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Vascular function in Neurofibromatosis 1 Jett, Kimberly Ann 2015

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VASCULAR FUNCTION IN NEUROFIBROMATOSIS I  by Kimberly Ann Jett  B.Sc., The University of Akron, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Medical Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  February 2015  © Kimberly Ann Jett, 2015 ii  Abstract  Neurofibromatosis 1 (NF1) is an autosomal dominant disorder with an estimated prevalence of 1/3000.  NF1 is characterized by multiple café-au-lait spots, iris hamartomas, and multiple nerve sheath tumors.  Patients may also present with heart disease, cerebrovascular disease, ischemia, or aneurysm.  Though well documented, vascular disease in NF1 patients remains poorly understood.    Previous in vitro studies suggest that endothelial and vascular smooth muscle function is altered in Nf1+/- mice; however, it is unknown how these alterations affect vascular function in vivo.  Haploinsufficiency for neurofibromin, the protein affected in patients with NF1, results in prolonged Ras hyperactivation.  We hypothesized that this may result in vascular endothelial dysfunction and impaired cardiac function.      To study this hypothesis we examined vascular function in Nf1+/- and control mice using wire myography.   Isometric force measurements in thoracic and abdominal aorta at 6-months of age were similar, with Nf1+/- mice demonstrating altered smooth muscle function, enhanced relaxation, and upregulation of the PI3K/Akt/eNOS pathway.  To determine if the alterations observed at 6 months of age remain stable or progress to a more dysfunctional state, we examined the abdominal aorta in older mice.  Interestingly, we observed increased contraction and reduced relaxation in 9-to-12 month old Nf1+/-mice compared to control littermates, indicative of endothelial dysfunction and progression to a more dysfunctional state.   iii   Vascular dysfunction is likely to impact cardiac performance, and Ras hyperactivation has also been linked to cardiac dysfunction.  We, therefore, used 2-dimensional echocardiography with color Doppler to measure cardiac function in Nf1+/- and control littermates.  We found that Nf1+/- mice have increased left ventricular wall thickness and reduced cardiac contractility.  We also observed alterations in cardiomyocyte organization in Nf1+/- animals.   The results presented in this thesis support the hypothesis that neurofibromin haploinsufficiency in Nf1+/- mice results in vascular endothelial and cardiac dysfunction.  Whether these findings extend to humans with NF1, who have the same genetic defect, is unknown, but if so, our observations may have important clinical implications.  The role of neurofibromin in other kinds of vascular disease needs to be studied, and the possibility that neurofibromin may provide a novel therapeutic target should be explored.    iv  Preface All of the work presented in this thesis was conducted at the University of British Columbia at the Child & Family Research Institute.  All projects and associated methods were approved by the University of British Columbia’s Research Ethics Board [certificate #A11-0117, certificate #A13-0050, and certificate #B11-0029]  A version of chapter 1 has been published.  Jett, K., & Friedman, J. M. (2010). Clinical and genetic aspects of neurofibromatosis 1. Genet Med, 12(1), 1-11.   Dr. Catherine Van Raamsdonk provided the murine colony on which the research in Chapters 3-5 was performed.    Chapter 3 and 4 are based on work that I conducted in Dr. Casey Van Breemen’s laboratory at the Child and Family Research Institute.  I was responsible for study design, developing protocols, conducting experiments, data analysis, and writing the manuscripts.  Dr. Mitra Esfandiarei supervised this work, helped with study design, developed protocols, and reviewed the manuscript.  Harleen Chohan and Darian Arman helped conduct some of the experiments and reviewed the manuscript.  Chapter 5 is based partly on work conducted in Dr. Glen Tibbits’ lab at the Child and Family Research Institute. I was responsible for study design, maintenance of the murine colony, conducting experiments, data analysis, and writing the manuscript. Ling Lee conducted 2-dimensional echocardiography with color Doppler and some of the v  data analysis.  Dr. Xiao-ye Sheng and Dr. Mitra Esfandiarei supervised this work and reviewed the manuscript.  Harleen Chohan helped with data analysis and reviewed the manuscript.  vi  Table of Contents  Abstract .......................................................................................................................... ii Preface .......................................................................................................................... iv Table of Contents ......................................................................................................... vi List of Tables ................................................................................................................. x List of Figures .............................................................................................................. xi List of Abbreviations .................................................................................................. xiv Acknowledgements .................................................................................................... xvi Dedication .................................................................................................................. xvii Chapter 1: Introduction ................................................................................................. 1 1.1 Clinical Description and Diagnosis of Neurofibromatosis I ................................ 1 1.2 Vascular Manifestations of NF1 ........................................................................ 5 1.2.1 NF1 Vasculopathy ...................................................................................... 6 1.2.1.1 Clinical Characteristics and Epidemiology .............................................. 6 1.2.1.2 Pathogenesis ......................................................................................... 8 1.2.1.3 Preliminary Studies of Vascular Disease in Patients With NF1 ............ 10 1.2.1.3.1 Flow-Mediated and Glyceryl-Trinitrate-Mediated Dilatation in NF1 Patients……………. ....................................................................... 10 1.2.1.3.2 Cardiovascular Risk Factors in NF1 Patients ................................. 11 1.2.2 Congenital Heart Defects ......................................................................... 13 1.2.2.1 Clinical Characteristics and Epidemiology ............................................ 13 1.2.2.2 Pathogenesis ....................................................................................... 14 vii  1.2.2.3 Preliminary Studies of Cardiac Structure and Function in NF1 Patients…………. ................................................................................... 14 1.3 Vascular Homeostasis .................................................................................... 16 1.4 Cardiac Function ............................................................................................. 21 1.5 Research Objectives ....................................................................................... 23 1.6 Hypotheses ..................................................................................................... 23 1.6.1 Vascular Function .................................................................................... 23 1.6.2 Cardiac Function and Structure ................................................................ 24 Chapter 2: Materials and Methods ............................................................................. 25 2.1 Experimental Animals and Aortic Segment Preparation ................................. 25 2.2 Buffer and Reagents ....................................................................................... 26 2.3 Measurement of Isometric Force .................................................................... 27 2.4 Measurement of Wall Stress and Elasticity ..................................................... 28 2.5 Assessment of Endothelial Function ............................................................... 29 2.6 Western Blotting .............................................................................................. 29 2.7 Assessment of Vessel Morphology - Trichrome and van Gieson Staining ...... 30 2.8 Echocardiography ........................................................................................... 31 2.9 Statistical Analysis .......................................................................................... 34 Chapter 3: Impairment of Vascular Function of the Thoracic Aorta in Nf1+/- Mice 35 3.1 Introduction ..................................................................................................... 35 3.2 Results ............................................................................................................ 35 3.2.1 Contractile Function of the Smooth Muscle in Nf1+/- Aorta ....................... 35 3.2.2 Endothelium-Dependent and Endothelium-Independent Vasodilation ..... 38 viii  3.2.3 The Role of Endogenous Nitric Oxide in Phenylephrine-Induced Vasoconstriction ....................................................................................... 42 3.2.4 Increased Expression and Activity of the PI3K/Akt/eNOS Signaling Pathway in Nf1+/- Aorta ............................................................................. 43 3.2.5 Structural Integrity of Aortic Wall .............................................................. 44 3.2.6 Aortic Response to Stress and Elasticity in Nf1+/-..................................... 46 3.3 Discussion ....................................................................................................... 48 Chapter 4: Endothelial Dysfunction in the Abdominal Aorta and Renal Arteries of Nf1+/- Mice ..................................................................................................................... 56 4.1 Introduction ..................................................................................................... 56 4.2 Results ............................................................................................................ 57 4.2.1 Contractile Function and Relaxation in 6-Month Old Mice ....................... 57 4.2.2 Contractile Function of the Smooth Muscle of 9 to 12-Month Old Nf1+/- Mice…………………………. ..................................................................... 59 4.2.3 Endothelium-Dependent and Endothelial-Independent Vasodilation ....... 61 4.2.4 The Role of Endogenous Nitric Oxide in Phenylephrine-Induced Vasoconstriction ....................................................................................... 65 4.3 Discussion ....................................................................................................... 66 Chapter 5: Echocardiographic Evidence of Cardiomyopathy in an Nf1+/- Mouse Model ............................................................................................................................ 70 5.1 Introduction ..................................................................................................... 70 5.2 Results ............................................................................................................ 72 5.3 Discussion ....................................................................................................... 79 ix  Chapter 6: General Discussion .................................................................................. 85 6.1 Summary......................................................................................................... 85 6.2 Vascular Function in Nf1+/- Mice...................................................................... 86 6.3 Cardiac Function ............................................................................................. 88 6.4 Conclusion ...................................................................................................... 89 Bibliography ................................................................................................................ 91       x  List of Tables  Table 5.1  Cardiac Structural Parameters in Control (Nf1+/+) and Nf1+/- Mice ................ 73 Table 5.2  Cardiac Functional Parameters in Control (Nf1+/+) and Nf1+/- Mice .............. 75  xi  List of Figures  Figure 1.1  Café-au-lait macules in a patient with NF1 ................................................... 2 Figure 1.2  Numerous subcutaneous neurofibromas, some of which are marked with arrows, in a man with NF1 ............................................................................................... 3 Figure 1.3  Numerous cutaneous neurofibromas of various sizes in an adult with NF1 . 3 Figure 1.4  Large diffuse plexiform neurofibroma of the right leg in a woman with NF1 . 4 Figure 1.5 Mechanisms involved in the regulation of vascular smooth muscle contraction.  The contractile response is initiated and maintained by extracellular Ca2+ influx through the VGCC, ROCC, or SOCC or through activation of receptor tyrosine kinases (RTK) and G-protein coupled receptors (GPCRs). ........................................... 18 Figure 1.6 A summary of the mechanisms regulating vascular smooth muscle contraction and relaxation. ............................................................................................ 20 Figure 2.1 Representative M-mode short-axis view of the mouse left ventricle. ........... 31 Figure 2.2 Representative B-mode short-axis view of the mouse left ventricle. ........... 32 Figure 2.3 Representative B-mode long-axis view of the mouse left ventricle. ............. 33 Figure 3.1 Phenylephrine-induced contraction in thoracic aorta from control and Nf1+/- mice at 6 months of age ................................................................................................ 37 Figure 3.2 Acetylcholine-induced relaxation in phenylephrine (20μM)-precontracted thoracic aorta from control and Nf1+/- mice at 6 months of age ..................................... 39 Figure 3.3 Sodium nitroprusside-induced relaxation in phenylephrine (20μM)-precontracted thoracic aorta from control and Nf1+/- mice at 6 months of age .............. 41 xii  Figure 3.4 Maximum force generated in response to 20 μM phenylephrine (PE) with and without pretreatment of 200 μM LNAME in the thoracic aorta from control and Nf1+/- mice at 6 months of age (values represent mean ± SEM). ............................................ 43 Figure 3.5 Protein Expression in thoracic aorta from control and Nf1+/- mice at 6 months of age ............................................................................................................................ 44 Figure 3.6  Representative histology sections in the aortic wall of control and Nf1+/- mice at 6 months of age ........................................................................................................ 45 Figure 3.7 Stress-strain curves from control and and Nf1+/- mice at 6 months of age (values represent means ± SEM) .................................................................................. 47 Figure 4.1 Agonist-induced contraction and relaxation in the abdominal aorta from control and Nf1+/- mice at 6 months of age .................................................................... 58 Figure 4.2 Phenylephrine-induced contraction in abdominal aorta from control and Nf1+/- mice at 9 to 12 months of age (values represent means ± SEM): ....................... 60 Figure 4.3 Acetylcholine-induced relaxation in phenylephrine (20μM)-precontracted abdominal aorta from control and Nf1+/- mice at 9 to 12 months of age (values represent mean ± SEM) ................................................................................................................ 62 Figure 4.4 Sodium nitroprusside-induced relaxation in phenylephrine (20μM)-precontracted abdominal aorta from control and Nf1+/- mice at 9 to 12 months of age (values represent means ± SEM) .................................................................................. 64 Figure 4.5 Maximum force generated in response to 20 μM phenylephrine (PE) with and without pretreatment of 200 μM LNAME in the abdominal aorta from control and Nf1+/- mice at 9 to 12 months of age (values represent means ± SEM). ....................... 65 Figure 5.1  Gross anatomy of the heart in control (Nf1+/+) and Nf1+/- mice .................... 76 xiii  Figure 5.2  Photomicrographs from 3 control and 3 Nf1+/- hearts (200x) showing alterations in myocyte orientation and increased myocyte size in the Nf1+/- mice. ........ 77 Figure 5.3 Photomicrograph (200x) from 6-month old Nf1+/- mouse with signs of myocardial infarction. .................................................................................................... 78 Figure 5.4  Morphometric analysis from cross-sections of the heart ............................ 79  xiv  List of Abbreviations  Neurofibromatosis 1 (NF1) Rat sarcoma (Ras)  Ras-GTPase activating protein (Ras-GAP) Ras-guanosine triphosphate (Ras-GTP)  Ras-guanosine diphosphate (Ras-GDP) Mitogen activated protein kinase (MAPK) Phosphatidylinositide 3-kinases (PI3K) Protein kinase B (PKB) (also known as Akt) Mammalian target of rapamycin (mTOR) Endothelial nitric oxide synthase (eNOS) Voltage gated cation channels (VGCCs) Receptor activated cation channels (ROCCs) Store operated cation channels (SOCCs)  Inositol-1, 4, 5-trisphosphate receptors (IP3R) Calcium (Ca2+) Myosin light chains (MLCs)  Myosin light chain kinase (MLCK) Myosin light chain phosphatase (MLCP) Potassium (K+) G-protein coupled receptors (GPCRs)  Phospholipase C isoform β (PLCβ)  xv  Ras homolog gene family, member A (RhoA) Protein kinase C (PKC) C-kinase activated protein phosphatase-1 inhibitor (CPI-17) Left ventricular internal dimension at end diastole (LVIDd) Left ventricular internal dimension at end systole (LVIDs) Left ventricular anterior wall thickness at end diastole (LVAWd) Left ventricular anterior wall thickness at end systole (LVAWs) Left ventricular posterior wall thickness at end diastole (LVPWd) Left ventricular posterior wall thickness at end systole (LVPWs) Left ventricular end-diastolic dimension (LVEDD) Left ventricular end-systolic dimension (LVESD)   Intraventricular septal thickness at end diastole (IVSd)  Intraventricular septal thickness at end systole (IVSs)  Pulse-wave (PW)  Isovolumic relaxation time (IVRT) Isovolumic contraction time (IVCT) Ejection time (ET)    xvi  Acknowledgements  It is with immense gratitude that I acknowledge the support of Dr. Jan Friedman, my supervisor.  Under his supervision I learned how to plan projects, write grants, and critically analyze scientific questions.  His enthusiasm for different research projects and their role in medicine has been contagious.    I would like to acknowledge my committee members and thank them for their continual feedback and questions, which helped me learn to think more critically.     I would also like to express my gratitude to Dr. Casey Van Breemen for allowing me to work in his laboratory and for teaching me the importance of the scientific method.    I am also extremely grateful and would like to acknowledge the help of Dr. Mitra Esfandiarei.  It was an honor to work with you, and I thank you for all of your teaching. Special thanks are owed to Harleen Chohan and Darian Arman who worked tirelessly on numerous projects and were invaluable during scientific discussions.         Lastly I thank:  Patricia Birch, for helping me navigate through numerous spreadsheets, being a sounding board during scientific discussions, and answering an endless number of questions; Cristina Dias, for being the best officemate I could ask for and a true friend; Isabel Filges for discussing numerous projects with me over coffee, drinks, and dinner; and Russell Chute for all you do and moral support during this process.   xvii  Dedication  Dedicated to my parents  Tim and Helen Jett who have always encouraged and supported me 1  Chapter 1: Introduction  1.1 Clinical Description and Diagnosis of Neurofibromatosis I Neurofibromatosis 1 (NF1) is a progressive autosomal dominant disorder that shows complete penetrance but a variable phenotype.  NF1 has an estimated prevalence of 1 in 3000 [1].  It is caused by heterozygous loss-of-function mutation of the NF1 gene and consequent haploinsufficiency for the protein product, neurofibromin [2, 3].    The most frequent clinical manifestations of NF1 are café-au-lait spots, iris hamartomas, and neurofibromas [1].  People with NF1 also frequently have learning disabilities [4], skeletal abnormalities [5, 6], cardiovascular disease [7], central nervous system tumors [8], and malignant peripheral nerve sheath tumors [9].    NF1 can be diagnosed in about half of all affected individuals within the first year of life, and in almost all affected individuals by eight years of age using established clinical criteria.  The criteria for diagnosis of NF1 were developed by an NIH Consensus Conference in 1987 [10] and are generally accepted for routine clinical use [11, 12].  According to the above-mentioned guideline, an individual who has two or more of the following features in the absence of another cause meets the diagnostic criteria for NF1:    Six or more  café-au-lait macules (Figure 1.1) over 5 mm in greatest diameter in prepubertal individuals or over 15 mm in greatest diameter in postpubertal individuals;  2   Two or more neurofibromas of any type (Figures 1.2 and 1.3) or one plexiform neurofibroma (Figure 1.4);   Freckling in the axillary or inguinal regions;   Optic glioma;   Two or more Lisch nodules (iris hamartomas);   A distinctive osseous lesion such as sphenoid dysplasia or tibial pseudarthrosis; or   A first-degree relative with NF1 as defined by the above criteria. Figure 1.1  Café-au-lait macules in a patient with NF1          3  Figure 1.2  Numerous subcutaneous neurofibromas, some of which are marked with arrows, in a man with NF1     Figure 1.3  Numerous cutaneous neurofibromas of various sizes in an adult with NF1       4  Figure 1.4  Large diffuse plexiform neurofibroma of the right leg in a woman with NF1     The majority of individuals with NF1 have germline mutations resulting in truncation of the gene product and loss of protein function [13, 14].  The NF1 gene is large, and reported mutations are highly varied.  Nonsense mutations, amino acid substitutions, deletions, insertions, intronic changes affecting splicing, alterations of the 3' un-translated region of the gene, and gross chromosomal rearrangements have all been reported [13, 15].  Deletion of the entire NF1 gene occurs in approximately 5% of patients and causes typical, although often severe, NF1 [16].    The product of the NF1 gene, neurofibromin, functions as a rat sarcoma (Ras) GTPase activating protein (Ras-GAP), which promotes the hydrolysis of active Ras-guanosine triphosphate (Ras-GTP) to inactive Ras-guanosine diphosphate (Ras-GDP) [17].  5  Tumor manifestations often involve loss of the normal NF1 allele in Schwann cells and a haploinsufficient cellular microenvironment [18], whereas non-tumor manifestations (e.g., learning disability) appear to result solely from haploinsufficiency [19].  Both haploinsufficiency and complete loss of neurofibromin lead to constitutive activation of Ras [20-22] and subsequent activation of downstream signaling pathways, including mitogen activated protein kinase (MAPK), phosphatidylinositide 3-kinases (PI3K), protein kinase B (PKB) (also known as Akt), and mammalian target of rapamycin (mTOR).  Activation of these pathways has a variety of cellular effects but generally stimulates cellular proliferation and survival [23].  1.2 Vascular Manifestations of NF1 Vascular manifestations are being described with increasing frequency in individuals with NF1, and are a significant cause of morbidity and mortality.  They are the 2nd leading cause of death in individuals with NF1 [24, 25].  However, the clinical importance of vascular complications in NF1 is under-recognized, and they remain one of the least studied features.  The most common clinical manifestation of vascular disease is hypertension, but vascular disease in individuals with NF1 is quite varied, and patients may have congenital heart defects, cardiomyopathy, or vasculopathy [7].  It is important to note that many patients with NF1-associated cardiovascular disease are asymptomatic, with the first indication of disease being a life-threatening event requiring treatment [26-28].  Involvement of multiple vessels or sites is common in patients with NF1, complicating screening and diagnosis [16].  As vascular disease is not commonly screened for in patients with NF1, the overall prevalence is not known.  Some reports 6  suggest that NF1 vasculopathy occurs in 2% of patients [29], while in others it is estimated to be as frequent as 19% [30].             1.2.1 NF1 Vasculopathy  1.2.1.1 Clinical Characteristics and Epidemiology NF1 vasculopathy refers to lesions in arteries and veins, which result in stenosis, occlusion, or rupture.  Affected individuals may have hypertension, cerebrovascular disease, ischemia, or hemorrhage [31-36].  Lesions are characterized by hyperplasia of the intimal and vascular smooth muscle layers.  A vascular lesion may rarely be associated with a neurofibroma that has invaded or compressed the vessel, but most lesions occur independently from neurofibromas.  When disease occurs in small vessels, stenosis usually develops as a result of intimal hyperplasia and nodular proliferation of smooth muscle cells within the vascular walls.  When disease occurs in large vessels, aneurysm is more common as intimal hyperplasia occurs in association with thinning of the media and elastin fragmentation [37].  However, stenosis has also been described in larger arteries and aneurysm has been described in smaller arteries [31, 33, 38].    Renal artery stenosis is the most common symptomatic form of NF1 vasculopathy, but stenosis and aneurysm of the aortic, carotid, intracerebral, mesenteric, subclavian and iliofemoral arteries have also been observed [16].  Coarctation (narrowing) of the aorta in patients with NF1 is often seen in association with renal artery stenosis.   In contrast 7  with the general population, coarctation of the aorta in NF1 patients is long and fusiform rather than abrupt and segmented [29, 39].  Some authors have suggested vascular lesions may develop more commonly in areas with turbulent blood flow, as they do in atherosclerosis [40, 41].  However, a recent study found that, like coarctation of the aorta in patients with NF1, many of the other vascular lesions in NF1 are also long and tapered and do not necessarily occur at the origin of the vessel [16].    The most serious manifestations of NF1 vasculopathy involve the arteries of the heart or brain and can have fatal consequences [7, 38, 42, 43].  Aneurysms and stenosis of the left main coronary artery and the left anterior descending coronary artery have been described in NF1 patients who required treatment for myocardial infarction [44-47].  Sudden death as a result of myocardial infarction has also been reported in children with NF1 [27].  However, the number of reports is very small and the prevalence of coronary artery disease in NF1 is not known.    NF1 patients may also be at higher risk to develop cardiomyopathy.  Several reports of left ventricular hypertrophy, hypertrophic cardiomyopathy [26, 48], and heart failure have been published.  Though the prevalence of cardiomyopathy in NF1 is unknown, early signs of disease and impaired cardiac function have been demonstrated in young NF1 patients.  Hypertension, which occurs in approximately 16% of young individuals with NF1 [49] and is more common in NF1 than in the general population, may contribute to these myocardial alterations or be a result of them [50].  We observed similar alterations in cardiac structure in a small cohort of unselected patients with NF1 8  [51] who had never previously been diagnosed with hypertension.  At this time, it is not clear if patients with early signs of myocardial hypertrophy will go on to develop significant cardiomyopathy.    Cerebrovascular abnormalities are present in approximately 2.5% of children with NF1 and are associated with stenosis or occlusion of the internal carotid, middle cerebral, or anterior cerebral artery.  Small telangiectatic vessels may form around the stenotic area and appear as a "puff of smoke" (moya-moya) on cerebral angiography [42].  Moya-moya develops about three times more often than expected in children with NF1 after cranial irradiation for primary brain tumours [52].  Ectatic vessels and intracranial aneurysms may also occur in individuals with NF1 and are more frequent than in the general population [53].    1.2.1.2 Pathogenesis Mechanisms underlying NF1 vasculopathy are not completely understood.  Neurofibromin is expressed in endothelial cells [54], vascular smooth muscle cells [55], mast cells, and macrophages [56].  Neurofibromin haploinsufficiency, which occurs in people with NF1, produces hyperactivation of the Ras pathway in bone marrow-derived cells (BMDCs), vascular smooth muscle cells, and endothelial cells, [57].    Research on neurofibromin haploinsufficient vascular smooth muscle cells and endothelial cells in vitro suggests that neurofibromin regulates cell proliferation and migration.  Li et al. [21] examined vascular smooth muscle cells from Nf1+/- mice and 9  found a two-fold increase in proliferation and migration in response to platelet-derived growth factor compared to cells from Nf1+/+ mice.  Similarly, Munchoff et al. [20] found increased proliferation and migration in response to vascular endothelial growth factor and fibroblast growth factor in endothelial cells from Nf1+/- mice in comparison to Nf1+/+ endothelial cells.  In both studies, the phenotype could be attributed to up-regulation of the Ras pathway.  Interestingly, baseline proliferation and migration are not significantly different between Nf1+/- and Nf1+/+ vascular smooth muscle or endothelial cells, suggesting that activation by upstream factors may be important and that neurofibromin haploinsufficiency alone is not sufficient to cause alterations in proliferation or haptotaxis.         These in vitro findings led some investigators to question whether the reduction or loss of neurofibromin causes vascular disease to develop under conditions of stress.  Occlusion of the carotid artery in Nf1+/- mice results in a proliferative injury response and neointimal hyperplasia in vivo, which is histologically similar to vascular lesions described in individuals with NF1 [57, 58].  Additionally, in response to fibroblast growth factor-induced ischemia, Nf1+/- mice have increased endothelial cell proliferation and migration in vivo, with local accumulation of macrophages and mast cells [59].    Recent evidence suggests that the main cell involved in the proliferative injury response observed in Nf1+/- mice is derived from bone marrow.  Neurofibromin haploinsufficiency in myeloid cells alone appears to be sufficient to augment the size of lesions when stenosis [60] or aneurysm [61] is induced by carotid artery ligation or angiotensin-II 10  infusion, respectively.  Additionally, increased concentrations of various cytokines, like interleukin 1β, interleukin-6, and fractalkine, which are associated with vascular disease in the general population [62], have been observed in Nf1+/- mice and in patients with NF1 [63].  1.2.1.3 Preliminary Studies of Vascular Disease in Patients With NF1 Inflammation and neointima proliferation are associated with vascular lesions and stenosis in individuals without NF1 [64-67] as a result of disrupted vessel dilation, contraction, or repair [67].  Similar mechanisms may operate in NF1, but vascular function has never been examined in patients with NF1.    1.2.1.3.1 Flow-Mediated and Glyceryl-Trinitrate-Mediated Dilatation in NF1 Patients Arterial flow-mediated vasodilation (FMD) and glyceryl-trinitrate-mediated dilation (NMD) are non-invasive tests that have been used to characterize vascular function in patients with atheroscolerosis [68] and other conditions that cause vasculopathy [69].  FMD tests the ability of the vascular endothelium to regulate vasomotor tone through the production of vasoactive substances, while NMD directly tests the ability of vascular smooth muscle to dilate [68, 70, 71].   In a preliminary clinical study, we used FMD and NMD to study four patients with NF1 and known NF1 vasculopathy and four patients with NF1 who had no history of cardiovascular disease [72].  All eight individuals were non-smokers, and none was 11  obese.  We found lower than expected FMD (less than 8% dilation) in all eight NF1 patients, including the four with no history of vascular disease. The older patients and those with known vascular disease appeared to have greater impairment of FMD than the younger patients and those without known vascular disease.   In contrast, NMD was in or within 10% of the normal range in all of the NF1 patients studied except one, a 61 year-old male with known atherosclerotic vascular disease as well as NF1 vasculopathy.    Low FMD is indicative of dysfunction of the vascular endothelium [70, 73].  The fact that we found evidence of endothelial dysfunction in all of the NF1 patients studied, including those who had no clinical symptoms of vascular disease, suggests that endothelial dysfunction may be a manifestation of neurofibromin haploinsufficiency even in the absence of NF1 vascular disease. On the other hand, the fact that FMD was lowest in patients with known NF1 vasculopathy suggests that impairment of FMD may also either result from or be predictive of symptomatic vasculopathy in people with NF1. In contrast, NMD, which is indicative of smooth muscle function, appears to be normal in patients with NF1 unless some other form of vasculopathy, such as atherosclerosis, is present.   1.2.1.3.2 Cardiovascular Risk Factors in NF1 Patients  Factors such as cigarette smoking, obesity, and hyperlipidemia are associated with increased risk of vascular disease in the general population, but the relationship of NF1 vasculopathy to such standard cardiovascular risk factors is completely unknown. In 12  another small-scale clinical study, we examined 20 young adults (18 to 37 years of age) with NF1 for abnormalities in endothelial and vascular function, pro-inflammatory monocytes, and biomarkers of vascular disease.  None of the patients examined had a known history of vascular disease.  Vascular disease status was assessed by means of clinical assessment and carotid ultrasound examination [74].    Consistent with earlier results, twelve of the 20 young NF1 patients examined demonstrated lower than expected FMD suggestive of endothelial dysfunction. Additionally, we found that more than 2/3 of these young NF1 patients had increased intima-media thickness of the carotid artery (defined as above the 75th percentile for their age and sex), which has been shown to be an early sign of vascular disease in other contexts [69, 70, 73, 75, 76].    Interestingly, all of the NF1 patients examined had at least one standard risk factor for cardiovascular disease:  high serum total cholesterol concentration was observed in 11/19 patients, high serum LDL cholesterol concentration in 8/20, high plasma homocysteine concentration in 3/20, low serum 25-hydroxyvitamin D concentration in 15/20, and low fasting serum insulin level in 9/20, high red blood cell count in 9/20, and high blood monocyte count in 7/20.  Blood glucose, calcium, triglyceride, HDL cholesterol, C reactive protein, and cystatin C measurements were within the normal range for all individuals.  From these results, it is clear that some patients with NF1 have altered vascular function and appear to be at increased risk for cardiovascular disease.  13  Why these alterations occur and how they actually affect future disease risk are unknown.   1.2.2 Congenital Heart Defects  1.2.2.1 Clinical Characteristics and Epidemiology Congenital heart defects are estimated to occur in 2% to 27% of individuals with NF1 [29, 50].  Lower estimates are based on the medical history of a large series of individuals with NF1 [29] and include only symptomatic patients, whereas higher estimates are based on screening young patients (4-19 years of age) with echocardiography [77].  The most common congenital heart defect reported in NF1 patients is pulmonic stenosis, but coarctation of the aorta and Tetralogy of Fallot also appear to be relatively frequent [29].  Additionally, mitral valve prolapse, valvar regurgitation, atrial septal defects, and atrial septal aneurysm have been observed on echocardiography [78].  Approximately half of the abnormalities reported on echocardiography were asymptomatic, so many anomalies are likely to be unrecognized clinically.    Large or whole gene deletions of the NF1 gene may account for some of the anomalies observed, as congenital heart defects appear to occur more frequently in patients with 1.4 Mb, 1.2 Mb, or 1 Mb deletions [51, 79].  However, large deletions are only found in a small percentage (5%) of NF1 patients [16] and are unlikely to account for the increase in frequency of congenital heart defects overall.   14   1.2.2.2 Pathogenesis The pathogenesis of congenital heart defects in patients with NF1 is not completely understood.  Most of the information available comes from mice with both Nf1 alleles deleted.  Complete loss of neurofibromin in mice results in severe malformation of the heart and is lethal by embryonic day 14.5 [22, 80].  Heart malformations in Nf1-/- mice include ventricular septal defects, outlet tract abnormalities, enlarged endocardial cushions, and thinned myocardium [81].    The phenotype appears to result primarily from alterations in the process of epithelial-mesenchymal transformation in endothelial cells [82].  During development, endothelial cells undergo epithelial-mesenchymal transformation and form the endocardial cushions.  In the heart, neurofibromin normally functions to down-regulate Ras and limit the expansion of cells within endocardial cushions.  Loss of neurofibromin results in hyperactivation of Ras and causes premature localization of Nfatc1 (a transcription factor required for cardiac development) to the nucleus [83].  The exact mechanism underlying premature localization of Nfatc1 in Nf1-/- embryos and the downstream effects that result in abnormal heart development are unknown.   1.2.2.3 Preliminary Studies of Cardiac Structure and Function in NF1 Patients As detailed in the previous sections, NF1 patients may present with a variety of congenital heart defects, including pulmonary stenosis, septal abnormalities, and hypertrophic cardiomyopathy [7].  The incidence of congenital heart defects may be 15  higher in patients with NF1 large or whole gene deletions.   Cardiac malformations were found in 11 of 61 evaluated patients with NF1 microdeletions in one series [79].  However, as the reported incidence of cardiac malformation in patients with NF1 (irrespective of their genotype) ranges from 2 to 27% [29, 50], and only 5% of this population would be expected to have microdeletions [16], it was not clear if the incidence of cardiac malformations actually is higher in NF1 patients with microdeletions.    To determine if cardiac abnormalities occur more frequently among NF1 patients with microdeletions than in those without microdeletions, we characterized cardiac features in 16 NF1 patients with microdeletions and 16 NF1 patients with other kinds of pathogenic mutations using 2-dimensional echocardiography.  Details of the method and results of this study have been reported [51].  Briefly, congenital heart defects were found in 2 of 16 patients with microdeletions and in none of the 16 NF1 patients without microdeletions.  The congenital heart defects were a ventricular septal defect in one patient and aortic stenosis another. Mild degrees of mitral insufficiency or aortic insufficiency were seen in two of the microdeletion patients, and evidence of hypertrophic cardiomyopathy were noted in three.    Interestingly, although congenital heart defects were more frequent in NF1 patients with microdeletions, alterations in echocardiographic parameters were found more often in patients without microdeletions.  In comparison to the reference population without NF1, thickness of the left posterior wall in diastole and of the interventricular septum were 16  significantly increased in patients without microdeletions (p<0.001 and p=0.001, respectively) but not in NF1 microdeletion patients.  These results provide evidence that patients with NF1 microdeletions do have an increased incidence of congenital heart defects but that NF1 patients with other kinds of pathogenic mutations often have alterations in myocardial structure.   1.3 Vascular Homeostasis  The vasculature continually responds to changes in blood pressure, inflammatory mediators, paracrine factors, hormones, and neurotransmitters in order to maintain vascular tone [84, 85].  Alterations in homeostasis lead to vascular remodeling, which may occur as part of normal adaptive or pathogenic processes [47, 64, 66, 86, 87].  Interactions between multiple cell types are required for this process, but the major contributors are vascular smooth muscle and endothelial cells [64, 88-90].  Vascular smooth muscle cells are responsible for the maintenance of vascular tone [91], while endothelial cells provide a permeable barrier and regulate smooth muscle contraction and relaxation [92, 93].    Vascular tone is primarily maintained through control of the intracellular calcium (Ca2+) concentration in vascular smooth muscle [94].  Calcium influx occurs through activation of voltage gated cation channels (VGCCs) [95, 96], receptor activated cation channels (ROCCs) [97, 98], and/or store operated cation channels (SOCCs) on the plasma membrane [81, 99-101], or through activation of ryanodine receptors and/or inositol- 1, 17  4, 5-trisphosphate receptors (IP3R) on the sarcoplasmic reticulum [102, 103].  As intracellular Ca2+ rises, it binds to calmodulin, which activates myosin light chain kinase (MLCK), causing phosphorylation of myosin light chains (MLCs) and enabling the interaction between myosin and actin [104-107].  Relaxation is initiated when myosin light chain phosphatase (MLCP) dephosphorylates MLCs, which prevents the interaction of myosin and actin [108-110].  The ratio of Ca2+-calmodulin activated MLCK to MLCP determines the level of phosphorylation of MLCs and the overall contractile state of vascular smooth muscle [111].  A variety of signaling pathways in both endothelial and smooth muscle cells affect the ratio of Ca2+-calmodulin activated MLCK to MLCP through both voltage-dependent and receptor mediated mechanisms [112-119] (Figure 1.5).  Voltage-dependent mechanisms require ion channels to open and close, changing the interior voltage and causing membrane depolarization [120-122].  Though multiple ions contribute to the membrane potential in vascular smooth muscle, the resting membrane potential is primarily controlled by the extracellular and intracellular potassium (K+) concentrations (i.e., the K+ equilibrium potential) [123, 124].  Increasing the extracellular K+ concentration decreases the activity of K+ channels, which prevents K+ efflux and causes membrane depolarization [120, 124-126].  Membrane depolarization activates VGCC, causing influx of Ca2+ and subsequent activation of MLCK [127].     18  Figure 1.5 Mechanisms involved in the regulation of vascular smooth muscle contraction.  The contractile response is initiated and maintained by extracellular Ca2+ influx through the VGCC, ROCC, or SOCC or through activation of receptor tyrosine kinases (RTK) and G-protein coupled receptors (GPCRs).       Similar to voltage-dependent mechanisms, receptor mediated mechanisms ultimately result in mobilization of intracellular Ca2+ following ligand activation [128].  Ligand binding to G-protein coupled receptors (GPCRs) activates the phospholipase C isoform β (PLCβ) and RhoA.  Activation of PLCβ generates inositol trisphosphate and diacylglycerol.  Inositol trisphosphate stimulates Ca2+ release from intracellular stores 19  and subsequently activates MLCK, while diacylglycerol activates protein kinase C signaling [98, 102, 129, 130].  Activation of Rho signaling leads to phosphorylation of the myosin binding subunit of MLCP and C-kinase activated protein phosphatase-1 inhibitor (CPI-17), reducing MLCP activity, increasing Ca2+ sensitivity, and resulting in increased contraction [97, 130, 131].    Endothelial cells regulate the above processes by producing vasocontracting and vasodilating factors in response to changes in membrane potential, neurotransmitters, and shear stress [132].  Nitric oxide, which is produced by endothelial nitric oxide synthase (eNOS), is one of the most potent vasodilators produced by endothelial cells.  eNOS is maintained in an inactive state in caveolae (invaginations of the plasma membrane) by caveolin-1 [133].  Activity of eNOS and subsequent production of nitric oxide are induced through the elevation of intracellular calcium and/or activation of the PI3K/Akt signaling cascade (Figure 1.6) [134-138].  Calcium directly activates calmodulin, enabling it to bind eNOS, which induces a conformational change required for enzyme activity and nitric oxide production.  PI3K does not directly bind calmodulin but recruits Akt to caveolae.  Akt then phosphorylates eNOS, which increases the affinity of calmodulin for eNOS and activates the enzyme even at low concentrations of calcium [137, 139]. Once produced, nitric oxide stimulates guanylyl cyclase in smooth muscle, resulting in activation of myosin light chain phosphatase and relaxation ([140, 141].    20  Figure 1.6 A summary of the mechanisms regulating vascular smooth muscle contraction and relaxation.     Endothelial cells help regulate vascular smooth muscle contraction by producing vasocontracting and vasodilating factors in response to changes in membrane potential, neurotransmitters secreted by axons, and shear stress.  In a manner similar to that shown in figure 1.5 for vascular smooth muscle, the activity of eNOS and endothelial 21  function depend largely on the regulation of intracellular Ca2+concentration in endothelial cells.    1.4 Cardiac Function Cardiac function is determined by the heart rate, myocardial contractility, initial myofiber length or the degree of myocardial distension (preload), and the force the ventricle must generate to eject blood from the heart (afterload) [142-144].  Alterations in any of these factors may affect cardiac function or output.  Cardiac output is defined as the volume of blood ejected from the heart on each beat.  It is measured on echocardiography by multiplying the stroke volume (the difference between the filled volume of the ventricle before contraction (the end-diastolic volume) and the remaining volume after ejection (the end-systolic volume)) by the heart rate [143, 145, 146].    Similar to vascular smooth muscle, myocardial contractility is largely controlled by intracellular Ca2+ through both electromechanical and pharmacomechanical mechanisms.  Alterations in myocardial contractility are independent of preload.  Increasing the contraction of the heart results in increased cardiac output, which may have pathological consequences [142, 147, 148].  Conversely, decreasing contraction may decrease cardiac output.  Contractility of the heart is frequently examined on echocardiography using the ejection fraction (calculated by dividing the stroke volume by the end-diastolic volume), as alterations in contraction also alter the force and pressure developed by the ventricle [145, 149, 150].    22   As the initial length of cardiomyocytes (the contractile cells in the heart) affects the number of possible interactions between actin and myosin, preload significantly affects cardiac function by affecting contractility.  Increasing the length of cardiomyocytes exposes more myosin binding sites and results in increased contraction compared to shorter cardiomyocytes [151, 152].  In vivo preload is defined as the passive ventricular wall stress at the end of diastole.  As it is not possible to measure preload directly in vivo, most people use the end-diastolic volume as an index of preload [153-157].    While myocardial contraction and preload are primarily a result of intrinsic cardiac function, the afterload is largely dependent on vascular tone.  In vivo afterload is defined as the total wall stress during systolic ejection.  Any factor that alters the ventricular pressure required during contraction will affect afterload [156-158].  Like preload, it is not possible to measure the afterload directly in vivo, and most people use the end-systolic volume as an index of afterload.  It is worth noting that in most cases the force the ventricle must generate to eject blood from the heart is proportional to the aortic pressure during ejection.  If aortic stenosis or peripheral resistance is present, the afterload increases [154, 159, 160].     23  1.5 Research Objectives The preliminary studies we performed in NF1 patients suggested that abnormalities of the vascular endothelium and heart may be frequent in people with NF1, even if they do not have clinical signs or symptoms of cardiovascular disease. The goal of my thesis research was to characterize vascular endothelial and cardiac function in Nf1+/- mice, a model of human neurofibromatosis 1, and to explore the pathogenic mechanisms responsible for any abnormalities observed. My objectives were to 1. Examine vascular function in Nf1+/- and control (Nf1+/+ littermates) mice using wire myography; 2. Determine if localized differences (e.g. differences in vessel size or shear stress) or age impact vascular function; and 3. Determine if cardiac function is altered in Nf1+/- and control (Nf1+/+ littermates) mice using 2-dimensional echocardiography with color Doppler.    1.6 Hypotheses  1.6.1 Vascular Function The exact role of neurofibromin in vascular function is not known.   As a negative regulator of the Ras pathway, haploinsufficiency of neurofibromin in vascular smooth muscle and endothelial cells causes hyperactivation of Ras following initial stimulation of the pathway by upstream activators [20, 21].  Ras activation primarily occurs through stimulation of receptor tyrosine kinases or G-protein signalling.  Hyperactivation of Ras, 24  as a result of neurofibromin haploinsufficiency, has been shown to affect the density or activity of ion channels in Schwann cells [161, 162], sensory neurons [163, 164], hippocampal neurons [165], and keratinocytes [166, 167].   The activity of ion channels in neurofibromin haploinsufficient vascular smooth muscle or endothelial cells has never been examined.  I hypothesize that neurofibromin haploinsufficiency affects vascular function by altering both vascular smooth muscle contraction and endothelial production of regulatory factors.    1.6.2 Cardiac Function and Structure Any vascular dysfunction in Nf1+/- animals is likely to impact cardiac function.  Additionally, Ras hyperactivation, as either a result of neurofibromin haploinsufficiency or constitutional expression of the Ras gene, is associated with various degrees of cardiac hypertrophy [168-171].  I hypothesize that neurofibromin haploinsufficiency affects cardiac function and structure in adult mice as a result of Ras hyperactivation.    25  Chapter 2: Materials and Methods  2.1 Experimental Animals and Aortic Segment Preparation All surgical procedures and animal care were conducted according to the Guidelines for Animal Experiments at the University of British Columbia and were approved by the UBC Animal Care Committee.  The NF1 mouse model was kindly provided by Dr. Catherine Van Raamsdonk (University of British Columbia, Vancouver, Canada) and has been described elsewhere [172].  Nf1+/Dsk9 (subsequently referred to as “Nf1+/-”) mice were maintained on a mixed (C3HeB/FeJ x C57BL/6) strain.    For the experiments described in chapter 3, Nf1+/- and control (Nf1+/+ littermate) mice were sacrificed using isoflurane followed by cervical dislocation at 6 to 7 months of age.  The thoracic aorta was isolated, cleaned of fat and connective tissue, and dissected into 2 mm segments to be used for wire myography.  For Western blotting and histochemistry, isolated aorta was flash-frozen in liquid nitrogen or fixed in 10% formalin, respectively.    For the experiments described in chapter 4, Nf1+/- and control (Nf1+/+ littermate) mice were sacrificed using isoflurane followed by cervical dislocation at 6 to 7 months of age or 9 to 12 months of age.  The abdominal aorta was isolated below the renal arteries and above the iliac arteries, cleaned of fat tissue, and dissected into 2 mm segments to be used for wire myography.  Likewise, the renal arteries were isolated, cleaned of fat tissue, and mounted on the wire myograph.   26   For the experiments described in chapter 5, Nf1+/- and control (Nf1+/+ littermate) mice were sacrificed using isoflurane followed by cervical dislocation at 6 to 7 months of age.  The heart was isolated and placed in ice-cold HEPES-PSS buffer (pH 7.4).  Within approximately 5 min, the heart was cleaned of fat tissue, weighed, and fixed in 10% formalin.  Following fixation, samples were dehydrated and embedded in paraffin.  Five-micrometer cross-sections were stained using standard hematoxylin and eosin (H&E) or Masson trichrome staining protocols (Sigma Aldrich, ON, Canada).  Sections were viewed and imaged using an Olympus BX61 upright light microscope and a Q Imaging Retiga Exi camera with InVivo 3.2.0 software (Media Cybernetics).  Computerized quantitative morphometric analysis was performed using Image-Pro® Plus software (Media Cybernetics).     2.2 Buffer and Reagents For all myography measurements, aortic or renal artery segments were perfused with HEPES-PSS buffer (pH 7.4) containing  10 mM HEPES, 6 mM glucose, 1.8 mM CaCl, 130 mM NaCl, 4 mM KCl, 4 mM NaHCO3 1.2 mM MgSO4,  1.2 mM KH2PO4, and 0.03 mM EDTA.  Nominal calcium-free buffer was identical to HEPES-PSS with the exception that CaCl2 was replaced with 0.1mM EGTA.  High K+ buffer was identical to HEPES-PSS buffer with the exception that it contained 74 mM NaCl and 60 mM KCl.  All pharmacological agonists and inhibitors used in this thesis were purchased from Sigma-Aldrich (Oakville, Ontario, Canada).  Primary antibodies for Akt, Akt 27  phosphorylated at threonine residue 308 (p-AktThr308), p44/42 MAPK (Erk1/2), p44/42 MAPK phosphorylated at threonine 202 and tyrosine 204 (p-Erk1/2), endothelial nitric oxide synthase (eNOS), and eNOS phosphorylated at serine reside 1179 (p-eNOSser1179) were purchased from Cell Signaling (Whitby, Ontario, Canada).  Alpha-smooth muscle actin and anti-rabbit conjugated secondary antibodies were purchased from Cell Signaling (Whitby, Ontario, Canada).    2.3 Measurement of Isometric Force Isometric force refers to the force generated by a muscle without corresponding changes in muscle length.  Thoracic aorta (2mm)(Chapter 3), abdominal aorta (2mm) (Chapter4), or renal artery segments (2mm) (Chapter 4) isolated from control and Nf1+/- mice were mounted isometrically in a small vessel myograph (A/S Danish Myotechnology, Aarthus N, Denmark), and generated force was measured as described previously [173].  For all experiments, the myograph chambers were kept at 37°C and bubbled continuously with 95% O2 + 5% CO2 in HEPES-PSS solution.  Optimal tension was determined by subjecting aortic segments to increasing resting tensions and subsequent stimulation with 60 mM KCl buffer.  To measure the isometric force, aortic segments were stretched to the optimal tension (6.0 mN for thoracic aorta in both control and Nf1+/- mice (Chapter 3) and 3.0 mN for the abdominal aorta and renal artery in both control and Nf1+/- mice (Chapter 4)) for 30 min before being challenged with 60 mM KCl.  Following washing with HEPES-PSS buffer, aortic segments were then contracted with cumulative applications (0.01-500 µM) of phenylephrine, and concentration-response curves were constructed.  The negative logarithm of the 28  concentration giving half-maximum response (pEC50) was determined by linear interpolation on the semi-logarithmic concentration-response curve.  2.4 Measurement of Wall Stress and Elasticity Stiffness of thoracic aortic segments (Chapter 3) was deduced from stress-strain curves as described by Mulvany and Warsaw [174].  Aortic rings (~2mm) were mounted in a small vessel myograph and stretched by increasing the distance between two stainless steel wires, which is equivalent to increasing the length of vascular smooth muscle cells.  At first, the distance between the wires was adjusted to L0, the length at which the vessel was not stretched.  The distance between wires was increased in 200-µm intervals, and the new length was denoted as “L”.  The developed force in mN was divided by the length of the blood vessel segment and thickness of the vessel wall to calculate wall stress (WS) in mN/mm2.  Thickness of the vessel was measured from hematoxylin and eosin (H&E) stained sections of thoracic aorta (described below).  Thickness was not significantly different between control and Nf1+/- mice (data not shown).  Wall stress was divided by delta L (∆L) over L0 in order to control for alterations in thickness that occur with the changing length of the vessel as it is stretched.  In order to control for alterations in thickness that occur with the changing length of the vessel as it is stretched, adjusted wall stress (aWS) was calculated as  where both L and Lo are calculated by L or Lo, respectively, minus the length when the wires are touching.  The procedure was repeated with step-wise increases in 29  circumferential length until the vessel was unable to maintain its tone.  This procedure was then repeated in a nominal calcium-free buffer solution containing 0.1 mM EGTA to eliminate smooth muscle cell contractility.  ∆L/L0 and wall stress were fitted to an exponential curve.    2.5 Assessment of Endothelial Function To study endothelium-dependent and independent relaxation (Chapters 3 and 4), vessel segments were pre-contracted with 20 µM phenylephrine and then subjected to cumulative applications (0.001-10 µM) of acetylcholine or sodium nitroprusside, respectively.  Concentration-response curves of both drugs were constructed.  In both cases, the pEC50 was assessed by linear interpolation on the semi-logarithmic concentration-response curve.  In some experiments, segments were pre-treated with 200 µM of Nω-Nitro-L-arginine methyl ester (L-NAME), a reversible inhibitor of nitric oxide synthase (NOS), prior to the application of phenylephrine (20 µM) or acetylcholine (500 nM).   2.6 Western Blotting Flash-frozen isolated thoracic aortas from control or Nf1+/- mice were homogenized in a pre-chilled stainless-steel mortar and pestle (Chapter 3).  The resulting tissue powder was mixed in 50 µl of ice-cold lysis buffer containing 50 mM pyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 5 mM ethylglycoltetraacetic acid (EGTA), 100 μM Na3VO4, 10 mM HEPES (pH 7.4), 0.1% Triton X-100, 10 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride.  Extracted 30  protein (40 µg) was fractionated by gel electrophoresis in 9% sodium dodecyl sulfate-polyacrylamide gels, transferred to nitrocellulose membranes, and then blocked for 1 hour with phosphate buffered saline containing 5% skim milk and 0.2% Tween-20.  Following overnight incubation at 4° C with specific primary antibody, the membrane was incubated with secondary antibody for 1 hour at room temperature.  Immunoblots were detected with an enhanced chemiluminescence detection system following the protocol provided by the manufacturer (Pierce Biotechnology, Rockford, IL, USA).  Protein band intensity was measured using the National Institutes of Health ImageJ software (version 1.44p).  Density values for proteins of interest were normalized to the level for control groups, which were arbitrarily set to 1.0-fold.     2.7 Assessment of Vessel Morphology - Trichrome and van Gieson Staining Aortic segments (Chapter 3) were formalin fixed and embedded in paraffin. Five-micrometer cross-sections were prepared.  Slides were stained with hematoxylin and eosin (H&E), Masson’s trichrome, or Verhoeff-Van Gieson stain.  Collagen staining was performed using Masson’s trichrome staining kit (Sigma Aldrich, ON, Canada), while elastin staining was performed using a Verhoeff-Van Gieson staining kit (Sigma-Aldrich, ON, Canada) according to the manufacturer’s standard procedure.  Sections were viewed and imaged using an Olympus BX61 upright light microscope and Q imaging Retiga Exi camera with InVivo 3.2.0 software (Media Cybernetics).  Computerized morphometric analysis was performed to determine the percent area of collagen and elastin staining using Image-Pro® Plus software (Media Cybernetics).    31  2.8 Echocardiography Transthoracic echocardiography (Chapter 5) was performed using a Vevo 2100 system (VisualSonics®, Toronto, ON, Canada) with a linear 40 MHz probe (MS550).  Briefly, 6-month-old male Nf1+/- mice and age-matched male Nf1+/+ littermates (controls) were anesthetized by inhalation of 1.5-2% isoflurane in oxygen to maintain a minimum heart rate of 400 beats per min.  Prior to echocardiography, chest hair was removed with Nair© (Church & Dwight Co.), and 5 ml of ultrasound gel (Aquasonic Clear®,Fairfield, NJ, USA) was applied to the chest.  Two-dimensional M-mode tracings (Figure 2.1) were recorded at the level of the papillary muscles.  Measurements were taken from 5 consecutive cardiac cycles and averaged.    Figure 2.1 Representative M-mode short-axis view of the mouse left ventricle.   The M-mode images show the left ventricle anterior wall thickness (LVAW), left ventricle posterior wall thickness (LVPW) throughout diastole and systole.  The y-axis indicates depth (millimeters) and the x-axis indicates time (milliseconds).      The follothe shordimensioanterior (LVPWd(LVEDD  Figure 2The antventricle The intewas mewave (Pdiastolicwing left vt-axis viewn at end wall thickn), posterio), and end-.2 Represeerior wall ( (LV), rirventriculaasured froW) Dopple filling waventricular s (Figure 2systole (Less at endr wall thicsystolic dimntative B-AW), posteght ventrir septal thm the longr flow of thes (E watructural a.2):  internVIDs), ante systole (LVkness at eension (LVmode shortrior wall (Pcle (RV),  ickness at -axis viewe mitral vaves) and pnd functional dimensirior wall tAWs), pond systoleESD).   -axis view W), intervand papend diasto (Figure 2.lve was useak velocal parameon at end hickness asterior wal (LVPWs)of the mouentricular sillary musle (IVSd) a3).  Two-ded to measity of late ters were diastole (Lt end diasl thickness , end-diasse left venteptal thickcles (PMnd at endimensionaure peak vdiastolic fmeasured VIDd), intetole (LVAat end diatolic dimenricle.   ness (IVS)) are sh systole (IVl guided pelocity of illing wave32 from rnal Wd), stole sion , left own.  Ss) ulse-early s (A 33  waves), isovolumic relaxation time (IVRT), isovolumic contraction time (IVCT), and ejection time (ET).  The E/A ratio was calculated from PW Doppler recordings.    Figure 2.3 Representative B-mode long-axis view of the mouse left ventricle.   The posterior wall (PW), intraventricular septal thickness (IVS), left ventricle (LV), right atrium (RV), left atrium (LV), and aorta (Ao) are shown.     The following values were calculated by the corresponding formulae:    End systolic volume (ESV) (µl) = 7 / [(2.4 + average LVIDs) x (average LVIDs)3] End diastolic volume (EDV) (µl) = 7 / [(2.4 + LVIDd) x (average LVIDd)3] Stroke volume (SV) (µl) = EDV – ESV Ejection fraction (EF) (%) = 100 * SV / EDV Fractional shortening (FS) (%) = 100 * [(average LVIDd - average LVIDs) / average LVIDd]  Cardiac output (CO) (ml/min) = (SV x heart rate) / 1000  34  LV mass (mg) = 0.842 * [(average diastolic diameter at outer wall)3 - (average diastolic diameter at inner wall)3] Myocardial performance index (MPI) = (IVCT+IVRT)/ET Velocity of circumferential fiber shortening (Vcf) = FS/ET.    2.9 Statistical Analysis In Chapters 3 and 4, data (except fold changes in figure 3.5) were reported as means ± SEM from at least three independent experiments.  Statistical analysis and construction of concentration-response curves were performed using GraphPad 5 Prism software (San Diego, Calif., USA).  Differences between control and Nf1+/- groups were analyzed by 2-tailed Student’s t-tests. Differences between stress-strain curves were analyzed by 2-way ANOVA.  Statistical significance was defined as p < 0.05.  In Chapter 5, echocardiographic data was normally distributed and was reported as means ± standard error of the mean (SEM).  Morphometric data were not normally distributed and so were reported as medians and interquartile ranges (IQR).  Statistical analysis was performed using GraphPad 5 Prism (San Diego, Calif., USA) and SPSS version 19.0 software (SPSS Inc., Chicago, Illinois, USA). Differences in echocardiographic parameters between control and Nf1+/- groups were analyzed by 2-tailed Student’s t tests, while differences in morphological data between control and Nf1 groups were analyzed by Mann Whitney U tests.  Statistical significance was defined as p ≤ 0.05.  35  Chapter 3: Impairment of Vascular Function of the Thoracic Aorta in Nf1+/- Mice   3.1 Introduction  In this chapter, I present a comprehensive examination of thoracic aortic function and structure in a mouse model of NF1.  We investigated the function of the smooth muscle and endothelial cell layers, and the integrity of the aortic wall.  We found the contractile function of the vascular smooth muscle in 6 month-old Nf1+/- mice to be markedly altered due to variations in endothelial function and enhanced calcium sensitivity in smooth muscle.  These findings appear to result from increased expression of phosphatidylinositide 3-kinases (PI3K) and the activation of Akt.    3.2 Results 3.2.1 Contractile Function of the Smooth Muscle in Nf1+/- Aorta Vasoconstriction is stimulated by calcium entry from the extracellular space and calcium release from the sarcoplasmic reticulum [118].  To study entry of calcium from the extracellular space, we examined vasoconstriction in response to KCl-induced depolarization, which increases intracellular calcium by altering membrane potential and activating voltage-operated calcium channels [89, 118].  The developed contractile force was significantly increased in Nf1+/- mice compared with control littermates (3.02 ± 0.15% in Nf1+/- and 2.44± 0.15% in controls; p= 0.01).   36  To study the more complex process of receptor-activated vasoconstriction involving the release of calcium from the sarcoplasmic reticulum and stimulation of calcium sensitivity [97, 110, 114, 175, 176], we examined force developed in response to phenylephrine, which increases intracellular calcium by stimulating the phospholipase C signaling cascade and activating inositol trisphosphate receptors (IP3Rs) on the sarcoplasmic reticulum [86, 177].  Phenylephrine-induced contraction was significantly reduced in Nf1+/- mice in comparison to control littermates (Figure 3.1).  The maximal force (Emax) generated in response to phenylephrine was significantly lower in Nf1+/- mice than in controls (Figures 3.1A and 3.1B). Additionally, in Nf1+/- thoracic aorta, the pEC50 values (50% effective concentration) for phenylephrine (5.78 ± 0.09) were significantly lower than in controls (6.46 ± 0.06), indicating a marked decrease in sensitivity to phenylephrine in Nf1+/- aorta (Figure 3.1C).             Figure 3mice at A) Conc10 nM, 5means ±means ±A) B) -90255075100125% PE-Induced Force.1 Phenyl6 months oentration–r0nM, 100  SEM) ; B) SEM); C) -8 -7Loephrine-indf age esponse cnM, 500 nM Maximal fpEC50 valu-6 -5 -g [PE]uced contrurve of phe, 1 µM, 5 orce (Emax)es for PE ( 4 -3Nf1+/Nf1+/-action in thnylephrineµM , 10 µM generatedvalues rep C) +oracic aor (PE) at th  50 µM ,  in responsresent meata from coe following100 µM (ve to PE (vns ± SEM)ntrol and N doses:   5alues reprealues repre. 37 f1+/-  nM, sent sent 38  3.2.2 Endothelium-Dependent and Endothelium-Independent Vasodilation To investigate the endothelial function in Nf1+/- mouse aorta, we measured endothelium-dependent vasodilation by generating concentration-response curves to acetylcholine.  Acetylcholine-induced relaxation was substantially increased in Nf1+/- mice in comparison to controls (Figure 3.2A).  The Emax of acetylcholine was significantly greater in Nf1+/- than control aorta (82.2 ± 14.6% in Nf1+/- and 53.2 ± 12.6% in controls) (Figure 3.2B).  The pEC50 of acetylcholine was significantly greater in Nf1+/- than control aorta (7.80 ± 0.12 in Nf1+/- and 7.12 ± 0.16 in controls) (Figure 3.2C), indicating a higher sensitivity to acetylcholine in the Nf1+/- aorta.    To test the involvement of nitric oxide in endothelium-dependent vasodilation, we assessed the response to acetylcholine in the presence of 200 μM L-NAME, an analog of arginine that inhibits nitric oxide production.  Pre-incubation with L-NAME abrogated the relaxation observed in both Nf1+/- and control vessels (Figure 3.2D), which demonstrates that the relaxation stimulated by acetylcholine is primarily due to nitric oxide production.         Figure thoracic A) Conc5mM, 10MaximapEC50 vresponsrepresenA)  B)  3.2 Acetylaorta fromentration–r nM, 50nMl force (Emaalues for Ae to 500nMt mean ± Scholine-ind control andesponse c, 100 nM,x) generateCh (values ACh witEM).  uced relax Nf1+/- micurve of ace 500 nM, 1d in respo represent h and with ation in pe at 6 montylcholine  µM, 5 µMnse to AChmean ± SEout pretreC) henylephrths of age:(ACh) at th (values re (values reM); D) Maatment of  ine (20μM  e followingpresent mepresent meximum forc200 μM L)-precontra doses:   1an ± SEMan ± SEMe generateNAME (va39 cted  nM, ); B) ); C) d in lues 40   D)     To study endothelium-independent vasodilation, we treated the aortic segments with sodium nitroprusside, a nitric oxide donor that bypasses endogenous nitric oxide production by the endothelium and acts directly on smooth muscle cells [178](Figure 3.3).  We did not observe a significant difference in the Emax of sodium nitroprusside between Nf1+/- and control mice (Figure 3.3A, 3.3B), but the pEC50, was significantly higher in Nf1+/- aorta (Figure 3.3C), indicating slightly increased sensitivity to nitric oxide in the vascular smooth muscle of Nf1+/- mice compared to controls.          Figure precontrA) Conc1 nM, 5mMaximapEC50 vaA) B) % SNP-induced Relaxation3.3 Sodiacted thoraentration–rM, 10 nMl force (Emalues for S-100255075100125um nitropcic aorta fresponse c, 50nM, 10x) generateNP (values-9 -8[russide-indom controurve of sod0 nM, 500d in respo represent      -7 -6SNP]uced relal and Nf1+/-ium nitropr nM, 1 µMnse to SNPmean ± SEC) -5 -4xation in  mice at 6 musside (SN(values rep (values reM). Nf1+/+Nf1+/-phenyleponths of aP) at the fresent mepresent m hrine (20ge:   ollowing doan ± SEM)ean ± SEM41 μM)-ses:   ;  B) ); C)  42   3.2.3 The Role of Endogenous Nitric Oxide in Phenylephrine-Induced Vasoconstriction As we showed that acetylcholine produced more relaxation in the thoracic aortas of Nf1+/- mice (Figure 3.2A & 3.2B) and this difference appears to be primarily related to nitric oxide production (Figure 3.2D), we further investigated the role of endogenous nitric oxide in regulating vasoconstriction by measuring the maximum percent contraction in response to 20 µM of phenylephrine, a selective α1-adrenergic receptor agonist, in the presence or absence of L-NAME (200 µM).  When nitric oxide synthesis is inhibited by L-NAME, the maximum amount of phenylephrine-induced contraction in Nf1+/- mice is significantly higher than in controls (Figure 3.4), suggesting that the endogenous level of nitric oxide production may be increased in Nf1+/- mice.  43  Figure 3.4 Maximum force generated in response to 20 μM phenylephrine (PE) with and without pretreatment of 200 μM LNAME in the thoracic aorta from control and Nf1+/- mice at 6 months of age (values represent mean ± SEM).   3.2.4 Increased Expression and Activity of the PI3K/Akt/eNOS Signaling Pathway in Nf1+/- Aorta Nitric oxide production is dependent on eNOS activity that is tightly regulated by the upstream phosphatidylinositide 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway.  Therefore, we measured the expression and activities of the components of the PI3K/Akt cascade in the freshly isolated aorta in both control and Nf1+/- mice.  Consistent with increased expression of eNOS and p-eNOSser1179, expression of PI3K and p-Aktser473 were also increased in Nf1+/- mice aorta (Figure 3.5), indicating that the observed increase in nitric oxide production in Nf1+/- aorta is via the PI3K/Akt pathway activation.  44  Figure 3.5 Protein Expression in thoracic aorta from control and Nf1+/- mice at 6 months of age A) Representative western blots; B) Bar graph demonstrating the fold change in protein expression between control and Nf1+/- mice.  All fold changes were scaled to the density of the α-actin loading controls.  A)                                                        B)                                                          3.2.5 Structural Integrity of Aortic Wall  Elastin and collagen provide elasticity and strength to the blood vessel wall.  Alterations in either of these functions may affect the extent of contraction and relaxation [179].  We studied the integrity of the aortic wall by investigating the structure and organization of elastin and collagen fibers.  Compared with controls, Nf1+/- aortic elastic fibers exhibited marked disorganization with some fragmentation (Figure 3.6A).  Furthermore, the total amount of elastin in Nf1+/- arteries was significantly greater than in controls (Figure 3.6B), while the amount of collagen in Nf1+/- arteries was similar to controls (Figure 3.6C & 3.6D).   pAkt Akt pErk Erk Pi3KeNOSpeNOS01234 Nf1+/+Nf1+/- Fold Change Figure 3at 6 monA) Elast(values demonsA)    B)    .6  Represths of age in (Van Gierepresent trating the                                     entative hi   son) stainimean ± Spercent are                                  stology secng; B) Bar EM); C) a of collag                                  tions in thegraph demcollagen (en (values                                    aortic walonstrating Trichrome)represent           B)           D)   l of control the percen staining; mean ± SE               and Nf1+/-t area of elD) Bar gM).   45 mice astin raph   46  3.2.6 Aortic Response to Stress and Elasticity in Nf1+/- Stress increases exponentially as blood vessels are stretched (i.e., as vessel diameter is increased) and depends on both the cellular (smooth muscle and endothelial cells) and extracellular (extracellular matrix and connective tissue) make-up of the vessel wall [174].  The effect of cellular components on wall stress was examined by measuring force development in the aortic segments in response to stretch in buffer containing physiological calcium.  To exclude the contribution of the smooth muscle during force development, the measurements were repeated in nominal calcium-free buffer.  Without the presence of calcium only the extracellular components of the vessel contribute to arterial wall stress.  In the presence of extracellular calcium, the slope of the stress-strain curve from 6-month old Nf1+/- mice was not significantly different from the age-matched control vessels (Figure 3.7A).  When the experiment was repeated using nominal calcium-free buffer containing the calcium chelator EGTA, the slope of the stress-strain curve was significantly steeper in Nf1+/- aorta (Figure 3.7B), indicating increased stiffness.  Under these conditions, the length at which the vessel is no longer able to maintain its tone and breaks is significantly lower in Nf1+/- mice than controls.         Figure (values A) physiA) 0200400600aWSB) 050010001500aWSC)  3.7 Stress-represent mological ca0 10 1strain curveans ± SElcium; B) n2 3L/Lo2 3L/Loes from coM): ominal calc44 ntrol and ium; C) BreNf1+/+Nf1+/- Nf1+/+- CalciumNf1+/-- Calciumand Nf1+/-aking forc free free mice at 6 e under nomonths ofminal calci47  age um.  48  3.3 Discussion The function of the smooth muscle and endothelial layers in NF1 vasculature has never been examined before, although abnormal function of these layers has been linked to atherosclerosis [67], aneurysm, and heart disease in the general population [65].  In the present study, we examined smooth muscle contractile function and endothelial relaxation in Nf1+/- and control (Nf1+/+) mice.  More specifically, we studied the ability of the endothelium to produce nitric oxide, the ability of the smooth muscle to respond to it, and protein expression of specific signaling molecules downstream from neurofibromin, which are required for proper regulation of eNOS enzymatic activity.  As alteration of these functions can affect vessel strength [180], we also quantified the elastin and collagen content of the vessel wall and produced stress-strain curves in the aorta.  We demonstrated that the alterations observed are associated with the upregulation of the PI3K/Akt/eNOS pathway, increased production of nitric oxide, and increased calcium sensitivity in the smooth muscle.  Our findings in Nf1+/- mice underscore the role of nitric oxide overproduction in neurofibromin haploinsufficient vasculature.    The pathology of vascular disease in NF1 is complex and involves all the components of the arterial wall and much of the circulating blood.  Upon ligation of the carotid artery, Nf1+/- mice demonstrate increased neointimal formation [57], which results from Nf1 haploinsufficiency in myeloid-derived cells [60].  Haploinsufficiency for neurofibromin in smooth muscle cells, endothelial cells, or both is not sufficient to cause neointimal formation but may contribute to it [63].  In response to multiple growth factors, neurofibromin haploinsufficiency in smooth muscle and endothelial cells results in 49  activated Ras/Erk signaling, leading to increased proliferation and migration [20, 21, 58, 181].  How these alterations affect the contractile state of the vessel is unknown.      This is the first study to examine relevant function of the vascular smooth muscle and endothelial layers in a model of NF1.  Normal vascular function depends on the contractile state of the vessel, which is tightly regulated [84, 85].  Contraction can be initiated through several mechanisms [118].  Depending on the mechanism used to induce vasoconstriction, the relative difference between control and Nf1+/- mice is altered.  Membrane depolarization induces greater contraction, while phenylephrine induces less contraction in the thoracic aorta of Nf1+/- mice than in controls.  This difference may be due, in part, to altered function of the endothelial cells.  KCl induces membrane depolarization of the smooth muscle and the endothelial cells [182], whereas phenylephrine only stimulates the smooth muscle cells, by acting on α1-adrenoceptors [139].  Due to the absence of voltage gated calcium channels in endothelial cells, depolarization of endothelial cells decreases the inward driving force on Ca2+ and therefore results in decreased intracellular calcium [182], which decreases the endogenous activity of eNOS and subsequent production of nitric oxide [137].  Although nitric oxide production was not directly examined in this set of experiments, our results suggest that Nf1+/- aortic segments may contract more than controls when KCl inhibits Ca2+ activation of eNOS, and less than controls when eNOS activity is not disrupted.  Therefore, we hypothesized that basal endogenous eNOS activity may be higher in Nf1+/- mice and thus promote relaxation.    50  To further examine this hypothesis, we studied endothelium-dependent relaxation using acetylcholine.  Acetylcholine induces endothelial production of nitric oxide, prostaglandins, and endothelial-derived relaxing factor.  Once produced, these factors act as second messengers to stimulate guanylyl cylcase in smooth muscle, leading to activation of myosin light chain phosphatase and subsequent muscle relaxation [183, 184].  Our results demonstrate that relaxation is increased in Nf1+/- mice compared to control littermates.  Since acetylcholine may induce multiple relaxing factors, we confirmed that the majority of relaxation in the thoracic aorta is a result of nitric oxide production [185] by using L-NAME, which directly blocks nitric oxide production.   As the relaxation observed in Nf1+/- mice may result when a higher concentration of nitric oxide is produced or when nitric oxide sensitivity is increased in the smooth muscle, we examined endothelium-independent relaxation using the nitric oxide donor, sodium nitroprusside.  The pEC50 for sodium nitroprusside was slightly higher in control littermates than in Nf1+/- littermates, while the Emax was not significantly different.  From these experiments, we conclude that the majority of alterations in relaxation observed in Nf1+/- mice are a result of increased nitric oxide production, but increased sensitivity may also play a small role.   Nitric oxide production in endothelial cells is due to eNOS activity [186].  eNOS is maintained in an inactive state by caveolin-1 [133].  Activity and subsequent nitric oxide production are induced through the elevation of intracellular calcium and/or activation of the PI3K/Akt signaling cascade [134-136].  Calcium directly activates calmodulin, 51  enabling it to bind eNOS, which induces a conformational change required for enzyme activity and nitric oxide production.  PI3K does not directly bind calmodulin but recruits Akt to caveolae.  Akt then phosphorylates eNOS and increases the affinity of calmodulin for eNOS [137, 139].    Augmented relaxation of the thoracic aorta in Nf1+/- mice appears to be related to up-regulation of the PI3K/Akt signaling cascade.  PI3K expression is increased in Nf1+/- thoracic aorta.  Consistent with this finding, increased expressed of PI3K has also been demonstrated in human endothelial cells isolated from NF1 patients [181].  Expanding on these results, we further demonstrated increased expression of the downstream effectors p-Aktthr308 and p-eNOSser1179 in Nf1+/- thoracic aorta, indicating that loss of neurofibromin affects eNOS activity.  Phosphorylation of eNOS increases the calcium sensitivity of the enzyme and stimulates nitric oxide production at basal levels of intracellular calcium, promoting the process of relaxation [137].  We thus conclude that phosphorylation of eNOS in Nf1+/- mice contributes to the relaxation observed by activating the enzyme causing increased nitric oxide production.     Since nitric oxide production is expected to reduce force development, we examined phenylephrine-induced contraction after blocking basal nitric oxide directly with L-NAME.  Blocking nitric oxide synthesis shifts the balance of relaxing and contracting factors towards contraction [115].  As expected, the maximum phenylephrine-induced contraction for both control and Nf1+/- mice is increased when L-NAME is present.  Interestingly, blocking nitric oxide production in Nf1+/- mice not only normalizes 52  contraction to that of controls but increases it above the control value.  This is consistent with the earlier KCL experiments, demonstrating increased contraction and confirms that blocking the synthesis of nitric oxide results in increased contraction in Nf1+/- mice.  Increased contraction in Nf1+/- aorta may be related to higher intracellular calcium or up-regulation of calcium sensitivity of smooth muscle contractile machinery [103, 114, 187].  Though neither the regulation of intracellular calcium nor ion channels has been examined in Nf1+/- vascular smooth muscle, hyperactivation of Ras is known to increase Rho signaling in Nf1+/- mice [188, 189].  Rho signaling has well-known effects on smooth muscle contraction and reduces myosin light chain phosphatase activity [110, 176].  This increases calcium-sensitivity of the smooth muscle, resulting in further increases in contraction [190], as we observed in Nf1+/- mice.  More information is required to confirm this observation and elucidate the exact mechanism.    As the alterations in relaxation and contraction outlined above may affect vascular wall structure, we examined morphology of the vessel wall as well as elastin and collagen content of the aorta.  Consistent with other reports, we did not detect vascular wall lesions or differences in wall thickness [57].  However, when we examined components of the extracellular matrix we found more elastin per unit area of artery in Nf1+/- mice versus controls, suggesting that Nf1+/- aorta may be more elastic than control aorta.    Nf1+/- elastin fibrils appeared disorganized with some fragmentation, which affects elasticity and causes the vessel to be susceptible to breakage.  Consistent with histology, stress-strain curves in Nf1+/- and control aortas demonstrated increased 53  stiffness in response to stretch in calcium-depleted (EGTA-chelated) buffer, which further confirms that the increased stiffness is due to properties of the extracellular matrix.  In physiological buffer, stress-strain curves in Nf1+/- and control aortas were not significantly different.  It has been reported that upon stretching, the calcium present in physiological buffer stimulates relaxation of smooth muscle through a PI3K-dependent mechanism [191, 192], while in the absence of calcium less relaxation is induced and differences in the extracellular matrix properties are evident.        How the alterations described in this study affect vascular disease in people with NF1 is not clear.  In the general population, properly regulated eNOS activity is associated with reduced risk of atherosclerosis [193, 194, 195{Hambrecht, 2003 #872, 196]} and has been shown to protect against left ventricular dysfunction [197].  These effects are likely a result of decreased endothelial apoptosis [198] and decreased expression of endothelial adhesion molecules [199-204].  Interestingly, in mice genetically engineered to overexpress eNOS, further increases in eNOS activity eliminate any beneficial effects of nitric oxide and augments disease  due to uncoupling of eNOS [205] and production of reactive oxygen species [204].  Excessive eNOS activity is associated with atherosclerotic lesions [206], pulmonary hypertension [207], cardiac hypertrophy [208], and myocardial infarction [205] in animal models.  As these phenotypes appear to occur with increased frequency in patients with NF1 and we have shown evidence suggesting eNOS activity is increased in a Nf1 mouse model eNOS may be important in vascular disease development in patients with NF1.     54  The increased eNOS activity and its effects on vascular function demonstrated in this study are consistent with other previously-described models.  Both caveolin-1 knockout (Cav-1-/-) mice [209] and SMAD3-deficient mice [210] demonstrate increased production of nitric oxide [209].  Similar to Nf1+/- mice, Cav-1-/- mice demonstrate an impaired response to phenylephrine, increased relaxation in response to acetylcholine, and increased eNOS activity in myographic assays of the aorta [209].  Interestingly, deletion of eNOS in Cav-1-/- mice induces pulmonary hypertension and right ventricular hypertrophy as a result of reactive oxygen species formation [207, 208, 211].  Though Nf1+/- mice have never been screened for pulmonary hypertension, which has been reported in NF1 patients, or hypertrophy, reactive oxygen species and inflammation characterize neointimal lesions and aneuryms induced in Nf1 models.  Also consistent with Nf1+/- mice, SMAD3-deficient mice demonstrate increased stiffness and degradation of elastin fibers.  In SMAD3-deficient mice, persistent nitric oxide production induces expression of matrix metalloproteinases 2 and 9, causing degradation of elastin fibers [210].    We have demonstrated that the haploinsufficiency of neurofibromin alters vascular function and affects eNOS activity.  Persistent production of nitric oxide has been shown to increase proliferation of smooth muscle and, as substrate availability decreases, result in the formation of reactive oxygen species and inflammation [184].  This research suggests eNOS uncoupling may contribute to vascular disease in NF1 patients.  More research is needed to determine how these findings extend to humans, but one implication may be that care should be taken in treating patients with compounds which 55  increase nitric oxide production unless careful supplementation with L-arginine and tetrahydrobiopterin (BH4) is also provided in order to avoid increased oxidative stress [205, 212-214].    56  Chapter 4: Endothelial Dysfunction in the Abdominal Aorta and Renal Arteries of Nf1+/- Mice  4.1 Introduction   Vascular lesions in different locations seem to have similar histological characteristics, suggesting all vascular lesions in NF1 share similar pathology [27, 215].  However, local differences (e.g. differences in vessel size or shear stress) are also likely to contribute the development of vascular lesions in NF1, as they do in the general population [216].    In the previous chapter, we demonstrated that alterations in vascular function occur in the thoracic aorta of 6-month old Nf1+/- mice and suggested that vascular disease in NF1 patients may result from eNOS uncoupling.  In this chapter, we examined the abdominal aorta and renal arteries in 6-month-old Nf1+/- mice to confirm that the alterations in vascular function in these animals are not limited to the thoracic aorta.  Additionally, we examined vascular function in 9-to 12 month-old- mice to determine if the alterations observed at 6 months of age remain stable, normalize, or progress.       57  4.2 Results   4.2.1 Contractile Function and Relaxation in 6-Month Old Mice As explained in the previous chapter, vasoconstriction can be stimulated by the release of calcium from the sarcoplasmic reticulum in vascular smooth muscle [118], while relaxation can be stimulated by the release of calcium from the sarcoplasmic reticulum in endothelial cells.  In order to determine if the contractile response of the abdominal aorta is similar to that of the thoracic aorta in 6-month old mice, we examined force developed in response to phenylephrine.  Consistent with findings in the thoracic aorta, phenylephrine-induced contraction was significantly reduced in the abdominal aorta of Nf1+/- mice in comparison to control littermates (2.55 ± 0.26 mN in Nf1+/- and 3.32 ± 0.15 mN in controls, p=0.02) (Figure 4.1A).  Similar results were also observed in the renal arteries (1.65 ± 0.29 mN in Nf1+/- and 3.02 ± 0.44 mN in controls, p=0.08).     To determine if endothelium-dependent relaxation in the abdominal aorta is similar to that in the thoracic aorta, we examined the maximum amount of relaxation developed following pre-contracting the aorta with phenylephrine.  Consistent with the thoracic aorta, acetylcholine-induced relaxation was significantly increased in Nf1+/- mice in comparison to controls (57.9 ± 3.3 mN in Nf1+/- and 46.5 ± 3.9 mN in controls, p=0.03) (Figure 4.1B).  Similar results were also observed in the renal arteries (72.10 ± 5.1 mN in Nf1+/- and 45.58 ± 9.1 in controls, p= 0.02)    58  Figure 4.1 Agonist-induced contraction and relaxation in the abdominal aorta from control and Nf1+/- mice at 6 months of age   A) Phenylephrine-induced contraction in abdominal aorta from control and Nf1+/- mice at 6 months of age (values represent mean ± SEM). B)  Acetylcholine-induced relaxation in phenylephrine (20μM)-precontracted abdominal aorta from control and Nf1+/- mice 6 months of age (values represent mean ± SEM).  A)   B)   59  4.2.2 Contractile Function of the Smooth Muscle of 9 to 12-Month Old Nf1+/- Mice As the abdominal aorta of 6-month old mice was not different from previous results in the thoracic aorta, we examined contractile function of the abdominal aorta in 9 to 12 month mice to determine if the alterations observed at 6-months remain stable, regress, or progress.  In the thoracic aorta, we demonstrated KCl-induced contraction was significantly higher in Nf1+/- mice than controls.  Consistent with this finding, the developed force in the abdominal aorta of 9- to 12-month old mice was significantly increased in Nf1+/- mice as well (1.14 ± 0.10% in Nf1+/- and 1.51 ± 0.10% in controls; p = 0.02).  We also measured phenylephrine-induced contraction, but in contrast to the thoracic aorta at 6 months of age, phenylephrine-induced contraction was significantly increased in Nf1+/- mice in comparison to control littermates (Figure 4.2).  The maximal force (Emax) generated in response to phenylephrine was significantly higher in Nf1+/- mice than in controls (Figure. 4.2A and 4.2B).  Additionally, in Nf1+/- abdominal aorta, the pEC50 values (50% effective concentration) for phenylephrine (6.27 ± 0.3) were significantly higher (p= 0.001) than in controls (6.03 ± 0.07), indicating a marked increase in sensitivity to phenylephrine in Nf1+/- aorta at 9-to 12 months of age (Figure 4.2C).        60   Figure 4.2 Phenylephrine-induced contraction in abdominal aorta from control and Nf1+/- mice at 9 to 12 months of age (values represent means ± SEM): A) Concentration–response curve of phenylephrine (PE) at the following doses:   5 nM, 10 nM, 50nM, 100 nM, 500 nM, 1 µM, 5 µM , 10 µM  50 µM , 100 µM ; B) Maximal force (Emax) generated in response to PE; C) pEC50 values for PE. A)  B)  C)    -10 -8 -6 -4 -2050100150 Nf1+/+Nf1+/-Log [PE]% PE-Induced Force61  4.2.3 Endothelium-Dependent and Endothelial-Independent Vasodilation  To study endothelium-dependent vasodilation, we generated concentration-response curves to acetylcholine.  Acetylcholine-induced relaxation was substantially decreased in Nf1+/- mice in comparison to controls (Figure 4.3A).  The Emax of acetylcholine was significantly lower in Nf1+/- than control aorta (42.3 ± 2.5 in Nf1+/- and 58.6 ± 5.0 in controls, p=0.002) (Figure 4.3B).  The pEC50 of acetylcholine was not significantly different between Nf1+/- and control abdominal aorta (7.62 ± 0.07 in Nf1+/- and 7.43 ± 0.08 in controls) (Figure. 4.3C) at 9-to 12 months of age, indicating similar sensitivity to acetylcholine in the Nf1+/- abdominal aorta.  In order to confirm that nitric oxide production is primarily responsible for the relaxation stimulated by acetylcholine, we assessed the acetylcholine response in the presence of 200 μM L-NAME, an analog of arginine that inhibits nitric oxide production.  Similar to the thoracic aorta at 6 months of age, pre-incubation with L-NAME abrogated the relaxation observed in both Nf1+/- and control vessels (Figure 4.3D) at 9-to 12 months of age.           62  Figure 4.3 Acetylcholine-induced relaxation in phenylephrine (20μM)-precontracted abdominal aorta from control and Nf1+/- mice at 9 to 12 months of age (values represent mean ± SEM):   A) Concentration–response curve of acetylcholine (ACh) at the following doses:  1 nM, 5mM, 10 nM, 50nM, 100 nM, 500 nM, 1 µM, 5 µM; B) Maximal force (Emax) generated in response to ACh; C) pEC50 values for ACh; D) Maximum force generated in response to 500nM ACh with and without pretreatment of 200 μM LNAME.  A)    B)   C)    -9 -8 -7 -6-20020406080 Nf1+/+Nf1+/-Log [ACh]% ACh- induced Relaxation63  D)   As in the previous chapter, to study endothelium-independent vasodilation, we treated abdominal aortic segments with sodium nitroprusside, a nitric oxide donor that bypasses endogenous nitric oxide production by the endothelium and acts directly on smooth muscle cells (Figure 4.4).  We did not observe a significant difference in the Emax (80.9± 2.1 in Nf1+/- and 85.9 ± 2.3 in controls) or pEC50 (7.43 ± 0.07 in Nf1+/- and 7.52 ± 0.05 in controls) of sodium nitroprusside between Nf1+/- and control mice at 9- to 12 months of age (Figure 4.4A & 4.4B), indicating that there is not an increase in the sensitivity to nitric oxide in the vascular smooth muscle of Nf1+/- mice compared to controls.      64  Figure 4.4 Sodium nitroprusside-induced relaxation in phenylephrine (20μM)-precontracted abdominal aorta from control and Nf1+/- mice at 9 to 12 months of age (values represent means ± SEM):   A) Concentration–response curve of sodium nitroprusside (SNP) at the following doses:  1 nM, 5mM, 10 nM, 50nM, 100 nM, 500 nM, 1 µM; B) Maximal force (Emax) generated in response to SNP; C) pEC50 values for SNP. A)  B)  C)   -9 -8 -7 -6 -5-20020406080100 Nf1+/+Nf1+/-Log [SNP]% SNP- induced Relaxation 4.2.4 TVasocoAs we abdominproductimeasurithe presmaximunot signendogen Figure 4and withNf1+/- m  he Rolenstriction demonstraal aorta oon, we invng the maxence and m amount ificantly difous levels.5 Maximout pretreice at 9 to 1 of Endted that f Nf1+/- micestigated timum perabsence ofof contractferent betw of nitric oxum force gatment of 22 months oogenous acetylcholie and thahe role of cent contra L-NAME ion increaseen controide are notenerated i00 μM LNf age (valuNitric One resultet this mayendogenouction in re(200 µM). ed in respol and Nf1+ significantn responseAME in thes represexide in d in decr be a resus nitric oxsponse to  In both conse to L-N/- mice (Fily different  to 20 μMe abdominnt means Phenylepeased rellt of reducide in vasophenylephntrol and AME, but gure 4.5), between g phenylepal aorta fr± SEM). hrine-Induaxation ined nitric oconstrictiorine (20 µMNf1+/- micethe increasuggestingroups.   hrine (PE) om control65 ced  the xide n by ) in , the se is  that with  and 66  4.3 Discussion  In the thoracic aorta of 6-month-old mice, we demonstrated that Nf1+/- mice have altered vascular function, which may result from enhanced activation of eNOS (Chapter 3).  The activation of eNOS with subsequent production of nitric oxide has been shown to protect against other kinds of vascular disease, but prolonged production of nitric oxide is associated with eNOS uncoupling and endothelial dysfunction.  At 6 months of age, Nf1+/- mice demonstrate reduced smooth muscle contraction and dysfunctional relaxation, but it was not clear if the alterations in function represented adaptive or pathologic processes in the Nf1+/- mouse model.    In order to further our understanding of vascular function in Nf1+/- mice, we examined two other types of blood vessels, the abdominal aorta and renal arteries, as well as abdominal aorta in older mice to determine if the alterations changed with age.  Similar to our previous findings in the thoracic aorta, we found decreased contraction and increased relaxation in the abdominal aorta and renal arteries of 6-month old mice.  Interestingly, we did not find the same alterations in 9 to 12 month old mice.  Instead, we observed a progression of endothelial dysfunction in older mice.  These findings suggest that enhanced activation of eNOS may contribute to vascular endothelial dysfunction as Nf1+/- mice age.    As demonstrated in the previous chapter, the reduction of neurofibromin in Nf1+/- mice affects function of the thoracic aorta.  We demonstrated marked alterations in the contractile function, variations in endothelial function, and enhanced calcium sensitivity 67  in vascular smooth muscle of the thoracic aorta.  Augmented relaxation in Nf1+/- mice appeared to be a result of upregulation of the PI3K/Akt/eNOS pathway affecting eNOS enzymatic activity in endothelial cells.  Similar functional alterations of the endothelium and smooth muscle were demonstrated in the abdominal aorta and renal arteries of 6-month old Nf1+/- mice, demonstrating that the changes observed are not restricted to a specific site.  Similarly, vascular disease in most patients with NF1 is not restricted to a specific site and usually affects multiple vessels.  Vascular lesions from different locations in people with NF1 appear to have similar histological characteristics, but many authors differentiate between small and large vessel disease.      As the response in the abdominal aorta appeared to be similar to that in the thoracic aorta of 6-month-old Nf1+/- mice, we examined vascular function in the abdominal aorta of 9-to 12-month old mice.  Similar to 6-month old mice, membrane depolarization induces greater contraction in 9-to 12-month old Nf1+/- mice than in controls.  However, in contrast to the findings in 6-month old mice, phenylephrine induced greater contraction in 9-to 12-month old Nf1+/- mice than in controls.  In 6-month old mice, phenylephrine-induced contraction was significantly reduced, which may be a result of enhanced production of nitric oxide in the endothelium.    The increased response to phenylephrine in 9-to 12-month old mice suggests that endothelial function in older Nf1+/- mice has decreased substantially in comparison to both 6-month old Nf1+/- mice and controls. In general, reduced production of vasodilators by the vascular endothelium contributes to increased contraction by shifting 68  the balance of factors promoting contraction and dilation to contraction.  Our results demonstrate that endothelial function is significantly reduced in Nf1+/- mice as endothelium-dependent relaxation was decreased, which suggests nitric oxide production is impaired.  Together, these findings suggest that the endothelial dysfunction has progressed in 9-to 12-month old Nf1+/- mice.  As it is possible that the increased contraction in Nf1+/- mice is a result of the smooth muscle not being responsive to nitric oxide, we directly examined the ability of the smooth muscle to relax using sodium nitroprusside.  We did not observe a significant difference in the relaxation between Nf1+/- and control mice at 9-to 12-months of age.  Therefore, we conclude that the increased phenylephrine-induced contraction observed is not due to a decreased ability of the smooth muscle to relax in 9-to 12-month old Nf1+/- mice.    In chapter 3 we suggested that neurofibromin haploinsufficiency may affect the calcium sensitivity of the smooth muscle since we observed increased contraction in Nf1+/- thoracic aorta when nitric oxide production was blocked.  In this chapter, we have shown that endothelial dysfunction in 9- to 12-month old Nf1+/- mice also results in increased contraction, which is consistent with our previous observation, but more information is required to elucidate more fully the role of neurofibromin in the contraction of smooth muscle.   69  Endothelial dysfunction is a characteristic feature of several different forms of vascular disease in the general population.  Growing evidence suggests that eNOS uncoupling is one of the primary mechanisms involved in the development of endothelial dysfunction [87, 213, 217-219].  eNOS uncoupling occurs when the ferrous-dioxygen complex within the enzyme becomes dissociated.  As a result, molecular oxygen is reduced to superoxide and nitric oxide is not produced.  eNOS function is highly dependent on the presence of BH4 and L-arginine.  Previous studies have demonstrated that overexpression of eNOS results in reduced levels of BH4 and leads to eNOS uncoupling [62].  The production of reactive oxygen species and reduced bioavailability of nitric oxide both result in endothelial activation [199, 201, 203, 220], which results in recruitment of inflammatory cells to the vascular wall and remodelling.    In the previous chapter, we demonstrated that neurofibromin haploinsufficiency affects eNOS activity and hypothesized that as substrate availability decreased, it may result in endothelial dysfunction.  In support of this hypothesis, we demonstrate here that endothelial dysfunction is present in the abdominal aorta of 9-to 12-month old Nf1+/- mice.  More research is needed to determine the exact mechanism by which endothelial dysfunction occurs in Nf1+/- mice and how these findings extend to humans with NF1. 70  Chapter 5: Echocardiographic Evidence of Cardiomyopathy in an Nf1+/- Mouse Model  5.1 Introduction As described in the introduction of this thesis, individuals with NF1 may present with various types of cardiovascular disease, including ventricular septal defects [29], coarctation of the aorta [221], left ventricular hypertrophy [26], myocardial infarction [27], pulmonary hypertension [222, 223], and aneurysm [28, 224-226].  Cardiovascular disease is associated with increased morbidity and premature mortality in people with NF1 [24, 25, 227].      Neurofibromin is required for proper development of the heart [83].  The effect of neurofibromin haploinsufficiency on cardiac development and function is not clear but may result in mild structural alterations [51].  On average, NF1 patients have higher posterior wall thickness and interventricular septal thickness measurements than individuals of the same age and sex without NF1 [51].  These alterations may, at least in part, be a result of hypertension, which has been estimated to occur in 16% of young patients with NF1 [49].  Young hypertensive NF1 patients have increased posterior wall thickness and atrial dimensions compared with normotensive NF1 patients and healthy subjects [50].  At this time, it is not clear whether the alterations found in asymptomatic patients have any long-term hemodynamic consequences.    71  Mouse models are an invaluable tool to determine what role neurofibromin and its downstream signaling partners play in cardiovascular disease.  Complete (homozygous) loss of neurofibromin is lethal in mouse embryos because of the development of interventricular septal defects, outlet tract abnormalities, enlarged endocardial cushions, and thinned myocardium [22, 80].  Evidence suggests that the primary cause of this phenotype is hyperactivation of Ras and premature localization of NFATc1 (a transcription factor required for cardiac development) to the nucleus of cardiac endothelial cells [54, 82].  Complete loss of neurofibromin in post-natal cardiomyocytes results in progressive hypertrophy, most likely due to Ras hyperactivation [171].  Interestingly, constitutional activation of H-Ras in mice also produces various degrees of hypertrophy [168, 228-230].  The severity of this hypertrophy is associated with the degree of Ras gene expression, but hypertrophic cardiomyopathy only develops after a certain threshold of Ras activation is met [169, 230].    Neurofibromin haploinsufficiency results in prolonged Ras hyperactivation [20, 21, 59, 231], but it is unclear how this affects the cardiovascular phenotype.  We used two-dimensional echocardiography with color Doppler and standard histological methods to study the role of neurofibromin haploinsufficiency in adult mice.  In this chapter, we demonstrate that Nf1+/- mice have increased left ventricular wall thickness and reduced cardiac contractility, which appear to result from alterations in cardiomyocyte organization.  We conclude that neurofibromin haploinsufficiency promotes alterations in cardiac structure and function.   72  5.2 Results  To evaluate myocardial structure and function, we performed two-dimensional echocardiography on Nf1+/- and control mice at 6 months of age.  M-mode images from Nf1+/- mice show modest but significant alterations in myocardial structure (Table 5.1).  Consistent with previous reports [57] in Nf1+/- mice, body weight (grams) was significantly different between control and Nf1+/- mice (48.81 ± 1.15 in controls versus 44.24 ± 1.36 in Nf1+/- mice; p= 0.02).  Of note, Nf1+/- mice demonstrate a significant increase in the thickness of the posterior wall of the left ventricle during both diastole and systole and a significant enlargement of internal diastolic and systolic dimensions.  Intraventricular septum and left ventricular anterior wall thicknesses were similar in Nf1+/- mice and controls.  LV mass was not significantly different in Nf1+/- and control mice.              73  Table 5.1  Cardiac Structural Parameters in Control (Nf1+/+) and Nf1+/- Mice  Cardiac Structural Parameter Nf1+/+ Nf1+/- p-value End-diastolic dimension, mm   0.09 ± 0.002 0.097 ± 0.002 0.02 End-systolic dimension, mm   0.06 ± 0.002   0.07 ± 0.002 0.002 End-diastolic volume, µl   1.77 ± 0.04   1.82 ± 0.07 0.55 End-systolic volume, µl   0.60 ± 0.05   0.80 ± 0.06 0.02 Diastolic Anterior Wall Thickness, mm 0.017 ± 0.001 0.018 ± 0.001 0.25 Systolic Anterior Wall Thickness, mm   0.02 ± 0.001   0.02 ± 0.002 0.49 Diastolic Posterior Wall Thickness, mm 0.016 ± 0.001 0.019 ± 0.001 0.05 Systolic Posterior Wall Thickness, mm 0.022 ± 0.001 0.026 ± 0.001 0.05 Diastolic Internal Dimension, mm 0.088 ± 0.001 0.096 ± 0.002 0.008 Systolic Internal Dimension, mm   0.06 ± 0.002   0.07 ± 0.002 0.002 Diastolic Intraventricular Septal Thickness, mm 0.017 ± 0.001 0.019 ± 0.001 0.39 Systolic Intraventricular Septal Thickness, mm 0.022 ± 0.001 0.022 ± 0.001 0.77 Values listed in each column are means ± SEM.  Bold numbers indicate p ≤ 0.05 and are considered to be statistically significant.    74  The alterations in structure were associated with impaired left ventricular contractile function in Nf1+/- mice (Table 5.2).  The ejection fraction and fractional shortening were both reduced, indicating systolic dysfunction.  Systolic dysfunction in Nf1+/- mice does not appear to be a secondary response as the velocity of circumferential shortening, which is independent of alterations in pre-load, was significantly reduced in Nf1+/- mice compared to controls.  Though a reduction in stroke volume was observed, the difference did not reach statistical significance (p = 0.07).  Consistent with a reduction in contractile performance, left ventricular end systolic diameter and volume were significantly reduced, while no change in diastolic diameter or volume were observed in Nf1+/- mice compared to controls.  Nf1+/- mice also demonstrated a significantly higher myocardial performance index compared to controls, indicating that ventricular filling of the heart may be impaired.                  75  Table 5.2  Cardiac Functional Parameters in Control (Nf1+/+) and Nf1+/- Mice  Cardiac Functional Parameter Nf1+/+ Nf1+/- p-value Heart Rate, beats/min    412 ± 4    447 ± 19 0.09 Stroke Volume, µl   1.14 ± 0.03   1.02 ± 0.05 0.07 Ejection Fraction, % 64.64 ± 2.24 56.48 ± 2.53 0.03 Fractional Shortening, % 35.35 ± 1.65 29.45 ± 1.64 0.02 Velocity of Circumferential  Shortening  37.34 ± 2.64 27.31 ± 2.11 0.02 Cardiac Output, ml/min   0.47 ± 0.02   0.49 ± 0.04 0.59 LV mass, mg   2.41 ± 0.12   2.30 ± 0.06 0.49 Myocardial Performance Index   0.62 ± 0.02 0.70 ± 0.02 0.03 Intraventricular Contraction Time   0.34 ± 0.02   0.34 ± 0.02 0.89 Intraventricular Relaxation Time   0.35 ± 0.01   0.37 ± 0.01 0.08 Ejection Time   0.95 ± 0.06   1.07 ± 0.07 0.17 Values listed in each column are mean ± SEM.  Bold numbers indicate p ≤ 0.05 and are considered to be statistically significant.  As shown in Figure 5.1, significant distortion in the shape of Nf1+/- hearts was observed.  Histological examination of cardiac cross-sections from Nf1+/- and control mice revealed mild to moderate alterations in myocyte architecture.  As demonstrated in Figure 5.2, myocytes from controls are well organized and run parallel to one another, while myocytes from Nf1+/- mice show alterations in orientation and disarray.  Pathologic  changesfragmenrarely, iinfarctioof the co Figure 5        , includintation, wern controls. n were obsntrols. .1  Gross ag enlarge observe Wavy myerved in thnatomy ofed nucleid in the mofibrils che right ven the heart i, perinucyocardia oaracteristictricle of twon control (N lear spacf Nf1+/- m of remode Nf1+/- micf1+/+) and ing, vacuice but noling followe (Figure 5Nf1+/- mice olization t, or only ing myoca.3) but in n76 and very rdial one  Figure alteratioAll micecontrolsin Nf1+/-        5.2  Photons in myoc were 6-mo, include emice. micrograpyte orientatnths of agnlarged nuhs from 3ion and ince.  Patholoclei, perinu control areased mygic changclear spacnd 3 Nf1+ocyte sizees, which ing, vacuo/- hearts (in the Nf1+are absentlization and200x) sho/- mice.    or very m fragment77 wing ild in ation    Figure myocard Consisteoverall npercenta         5.3 Photoial infarctiont with theumber of nge of fibromicrographn.   changes uclei, the psis per field (200x) fr observed,ercentage were signom 6-mon morphome of interstitificantly incth old Nftric analysial space inreased in N1+/- mouseis demons the myocaf1+/- mice  with signtrated thardium, and(Figure 5.478 s of t the  the ).   Figure 5Bar grapinterstitiaof Nf1+/- a)          c)  5.3 DisThe roleit was n.4  Morphohs demonl space peand contro                 cussion  of neurofibot clear whmetric anastrate a) thr field andl mice. Val                 romin in hat effect, iflysis from e overall n c) the perues repres                             eart develo any, haplocross-sectiumber of ncentage ofent median               b       pment is winsufficienons of the uclei per f fibrosis pe ± IQR. ) ell known cy for neurheart ield, b) ther field in t[83], but profibromin h percentaghe myocar ior to this sad in the 79 e of dium tudy adult 80  heart.  When patients with NF1 are examined with echocardiography, the average relative wall thickness of the heart is increased [51], suggesting that loss of one neurofibromin allele is sufficient to affect structure of the heart.  We hypothesized that adult Nf1+/- mice may also demonstrate alterations in cardiac morphology as a result of neurofibromin haploinsufficiency.    To test this hypothesis, we examined cardiac structure and function in 6-month-old Nf1+/- and control mice.  Using two-dimensional echocardiography, we found that Nf1+/- mice have increased wall thickness and reduced contractility in the heart.  Furthermore, histological sections from Nf1+/- mice demonstrated alterations in myocyte orientation and signs suggestive of localized infarction.  At this time, it is not clear if the histological changes are a cause or manifestation of the cardiomyopathy observed.  However, our findings demonstrate that neurofibromin haploinsufficiency can result in alterations in the adult mouse heart and suggest that alterations in neurofibromin may contribute to the development of heart disease.      In the heart, neurofibromin functions as a GTP-ase activating protein, which downregulates Ras following activation of receptor tyrosine kinases and G-protein coupled receptors [83].  Loss of neurofibromin in mature cardiomyocytes results in progressive hypertrophy, fibrosis, and reactivation of the normally arrested cell cycle [171].  In the absence of neurofibromin, several authors have demonstrated that stimulation by various agonists results in prolonged activation of Ras [20, 56, 58, 232, 233].  We hypothesize similar mechanisms occur in patients with NF1 as they appear to 81  be more susceptible to cardiac hypertrophy, myocardial infarction and heart failure, [29, 30, 234, 235] but further studies are needed to confirm this.    Unfortunately, available studies have had insufficient power to detect whether many of these abnormalities occur more frequently in patients with NF1 than in the general population [29].  Of note, flow defects, the most common of which is pulmonic stenosis, do occur more frequently in patients with NF1 but are proposed to result from abnormalities in embryonic intra-cardiac hemodynamics [29, 223].  It is unclear if hemodynamic alterations in adults with NF1 also contribute to heart disease.  Though it has been recognized that neurofibromin is required for cardiac development for many years, most studies in animal models have assumed that neurofibromin haploinsufficiency is not sufficient to result in a cardiac phenotype, as Nf1+/- mice do not develop overt cardiovascular abnormalities.  In this study, we show that adult Nf1+/- mice do have a cardiovascular phenotype. We demonstrated structural alterations, including increased posterior wall thickness and internal dimension, which are associated with systolic and diastolic dysfunction [236, 237].  This finding is consistent with previous studies, which showed that cardiomyocyte-specific deletion of both neurofibromin alleles [171] and constitutional activation of H-Ras [168, 228, 238] in mice resulted in hypertrophic dilated cardiomyopathy of various degrees.  In contrast to mice with cardiomyocyte-specific deletion of neurofibromin [171], however, Nf1+/- mice did not demonstrate hypertrophic cardiomyopathy, as the thickness of the interventricular septal 82  wall was similar to controls.  However, there was a trend towards higher left ventricular mass and localized areas of hypertrophy were observed on histological examination.    Little information is known about the role of neurofibromin in contractile abnormalities.  We observed a global decrease in left ventricular contractility in Nf1+/- mice when compared to controls, suggesting that neurofibromin may have a role in contractility [236].  Contractility of myocytes is dependent on proper regulation of excitation and contraction [148, 239, 240].  Constitutional expression of Ras is known to affect sarcoplasmic reticulum calcium release, causing myocyte disarray and affecting systolic function [239, 241, 242].  Although we did not examine sarcoplasmic reticulum calcium handling in this study, Nf1+/- mice demonstrated localized alterations in myocyte orientation, perinuclear spacing, and vacuolization, which are similar to those seen in mice with constitutional overexpression of Ras [243].  Therefore, it is possible that hyperactivation of Ras in neurofibromin haploinsufficient mice also affects calcium handling and is responsible for the reduced contractility observed in Nf1+/- mice.  This possibility merits further examination.    Remodeled areas of myocardial infarction, which may also affect contractility [244], were observed in the right ventricles of some Nf1+/- mice.  Though myocardial infarction has not previously been reported in Nf1+/- mice, NF1 patients with myocardial infarction have been reported [26, 27, 245].  However, it is worth noting that in NF1 patients, myocardial infarction may be a result of NF1 vasculopathy [27].  Further research is 83  required to determine the exact role of neurofibromin in the regulation of cardiomyocyte contractility.      The cardiovascular phenotype observed here in Nf1+/- mice is reminiscent of in vivo models that constitutionally express Ras [169, 238, 243].  Constitutional expression of H-Ras in mice causes variable amounts of myocyte disarray, hypertrophy, and diastolic dysfunction [169, 170, 243].  Phenotypic variability appears to be related to differences in transgene expression.  Mice with very low expression of p21ras under the control of the ventricle-specific myosin light chain 2V (MLC-2V) promoter demonstrate increased ventricular mass and normal myocyte orientation, while mice with higher transgene expression demonstrate a hypertrophic phenotype [169].  The phenotype we observed in Nf1+/- mice is less severe than that in mice with constitutional activation of H-Ras, which may be partially explained by the duration of Ras activation, as demonstrated in a recent study utilizing a tet-on-off system [230].  Alterations in myocyte orientation and size were observed in this model after just two weeks of Ras activation and became progressively more severe the longer Ras activation was induced [230].  In our study of Nf1+/- mice, we also observed mild-to-moderate alterations in myocyte architecture, which resembled the lesions seen after 2-4 weeks of Ras activation in the tet-on-off model.    Prolonged activation of Ras requires an initial activation by upstream signals in neurofibromin haploinsufficient vascular smooth muscle [58], endothelial cells [20], mast 84  cells [246] and epicardial cells [232].  Further perturbation of Ras and its downstream effectors is likely to result in more severe lesions as cardiomyocyte-specific deletion of both neurofibromin alleles in mice causes progressive hypertrophic lesions [171].  More research is needed to determine which factors contribute to the development of cardiomyopathy in NF1.     85  Chapter 6: General Discussion  6.1 Summary  Vascular disease is an important complication of NF1.  NF1 patients may present with stenosis, aneurysm, or hemorrhage of various arteries including, but not limited to, those in the heart and brain [7].  The average life expectancy of patients with NF1 is reduced, partially as a result of vascular disease [24, 25, 227].    Previous models examining vascular smooth muscle and endothelial cells in vitro have provided valuable insight into the role of neurofibromin in cellular proliferation and migration [20, 21].  Inducing the development of vascular lesions in Nf1+/- mice further suggested that neurofibromin haploinsufficiency may promote the development of vascular lesions but does not fully explain their development, as the lesions are induced [57, 60, 61, 63, 247].  In humans, many other kinds of vascular disease result from alterations in vascular function [65, 67, 92, 214, 219, 220, 248-250], but vascular function has never been examined in Nf1+/- mice.    Normal vascular function depends on the activity of the smooth muscle and endothelial cells in blood vessels [92, 127].  I used wire myography to examine smooth muscle contraction and relaxation, histology to examine vessel structure, and protein expression to examine signaling mechanisms that regulate the contractile state of the vasculature.  I also investigated cardiac structure and function in order to determine if the alterations in vascular function impact cardiac function in Nf1+/- animals.    86  6.2 Vascular Function in Nf1+/- Mice The alterations in contraction and relaxation that I observed in Nf1+/- mice (Chapter 3 and Chapter 4) suggest that neurofibromin haploinsufficiency has a strong role in maintaining normal vascular function.  This is the first study that has examined vascular function in Nf1+/- mice.  Using a wire myograph enabled me to study the interaction between the endothelial and smooth muscle cell layers as well as examine them independently of one another.    The observation that vascular dysfunction can be observed in multiple vessels at 6-months of age in Nf1+/- mice is significant for two reasons.  First, Nf1+/- mice were not thought to be an accurate representation of vascular disease in NF1 patients as they do not spontaneously develop vascular disease.  Our results suggest that Nf1+/- mice do have a vascular phenotype, but, consistent with previous reports [22, 80], we did not observe vascular lesions.  Second, NF1 patients with vascular lesions tend to have involvement of multiple vessels [16].  As the results were similar between different arteries in 6-month old Nf1+/- mice and were very similar from animal to animal, localized differences are not necessary for the development of vascular dysfunction in these animals.  The evidence suggests that neurofibromin haploinsufficiency causes vascular dysfunction as a result of altered signaling in smooth muscle and/or endothelial cells. We found no indication that somatic loss of the normal Nf1 allele (i.e., “a second hit”) is necessary for vascular dysfunction to occur.  87  How the vascular dysfunction we observed in 6-month old Nf1+/-mice contributes to the development of vascular lesions is not known.  Recent studies have demonstrated that neointimal formation [60] and aneurysm [61] in Nf1+/- mice require neurofibromin haploinsufficiency in myeloid cells.    In both studies recruitment of Nf1+/- myeloid-derived cells to the vascular wall and subsequent production of pro-inflammatory cytokines and mediators is thought to cause lesion formation.  Though these models demonstrate the factors required for lesion formation, the lesions observed are induced.  Carotid artery ligation is used to induce neointima lesion formation, while angiotensin-II infusion is used to induce the development of abdominal aortic aneurysm.  Ligation of the carotid artery results in recruitment of pro-inflammatory monocytes to the vessel wall, causing secretion of pro- inflammatory cytokines and proliferation of vascular smooth muscle [63].  Angiotensin-II stimulates the production of matrix metalloproteinases, causing degradation of the vessel wall and inflammatory cell recruitment [249, 251, 252].  Both of these methods result in severe injury to the vascular wall and are not comparable to physiological stressors.        Though vascular lesion development was not examined in this thesis, the results presented in Chapters 3 and 4 may provide some insight into the initial processes stimulating the formation of such lesions.   The increased production of nitric oxide we found in Nf1+/- aorta (Chapter 3) suggests that neurofibromin haploinsufficiency may increase the risk for eNOS uncoupling and subsequent oxidative injury.  The alteration in contraction and relaxation observed in the abdominal aortas of Nf1+/- mice between 9-to-12 months of age (Chapter 4) supports the hypothesis that increased production of 88  nitric oxide can progress to eNOS uncoupling, as evidenced by the decreased relaxation observed.  As described previously, eNOS uncoupling results in the production of reactive oxygen species and endothelial activation [46, 141, 203, 218, 219, 253, 254], a term used to describe the increased expression of adhesion molecules and inflammatory cell recruitment [202, 203, 255].  Though this is only one potential mechanism, it may help explain how vascular lesions develop in NF1 and merits further exploration.    6.3 Cardiac Function In vivo analysis of the function and structure of the heart in Nf1+/- mice (Chapter 5) provides evidence that neurofibromin haploinsufficiency affects cardiac contractility.  By using 2-dimensional echocardiography, I was able to examine systolic and diastolic cardiac function as well as mitral valve function in Nf1+/- mice and control littermates.  An advantage of this method is that functional alterations that range from mild to more severe can be observed.    Though modest, the alterations in cardiac function I observed in Nf1+/- mice are significant and support the hypothesis that Ras activation as a result of neurofibromin haploinsufficiency affects cardiac function.  The activation of Ras in cardiomyocytes has been shown to result in reduced contractility and hypertrophy in a dose-dependent manner.  The degree of Ras activation has been shown to play a role in phenotypic variability observed in these models [169, 238, 256].   89  Nf1+/- models may be an ideal system to study the role of Ras in the heart as neurofibromin haploinsufficiency promotes Ras hyperactivation, the extent of which can be modified by several different upstream factors [20, 232].  We found Nf1+/- mice have increased left ventricular wall thickness and decreased global cardiac contractility.  As more severe lesions are observed in mice with cardiomyocyte-specific deletion of both neurofibromin alleles [171], further perturbation of Ras and its downstream effectors may influence the severity of heart disease in Nf1 mouse models.  Conditional knockout of both Nf1 alleles in post-natal cardiomyocytes activates Ras sufficiently to cause progressive hypertrophic cardiomyopathy [171].  Further activation of Ras in Nf1+/- mice may result in more severe alterations.  Further studies should explore this possibility and an examination of older mice may be beneficial.    Neurofibromin haploinsufficiency affects relaxation and contraction of vascular smooth muscle (Chapters 3 and 4) and also affects global cardiac contractility (Chapter 5).  As we found evidence for differences in the regulation of calcium in vascular smooth muscle (Chapter 3) and calcium is also responsible for the contractile state of cardiac smooth muscle, similar differences in the regulation of contraction and relaxation may be observed in cardiomyocytes and should be explored in future studies.  6.4 Conclusion The results presented in this thesis support the hypothesis that neurofibromin haploinsufficiency in Nf1+/- mice results in vascular endothelial and cardiac dysfunction.  Whether these findings extend to humans with NF1, who have the same genetic defect, 90  is unknown, but, if so, our observations may have important clinical implications.  The role of neurofibromin in other kinds of vascular disease needs to be studied, and the possibility that neurofibromin may provide a novel therapeutic target in cardiovascular disease more generally should be explored.   91  Bibliography  1. Jett, K. and J.M. 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