Luận văn thạc sĩ inhibition of the akt1 mtorc1 axis alters venous remodeling to improve arteriovenous fistula patency

Yale University EliScholar – A Digital Platform for Scholarly Publishing at Yale Yale Medicine Thesis Digital Library School of Medicine January 2019 Inhibition Of The Akt1-Mtorc1 Axis Alters Venous Remodeling To Improve Arteriovenous Fistula Patency Arash Fereydooni Follow this and additional works at: https://elischolar.library.yale.edu/ymtdl Recommended Citation Fereydooni, Arash, "Inhibition Of The Akt1-Mtorc1 Axis Alters Venous Remodeling To Improve Arteriovenous Fistula Patency" (2019) Yale Medicine Thesis Digital Library 3899 https://elischolar.library.yale.edu/ymtdl/3899 This Open Access Thesis is brought to you for free and open access by the School of Medicine at EliScholar – A Digital Platform for Scholarly Publishing at Yale It has been accepted for inclusion in Yale Medicine Thesis Digital Library by an authorized administrator of EliScholar – A Digital Platform for Scholarly Publishing at Yale For more information, please contact elischolar@yale.edu Inhibition of the Akt1-mTORC1 Axis Alters Venous Remodeling to Improve Arteriovenous Fistula Patency A Thesis Submitted to the Yale University School of Medicine in Partial Fulfillment of the Requirements for the Degree of Doctor of Medicine and Master of Health Sciences By Arash Fereydooni 2020 Abstract Arteriovenous fistulae (AVF) are the most common access created for hemodialysis, but up to 60% not sustain dialysis within a year, suggesting a need to improve AVF maturation and patency In a mouse AVF model, Akt1 regulates fistula wall thickness and diameter We hypothesized that inhibition of the Akt1-mTORC1 axis alters venous remodeling to improve AVF patency Daily intraperitoneal injections of rapamycin reduced AVF wall thickness with no change in diameter Rapamycin decreased smooth muscle cell (SMC) and macrophage proliferation; rapamycin also reduced both M1 and M2 type macrophages AVF in mice treated with rapamycin had reduced Akt1 and mTORC1 but not mTORC2 phosphorylation Depletion of macrophages with clodronate-containing liposomes was also associated with reduced AVF wall thickness and both M1- and M2-type macrophages; however, AVF patency was reduced Rapamycin was associated with improved long-term patency, enhanced early AVF remodeling and sustained reduction of SMC proliferation These results suggest that rapamycin improves AVF patency by reducing early inflammation and wall thickening while attenuating the Akt1-mTORC1 signaling pathway in SMC and macrophages Macrophages are associated with AVF wall thickening and M2-type macrophages may play a mechanistic role in AVF maturation Rapamycin is a potential translational strategy to improve AVF patency Acknowledgements I am eternally indebted to my incredible mentor, Professor Alan Dardik, for his constant support and insight; he has served as an inspiring role model and showed me what it means to be a successful surgeon-scientist He has invested in my career and given me opportunities I did not deserve I am grateful to my colleagues at Dardik Lab for their help, particularly Dr Jolanta Gorecka for her teamwork and willingness serve as a valuable sounding board I would like to also thank my clinical mentors, Dr Cassius Ochoa Chaar and Dr Naiem Nassiri, for showing me what it means to be excellent academic surgeons, to deliver the best comprehensive care to our patients, and not to be afraid to push the envelope and advance the field of vascular surgery Drs Julia Chen, Christine Deyholos, Anand Brahmandam, Robert Botta, Jason Chin and Kristine Orion, I sincerely appreciate your teaching, mentorship and friendship Dr Raul Guzman, thank you for your leadership, support and encouragement I would like to thank the Howard Hughes Medical Institute, the Society for Vascular Surgery and the American Heart Association for funding my research at Dardik Lab I would also like to thank the Office of Student Research for their support with my research endeavors throughout medical school Most importantly, my journey to become a surgeon-scientist would not be possible without the sacrifices of my parents, Alireza and Naimeh, who unrooted their lives and immigrated to the United States ten years ago to provide my sisters and me with better educational opportunities This work is dedicated to them Table of Contents Introduction……………………………………………………………………………………………………………1 1.1 Poor Clinical Outcomes in Arteriovenous Fistulae Utilization……………………….1 1.2 Mechanisms of Fistula Maturation and Failure…………………………………………….1 1.3 Akt1 signaling in AVF maturation…………………………………………………………………4 Statement of Purpose and Aims…………………………………………………………………………… 2.1 Statement of Purpose 2.2 Aims Methods…………………………………………………………………………………………………………………7 3.1 Study Approval…………………………………………………………………………………………….7 3.2 Infrarenal aorto-caval fistula……………………………………………………………………… 3.3 Confirmation of fistula patency and measurement of fistula dilation………… 3.4 Histology.…………………………………………………………………………………………………….8 3.5 Immunohistochemistry and Immunofluorescence……………………………………….8 3.6 Western Blot.……………………………………………………………………………………….……10 3.7 Rapamycin and clodronate treatment………………………………………………… ……11 3.8 Adenovirus treatment……………………………………………………………………………… 12 3.9 Statistics.……………………………….………………………………………………………………… 12 Results………………………………………………………………………………………………………………….13 4.1 Reduced AVF wall thickness, extracellular matrix deposition, SMC and macrophages with rapamycin………………………………………………… ………………………13 4.2 Reduced M1- and M2-type macrophages with rapamycin…………………………15 4.3 Reduced Akt1 and mTORC1 but not mTORC2 phosphorylation with rapamycin………………………………………………… ………………………………….…………………17 4.4 Macrophage depletion is associated with reduced AVF wall thickness and patency ………………………………………………… ………………………………….……………………24 4.5 Rapamycin treatment is associated with reduced AVF wall thickness but increased AVF patency ………………………… ………………………………….……………………26 4.6 Rapamycin enhances early AVF remodeling to improve patency……………….27 Discussion…………………………………………………………………………………………………………….31 Conclusion…………………………………………………………………………………………………………….36 References……………………………………………………………………………………………………………37 Appendix………………………………………………………………………………………………………………42 1 Introduction 1.1 Poor Clinical Outcomes in Arteriovenous Fistulae Utilization Veins are frequently exposed to arterial environment by surgeons when creating arteriovenous fistulae (AVF) for hemodialysis access in end-stage renal disease (ESRD) With over half a million people affected by ESRD in the United States and a mortality of approximately 88,000 people each year, the incidence of ESRD requiring therapy is over 100,000 new cases a year.1 An AVF, which joins a vein directly to the artery is the preferred mode of hemodialysis access with demonstrated superior long-term results compared to prosthetic grafts and catheter access.2 Despite the known superiority, AVF are still far from perfect; they must mature, e.g dilate, thicken and increase flow prior to use However AVF can fail to mature in ~30% of cases3 and even if matured correctly, primary AVF failure occurs in ~35-40% in just the first year.4 These poor clinical results of AVF reflect our imperfect understanding of how the vein adapts to the arterial environment and clearly shows that our knowledge gap creates an unmet medical need for novel approaches to enhance venous adaptation.4-6 The Society of Vascular Surgery recently published enhancing AVF maturation and durability as one of its highest and most critical clinical research priorities.7 1.2 Mechanisms of Fistula Maturation and Failure Following AVF creation, the vein is exposed to a high flow and shear stress, low pressure arterial environment, leading to “maturation” of both the arterial inflow and venous outflow segments – a process necessary to sustain the high flow rates required for a successful dialysis session Adaptation of the vein to the increased flow and shear stress requires dilation and outward remodeling of the venous wall This process is accomplished by a delicate balance of extracellular matrix (ECM) remodeling, inflammation, growth factor secretion, and cell adhesion molecule upregulation in all three layers of the venous wall.8-11 During fistula maturation, the ECM of the venous limb exhibits changes as an adaptive response to the “arterialized” environment.12 These changes can be categorized in to three temporal phases; early phase (breakdown), transition phase (reorganize) and late phase (rebuild) The early phase is characterized by an increased ratio of matrix metalloproteinase (MMP) to tissue inhibitor of metalloproteinase (TIMP), which results in degradation of collagen and elastin scaffolds, allowing for easier cell migration during the transition and late phases Reorganization of scaffolds and rebuilding of the ECM with larger non-collagenous and glycoproteins such as fibronectin occur after the breakdown phase to allow for complete fistula maturation.13 While ECM degradation is regulated by MMP, its deposition is modulated by transforming growth factor-β (TGF-β).14 Diverse cell types in the venous wall, such as endothelial cells (EC), smooth muscle cells (SMC), and inflammatory cells produce TGF-β and its expression is upregulated during both early and late phases of AVF maturation While local inflammation of the vessel wall is necessary for successful fistula maturation, elevated systemic inflammatory markers predict fistula failure.9,15 Locally, macrophages and T-cells play an important role in AVF maturation, with maturation being promoted by M2 type macrophage and a lack of T cell activity resulting in AVF maturation failure Furthermore, presence of CD4+ T-cells in mature AVF coincides with the presence of macrophages, and the absence of mature T-cells results in reduced macrophage infiltration.16,17 Systemic inflammation has been shown to negatively correlate with AVF maturation, and higher levels of C-relative protein increase the risk of AVF failure Further, prednisolone, a drug with anti-inflammatory properties, enhances venous outward remodeling.18 Use of paclitaxel, a chemotherapeutic and immunosuppressive agent, during drug-coated balloon angioplasty leads to inhibition of neointimal hyperplasia (NIH) and has shown encouraging 6-month patency rates.19-21 However, increased infection rates have become a major concern for paclitaxel use in AVF.22 Successful AVF maturation relies on venous wall thickening and outward remodeling in order to support flow rates required for successful hemodialysis AVF failure occurs via distinct mechanisms; early fistula failure occurs secondary to lack of outward remodeling or wall thickening, while late failure occurs as a result of development of NIH and impaired outward remodeling in a previously functional conduit.23 Unfortunately primary maturation and patency rates of AVF remain low Up to 60% of AVF fail to mature by months after creation, and literature shows primary patency rates of 60% at year and 51% at years, with secondary patency rates of 71% at year and 64% at years.5,24,25 Factors such as diabetes mellitus, peripheral vascular disease, congestive heart failure, and older age are poor prognostic factors for successful AVF placement.26 Furthermore, studies have demonstrated prolonged maturation time, decreased patency, and increased early thrombosis of AVF in female patients, differences not accounted for by smaller vein size in females.27-29 1.3 Akt1 signaling in AVF maturation Erythropoietin-producing hepatocellular carcinoma (Eph) receptors with ephrins, their ligands, play an essential role in vascular development and determine arterial versus venous identities.30,31 Eph receptor activation leads to downstream signaling via the PI3K-Akt pathway, resulting in cell migration and proliferation, functions critical for venous remodeling.32,33 Specifically, Eph-B4 modulates adaptation and AVF maturation with distinct patterns of altered vessel identity.34-36 During successful AVF maturation, the venous limb gains expression of ephrin-B2 and has increased Eph-B4 expression, relative to control veins, suggesting acquisition of dual arterial-venous identity.12 Although the route of ephrin-B2 signaling during AVF maturation remains unknown, it must be membrane bound and circulating endothelial progenitor cells can be a source.37 In vivo, Eph-B4 activation attenuates Akt1 phosphorylation leading to reduced venous wall thickening, reduced outward remodeling and improved long-term patency rates This was corroborated with constitutively active-Akt1 studies which lead to increased venous wall thickening and dominant negative-Akt1 studies which lead to reduced outward remodeling.36 Therefore, it is proposed that Eph-B4 can regulate venous remodeling via an Akt1-mediated mechanism.36 Moreover, Akt1 expression is upregulated during venous remodeling, both during vein graft adaptation,38 as well as during AVF maturation, a consistent response to two different hemodynamic environments;36 during AVF maturation, Akt1 regulates both venous wall thickening as well as dilation.36 Mammalian target of rapamycin (mTOR) is a key regulatory protein that integrates signals from several pathways including the Akt1 pathway to modulate 36 Conclusion In conclusion, rapamycin improves AVF patency and early venous remodeling while reducing wall thickening and early inflammation These effects are associated with reduced Akt1-mTORC1 signaling in macrophages and SMC during the early maturation phase and sustained reduction in SMC during the late maturation phase Macrophages are essential for AVF remodeling and M2 macrophages may have a mechanistic role in AVF maturation The mTORC1 pathway is a key regulator of AVF maturation and its inhibition with rapamycin may be a translational strategy to improve AVF patency 37 References Collins AJ, Foley RN, Chavers B, et al 'United States Renal Data System 2011 Annual Data Report: Atlas of chronic kidney disease & 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