Elucidation of the Pathogenesis

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Elucidation of the Pathogenesis ELUCIDATION OF THE PATHOGENESIS OF TGF-BETA VASCULOPATHIES IDENTIFIES NOVEL THERAPEUTIC STRATEGIES by Jefferson J. Doyle A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland October 2015 © 2015 Jefferson J. Doyle All Rights Reserved ABSTRACT Marfan syndrome (MFS) is one of several related disorders that is driven by increased TGFβ signaling. While TGFβ can stimulate both canonical (Smad2/3) and noncanonical (MAPK; ERK, JNK, p38) cascades, this work has identified a novel and critical role for ERK1/2 activation in aortic aneurysm pathogenesis in Marfan mice, while inhibition of ERK1/2 activation represents a novel therapeutic strategy for the disorder. Angiotensin II influences aortic growth in Marfan mice, but the contribution of its type 1 receptor (AT1R) and type 2 receptor (AT2R) was unknown. This has direct clinical relevance, as the relative therapeutic merits of selective AT1R blockade using losartan versus inhibition of signaling through both receptors using the ACE inhibitor enalapril remained unknown. We find that losartan shows superior efficacy to enalapril in Marfan mice, this is mediated by their differential effects on ERK1/2 activation, and losartan’s full protective effect requires intact AT2R signaling. Calcium channel blockers (CCBs) are used in Marfan patients, although evidence for their efficacy is limited. Surprisingly, we find that CCBs accelerate aortic growth, dissection and premature lethality in Marfan mice, while Marfan patients taking CCBs show increased odds for aortic dissection and need for aortic surgery. CCBs enhance ERK1/2 activation via PKCβ, while PKCβ inhibition using enzastaurin or the clinically available agent hydralazine rescues aortic growth and ERK1/2 activation in Marfan mice, and hence represents a novel therapeutic strategy for the disorder. Finally, we have identified mutations in the proto-oncogene SKI, a repressor of TGFβ, as the cause of Shprintzen Goldberg syndrome, and mutations in TGFB2 ligand as the cause of a Loeys-Dietz like syndrome. We have also found strain-specific differences ii in aortic aneurysm severity between Marfan mice on C57BL/6J and 129S6 backgrounds, and have identified likely causal genes in QTLs on chromosomes 5 and 11 that link with severe disease, reach genome wide significance, and show additive epistasis. In summary, this work has sought to enhance our understanding of disease pathogenesis in MFS and related conditions, to elucidate a series of novel genetic and pharmacological modifiers of disease progression, and to identify a number of novel therapeutic strategies as a result. Advisor: Harry C. Dietz, M.D. Reader: Daniel P. Judge, M.D. Professor Associate Professor iii PREFACE I am indebted to many individuals for their contributions to my thesis. First and foremost, I would like to thank my advisor, Hal Dietz, for his support, guidance and generosity. I could not have asked for more in a PhD mentor, and I am deeply grateful for the opportunities he created for me during my time in the lab. I would like to thank many members of the Dietz laboratory, past and present, who contributed to my time in the lab. Special mention must go to: Sara Cooke, for her tireless work as lab manager that allows the lab to function on a day-to-day basis; Deb Churchill, for all her administrative assistance; Mark Lindsay, who was always willing to spare time to listen and provide advice; Jen Habashi, for allowing me to be part of the AT2R story; Nicole Wilson, for her frequent help and banter; Loretha Myers, for her assistance with drug waters and other tasks; finally, my brother Alex, whose friendship and camaraderie during our time in the lab was one of the highlights of my time there. I would like to thank all my Thesis Committee, David Kass (Chair), Ronni Cohn, Daniel Judge, and Albert Jun, for their guidance. I must also offer huge thanks to the CMM program, not only for having the faith to accept me in the first place, but for their flexibility, understanding and immeasurable support thereafter. I am also deeply grateful to the National Marfan Foundation (NMF), who generously provided me with four years of financial support during my PhD, and who always made me feel very welcome at the multiple conferences and charity events that I had the pleasure to attend as their Fellow. For their encouragement, support and affection, I must thank my parents, Teresa and Mike. Finally, I would like to dedicate this work to Pat, Bernard, and above all Terry, who would have loved to have had the opportunity to read this in person. iv TABLE OF CONTENTS Abstract ii Preface iv Table of Contents v List of Tables vi List of Figures vii Chapter 1 Introduction: Marfan Syndrome and Related Disorders 1 Chapter 2 ERK Activation in Marfan Syndrome Mice and its Role in 66 Explaining the Differential Therapeutic Efficacies of AT1R Blockers and ACE Inhibitors in the Disorder Chapter 3 A Deleterious Gene-By-Environment Interaction Imposed by 90 Calcium Channel Blockers in Marfan Syndrome and Related Disorders Chapter 4 Elucidating the Genetic Basis of Marfan-Related Conditions: 125 Shprintzen Goldberg & Loeys Dietz-Like Syndromes Chapter 5 Elucidation of Major Genetic Modifiers of Aortic Disease in 137 Marfan Syndrome Mice Identifies Novel Therapeutic Strategies Chapter 6 Discussion 167 Chapter 7 Materials and Methods 211 References 223 Appendices 248 Curriculum Vitae 330 v LIST OF TABLES 1-1 Current Diagnostic Criteria for Marfan Syndrome 3 1-2 Systemic Feature Scoring in Marfan Syndrome 4 1-3 Current Criteria for Alternative Diagnoses to Marfan Syndrome 6 3-1 GenTAC: Effect of CCBs on Aortic Disease in Patients with Marfan 119 Syndrome & Related Inherited Thoracic Aortic Aneurysm Syndromes vi LIST OF FIGURES 1-1 Domain Organization of Fibrillin-1, Fibrillin-2 and LTBP-1 24 1-2 A Representative cbEGF-like Domain from Fibrillin-1 26 2-1 Western blot of the ascending aorta in WT and Marfan mice 68 2-2 Western blot of the descending thoracic aorta in WT and Marfan mice 69 2-3 Western blot of placebo-, TGFNAb- and losartan-treated Marfan mice 70 2-4 Aortic root growth in RDEA119-treated WT and Marfan mice 71 2-5 Western blot of RDEA119-treated WT and Marfan mice 71 2-6 Final weight of placebo- and RDEA119-treated WT and Marfan mice 72 2-7 Angiotensin signaling and its effect on the TGFβ signaling cascade 73 2-8 Aortic root diameter in AT2KO WT and Marfan mice 75 2-9 Ascending aortic growth in AT2KO WT and Marfan mice 76 2-10 Kaplan-Meier survival curve in AT2KO WT and Marfan mice 77 2-11 Aortic histology in AT2KO WT and Marfan mice 77 2-12 Aortic architecture score in AT2KO WT and Marfan mice 78 2-13 Western blot of ERK & Smad in the ascending aorta of AT2KO mice 79 2-14 Western blot of JNK & p38 in the ascending aorta of AT2KO mice 79 2-15 Systolic blood pressure in placebo-, losartan- and enalapril-treated mice 80 2-16 Aortic root growth in placebo-, losartan- and enalapril-treated mice 81 2-17 Aortic architecture in placebo-, losartan- and enalapril-treated mice 82 2-18 Western blot of ERK & Smad in losartan- and enalapril-treated mice 83 vii 2-19 Western blot of JNK & p38 in losartan- and enalapril-treated mice 84 2-20 Aortic root growth in placebo- and losartan-treated AT2KO mice 85 2-21 Western blot of ERK in placebo- and losartan-treated AT2KO mice 86 2-22 Western blot of Smad, JNK & p38 in losartan-treated AT2KO mice 86 2-23 Diagram of the effects of AngII on TGF signaling in Marfan mice 87 3-1 Blood pressure in placebo-, losartan- and amlodipine-treated mice 91 3-2 Aortic root and ascending aortic growth in amlodipine-treated mice 92 3-3 Kaplan-Meier survival curve in amlodipine-treated mice 93 3-4 Latex images of the ascending aorta in amlodipine-treated mice 93 3-5 Ascending aortic histology in amlodipine-treated mice 94 3-6 Ascending aortic architecture score in amlodipine-treated mice 95 3-7 Descending thoracic aortic histology in amlodipine-treated mice 95 3-8 Descending thoracic aortic architecture score in amlodipine-treated mice 96 3-9 Aortic root and ascending growth in low-dose amlodipine-treated mice 97 3-10 Aortic root and ascending aortic growth in verapamil-treated mice 97 3-11 Latex images of the ascending aorta in verapamil-treated mice 98 3-12 Ascending aortic histology in verapamil-treated mice 98 3-13 Ascending aortic architecture score in verapamil-treated mice 99 3-14 Descending thoracic aortic histology in verapamil-treated mice 99 3-15 Descending thoracic aortic architecture score in verapamil-treated mice 100 3-16 Western blot of Smad3 and ERK in amlodipine-treated mice 101 3-17 Ascending aortic growth in amlodipine/RDEA119-treated mice 101 viii 3-18 Kaplan-Meier survival curve in amlodipine/RDEA119-treated mice 102 3-19 Latex images & aortic histology in amlodipine/RDEA119-treated mice 102 3-20 Aortic architecture score in amlodipine/RDEA119-treated mice 103 3-21 Western blot of Smad3 and ERK in amlodipine/RDEA119-treated mice 103 3-22 Ascending aortic growth in amlodipine/losartan-treated mice 104 3-23 Western blot of ERK in amlodipine/losartan-treated mice 105 3-24 Western blot of PKCβ and ERK in TGFβ NAb-treated mice 106 3-25 Western blot of PKCβ and PLCγ in amlodipine/losartan-treated mice 107 3-26 Ascending aortic growth in amlodipine/enzastaurin-treated mice 108 3-27 Latex images of the aorta in amlodipine/enzastaurin-treated mice 109 3-28 Ascending aortic histology in amlodipine/enzastaurin-treated mice 109 3-29 Aortic architecture score in amlodipine/enzastaurin-treated
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