TSG-6: AN INDUCIBLE MEDIATOR OF PARACRINE ANTI-INFLAMMATORY AND MYELOPROTECTIVE EFFECTS OF ADIPOSE STEM CELLS Jie Xie Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Cellular & Integrative Physiology, Indiana University December 2012 Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy Keith L March, M.D., Ph.D., Chair Hal E Broxmeyer, Ph.D Doctoral Committee Irina Petrache, M.D November 13, 2012 Matthias A Clauss, Ph.D   ii DEDICATION This work is dedicated to my wife Ru, my parents Haiyuan and Yue, and my mentor Keith March and his wife Sarah To my beloved wife Ru, thank you for indulging my obsession with research and endless hours in the lab With my less than minimum income, I cannot imagine the stress, difficulties, and frustrations you have fought against and overcome throughout the years to make ends meet, and make our home a harbor of comfort when I needed it most Your unconditional love is the fire in my heart that keeps me warm on rainy days and gives me strength and courage to stand up against any challenge in front To Mom and Dad, thank you for instilling in me the faith in hardworking, honesty, and self-discipline, and for supporting my pursuit of a wildest dream I know how hard it is to see other people’s children at home with the family during spring festival while your only son is thousands of miles away I want you to know you are also part of this achievement To Sarah, with six children, two horses, two dogs, one cat in the house, you must have used some magic so that Keith could be there when I needed his guidance most To Keith, I truly enjoyed those sleepless nights in your basement before deadlines and early morning discussions at Starbucks Thank you for caring not only my research but also me as a person, and for always respecting, encouraging, and supporting my interest, ideas, and decisions   iii ACKNOWLEDGEMENTS My journey to a PhD degree in a country, which I have yet to learn about its society and culture, has truly been an adventure filled with surprises, challenges, victories, defeats, and, above all, unforgettable memories In retrospect, I feel blessed and grateful that I have come across many wonderful companions along this bumpy road Knowing and working with my idol, mentor, and friend Dr Keith March in the past four years is an experience I will hold dear in my life I owe my deepest gratitude to him for seeing the best in me and bringing them into blossom Thanks to his exceptional mentorship, my training extends far beyond bench side to other crucial aspects of science: grant application, peer paper review, scientific writing, academic communication and networking, research collaboration, and teaching Following his footsteps, I had a fascinating, exciting, and fruitful journey in stem cell research My gratitude also goes to other mentors in my research committee, Dr Hal Broxmeyer, Dr Irina Petrache, and Dr Matthias Clauss I owe them my earnest gratitude for holding their standards high and always inspiring and cultivating independence, critical thinking, and open mindedness in my training I thank them for devoting their enthusiasm and expertise to my research and leading me to one after another exciting scientific discoveries I also would like to thank current and former members of the ICVBM, especially of March, Clauss, Petrache, Broxmeyer, Murphy, Srour, Rosen, and Gangaraju Labs, as well as my off-campus collaborators: Dr Darwin Prockop at   iv Texas A&M Health Science Center, Dr Katalin Mikecz at Rush University, and Dr Mikhail Kolonin at University of Texas Health Science Center We work together like one big family My research would not have reached its fullest potential without help from each and every one of you I will cherish all the tears and laughter we have shared over the years I also feel grateful to all other faculties and staff in the graduate program, especially Monica Henry, Dr Joseph Bidwell, Dr Simon Rhodes, Dr Mervin Yoder, Dr Stephen Trippel, Dr Patricia Gallagher, Dr Johnathan Tune, and Dr Michael Sturek They cheer for my progress and caution me of pitfalls in front They are my guardian angels I also thank American Heart Association and Cryptic Masons Medical Foundation for generously supporting my research at this hard economic time I also feel blessed to have met all the friends in Indy over the years, whose companionship I thoroughly enjoyed Your friendship has fulfilled my life with plenty of joy, happiness, and thankfulness   v ABSTRACT Jie Xie TSG-6: AN INDUCIBLE MEDIATOR OF PARACRINE ANTI-INFLAMMATORY AND MYELOPROTECTIVE EFFECTS OF ADIPOSE STEM CELLS Tumor necrosis factor-induced protein (TSG-6) has been shown to mitigate inflammation Its presence in the secretome of adipose stem / stromal cells (ASC) and its role in activities of ASC have been overlooked This thesis described for the first time the release of TSG-6 from ASC, and its modulation by endothelial cells It also revealed that protection of endothelial barrier function was a novel mechanism underlying the anti-inflammatory activity of both ASC and TSG-6 Moreover, TSG-6 was found to inhibit mitogen-activated lymphocyte proliferation, extending the understanding of its pleiotropic effects on major cell populations involved in inflammation Next, enzyme-linked immunosorbent assays (ELISA) were established to quantify secretion of TSG-6 from human and murine ASC To study the importance of TSG-6 to specific activities of ASC, TSG-6 was knocked down in human ASC by siRNA Murine ASC from TSG-6-/- mice were isolated and the down-regulation of TSG-6 was verified by ELISA The subsequent attempt to determine the efficacy of ASC in ameliorating ischemic limb necrosis and the role of TSG-6, however, was hampered by the highly variable ischemic tissue necrosis in the BALB/c mouse strain   vi Afterwards in a mouse model of cigarette smoking (CS), in which inflammation also plays an important role, it was observed, for the first time, that 3-day CS exposure caused an acute functional exhaustion and cell cycle arrest of hematopoietic progenitor cells; and that 7-week CS exposure led to marked depletion of phenotypic bone marrow stem and progenitor cells (HSPC) Moreover, a dynamic crosstalk between human ASC and murine host inflammatory signals was described, and specifically TSG-6 was identified as a necessary and sufficient mediator accounting for the activity of the ASC secretome to ameliorate CS-induced myelotoxicity These results implicate TSG6 as a key mediator for activities of ASC in mitigation of inflammation and protection of HSPC from the myelotoxicity of cigarette smoke They also prompt the notion that ASC and TSG-6 might potentially play therapeutic roles in other scenarios involving myelotoxicity Keith L March, M.D., Ph.D., Chair     vii TABLE OF CONTENTS LIST OF TABLES x   LIST OF FIGURES xi   LIST OF ABBREVIATIONS xiii   Chapter Introduction   1.1 Inflammation and Mesenchymal Stem Cells   1.2 Tumor Necrosis Factor-Stimulated Gene (TSG-6)   1.3 Cigarette Smoking-induce Myelotoxicity as an Inflammatory Disease Model 12   1.4 Mesenchymal Stem Cells and Hematopoiesis 14   Chapter Inflammation Control through TSG-6: Dynamics at the Perivascular Niche 16   2.1 Introduction 16   2.2 Materials and Methods 17   2.3 Results 22   2.4 Conclusion 37   2.5 Discussion 38   Chapter Quantitation of TSG-6 Secretion from ASC and Investigation of TSG-6 Role in Rescuing Hindlimb Ischemia 43   3.1 Introduction 43   3.2 Materials and Methods 44   3.3 Results 52   3.4 Conclusion 70   3.5 Discussion 70   Chapter Rejuvenation of Smoking-induced Bone Marrow Progenitor Exhaustion: Moving from Adipose-Derived Stem Cells to Their Secretome 72 4.1 Introduction 72   4.2 Materials and Methods 73   4.3 Results 82   4.4 Conclusion 105   4.5 Discussion 106     viii Chapter Future Directions 110   5.1 Anti-inflammatory Aspect of ASC & TSG-6 110   5.2 Myeloprotective Aspect of ASC & TSG-6 111   References 114   Curriculum Vitae   ix LIST OF TABLES Table The sequences of the polymerase chain reaction (PCR) amplification primers 81   x 59 60 61 62 63 64 65 66 67 68 69 70 71 72   Cao TV, La M, Getting SJ, Day AJ, Perretti M Inhibitory effects of tsg-6 link module on leukocyte-endothelial cell interactions in vitro and in vivo Microcirculation 2004;11:615-624 Lee J, Taneja V, Vassallo R Cigarette smoking and inflammation: Cellular and molecular mechanisms Journal of dental research 2012;91:142-149 Erhardt L Cigarette smoking: An undertreated risk factor for cardiovascular disease Atherosclerosis 2009;205:23-32 Csiszar A, Podlutsky A, Wolin MS, Losonczy G, Pacher P, Ungvari Z Oxidative stress and accelerated vascular aging: Implications for cigarette smoking Frontiers in bioscience : a journal and virtual library 2009;14:3128-3144 Patel RR, Ryu JH, Vassallo R 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and systemic injury induced by cigarette smoking Am J Respir Crit Care Med 2011;183:215225 Braber S, Henricks PA, Nijkamp FP, Kraneveld AD, Folkerts G Inflammatory changes in the airways of mice caused by cigarette smoke exposure are only partially reversed after smoking cessation Respir Res 2010;11:99 Grassi D, Desideri G, Ferri L, Aggio A, Tiberti S, Ferri C Oxidative stress and endothelial dysfunction: Say no to cigarette smoking! Current pharmaceutical design 2010;16:2539-2550 Cacciola RR, Guarino F, Polosa R Relevance of endothelial-haemostatic dysfunction in cigarette smoking Current medicinal chemistry 2007;14:1887-1892 126 156 157 158 159 160 161 162 163 164 165   Michael Pittilo R Cigarette smoking, endothelial injury and cardiovascular disease International journal of experimental pathology 2000;81:219-230 Rohrabaugh SL, Hangoc G, Kelley MR, Broxmeyer HE Mad2 haploinsufficiency protects hematopoietic progenitor cells subjected to cell-cycle stress in vivo and to inhibition of redox function of ape1/ref-1 in vitro Experimental hematology 2011;39:415-423 Hagiwara E, Takahashi KI, Okubo T, Ohno S, Ueda A, Aoki A, Odagiri S, Ishigatsubo Y Cigarette smoking depletes cells spontaneously secreting th(1) cytokines in the human airway Cytokine 2001;14:121-126 Hersey P, Prendergast D, Edwards A Effects of cigarette smoking on the immune system Follow-up studies in normal subjects after cessation of smoking The Medical journal of Australia 1983;2:425-429 Griffin MD, Ritter T, Mahon BP Immunological aspects of allogeneic mesenchymal stem cell therapies Human gene therapy 2010;21:16411655 Marconi S, Castiglione G, Turano E, Bissolotti G, Angiari S, Farinazzo A, Constantin G, Bedogni G, Bedogni A, Bonetti B Human adipose-derived mesenchymal stem cells systemically injected promote peripheral nerve regeneration in the mouse model of sciatic crush Tissue engineering Part A 2012;18:1264-1272 Merfeld-Clauss S, Gollahalli N, March KL, Traktuev DO Adipose tissue progenitor cells directly interact with endothelial cells to induce vascular network formation Tissue Eng Part A 2010 Terashima T, Wiggs B, English D, Hogg JC, van Eeden SF The effect of cigarette smoking on the bone marrow Am J Respir Crit Care Med 1997;155:1021-1026 Sakalihasan N, Delvenne P, Nusgens BV, Limet R, Lapiere CM Activated forms of mmp2 and mmp9 in abdominal aortic aneurysms Journal of vascular surgery 1996;24:127-133 Xiong W, MacTaggart J, Knispel R, Worth J, Persidsky Y, Baxter BT Blocking tnf-alpha attenuates aneurysm formation in a murine model J Immunol 2009;183:2741-2746 127 Curriculum Vitae Jie Xie Education/Training 2008-2012 Ph.D., Major: Cellular & Integrative Physiology Minor: Health Sciences Indiana University, Indianapolis, IN Advisor: Keith L March, M.D., Ph.D 2003-2008 M.D Peking Union Medical College, Beijing, China 2000-2003 Premedicine Peking University, Beijing, China Memberships 2009-Present American Physiological Society 2010-Present International Federation for Adipose Therapeutics and Science 2011-Present Indiana Physiological Society 2011-Present Society for Experimental Biology and Medicine Professional Service 2011 Reviewer for Journal of Leukocyte Biology Teaching 2011 Mentor for rotation Senior Undergraduate Student (Krista Podell, University of Indianapolis, IN) 2011 Mentor for Biomedical Engineering Senior Design Group (Alisa Beal, etc Purdue University, IN)       2010 Mentor for rotation Medical Student (Jennifer Bell, Lake Erie College of Osteopathic Medicine, FL) Assisted Grant Writing 2012 Applicant: Michael P Murphy, M.D.; Keith L March, M.D., Ph.D Title Attenuation of Abdominal aortic aneurysm with mesenchymal stem cells Funding R01, National Institute of Health Outcome Unknown 2012 Applicant: Keith L March, M.D., Ph.D Title Restoration of Bone Marrow Hematopoietic Function Impaired by Cigarette Smoking: Novel Therapy Based on Adipose Stromal Cells and the Secreted Factor TSG6 Funding Bone Marrow Failure Research Program Idea Award, Department of Defense Outcome Unfunded 2011 Applicant: Keith L March, M.D., Ph.D Title Impact of Cigarette Smoking on Mobilization of Bone Marrow   Stem and Progenitor Cells and Possible Treatment Funding Hypothesis Development Award, Department of Defense Outcome Unfunded 2011 Applicant: Irina Petrache, M.D.; Keith L March, M.D., Ph.D Title Direct and Bone-Marrow Mediated Effects of Adipose Stem Cells in Emphysema Funding R01, National Institute of Health Outcome Funded       2011 Applicant: Keith L March, M.D., Ph.D Title Adipose-derived Stem Cells Inexpensively Produce a Cocktail of Novel and Broad-Spectrum Anti-pathogenic Agents Funding Grand Challenges Explorations, Bill & Melinda Gates Foundation Outcome Unfunded 2011 Applicant: Rajashekhar Gangaraju, Ph.D.; Keith L March, M.D., Ph.D Title Neuronal Repair and Vascular Stabilization with Adipose Stromal Cells Funding Young Investigator Grants, Alcon Research Institute Outcome Unfunded 2009 Applicant: Keith L March, M.D., Ph.D Title Vasculogenic Potential of Adipose Stromal Cells: Provision of Tissue Perfusion and Islet Survival in Health, Aging, and Diabetes Funding VA Merit Review Grant, Department of Veterans Affairs Outcome Funded 2009 Applicant: Keith L March, M.D., Ph.D Title Adipose Stromal Cells as a Novel Therapy for Ionizing Radiation Toxicity: Evaluation in Murine Radiation-induced Bone Marrow Aplasia Funding Research Support Funds Grant, IUPUI Office of the Vice Chancellor for Research Outcome     Unfunded Grant Support 2010-2012 PI: Jie Xie, M.D Mentor: Keith L March, M.D., Ph.D Title Cigarette Smoking effects on Marrow Stromal Cells and Angiogenesis: Modulation by Adipose Stromal Cell Transplantation Funding Predoctoral Fellowship (11PRE5730005) American Heart Association Midwest Affiliation Awards And Honors 2012 Caroline tum Suden/Hellebrand Professional Opportunity Award American Physiological Society 2012 The Kenneth L Barker Young Investigator Award Society for Experimental Biology and Medicine 2012 Research Day Poster Award Indiana University 2011 Graduate Student Travel Award Indiana University 2008 Biomedical Gateway Fellowship Indiana University 2005 Outstanding Student Award Peking Union Medical College Conference Presentations Podium Presentation: Correcting Smoke-Induced Bone Marrow Dysfunction: Moving from Adipose-Derived Stem Cells to Their Secretome Jie Xie, Dongni Feng, Todd G Cook, Mary J Van Demark, Kelly S Schweitzer, Brian H Johnstone, Irina Petrache, Hal E Broxmeyer, Keith L March 10th Annual Symposium on Adipose Stem Cells and Clinical Applications of Adipose Tissue; Quebec, Canada, 2012   Poster: Human adipose-derived stem cells attenuate cigarette smoke induced bone marrow hypoplasia via secretion of anti-inflammatory cytokine TSG6 Jie Xie, Kelly S Schweitzer, Brian H Johnstone, Todd G Cook, Dongni Feng, Marjorie E Albrecht, Yong Gao, Matthew J Justice, Scott H Cooper, Hal E Broxmeyer, Irina Petrache, Keith L March Annual Experimental Biology Meeting; San Diego, 2012 Podium Presentation: Hepatocyte Growth Factor / c-Met Receptor Autocrine Loop is Essential for the Resistance of Adipose-Derived Stem Cells to Reactive Oxygen Species Jie Xie, Brian H Johnstone, Dongni Feng, Keith L March 9th Annual Symposium on Adipose Stem Cells and Clinical Applications of Adipose Tissue; Miami, 2011 Podium Presentation: Human Adipose-Derived Stem Cells Protect Against Cigarette-Smoke Induced Bone Marrow Hypoplasia through Paracrine Factors Jie Xie, Kelly S Schweitzer, Brian H Johnstone, Todd G Cook, Dongni Feng, Marjorie E Albrecht, Yong Gao, Matthew J Justice, Scott H Cooper, Hal E Broxmeyer, Irina Petrache, Keith L March 9th Annual Symposium on Adipose Stem Cells and Clinical Applications of Adipose Tissue; Miami, 2011 Abstract: Protection of mouse pancreatic islet viability from hypoxia induced injury by adipose derived-stromal cells conditioned media Dongni Feng, Jie Xie, Brian H Johnstone, Dmitry O Traktuev, Carmella Evans-Molina, Keith L March 9th Annual Symposium on Adipose Stem Cells and Clinical Applications of Adipose Tissue; Miami, 2011 Poster: Secretion of Anti-inflammatory Protein TSG6 from Adipose Stromal Cells is Suppressed by Neighboring Endothelial Cells Jie Xie, Ru Yi, Brian H Johnstone, Dmitry O Traktuev, Stephanie Merfeld-Clauss, Keith L March 1st Annual Indiana Physiological Society Meeting; Indianapolis, 2010     Patent Jie Xie and Keith L March Protection Against Chemical-Induced or Other (i.e., Endogenous) Myelosuppression Based on the Protein TSG6, Adipose Stromal Cells (ASC), or ASC Conditioned Medium April, 2012 (IURTC 12171) Publications Xie J, Broxmeyer HE, Feng D, Van Demark M, Yi R, Cook TG, Chitteti BR, Barwinska D, Traktuev DO, Johnstone BH, Srour EF, Petrache I, March KL Rejuvenation of Smoking-induced Murine Bone Marrow Progenitor Exhaustion: Moving from Adipose-Derived Stem Cells to Their Secretome (In preparation) Xie J, Yi R, Jester A, Feng D, Traktuev D, Johnstone BH, Murphy MP, Clauss MA, March KL Inflammation Control through TSG6: Dynamics at the Perivascular Niche (In preparation) Xie J, Feng D, Deuter-Reinhard M, Johnstone BH, March KL Hepatocyte Growth Factor / c-Met Receptor Autocrine Loop is Essential for the Survival and Potency of Adipose-Derived Stem Cells (In preparation) Hong SJ, Rogers PI, Kihlken J, Warfel J, Bull C, Deuter-Reinhard M, Feng D, Xie J, Choy JS, Kyle A, Merfeld-Clauss S, Johnstone BH, Traktuev DO, Chen P, Lindner JR, March KL Intravenous Xenogeneic Transplantation of Human Adipose-Derived Stem Cells Improves Left Ventricular Function and Microvascular Integrity in Swine Myocardial Infarction Model (In revision)   ... days and gives me strength and courage to stand up against any challenge in front To Mom and Dad, thank you for instilling in me the faith in hardworking, honesty, and self-discipline, and for... mean ± standard error (SEM) Comparisons between two groups were made with the use of analysis of variance (ANOVA) and Newman-Keuls post-hoc analysis A p value of < 0.05 was considered significant... whose companionship I thoroughly enjoyed Your friendship has fulfilled my life with plenty of joy, happiness, and thankfulness   v ABSTRACT Jie Xie TSG-6: AN INDUCIBLE MEDIATOR OF PARACRINE ANTI-INFLAMMATORY