified using second harmonic generation and multiphoton excitation fluorescence. Journal of structural biology. 171:189-196.  Abraham, T., D. Kayra, B. McManus, and A. Scott. 2012. Quantitative assessment of forward and backward second harmonic three dimensional images of collagen Type I matrix remodeling in a stimulated cellular environment. Journal of structural biology. 180:17-25.  Abraham, T., S. Wadsworth, J.M. Carthy, D.V. Pechkovsky, and B. McManus. 2011. Minimally invasive imaging method based on second harmonic generation and multiphoton excitation fluorescence in translational respiratory research. Respirology (Carlton, Vic.). 16:22-33.  Agmon, Y., B.K. Khandheria, I. Meissner, G.L. Schwartz, J.D. Sicks, A.J. Fought, W.M. O'Fallon, D.O. Wiebers, and A.J. Tajik. 2003. Is aortic dilatation an atherosclerosis-related process? Clinical, laboratory, and transesophageal echocardiographic correlates of thoracic aortic dimensions in the population with implications for thoracic aortic aneurysm formation. Journal of the American College of Cardiology. 42:1076-1083.  Agren, M.S. 1994. Gelatinase activity during wound healing. The British journal of dermatology. 131:634-640.  Ailawadi, G., B.S. Knipp, G. Lu, K.J. Roelofs, J.W. Ford, K.K. Hannawa, K. Bishop, P. Thanaporn, P.K. Henke, J.C. Stanley, and G.R. Upchurch, Jr. 2003. A nonintrinsic regional basis for increased infrarenal aortic MMP-9 expression and activity. Journal of vascular surgery. 37:1059-1066.  Albo, D., D.H. Berger, J. Vogel, and G.P. Tuszynski. 1999. Thrombospondin-1 and transforming growth factor beta-1 upregulate plasminogen activator inhibitor type 1 in pancreatic cancer. Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract. 3:411-417.  Allaire, E., R. Forough, M. Clowes, B. Starcher, and A.W. Clowes. 1998. Local overexpression of TIMP-1 prevents aortic aneurysm degeneration and rupture in a rat model. The Journal of clinical investigation. 102:1413-1420.  Amadeu, T.P., A.S. Braune, L.C. Porto, A. Desmouliere, and A.M. Costa. 2004. Fibrillin-1 and elastin are differentially expressed in hypertrophic scars and keloids. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 12:169-174.  Ammash, N.M., T.M. Sundt, and H.M. Connolly. 2008. Marfan syndrome-diagnosis and management. Current problems in cardiology. 33:7-39.  Arakaki, P.A., M.R. Marques, and M.C. Santos. 2009. MMP-1 polymorphism and its relationship to pathological processes. Journal of biosciences. 34:313-320.  Arnlov, J., E. Ingelsson, U. Riserus, B. Andren, and L. Lind. 2004. Myocardial performance index, a Doppler-derived index of global left ventricular function, predicts congestive heart failure in elderly men. European heart journal. 25:2220-2225.  Asmar, R., A. Benetos, J. Topouchian, P. Laurent, B. Pannier, A.M. Brisac, R. Target, and B.I. Levy. 1995. Assessment of arterial distensibility by automatic pulse wave velocity measurement. Validation and clinical application studies. Hypertension (Dallas, Tex. : 1979). 26:485-490.  Avolio, A.P., S.G. Chen, R.P. Wang, C.L. Zhang, M.F. Li, and M.F. O'Rourke. 1983. Effects of aging on changing arterial compliance and left ventricular load in a northern Chinese urban community. Circulation. 68:50-58.  91  Bahk, T.J., M.D. Daniels, J.S. Leon, K. Wang, and D.M. Engman. 2008. Comparison of angiotensin converting enzyme inhibition and angiotensin II receptor blockade for the prevention of experimental autoimmune myocarditis. International journal of cardiology. 125:85-93.  Baramova, E., and J.M. Foidart. 1995. Matrix metalloproteinase family. Cell biology international. 19:239-242.  Beighton, P., A. de Paepe, D. Danks, G. Finidori, T. Gedde-Dahl, R. Goodman, J.G. Hall, D.W. Hollister, W. Horton, V.A. McKusick, and et al. 1988. International Nosology of Heritable Disorders of Connective Tissue, Berlin, 1986. American journal of medical genetics. 29:581-594.  Bewersdorf, J., R. Pick, and S.W. Hell. 1998. Multifocal multiphoton microscopy. Optics letters. 23:655-657.  Bierie, B., and H.L. Moses. 2010. Transforming growth factor beta (TGF-beta) and inflammation in cancer. Cytokine and growth factor reviews. 21:49-59.  Birkedal-Hansen, H. 1995. Proteolytic remodeling of extracellular matrix. Current opinion in cell biology. 7:728-735.  Blanchette, F., R. Day, W. Dong, M.H. Laprise, and C.M. Dubois. 1997. TGFbeta1 regulates gene expression of its own converting enzyme furin. The Journal of clinical investigation. 99:1974-1983.  Bock, P., and L. Stockinger. 1984. Light and electron microscopic identification of elastic, elaunin and oxytalan fibers in human tracheal and bronchial mucosa. Anatomy and embryology. 170:145-153.  Boileau, C., D.C. Guo, N. Hanna, E.S. Regalado, D. Detaint, L. Gong, M. Varret, S.K. Prakash, A.H. Li, H. d'Indy, A.C. Braverman, B. Grandchamp, C.S. Kwartler, L. Gouya, R.L. Santos-Cortez, M. Abifadel, S.M. Leal, C. Muti, J. Shendure, M.S. Gross, M.J. Rieder, A. Vahanian, D.A. Nickerson, J.B. Michel, G. Jondeau, and D.M. Milewicz. 2012. TGFB2 mutations cause familial thoracic aortic aneurysms and dissections associated with mild systemic features of Marfan syndrome. Nature genetics. 44:916-921.  Booher, A.M., and K.A. Eagle. 2011. Diagnosis and management issues in thoracic aortic aneurysm. American heart journal. 162:38-46 e31.  Bradley, T.J., J.E. Potts, M.T. Potts, A.M. DeSouza, and G.G. Sandor. 2005. Echocardiographic Doppler assessment of the biophysical properties of the aorta in pediatric patients with the Marfan syndrome. The American journal of cardiology. 96:1317-1321.  Brenneisen, P., K. Briviba, M. Wlaschek, J. Wenk, and K. Scharffetter-Kochanek. 1997. Hydrogen peroxide (H2O2) increases the steady-state mRNA levels of collagenase/MMP-1 in human dermal fibroblasts. Free radical biology and medicine. 22:515-524.  Brenneisen, P., J. Oh, M. Wlaschek, J. Wenk, K. Briviba, C. Hommel, G. Herrmann, H. Sies, and K. Scharffetter-Kochanek. 1996. Ultraviolet B wavelength dependence for the regulation of two major matrix-metalloproteinases and their inhibitor TIMP-1 in human dermal fibroblasts. Photochemistry and photobiology. 64:649-657.  Brinckerhoff, C.E., and L.M. Matrisian. 2002. Matrix metalloproteinases: a tail of a frog that became a prince. Nature reviews. Molecular cell biology. 3:207-214.  Burns, F.R., M.S. Stack, R.D. Gray, and C.A. Paterson. 1989. Inhibition of purified collagenase from alkali-burned rabbit corneas. Investigative ophthalmology & visual science. 30:1569-1575.  Canadas, V., I. Vilacosta, I. Bruna, and V. Fuster. 2010. Marfan syndrome. Part 1: pathophysiology and diagnosis. Nature reviews. Cardiology. 7:256-265.  Centonze, V.E., and J.G. White. 1998. Multiphoton excitation provides optical sections from deeper within 92  scattering specimens than confocal imaging. Biophysical journal. 75:2015-2024.  Chang, K.M., N.S. Ramamurthy, T.F. McNamara, R.T. Evans, B. Klausen, P.A. Murray, and L.M. Golub. 1994. Tetracyclines inhibit Porphyromonas gingivalis-induced alveolar bone loss in rats by a non-antimicrobial mechanism. Journal of periodontal research. 29:242-249.  Chatrath, R., L.M. Beauchesne, H.M. Connolly, V.V. Michels, and D.J. Driscoll. 2003. Left ventricular function in the Marfan syndrome without significant valvular regurgitation. The American journal of cardiology. 91:914-916.  Chen, J., S. Zhuo, X. Jiang, X. Zhu, L. Zheng, S. Xie, B. Lin, and H. Zeng. 2011. Multiphoton microscopy study of the morphological and quantity changes of collagen and elastic fiber components in keloid disease. Journal of biomedical optics. 16:051305.  Chin, G., T. Thigpin, K. Perrin, L. Moldawer, and G. Schultz. 2003. Treatment of chronic ulcers in diabetic patients with a topical metalloproteinase inhibitor, doxycycline. Wounds-a Compendium of Clinical Research and Practice. 15:315-323.  Chiu, A.T., W.F. Herblin, D.E. McCall, R.J. Ardecky, D.J. Carini, J.V. Duncia, L.J. Pease, P.C. Wong, R.R. Wexler, A.L. Johnson, and et al. 1989. Identification of angiotensin II receptor subtypes. Biochemical and biophysical research communications. 165:196-203.  Chung, A.W., K. Au Yeung, G.G. Sandor, D.P. Judge, H.C. Dietz, and C. van Breemen. 2007a. Loss of elastic fiber integrity and reduction of vascular smooth muscle contraction resulting from the upregulated activities of matrix metalloproteinase-2 and -9 in the thoracic aortic aneurysm in Marfan syndrome. Circulation research. 101:512-522.  Chung, A.W., H. Luo, T. Tejerina, C. van Breemen, and E.B. Okon. 2007b. Enhanced cell cycle entry and mitogen-activated protein kinase-signaling and downregulation of matrix metalloproteinase-1 and -3 in human diabetic arterial vasculature. Atherosclerosis. 195:e1-8.  Chung, A.W., H.H. Yang, M.W. Radomski, and C. van Breemen. 2008. Long-term doxycycline is more effective than atenolol to prevent thoracic aortic aneurysm in marfan syndrome through the inhibition of matrix metalloproteinase-2 and -9. Circulation research. 102:e73-85.  Coe, M., and S.H. Silvers. 1940. Ehlers-Danlos syndrome (cutis hyperelastica). American Journal of Diseases of Children. 59:129-135.  Colnot, C., Z. Thompson, T. Miclau, Z. Werb, and J.A. Helms. 2003. Altered fracture repair in the absence of MMP9. Development (Cambridge, England). 130:4123-4133.  Coussens, L.M., B. Fingleton, and L.M. Matrisian. 2002. Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science (New York, N.Y.). 295:2387-2392.  Cruickshank, K., L. Riste, S.G. Anderson, J.S. Wright, G. Dunn, and R.G. Gosling. 2002. Aortic pulse-wave velocity and its relationship to mortality in diabetes and glucose intolerance: an integrated index of vascular function? Circulation. 106:2085-2090.  Cui, J.Z. 2016. Investigation of effects of losartan, mild aerobic exercise and their combination on Loeys-Dietz syndrome-associated aortic aneurysm in mice. University of British Columbia.  Cui, J.Z., A.Y. Tehrani, K.A. Jett, P. Bernatchez, C. van Breemen, and M. Esfandiarei. 2014. Quantification of aortic and cutaneous elastin and collagen morphology in Marfan syndrome by multiphoton microscopy. Journal of structural biology. 187:242-253.  Curci, J.A., S. Liao, M.D. Huffman, S.D. Shapiro, and R.W. Thompson. 1998a. Expression and localization of macrophage elastase (matrix metalloproteinase-12) in abdominal aortic aneurysms. The Journal of clinical investigation. 102:1900-1910.  Curci, J.A., D. Petrinec, S. Liao, L.M. Golub, and R.W. Thompson. 1998b. Pharmacologic suppression of 93  experimental abdominal aortic aneurysms: acomparison of doxycycline and four chemically modified tetracyclines. Journal of vascular surgery. 28:1082-1093.  Das, B.B., A.L. Taylor, and A.T. Yetman. 2006. Left ventricular diastolic dysfunction in children and young adults with Marfan syndrome. Pediatric cardiology. 27:256-258.  Davis, V., R. Persidskaia, L. Baca-Regen, Y. Itoh, H. Nagase, Y. Persidsky, A. Ghorpade, and B.T. Baxter. 1998. Matrix metalloproteinase-2 production and its binding to the matrix are increased in abdominal aortic aneurysms. Arteriosclerosis, thrombosis, and vascular biology. 18:1625-1633.  De Backer, J.F., D. Devos, P. Segers, D. Matthys, K. Francois, T.C. Gillebert, A.M. De Paepe, and J. De Sutter. 2006. Primary impairment of left ventricular function in Marfan syndrome. International journal of cardiology. 112:353-358.  De Paepe, A., R.B. Devereux, H.C. Dietz, R.C. Hennekam, and R.E. Pyeritz. 1996. Revised diagnostic criteria for the Marfan syndrome. American journal of medical genetics. 62:417-426.  Denk, W. 1994. Two-photon scanning photochemical microscopy: mapping ligand-gated ion channel distributions. Proceedings of the National Academy of Sciences of the United States of America. 91:6629-6633.  Denk, W., K.R. Delaney, A. Gelperin, D. Kleinfeld, B.W. Strowbridge, D.W. Tank, and R. Yuste. 1994b. Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy. Journal of neuroscience methods. 54:151-162.  Denk, W., and K. Svoboda. 1997. Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron. 18:351-357.  Denk, W., and W.W. Webb. 1990. Optical measurement of picometer displacements of transparent microscopic objects. Applied optics. 29:2382-2391.  Derynck, R., J.A. Jarrett, E.Y. Chen, D.H. Eaton, J.R. Bell, R.K. Assoian, A.B. Roberts, M.B. Sporn, and D.V. Goeddel. 1985. Human transforming growth factor-beta complementary DNA sequence and expression in normal and transformed cells. Nature. 316:701-705.  Di Lascio, N., F. Stea, C. Kusmic, R. Sicari, and F. Faita. 2014. Non-invasive assessment of pulse wave velocity in mice by means of ultrasound images. Atherosclerosis. 237:31-37.  Dietrich, W.F., E.S. Lander, J.S. Smith, A.R. Moser, K.A. Gould, C. Luongo, N. Borenstein, and W. Dove. 1993. Genetic identification of Mom-1, a major modifier locus affecting Min-induced intestinal neoplasia in the mouse. Cell. 75:631-639.  Dietz, H.C., G.R. Cutting, R.E. Pyeritz, C.L. Maslen, L.Y. Sakai, G.M. Corson, E.G. Puffenberger, A. Hamosh, E.J. Nanthakumar, S.M. Curristin, and et al. 1991a. Marfan syndrome caused by a recurrent de novo missense mutation in the fibrillin gene. Nature. 352:337-339.  Dietz, H.C., R.E. Pyeritz, B.D. Hall, R.G. Cadle, A. Hamosh, J. Schwartz, D.A. Meyers, and C.A. Francomano. 1991b. The Marfan syndrome locus: confirmation of assignment to chromosome 15 and identification of tightly linked markers at 15q15-q21.3. Genomics. 9:355-361.  Dimitrow, E., I. Riemann, A. Ehlers, M.J. Koehler, J. Norgauer, P. Elsner, K. Konig, and M. Kaatz. 2009a. Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis. Experimental dermatology. 18:509-515.  Dimitrow, E., M. Ziemer, M.J. Koehler, J. Norgauer, K. Konig, P. Elsner, and M. Kaatz. 2009b. Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma. The Journal of investigative dermatology. 129:1752-1758.  Doyle, A.J., J.J. Doyle, S.L. Bessling, S. Maragh, M.E. Lindsay, D. Schepers, E. Gillis, G. Mortier, T. Homfray, K. Sauls, R.A. Norris, N.D. Huso, D. Leahy, D.W. Mohr, M.J. Caulfield, A.F. Scott, A. 94  Destree, R.C. Hennekam, P.H. Arn, C.J. Curry, L. Van Laer, A.S. McCallion, B.L. Loeys, and H.C. Dietz. 2012. Mutations in the TGF-beta repressor SKI cause Shprintzen-Goldberg syndrome with aortic aneurysm. Nature genetics. 44:1249-1254.  Dubois, C.M., M.H. Laprise, F. Blanchette, L.E. Gentry, and R. Leduc. 1995. Processing of transforming growth factor beta 1 precursor by human furin convertase. The Journal of biological chemistry. 270:10618-10624.  Dzau, V.J. 1986. Significance of the vascular renin-angiotensin pathway. Hypertension (Dallas, Tex. : 1979). 8:553-559.  Egeblad, M., and Z. Werb. 2002. New functions for the matrix metalloproteinases in cancer progression. Nature reviews. Cancer. 2:161-174.  El-Hamamsy, I., and M.H. Yacoub. 2009. Cellular and molecular mechanisms of thoracic aortic aneurysms. Nature reviews. Cardiology. 6:771-786.  Evangelista, A., G. Avegliano, R. Aguilar, H. Cuellar, A. Igual, T. Gonzalez-Alujas, J. Rodriguez-Palomares, P. Mahia, and D. Garcia-Dorado. 2010. Impact of contrast-enhanced echocardiography on the diagnostic algorithm of acute aortic dissection. European heart journal. 31:472-479.  Faivre, L., G. Collod-Beroud, B.L. Loeys, A. Child, C. Binquet, E. Gautier, B. Callewaert, E. Arbustini, K. Mayer, M. Arslan-Kirchner, A. Kiotsekoglou, P. Comeglio, N. Marziliano, H.C. Dietz, D. Halliday, C. Beroud, C. Bonithon-Kopp, M. Claustres, C. Muti, H. Plauchu, P.N. Robinson, L.C. Ades, A. Biggin, B. Benetts, M. Brett, K.J. Holman, J. De Backer, P. Coucke, U. Francke, A. De Paepe, G. Jondeau, and C. Boileau. 2007. Effect of mutation type and location on clinical outcome in 1,013 probands with Marfan syndrome or related phenotypes and FBN1 mutations: an international study. American journal of human genetics. 81:454-466.  Fedak, P.W., S. Verma, T.E. David, R.L. Leask, R.D. Weisel, and J. Butany. 2002. Clinical and pathophysiological implications of a bicuspid aortic valve. Circulation. 106:900-904.  Feigenbaum, H. 1996. Evolution of echocardiography. Circulation. 93:1321-1327.  Freestone, T., R.J. Turner, A. Coady, D.J. Higman, R.M. Greenhalgh, and J.T. Powell. 1995. Inflammation and matrix metalloproteinases in the enlarging abdominal aortic aneurysm. Arteriosclerosis, thrombosis, and vascular biology. 15:1145-1151.  Galis, Z.S., C. Johnson, D. Godin, R. Magid, J.M. Shipley, R.M. Senior, and E. Ivan. 2002. Targeted disruption of the matrix metalloproteinase-9 gene impairs smooth muscle cell migration and geometrical arterial remodeling. Circulation research. 91:852-859.  Gallo, E.M., D.C. Loch, J.P. Habashi, J.F. Calderon, Y. Chen, D. Bedja, C. van Erp, E.E. Gerber, S.J. Parker, K. Sauls, D.P. Judge, S.K. Cooke, M.E. Lindsay, R. Rouf, L. Myers, C.M. ap Rhys, K.C. Kent, R.A. Norris, D.L. Huso, and H.C. Dietz. 2014. Angiotensin II-dependent TGF-beta signaling contributes to Loeys-Dietz syndrome vascular pathogenesis. The Journal of clinical investigation. 124:448-460.  Gao, S., D. Ho, D.E. Vatner, and S.F. Vatner. 2011. Echocardiography in Mice. Current protocols in mouse biology. 1:71-83.  Gentry, L.E., and B.W. Nash. 1990. The pro domain of pre-pro-transforming growth factor beta 1 when independently expressed is a functional binding protein for the mature growth factor. Biochemistry. 29:6851-6857.  Gerstenblith, G., J. Frederiksen, F.C. Yin, N.J. Fortuin, E.G. Lakatta, and M.L. Weisfeldt. 1977. Echocardiographic assessment of a normal adult aging population. Circulation. 56:273-278.  Godfrey, M., V. Menashe, R.G. Weleber, R.D. Koler, R.H. Bigley, E. Lovrien, J. Zonana, and D.W. Hollister. 1990a. Cosegregation of elastin-associated microfibrillar abnormalities with the Marfan 95  phenotype in families. American journal of human genetics. 46:652-660.  Godfrey, M., S. Olson, R.G. Burgio, A. Martini, M. Valli, G. Cetta, H. Hori, and D.W. Hollister. 1990b. Unilateral microfibrillar abnormalities in a case of asymmetric Marfan syndrome. American journal of human genetics. 46:661-671.  Golub, L.M., H.M. Lee, M.E. Ryan, W.V. Giannobile, J. Payne, and T. Sorsa. 1998. Tetracyclines inhibit connective tissue breakdown by multiple non-antimicrobial mechanisms. Advances in dental research. 12:12-26.  Golub, L.M., T. Sorsa, H.M. Lee, S. Ciancio, D. Sorbi, N.S. Ramamurthy, B. Gruber, T. Salo, and Y.T. Konttinen. 1995. Doxycycline inhibits neutrophil (PMN)-type matrix metalloproteinases in human adult periodontitis gingiva. Journal of clinical periodontology. 22:100-109.  Gosling, R.G., and M.M. Budge. 2003. Terminology for describing the elastic behavior of arteries. Hypertension (Dallas, Tex. : 1979). 41:1180-1182.  Goumans, M.J., Z. Liu, and P. ten Dijke. 2009. TGF-beta signaling in vascular biology and dysfunction. Cell research. 19:116-127.  Groenink, M., A. de Roos, B.J. Mulder, B. Verbeeten, Jr., J. Timmermans, A.H. Zwinderman, J.A. Spaan, and E.E. van der Wall. 2001. Biophysical properties of the normal-sized aorta in patients with Marfan syndrome: evaluation with MR flow mapping. Radiology. 219:535-540.  Gross, J., and C.M. Lapiere. 1962. Collagenolytic activity in amphibian tissues: a tissue culture assay. Proceedings of the National Academy of Sciences of the United States of America. 48:1014-1022.  Habashi, J.P., J.J. Doyle, T.M. Holm, H. Aziz, F. Schoenhoff, D. Bedja, Y. Chen, A.N. Modiri, D.P. Judge, and H.C. Dietz. 2011. Angiotensin II type 2 receptor signaling attenuates aortic aneurysm in mice through ERK antagonism. Science (New York, N.Y.). 332:361-365.  Habashi, J.P., D.P. Judge, T.M. Holm, R.D. Cohn, B.L. Loeys, T.K. Cooper, L. Myers, E.C. Klein, G. Liu, C. Calvi, M. Podowski, E.R. Neptune, M.K. Halushka, D. Bedja, K. Gabrielson, D.B. Rifkin, L. Carta, F. Ramirez, D.L. Huso, and H.C. Dietz. 2006. Losartan, an AT1 antagonist, prevents aortic aneurysm in a mouse model of Marfan syndrome. Science (New York, N.Y.). 312:117-121.  Hartley, C.J., G.E. Taffet, L.H. Michael, T.T. Pham, and M.L. Entman. 1997. Noninvasive determination of pulse-wave velocity in mice. The American journal of physiology. 273:H494-500.  Hayashi, H., and T. Sakai. 2012. Biological Significance of Local TGF-beta Activation in Liver Diseases. Frontiers in physiology. 3:12.  Helmchen, F., and W. Denk. 2002. New developments in multiphoton microscopy. Current opinion in neurobiology. 12:593-601.  Helmchen, F., and W. Denk. 2005. Deep tissue two-photon microscopy. Nature methods. 2:932-940.  Hijova, E. 2005. Matrix metalloproteinases: their biological functions and clinical implications. Bratislavske lekarske listy. 106:127-132.  Hirata, K., F. Triposkiadis, E. Sparks, J. Bowen, C.F. Wooley, and H. Boudoulas. 1991. The Marfan syndrome: abnormal aortic elastic properties. Journal of the American College of Cardiology. 18:57-63.  Hucthagowder, V., N. Sausgruber, K.H. Kim, B. Angle, L.Y. Marmorstein, and Z. Urban. 2006. Fibulin-4: a novel gene for an autosomal recessive cutis laxa syndrome. American journal of human genetics. 78:1075-1080.  Hughes, S.M., L.J. Dixon, and G.E. McVeigh. 2004. Arterial stiffness and pulse wave velocity: problems with terminology. Circulation. 109:e3; author reply e3. 96   Iwai, N., and T. Inagami. 1992. Identification of two subtypes in the rat type I angiotensin II receptor. FEBS letters. 298:257-260.  Jarrett, A. 1974. Ageing of the dermis. In: The Physiology and Pathophysiology of the Skin. Academic Press, London, UK.  Jeremy, R.W., H. Huang, J. Hwa, H. McCarron, C.F. Hughes, and J.G. Richards. 1994. Relation between age, arterial distensibility, and aortic dilatation in the Marfan syndrome. The American journal of cardiology. 74:369-373.  Jones, E.S., M.J. Black, and R.E. Widdop. 2004. Angiotensin AT2 receptor contributes to cardiovascular remodelling of aged rats during chronic AT1 receptor blockade. Journal of molecular and cellular cardiology. 37:1023-1030.  Judge, D.P., N.J. Biery, D.R. Keene, J. Geubtner, L. Myers, D.L. Huso, L.Y. Sakai, and H.C. Dietz. 2004. Evidence for a critical contribution of haploinsufficiency in the complex pathogenesis of Marfan syndrome. The Journal of clinical investigation. 114:172-181.  Judge, D.P., and H.C. Dietz. 2005. Marfan's syndrome. Lancet (London, England). 366:1965-1976.  Keane, M.G., and R.E. Pyeritz. 2008. Medical management of Marfan syndrome. Circulation. 117:2802-2813.  Kielty, C.M., M.J. Sherratt, A. Marson, and C. Baldock. 2005. Fibrillin microfibrils. Advances in protein chemistry. 70:405-436.  Kim, E.M., and O. Hwang. 2011. Role of matrix metalloproteinase-3 in neurodegeneration. Journal of neurochemistry. 116:22-32.  Kim, M., M.J. Roman, M.C. Cavallini, J.E. Schwartz, T.G. Pickering, and R.B. Devereux. 1996. Effect of hypertension on aortic root size and prevalence of aortic regurgitation. Hypertension (Dallas, Tex. : 1979). 28:47-52.  Kiotsekoglou, A., J.C. Moggridge, B.H. Bijnens, V. Kapetanakis, F. Alpendurada, M.J. Mullen, S. Saha, D.K. Nassiri, J. Camm, G.R. Sutherland, and A.H. Child. 2009. Biventricular and atrial diastolic function assessment using conventional echocardiography and tissue-Doppler imaging in adults with Marfan syndrome. European journal of echocardiography : the journal of the Working Group on Echocardiography of the European Society of Cardiology. 10:947-955.  Kiotsekoglou, A., J.C. Moggridge, S.K. Saha, V. Kapetanakis, M. Govindan, F. Alpendurada, M.J. Mullen, J. Camm, G.R. Sutherland, B.H. Bijnens, and A.H. Child. 2011. Assessment of aortic stiffness in marfan syndrome using two-dimensional and Doppler echocardiography. Echocardiography (Mount Kisco, N.Y.). 28:29-37.  Koehler, M.J., A. Preller, P. Elsner, K. Konig, U.C. Hipler, and M. Kaatz. 2012. Non-invasive evaluation of dermal elastosis by in vivo multiphoton tomography with autofluorescence lifetime measurements. Experimental dermatology. 21:48-51.  Koehler, M.J., A. Preller, N. Kindler, P. Elsner, K. Konig, R. Buckle, and M. Kaatz. 2009. Intrinsic, solar and sunbed-induced skin aging measured in vivo by multiphoton laser tomography and biophysical methods. Skin research and technology : official journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging (ISSI). 15:357-363.  Konig, K., A. Ehlers, F. Stracke, and I. Riemann. 2006. In vivo drug screening in human skin using femtosecond laser multiphoton tomography. Skin pharmacology and physiology. 19:78-88.  Kroner, E.S., A.J. Scholte, P.J. de Koning, P.J. van den Boogaard, L.J. Kroft, R.J. van der Geest, Y. Hilhorst-Hofstee, H.J. Lamb, H.M. Siebelink, B.J. Mulder, M. Groenink, T. Radonic, E.E. van der 97  Wall, A. de Roos, J.H. Reiber, and J.J. Westenberg. 2013. MRI-assessed regional pulse wave velocity for predicting absence of regional aorta luminal growth in marfan syndrome. International journal of cardiology. 167:2977-2982.  Lakatta, E.G., and D. Levy. 2003. Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: Part I: aging arteries: a "set up" for vascular disease. Circulation. 107:139-146.  Lalchandani, S., and M. Wingfield. 2003. Pregnancy in women with Marfan's Syndrome. European journal of obstetrics, gynecology, and reproductive biology. 110:125-130.  Lauhio, A., Y.T. Konttinen, T. Salo, H. Tschesche, D. Nordstrom, J. Lahdevirta, L.M. Golub, and T. Sorsa. 1994. The in vivo effect of doxycycline treatment on matrix metalloproteinases in reactive arthritis. Annals of the New York Academy of Sciences. 732:431-432.  Laurent, S., J. Cockcroft, L. Van Bortel, P. Boutouyrie, C. Giannattasio, D. Hayoz, B. Pannier, C. Vlachopoulos, I. Wilkinson, and H. Struijker-Boudier. 2006. Expert consensus document on arterial stiffness: methodological issues and clinical applications. European heart journal. 27:2588-2605.  Laurent, S., S. Katsahian, C. Fassot, A.I. Tropeano, I. Gautier, B. Laloux, and P. Boutouyrie. 2003. Aortic stiffness is an independent predictor of fatal stroke in essential hypertension. Stroke. 34:1203-1206.  Lavoie, P., G. Robitaille, M. Agharazii, S. Ledbetter, M. Lebel, and R. Lariviere. 2005. Neutralization of transforming growth factor-beta attenuates hypertension and prevents renal injury in uremic rats. Journal of hypertension. 23:1895-1903.  Lawrence, D.A. 1996. Transforming growth factor-beta: a general review. European cytokine network. 7:363-374.  Lee, L., J.Z. Cui, M. Cua, M. Esfandiarei, X. Sheng, W.A. Chui, M.H. Xu, M.V. Sarunic, M.F. Beg, C. van Breemen, G.G. Sandor, and G.F. Tibbits. 2016. Aortic and Cardiac Structure and Function Using High-Resolution Echocardiography and Optical Coherence Tomography in a Mouse Model of Marfan Syndrome. PloS one. 11:e0164778.  Leloup, A.J., P. Fransen, C.E. Van Hove, M. Demolder, G.W. De Keulenaer, and D.M. Schrijvers. 2014. Applanation tonometry in mice: a novel noninvasive technique to assess pulse wave velocity and arterial stiffness. Hypertension (Dallas, Tex. : 1979). 64:195-200.  Levenson, D. 2010. New guidelines for diagnosis of Marfan and Loey-Dietz syndromes. American journal of medical genetics. Part A. 152A:fmvii-fmviii.  Levitt, J.M., M.E. McLaughlin-Drubin, K. Munger, and I. Georgakoudi. 2011. Automated biochemical, morphological, and organizational assessment of precancerous changes from endogenous two-photon fluorescence images. PloS one. 6:e24765.  Lillie, M.A., G.J. David, and J.M. Gosline. 1998. Mechanical role of elastin-associated microfibrils in pig aortic elastic tissue. Connective tissue research. 37:121-141.  Lillie, R. 1965. Histopathologic Technic and Practical Histochemistry. McGraw-Hill, New York.  Lim, D.S., S. Lutucuta, P. Bachireddy, K. Youker, A. Evans, M. Entman, R. Roberts, and A.J. Marian. 2001. Angiotensin II blockade reverses myocardial fibrosis in a transgenic mouse model of human hypertrophic cardiomyopathy. Circulation. 103:789-791.  Lima, B.L., E.J. Santos, G.R. Fernandes, C. Merkel, M.R. Mello, J.P. Gomes, M. Soukoyan, A. Kerkis, S.M. Massironi, J.A. Visintin, and L.V. Pereira. 2010. A new mouse model for marfan syndrome presents phenotypic variability associated with the genetic background and overall levels of Fbn1 expression. PloS one. 5:e14136.  98  Lin, C.S., and C.H. Pan. 2008. Regulatory mechanisms of atrial fibrotic remodeling in atrial fibrillation. Cellular and molecular life sciences : CMLS. 65:1489-1508.  Lin, S.J., S.H. Jee, and C.Y. Dong. 2007. Multiphoton microscopy: a new paradigm in dermatological imaging. European journal of dermatology : EJD. 17:361-366.  Lindeman, J.H., H. Abdul-Hussien, J.H. van Bockel, R. Wolterbeek, and R. Kleemann. 2009. Clinical trial of doxycycline for matrix metalloproteinase-9 inhibition in patients with an abdominal aneurysm: doxycycline selectively depletes aortic wall neutrophils and cytotoxic T cells. Circulation. 119:2209-2216.  Lindsay, M.E., D. Schepers, N.A. Bolar, J.J. Doyle, E. Gallo, J. Fert-Bober, M.J. Kempers, E.K. Fishman, Y. Chen, L. Myers, D. Bjeda, G. Oswald, A.F. Elias, H.P. Levy, B.M. Anderlid, M.H. Yang, E.M. Bongers, J. Timmermans, A.C. Braverman, N. Canham, G.R. Mortier, H.G. Brunner, P.H. Byers, J. Van Eyk, L. Van Laer, H.C. Dietz, and B.L. Loeys. 2012. Loss-of-function mutations in TGFB2 cause a syndromic presentation of thoracic aortic aneurysm. Nature genetics. 44:922-927.  Liu, J., W. Xiong, L. Baca-Regen, H. Nagase, and B.T. Baxter. 2003. Mechanism of inhibition of matrix metalloproteinase-2 expression by doxycycline in human aortic smooth muscle cells. Journal of vascular surgery. 38:1376-1383.  Lluri, G., and D.M. Jaworski. 2005. Regulation of TIMP-2, MT1-MMP, and MMP-2 expression during C2C12 differentiation. Muscle & nerve. 32:492-499.  Loeys, B.L., J. Chen, E.R. Neptune, D.P. Judge, M. Podowski, T. Holm, J. Meyers, C.C. Leitch, N. Katsanis, N. Sharifi, F.L. Xu, L.A. Myers, P.J. Spevak, D.E. Cameron, J. De Backer, J. Hellemans, Y. Chen, E.C. Davis, C.L. Webb, W. Kress, P. Coucke, D.B. Rifkin, A.M. De Paepe, and H.C. Dietz. 2005. A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2. Nature genetics. 37:275-281.  Loeys, B.L., H.C. Dietz, A.C. Braverman, B.L. Callewaert, J. De Backer, R.B. Devereux, Y. Hilhorst-Hofstee, G. Jondeau, L. Faivre, D.M. Milewicz, R.E. Pyeritz, P.D. Sponseller, P. Wordsworth, and A.M. De Paepe. 2010. The revised Ghent nosology for the Marfan syndrome. Journal of medical genetics. 47:476-485.  Loeys, B.L., U. Schwarze, T. Holm, B.L. Callewaert, G.H. Thomas, H. Pannu, J.F. De Backer, G.L. Oswald, S. Symoens, S. Manouvrier, A.E. Roberts, F. Faravelli, M.A. Greco, R.E. Pyeritz, D.M. Milewicz, P.J. Coucke, D.E. Cameron, A.C. Braverman, P.H. Byers, A.M. De Paepe, and H.C. Dietz. 2006. Aneurysm syndromes caused by mutations in the TGF-beta receptor. The New England journal of medicine. 355:788-798.  Longo, G.M., W. Xiong, T.C. Greiner, Y. Zhao, N. Fiotti, and B.T. Baxter. 2002. Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms. The Journal of clinical investigation. 110:625-632.  Lopez, A.R., J. Cook, P.L. Deininger, and R. Derynck. 1992. Dominant negative mutants of transforming growth factor-beta 1 inhibit the secretion of different transforming growth factor-beta isoforms. Molecular and cellular biology. 12:1674-1679.  Malhotra, A., and P.L. Westesson. 2009. Loeys-Dietz syndrome. Pediatric radiology. 39:1015.  Manabe, T., K. Imoto, K. Uchida, C. Doi, and Y. Takanashi. 2004. Decreased tissue inhibitor of metalloproteinase-2/matrix metalloproteinase ratio in the acute phase of aortic dissection. Surgery today. 34:220-225.  Marfan, A. 1896. Un cas de déformation congénitale des quatre membres, plus prononcée aux extrémités, caractérisée par l´allongement des os avec un certain degré d´amincissement [French]. Bull. Mem. Soc. Med. Hop (Paris). 13, 220–226.  Mariko, B., M. Pezet, B. Escoubet, S. Bouillot, J.P. Andrieu, B. Starcher, D. Quaglino, M.P. Jacob, P. 99  Huber, F. Ramirez, and G. Faury. 2011. Fibrillin-1 genetic deficiency leads to pathological ageing of arteries in mice. The Journal of pathology. 224:33-44.  Marque, V., P. Kieffer, B. Gayraud, I. Lartaud-Idjouadiene, F. Ramirez, and J. Atkinson. 2001. Aortic wall mechanics and composition in a transgenic mouse model of Marfan syndrome. Arteriosclerosis, thrombosis, and vascular biology. 21:1184-1189.  Massague, J. 1990. The transforming growth factor-beta family. Annual review of cell biology. 6:597-641.  Masters, B.R., P.T. So, and E. Gratton. 1997. Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin. Biophysical journal. 72:2405-2412.  Matrisian, L.M. 1990. Metalloproteinases and their inhibitors in matrix remodeling. Trends in genetics : TIG. 6:121-125.  Matrisian, L.M. 1992. The matrix-degrading metalloproteinases. BioEssays : news and reviews in molecular, cellular and developmental biology. 14:455-463.  Mauch, C., T. Krieg, and E.A. Bauer. 1994. Role of the extracellular matrix in the degradation of connective tissue. Archives of dermatological research. 287:107-114.  Meijboom, L.J., J. Timmermans, J.P. van Tintelen, G.J. Nollen, J. De Backer, M.P. van den Berg, G.H. Boers, and B.J. Mulder. 2005. Evaluation of left ventricular dimensions and function in Marfan's syndrome without significant valvular regurgitation. The American journal of cardiology. 95:795-797.  Milewicz, D.M., H.C. Dietz, and D.C. Miller. 2005. Treatment of aortic disease in patients with Marfan syndrome. Circulation. 111:e150-157.  Mitchell, G.F., H. Parise, E.J. Benjamin, M.G. Larson, M.J. Keyes, J.A. Vita, R.S. Vasan, and D. Levy. 2004. Changes in arterial stiffness and wave reflection with advancing age in healthy men and women: the Framingham Heart Study. Hypertension (Dallas, Tex. : 1979). 43:1239-1245.  Mitchell, J., and C. Schwartz. 1965. Arterial Disease FA Davis Co. Philadelphia. PA.  Mizuguchi, T., G. Collod-Beroud, T. Akiyama, M. Abifadel, N. Harada, T. Morisaki, D. Allard, M. Varret, M. Claustres, H. Morisaki, M. Ihara, A. Kinoshita, K. Yoshiura, C. Junien, T. Kajii, G. Jondeau, T. Ohta, T. Kishino, Y. Furukawa, Y. Nakamura, N. Niikawa, C. Boileau, and N. Matsumoto. 2004. Heterozygous TGFBR2 mutations in Marfan syndrome. Nature genetics. 36:855-860.  Mizuguchi, T., and N. Matsumoto. 2007. Recent progress in genetics of Marfan syndrome and Marfan-associated disorders. Journal of human genetics. 52:1-12.  Mochizuki, S., B. Brassart, and A. Hinek. 2002. Signaling pathways transduced through the elastin receptor facilitate proliferation of arterial smooth muscle cells. The Journal of biological chemistry. 277:44854-44863.  Mohan, R., S.K. Chintala, J.C. Jung, W.V. Villar, F. McCabe, L.A. Russo, Y. Lee, B.E. McCarthy, K.R. Wollenberg, J.V. Jester, M. Wang, H.G. Welgus, J.M. Shipley, R.M. Senior, and M.E. Fini. 2002. Matrix metalloproteinase gelatinase B (MMP-9) coordinates and effects epithelial regeneration. The Journal of biological chemistry. 277:2065-2072.  Mosorin, M., J. Juvonen, F. Biancari, J. Satta, H.M. Surcel, M. Leinonen, P. Saikku, and T. Juvonen. 2001. Use of doxycycline to decrease the growth rate of abdominal aortic aneurysms: a randomized, double-blind, placebo-controlled pilot study. Journal of vascular surgery. 34:606-610.  Mukoyama, M., M. Nakajima, M. Horiuchi, H. Sasamura, R.E. Pratt, and V.J. Dzau. 1993. Expression cloning of type 2 angiotensin II receptor reveals a unique class of seven-transmembrane receptors. The Journal of biological chemistry. 268:24539-24542.  100  Munger, J.S., J.G. Harpel, P.E. Gleizes, R. Mazzieri, I. Nunes, and D.B. Rifkin. 1997. Latent transforming growth factor-beta: structural features and mechanisms of activation. Kidney international. 51:1376-1382.  Murphy, T.J., R.W. Alexander, K.K. Griendling, M.S. Runge, and K.E. Bernstein. 1991. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature. 351:233-236.  Nagase, H., R. Visse, and G. Murphy. 2006. Structure and function of matrix metalloproteinases and TIMPs. Cardiovascular research. 69:562-573.  Nagase, H., and J.F. Woessner, Jr. 1999. Matrix metalloproteinases. The Journal of biological chemistry. 274:21491-21494.  Nagashima, H., Y. Sakomura, Y. Aoka, K. Uto, K. Kameyama, M. Ogawa, S. Aomi, H. Koyanagi, N. Ishizuka, M. Naruse, M. Kawana, and H. Kasanuki. 2001. Angiotensin II type 2 receptor mediates vascular smooth muscle cell apoptosis in cystic medial degeneration associated with Marfan's syndrome. Circulation. 104:I282-287.  Nataatmadja, M., J. West, and M. West. 2006. Overexpression of transforming growth factor-beta is associated with increased hyaluronan content and impairment of repair in Marfan syndrome aortic aneurysm. Circulation. 114:I371-377.  Neptune, E.R., P.A. Frischmeyer, D.E. Arking, L. Myers, T.E. Bunton, B. Gayraud, F. Ramirez, L.Y. Sakai, and H.C. Dietz. 2003. Dysregulation of TGF-beta activation contributes to pathogenesis in Marfan syndrome. Nature genetics. 33:407-411.  Ng, C.M., A. Cheng, L.A. Myers, F. Martinez-Murillo, C. Jie, D. Bedja, K.L. Gabrielson, J.M. Hausladen, R.P. Mecham, D.P. Judge, and H.C. Dietz. 2004. TGF-beta-dependent pathogenesis of mitral valve prolapse in a mouse model of Marfan syndrome. The Journal of clinical investigation. 114:1586-1592.  Nichols, W., and M. O’Rourke. 2005. McDonald’s Blood Flow in Arteries 5th edn (London: Arnold).  Nichols, W.W., M.F. O'Rourke, A.P. Avolio, T. Yaginuma, J.P. Murgo, C.J. Pepine, and C.R. Conti. 1985. Effects of age on ventricular-vascular coupling. The American journal of cardiology. 55:1179-1184.  Nollen, G.J., M. Groenink, J.G. Tijssen, E.E. Van Der Wall, and B.J. Mulder. 2004. Aortic stiffness and diameter predict progressive aortic dilatation in patients with Marfan syndrome. European heart journal. 25:1146-1152.  O'Rourke, M., A. Avolio, P. Lauren, and J. Yong. 1987. Age-related-changes of elastin lamellae in the human thoracic aorta, pp. A53-A53 Journal of the American College of Cardiology, Vol. 9. Elsevier Science Inc 655 Avenue of the Americas, New York, NY 10010.  O'Rourke, M.F., J.A. Staessen, C. Vlachopoulos, D. Duprez, and G.E. Plante. 2002. Clinical applications of arterial stiffness; definitions and reference values. American journal of hypertension. 15:426-444.  Oheim, M., E. Beaurepaire, E. Chaigneau, J. Mertz, and S. Charpak. 2001. Two-photon microscopy in brain tissue: parameters influencing the imaging depth. Journal of neuroscience methods. 111:29-37.  Okamura, H., L.J. Pisani, A.R. Dalal, F. Emrich, B.A. Dake, M. Arakawa, D.C. Onthank, R.R. Cesati, S.P. Robinson, M. Milanesi, G. Kotek, H. Smit, A.J. Connolly, H. Adachi, M.V. McConnell, and M.P. Fischbein. 2014. Assessment of elastin deficit in a Marfan mouse aneurysm model using an elastin-specific magnetic resonance imaging contrast agent. Circulation. Cardiovascular imaging. 7:690-696.  Oliver, J.J., and D.J. Webb. 2003. Noninvasive assessment of arterial stiffness and risk of atherosclerotic events. Arteriosclerosis, thrombosis, and vascular biology. 23:554-566. 101   Page-McCaw, A., A.J. Ewald, and Z. Werb. 2007. Matrix metalloproteinases and the regulation of tissue remodelling. Nature reviews. Molecular cell biology. 8:221-233.  Pelton, R.W., M.D. Johnson, E.A. Perkett, L.I. Gold, and H.L. Moses. 1991. Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 mRNA and protein in the murine lung. American journal of respiratory cell and molecular biology. 5:522-530.  Pelton, R.W., and H.L. Moses. 1990. The beta-type transforming growth factor. Mediators of cell regulation in the lung. The American review of respiratory disease. 142:S31-35.  Pereira, L., K. Andrikopoulos, J. Tian, S.Y. Lee, D.R. Keene, R. Ono, D.P. Reinhardt, L.Y. Sakai, N.J. Biery, T. Bunton, H.C. Dietz, and F. Ramirez. 1997. Targetting of the gene encoding fibrillin-1 recapitulates the vascular aspect of Marfan syndrome. Nature genetics. 17:218-222.  Pereira, L., S.Y. Lee, B. Gayraud, K. Andrikopoulos, S.D. Shapiro, T. Bunton, N.J. Biery, H.C. Dietz, L.Y. Sakai, and F. Ramirez. 1999. Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1. Proceedings of the National Academy of Sciences of the United States of America. 96:3819-3823.  Perry, H.D., and L.M. Golub. 1985. Systemic tetracyclines in the treatment of noninfected corneal ulcers: a case report and proposed new mechanism of action. Annals of ophthalmology. 17:742-744.  Petroll, W.M. 2006. Differential interference contrast and confocal reflectance imaging of collagen organization in three-dimensional matrices. Scanning. 28:305-310.  Pilcher, B.K., M. Wang, X.J. Qin, W.C. Parks, R.M. Senior, and H.G. Welgus. 1999. Role of matrix metalloproteinases and their inhibition in cutaneous wound healing and allergic contact hypersensitivity. Annals of the New York Academy of Sciences. 878:12-24.  Pyeritz, R.E. 2000. The Marfan syndrome. Annual review of medicine. 51:481-510.  Rabben, S.I., N. Stergiopulos, L.R. Hellevik, O.A. Smiseth, S. Slordahl, S. Urheim, and B. Angelsen. 2004. An ultrasound-based method for determining pulse wave velocity in superficial arteries. Journal of biomechanics. 37:1615-1622.  Rahbar, E., J. Weimer, H. Gibbs, A.T. Yeh, C.D. Bertram, M.J. Davis, M.A. Hill, D.C. Zawieja, and J.E. Moore, Jr. 2012. Passive pressure-diameter relationship and structural composition of rat mesenteric lymphangions. Lymphatic research and biology. 10:152-163.  Ramamurthy, N.S., A.J. Kucine, S.A. McClain, T.F. McNamara, and L.M. Golub. 1998. Topically applied CMT-2 enhances wound healing in streptozotocin diabetic rat skin. Advances in dental research. 12:144-148.  Rhoades R, B.D. 2013. Medical physiology: principles for clinical medicine. 4th ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins; xvi, 819 p. p.  Richard, M., P. Broquet, E. Vignon, M.J. Peschard, J.P. Carret, and P. Louisot. 1991. Calmodulin-dependent collagenase and proteoglycanase activities in chondrocytes from human osteoarthritic cartilage. Biochemical and biophysical research communications. 174:1204-1207.  Roach, M.R., and A.C. Burton. 1957. The reason for the shape of the distensibility curves of arteries. Canadian journal of biochemistry and physiology. 35:681-690.  Rodriguez-Pla, A., J.A. Bosch-Gil, J. Rossello-Urgell, P. Huguet-Redecilla, J.H. Stone, and M. Vilardell-Tarres. 2005. Metalloproteinase-2 and -9 in giant cell arteritis: involvement in vascular remodeling. Circulation. 112:264-269.  Rodriguez-Vita, J., E. Sanchez-Lopez, V. Esteban, M. Ruperez, J. Egido, and M. Ruiz-Ortega. 2005. Angiotensin II activates the Smad pathway in vascular smooth muscle cells by a transforming 102  growth factor-beta-independent mechanism. Circulation. 111:2509-2517.  Roman, M.J., R.B. Devereux, R. Kramer-Fox, and M.C. Spitzer. 1989. Comparison of cardiovascular and skeletal features of primary mitral valve prolapse and Marfan syndrome. The American journal of cardiology. 63:317-321.  Roman, M.J., S.E. Rosen, R. Kramer-Fox, and R.B. Devereux. 1993. Prognostic significance of the pattern of aortic root dilation in the Marfan syndrome. Journal of the American College of Cardiology. 22:1470-1476.  Rosenberg, G.A. 2009. Matrix metalloproteinases and their multiple roles in neurodegenerative diseases. The Lancet. Neurology. 8:205-216.  Roth, D.M., J.S. Swaney, N.D. Dalton, E.A. Gilpin, and J. Ross, Jr. 2002. Impact of anesthesia on cardiac function during echocardiography in mice. American journal of physiology. Heart and circulatory physiology. 282:H2134-2140.  Roy, R., J. Yang, and M.A. Moses. 2009. Matrix metalloproteinases as novel biomarkers and potential therapeutic targets in human cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 27:5287-5297.  Rubin, E.a.J. 1999. Pathology. 3rd. ed. Philadelphia, Pa: Lippincott-Raven.  Ruska, E. 1987. Nobel lecture. The development of the electron microscope and of electron microscopy. Bioscience reports. 7:607-629.  Rybczynski, M., D.H. Koschyk, M.A. Aydin, P.N. Robinson, T. Brinken, O. Franzen, J. Berger, T. Hofmann, T. Meinertz, and Y. von Kodolitsch. 2007. Tissue Doppler imaging identifies myocardial dysfunction in adults with Marfan syndrome. Clinical cardiology. 30:19-24.  Safar, M.E., B.I. Levy, and H. Struijker-Boudier. 2003. Current perspectives on arterial stiffness and pulse pressure in hypertension and cardiovascular diseases. Circulation. 107:2864-2869.  Saharinen, J., J. Taipale, and J. Keski-Oja. 1996. Association of the small latent transforming growth factor-beta with an eight cysteine repeat of its binding protein LTBP-1. The EMBO journal. 15:245-253.  Sakalihasan, N., P. Delvenne, B.V. Nusgens, R. Limet, and C.M. Lapiere. 1996. Activated forms of MMP2 and MMP9 in abdominal aortic aneurysms. Journal of vascular surgery. 24:127-133.  Sandor, G.G., M.H. Alghamdi, L.A. Raffin, M.T. Potts, L.D. Williams, J.E. Potts, M. Kiess, and C. van Breemen. 2015. A randomized, double blind pilot study to assess the effects of losartan vs. atenolol on the biophysical properties of the aorta in patients with Marfan and Loeys-Dietz syndromes. International journal of cardiology. 179:470-475.  Sasaki, K., Y. Yamano, S. Bardhan, N. Iwai, J.J. Murray, M. Hasegawa, Y. Matsuda, and T. Inagami. 1991. Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor. Nature. 351:230-233.  Sasamura, H., L. Hein, J.E. Krieger, R.E. Pratt, B.K. Kobilka, and V.J. Dzau. 1992. Cloning, characterization, and expression of two angiotensin receptor (AT-1) isoforms from the mouse genome. Biochemical and biophysical research communications. 185:253-259.  Savolainen, A., L. Nisula, P. Keto, P. Hekali, M. Viitasalo, I. Kaitila, and M. Kupari. 1994. Left ventricular function in children with the Marfan syndrome. European heart journal. 15:625-630.  Schieffer, B., C. Bunte, J. Witte, K. Hoeper, R.H. Boger, E. Schwedhelm, and H. Drexler. 2004. Comparative effects of AT1-antagonism and angiotensin-converting enzyme inhibition on markers of inflammation and platelet aggregation in patients with coronary artery disease. Journal of the American College of Cardiology. 44:362-368.  103  Schlatmann, T.J., and A.E. Becker. 1977. Pathogenesis of dissecting aneurysm of aorta. Comparative histopathologic study of significance of medial changes. The American journal of cardiology. 39:21-26.  Schlondorff, D., and J. Satriano. 1985. Interactions with calmodulin: potential mechanism for some inhibitory actions of tetracyclines and calcium channel blockers. Biochemical pharmacology. 34:3391-3393.  Schlunegger, M.P., and M.G. Grutter. 1992. An unusual feature revealed by the crystal structure at 2.2 A resolution of human transforming growth factor-beta 2. Nature. 358:430-434.  Seedor, J.A., H.D. Perry, T.F. McNamara, L.M. Golub, D.F. Buxton, and D.S. Guthrie. 1987. Systemic tetracycline treatment of alkali-induced corneal ulceration in rabbits. Archives of ophthalmology (Chicago, Ill. : 1960). 105:268-271.  Segarini, P.R. 1993. TGF-beta receptors: a complicated system of multiple binding proteins. Biochimica et biophysica acta. 1155:269-275.  Segura, A.M., R.E. Luna, K. Horiba, W.G. Stetler-Stevenson, H.A. McAllister, Jr., J.T. Willerson, and V.J. Ferrans. 1998. Immunohistochemistry of matrix metalloproteinases and their inhibitors in thoracic aortic aneurysms and aortic valves of patients with Marfan's syndrome. Circulation. 98:II331-337; discussion II337-338.  Shlopov, B.V., G.N. Smith, Jr., A.A. Cole, and K.A. Hasty. 1999. Differential patterns of response to doxycycline and transforming growth factor beta1 in the down-regulation of collagenases in osteoarthritic and normal human chondrocytes. Arthritis and rheumatism. 42:719-727.  Shores, J., K.R. Berger, E.A. Murphy, and R.E. Pyeritz. 1994. Progression of aortic dilatation and the benefit of long-term beta-adrenergic blockade in Marfan's syndrome. The New England journal of medicine. 330:1335-1341.  Siemonsma, M.A., I.H. de Hingh, B.M. de Man, R.M. Lomme, A.A. Verhofstad, and T. Hendriks. 2003. Doxycycline improves wound strength after intestinal anastomosis in the rat. Surgery. 133:268-276.  Smith, G.N., Jr., E.A. Mickler, K.A. Hasty, and K.D. Brandt. 1999. Specificity of inhibition of matrix metalloproteinase activity by doxycycline: relationship to structure of the enzyme. Arthritis and rheumatism. 42:1140-1146.  So, P.T., C.Y. Dong, B.R. Masters, and K.M. Berland. 2000. Two-photon excitation fluorescence microscopy. Annual review of biomedical engineering. 2:399-429.  Stechmiller, J., L. Cowan, and G. Schultz. 2010. The role of doxycycline as a matrix metalloproteinase inhibitor for the treatment of chronic wounds. Biological research for nursing. 11:336-344.  Steenport, M., K.M. Khan, B. Du, S.E. Barnhard, A.J. Dannenberg, and D.J. Falcone. 2009. Matrix metalloproteinase (MMP)-1 and MMP-3 induce macrophage MMP-9: evidence for the role of TNF-alpha and cyclooxygenase-2. Journal of immunology (Baltimore, Md. : 1950). 183:8119-8127.  Sternlicht, M.D., and Z. Werb. 2001. How matrix metalloproteinases regulate cell behavior. Annual review of cell and developmental biology. 17:463-516.  Streuli, C. 1999. Extracellular matrix remodelling and cellular differentiation. Current opinion in cell biology. 11:634-640.  Sutton-Tyrrell, K., S.S. Najjar, R.M. Boudreau, L. Venkitachalam, V. Kupelian, E.M. Simonsick, R. Havlik, E.G. Lakatta, H. Spurgeon, S. Kritchevsky, M. Pahor, D. Bauer, and A. Newman. 2005. Elevated aortic pulse wave velocity, a marker of arterial stiffness, predicts cardiovascular events in well-functioning older adults. Circulation. 111:3384-3390. 104   Suzuki, M., D. Kayra, W.M. Elliott, J.C. Hogg, and T. Abraham. 2012. Second harmonic generation microscopy differentiates collagen type I and type III in diseased lung tissues. In SPIE BiOS. International Society for Optics and Photonics. 82263F-82263F-82269.  Svoboda, K., and S.M. Block. 1994. Biological applications of optical forces. Annual review of biophysics and biomolecular structure. 23:247-285.  Tamargo, R.J., R.A. Bok, and H. Brem. 1991. Angiogenesis inhibition by minocycline. Cancer research. 51:672-675.  Tamarina, N.A., W.D. McMillan, V.P. Shively, and W.H. Pearce. 1997. Expression of matrix metalloproteinases and their inhibitors in aneurysms and normal aorta. Surgery. 122:264-271; discussion 271-262.  Tang, S., T.B. Krasieva, Z. Chen, G. Tempea, and B.J. Tromberg. 2006. Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy. Journal of biomedical optics. 11:020501.  Ten Dijke, P., K.K. Iwata, C. Goddard, C. Pieler, E. Canalis, T.L. McCarthy, and M. Centrella. 1990. Recombinant transforming growth factor type beta 3: biological activities and receptor-binding properties in isolated bone cells. Molecular and cellular biology. 10:4473-4479.  Tezvergil-Mutluay, A., K.A. Agee, T. Hoshika, M. Carrilho, L. Breschi, L. Tjaderhane, Y. Nishitani, R.M. Carvalho, S. Looney, F.R. Tay, and D.H. Pashley. 2010. The requirement of zinc and calcium ions for functional MMP activity in demineralized dentin matrices. Dental materials : official publication of the Academy of Dental Materials. 26:1059-1067.  Thompson, R.W., D.R. Holmes, R.A. Mertens, S. Liao, M.D. Botney, R.P. Mecham, H.G. Welgus, and W.C. Parks. 1995. Production and localization of 92-kilodalton gelatinase in abdominal aortic aneurysms. An elastolytic metalloproteinase expressed by aneurysm-infiltrating macrophages. The Journal of clinical investigation. 96:318-326.  Tozzi, P., A. Corno, and D. Hayoz. 2000. Definition of arterial compliance. Re: Hardt et al., "Aortic pressure-diameter relationship assessed by intravascular ultrasound: experimental validation in dogs.". American journal of physiology. Heart and circulatory physiology. 278:H1407.  Tsuji, T., and T. Hamada. 1981. Age-related changes in human dermal elastic fibres. The British journal of dermatology. 105:57-63.  Underwood, J. 2000. General and Systematic Pathology. 3rd ed. Edinburgh, UK: Churchill Livingstone.  Valdez-Jasso, D., D. Bia, Y. Zocalo, R.L. Armentano, M.A. Haider, and M.S. Olufsen. 2011. Linear and nonlinear viscoelastic modeling of aorta and carotid pressure-area dynamics under in vivo and ex vivo conditions. Annals of biomedical engineering. 39:1438-1456.  Vasan, R.S., M.G. Larson, and D. Levy. 1995. Determinants of echocardiographic aortic root size. The Framingham Heart Study. Circulation. 91:734-740.  Vincenti, M.P., and C.E. Brinckerhoff. 2002. Transcriptional regulation of collagenase (MMP-1, MMP-13) genes in arthritis: integration of complex signaling pathways for the recruitment of gene-specific transcription factors. Arthritis research. 4:157-164.  Virmani, R., A.P. Avolio, W.J. Mergner, M. Robinowitz, E.E. Herderick, J.F. Cornhill, S.Y. Guo, T.H. Liu, D.Y. Ou, and M. O'Rourke. 1991. Effect of aging on aortic morphology in populations with high and low prevalence of hypertension and atherosclerosis. Comparison between occidental and Chinese communities. The American journal of pathology. 139:1119-1129.  Vlachopoulos, C. 2012. Progress towards identifying biomarkers of vascular aging for total cardiovascular risk prediction. Journal of hypertension. 30 Suppl:S19-26. 105   Vlachopoulos, C., N. Alexopoulos, and C. Stefanadis. 2010. Aortic stiffness: prime time for integration into clinical practice? Hellenic journal of cardiology : HJC = Hellenike kardiologike epitheorese. 51:385-390.  Wang, L., Z.G. Zhang, R.L. Zhang, S.R. Gregg, A. Hozeska-Solgot, Y. LeTourneau, Y. Wang, and M. Chopp. 2006. Matrix metalloproteinase 2 (MMP2) and MMP9 secreted by erythropoietin-activated endothelial cells promote neural progenitor cell migration. The Journal of neuroscience : the official journal of the Society for Neuroscience. 26:5996-6003.  Westenberg, J.J., A.J. Scholte, Z. Vaskova, R.J. van der Geest, M. Groenink, G. Labadie, P.J. van den Boogaard, T. Radonic, Y. Hilhorst-Hofstee, B.J. Mulder, L.J. Kroft, J.H. Reiber, and A. de Roos. 2011. Age-related and regional changes of aortic stiffness in the Marfan syndrome: assessment with velocity-encoded MRI. Journal of magnetic resonance imaging : JMRI. 34:526-531.  Whitebread, S., M. Mele, B. Kamber, and M. de Gasparo. 1989. Preliminary biochemical characterization of two angiotensin II receptor subtypes. Biochemical and biophysical research communications. 163:284-291.  Whiteman, P., and P.A. Handford. 2003. Defective secretion of recombinant fragments of fibrillin-1: implications of protein misfolding for the pathogenesis of Marfan syndrome and related disorders. Human molecular genetics. 12:727-737.  Williams, A., S. Davies, A.G. Stuart, D.G. Wilson, and A.G. Fraser. 2008. Medical treatment of Marfan syndrome: a time for change. Heart (British Cardiac Society). 94:414-421.  Williams, J.A., B.L. Loeys, L.U. Nwakanma, H.C. Dietz, P.J. Spevak, N.D. Patel, K. Francois, J. DeBacker, V.L. Gott, L.A. Vricella, and D.E. Cameron. 2007a. Early surgical experience with Loeys-Dietz: a new syndrome of aggressive thoracic aortic aneurysm disease. The Annals of thoracic surgery. 83:S757-763; discussion S785-790.  Williams, R., A. Needles, E. Cherin, Y.Q. Zhou, R.M. Henkelman, S.L. Adamson, and F.S. Foster. 2007b. Noninvasive ultrasonic measurement of regional and local pulse-wave velocity in mice. Ultrasound in medicine & biology. 33:1368-1375.  Wrana, J.L. 1998. TGF-beta receptors and signalling mechanisms. Mineral and electrolyte metabolism. 24:120-130.  Wrana, J.L., L. Attisano, J. Carcamo, A. Zentella, J. Doody, M. Laiho, X.F. Wang, and J. Massague. 1992. TGF beta signals through a heteromeric protein kinase receptor complex. Cell. 71:1003-1014.  Yang, Y., E. Candelario-Jalil, J.F. Thompson, E. Cuadrado, E.Y. Estrada, A. Rosell, J. Montaner, and G.A. Rosenberg. 2010. Increased intranuclear matrix metalloproteinase activity in neurons interferes with oxidative DNA repair in focal cerebral ischemia. Journal of neurochemistry. 112:134-149.  Yang, Y., and G.A. Rosenberg. 2011. MMP-mediated disruption of claudin-5 in the blood-brain barrier of rat brain after cerebral ischemia. Methods in molecular biology (Clifton, N.J.). 762:333-345.  Yaroslavsky, A.N., P.C. Schulze, I.V. Yaroslavsky, R. Schober, F. Ulrich, and H.J. Schwarzmaier. 2002. Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range. Physics in medicine and biology. 47:2059-2073.  Yildiz, M., B. Sahin, and A. Sahin. 2006. Acute effects of oral melatonin administration on arterial distensibility, as determined by carotid-femoral pulse wave velocity, in healthy young men. Experimental and clinical cardiology. 11:311-313.  Yu, L.P., Jr., G.N. Smith, Jr., K.A. Hasty, and K.D. Brandt. 1991. Doxycycline inhibits type XI collagenolytic activity of extracts from human osteoarthritic cartilage and of gelatinase. The Journal of rheumatology. 18:1450-1452.  106  Yu, Q., and I. Stamenkovic. 2000. Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis. Genes & development. 14:163-176.  Zangwill, S.D., M.D. Brown, C.R. Bryke, J.R. Cava, and A.D. Segura. 2006. Marfan syndrome type II: there is more to Marfan syndrome than fibrillin 1. Congenital heart disease. 1:229-232.  Zhang, Y., D.L. DeWitt, T.B. McNeely, S.M. Wahl, and L.M. Wahl. 1997. Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases. The Journal of clinical investigation. 99:894-900.  Zhou, F., G.Y. Li, Z.Z. Gao, J. Liu, T. Liu, W.R. Li, W.S. Cui, G.Y. Bai, and Z.C. Xin. 2012. The TGF-beta1/Smad/CTGF pathway and corpus cavernosum fibrous-muscular alterations in rats with streptozotocin-induced diabetes. Journal of andrology. 33:651-659.  Zipfel, W.R., R.M. Williams, R. Christie, A.Y. Nikitin, B.T. Hyman, and W.W. Webb. 2003. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proceedings of the National Academy of Sciences of the United States of America. 100:7075-7080.                                   107  Appendix A: Publications and Abstracts  Cui, J. Z., Harris, K. C. (Co-First Author), Raffin, L. A., Hollander, Z., Potts, J. E., De Souza, A., Paine, H., McManus, B.M., van Breemen, C., Esfandiarei, M., and Sandor, G. G. (Co-Senior Author) "Evaluation of the association between aortic dimensions, biophysical properties and plasma biomarkers in children and adult patients with Marfan Syndrome." (2017): Manuscript in preparation.  Cui, J. Z., Lee, L. L. (Co-First Author), Sheng, X., Sandor, G. G., van Breemen, C., Tibbits, G. F., and Esfandiarei, M. (Co-Senior Author) "Evaluation of the protective effects of long-term doxycycline treatment on the progression of Marfan-associated aortic aneurysm by high-resolution ultrasound imaging and on the ultrastructure of Marfan mice aorta by electron microscopy." (2017): Manuscript in preparation.  Gibson, C.P., Nielsen, C., Alex, R., Cooper, K., Farney, M., Gaufin, D., Cui, J. Z., van Breemen, C., Broderick, T.L., Vallejo-Elias, J. and Esfandiarei, M. "Mild aerobic exercise blocks elastin fiber fragmentation and aortic dilatation in a mouse model of Marfan syndrome associated aortic aneurysm." Journal of Applied Physiology (2017): jap-00132.  Lee, L., Cui, J. Z., Cua, M., Esfandiarei, M., Sheng, X., Chui, W.A., Xu, M.H., Sarunic, M.V., Beg, M.F., van Breemen, C., Sandor, G.G., and Tibbits, G. F. "Aortic and Cardiac Structure and Function Using High-Resolution Echocardiography and Optical Coherence Tomography in a Mouse Model of Marfan Syndrome." PloS one 11.11 (2016): e0164778.  Cui, J. Z., Lee, L. L., Sheng, X., Sandor, G. G., Van Breemen, C., Tibbits, G. F., & Esfandiarei, M. "Longitudinal study of long-term effects of doxycycline on cardiac structure and functions using high-frequency, high-resolution ultrasound imaging in a mouse model of Marfan syndrome." European Journal of Heart Failure (2016). Vol. 18. 111 River St., Hoboken 07030-5774, NJ USA: WILEY-BLACKWELL.  Cui, J. Z., Lee, L., Sheng, X., Tibbits, G. F., van Breemen, C., & Esfandiarei, M. "Evaluation of the protective effects of long-term doxycycline treatment on progression of Marfan-associated aortic aneurysm by high-resolution ultrasound imaging." Circulation (2015): 132.Suppl 3 A14680-A14680.  Jett, K., Cui, J. Z., Chohan, H., Tehrani, A., Friedman, J., van Breemen, C., & Esfandiarei, M. "Impairment of Vascular Function in a Mouse Model of Neurofibromatosis Type 1." The FASEB Journal (2015): 29 (1 Supplement), 638-4.  Jett, K., Cui, J. Z., Chohan, H., Friedman, J., van Breemen, C., & Esfandiarei, M. "Dysfunctions of the Abdominal Aorta and Renal Arteries in a Mouse Model of Neurofibromatosis Type 1." The FASEB Journal (2015): 29 (1 Supplement), 629-17.  Cui, J. Z., Tehrani, A. Y., Jett, K. A., Bernatchez, P., van Breemen, C., & Esfandiarei, M. "Quantification of aortic and cutaneous elastin and collagen morphology in Marfan syndrome by multiphoton microscopy." Journal of structural biology 187.3 (2014): 242-253.  Craig, E. L., Zhao, B., Cui, J. Z., Novalen, M., Miksys, S., & Tyndale, R. F. "Nicotine pharmacokinetics in rats is altered as a function of age, impacting the interpretation of animal model data." Drug Metabolism and Disposition (2014): 42(9), 1447-1455.  Jett, K., Cui, J. Z., Chohan, H., Arman, D., Tehrani, A., Friedman, J., Van Breemen, C., & Esfandiarei, M. "Impairment of aortic structure and function in a mouse model of neurofibromatosis type 1." Proceedings of The Physiological Society. (2013): Proc 37th IUPS, PCD417.    108  Appendix B: Conference Presentations American Heart Association (AHA) Scientific Sessions 2017, November 11-15, 2017. Anaheim, CA, U.S. European Society of Cardiology (ESC) Heart Failure 2016 Congresses, May 21-24, 2016. Florence, Italy. American Heart Association (AHA) Scientific Sessions 2015, November 06-11, 2015. Orlando, FL, U.S. ($1,000 USD BCVS International Travel Grant was awarded). The International Microscopy (IMC) 2014 - 18th International Microscopy Congress, September 07-12, 2014. Prague, Czech Republic.  Canadian Marfan Association (CMA) 2013 Marfan Conference, September 27-28, 2013. Vancouver, BC. 

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.24.1-0357258/manifest

Comment

Related Items