Regulation and function of heparanase in the heart by FULONG WANG B.Sc., Southeast University, 2009 M.Sc., University of Chinese Academy of Sciences, 2013 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pharmaceutical Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2018 © Fulong Wang, 2018 The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Regulation and function of heparanase in the heart submitted Fulong Wang in partial fulfillment of the requirements by for the Doctor of Philosophy degree of in Pharmaceutical Sciences Examining Committee: Brian Rodrigues, Pharmaceutical Sciences Supervisor Dan Luciani, Faculty of Medicine Supervisory Committee Member Bruce Verchere, Faculty of Medicine Supervisory Committee Member Lucy Marzban University Examiner Angela Devlin University Examiner Additional Supervisory Committee Members: David Granville, Faculty of Medicine Supervisory Committee Member Corey Nislow, Pharmaceutical Sciences Supervisory Committee Member ii Abstract Enzymatically-active heparanase (HepA) has been implicated as an essential metabolic adaptation in the heart following diabetes. However, the regulation of the enzymatically- inactive heparanase (HepL) remain poorly understood. We hypothesized that in response to high glucose (HG) and secretion of HepL from the endothelial cell (EC), HepL uptake and function can protect the cardiomyocyte by modifying its cell death signature. HG promoted both HepL and HepA secretion from EC, with subsequent uptake of HepL into cardiomyocytes. This occurred through a low-density lipoprotein receptor-related protein 1 (LRP1) dependent mechanism, as LRP1 inhibition significantly reduced uptake. Exogenous addition of HepL to rat cardiomyocytes produced a dramatically altered expression of apoptosis-related genes, and protection against HG and H2O2 induced cell death. Cardiomyocytes from acutely diabetic rats demonstrated a robust increase in LRP1 expression and levels of heparanase, a pro-survival gene signature, and limited evidence of cell death, observations that were not apparent following chronic and progressive diabetes. We also tested if overexpression of heparanase can protect the heart against chemically induced or ischemia/reperfusion (I/R) injury. Mice overexpressing heparanase (Hep-tg) displayed physiological cardiac hypertrophy and changes in expression of genes related to the stress response, immune response, cell death, and development. These transcriptomic alterations were associated with promotion of unfolded protein response (UPR), autophagy, and oxidative stress resistance in a pro-survival direction. The UPR activation was adaptive and not apoptotic, and together with mTOR inhibition, induced autophagy. Subjecting wild type mice to thapsigargin evoked a transition from adaptive iii to apoptotic UPR, an effect attenuated in Hep-tg hearts. When exposed to I/R, infarct size and apoptosis were significantly lower in the Hep-tg heart, an effect reversed by inhibitors of UPR and autophagy. Our results highlight EC-to-cardiomyocyte transfer of heparanase to modulate the cardiomyocyte cell death signature. This mechanism was observed in the acutely diabetic heart, and its interruption following chronic diabetes may contribute towards the development of diabetic cardiomyopathy. Moreover, we established that the mechanisms by which heparanase promotes cell survival in cancer could be uniquely beneficial to the heart and exploited as a therapeutic target for the treatment of heart disease. iv Lay Summary Heparanase is an enzyme that contributes to the survival of cancer cells under harsh and stressful conditions like radioactive-therapy and chemotherapy and thus contributes towards the aggressiveness of cancer development. The heart cells, contrary to cancer cells, are vulnerable to different types of stresses. This, combined with the limited capability of this organ to regenerate new cells following cell death, leads to the prevalence of heart diseases. We explored the possibility of utilizing the properties of this enzyme to protect cardiac cells against cell death. We found out that heparanase, using its properties to promote cell survival, protected the heart cells against multiple stresses frequently seen in patients with ischemia and diabetes induced heart diseases, in both cell experiments and animal studies. This research could help devise new strategies to combat heart diseases. v Preface This thesis is written by F. Wang and reviewed by Dr. Rodrigues. All the experiments in this thesis were designed and conceived by F. Wang under the supervision of Dr. Rodrigues. Studies in chapter 3 has been previously published (F. Wang, J. Jia, N. Lal, D. Zhang, A. P. Chiu, A. Wan, I. Vlodavsky, B. Hussein, B. Rodrigues. 2016. High glucose facilitated endothelial heparanase transfer to the cardiomyocyte modifies its cell death signature. Cardiovascular Research). F. Wang designed all the experiments and wrote the manuscript with Dr. Rodrigues. D. Zhang, B. Hussein, J. Jia, N. Lal, R. Shang, A. Wan, Y. Wang, and AP. Chiu participated in data analysis and manuscript editing. Dr. Vlodavsky provided the recombinant heparanase. Data in chapter 4 is currently submitted (Fulong Wang, Yanzhi Carolyn Jia, Dahai Zhang, Boris Trinajstic, Jocelyn Jia, Nathaniel Lal, Karn Puri, Rui Shang, Stephane Flibotte, Sunita Sinha, Purvi Trivedi, Dipsikha Biswas, Kathleen Macleod, Corey Nislow, Israel Vlodavsky, Thomas Pulinilkunnil, Bahira Hussein, and Brian Rodrigues. 2018. Heparanase Overexpression Protects Mouse Heart against Chemical or Ischemia/Reperfusion Injury. Submitted). D. Zhang, B. Hussein, J. Jia, N. Lal, R. Shang, A. Wan, Y. Wang, and AP. Chiu contributed in editing the manuscript. S. Flibotte and S. Sinha performed the RNAseq of ventricle and analyzed the data. YZ-C. Jia performed the echocardiology. Dr. Vlodavsky provided the heparanase transgenic mice. Dr. Pulinilkunnil assisted in conceiving some of the experiments and did part of the ischemia/reperfusion experiments. B. Hussein provided assistance with monitoring the animals, technical support and editing the manuscript. The study conforms to the guide for the care and use of laboratory animals published by the US National Institutes of Health, and was approved vi by the Animal Care Committee in the University of British Columbia (Certificate No. A13- 0250). vii Table of Contents Abstract ........................................................................................................................................... iii Lay Summary .................................................................................................................................... v Preface ............................................................................................................................................ vi Table of Contents .......................................................................................................................... viii List of Tables ................................................................................................................................... xi List of Figures ................................................................................................................................. xii List of Abbreviations and Acronyms ............................................................................................ xiv Acknowledgements .................................................................................................................... xviii Chapter 1: Introduction .................................................................................................................. 1 1.1 Heparan sulfate proteoglycans (HSPGs) ..................................................................... 1 1.2 Heparanase .................................................................................................................. 2 1.3 Heart disease ............................................................................................................... 5 1.3.1 Heart disease ............................................................................................... 5 1.3.2 Diabetic cardiomyopathy (DCM) ................................................................. 6 1.3.3 Ischemic heart diseases ............................................................................... 6 1.4 Unfolded protein response (UPR) and heart disease ................................................. 9 1.4.1 UPR ............................................................................................................... 9 1.4.1 UPR and heart diseases ............................................................................. 12 1.5 Heart disease and autophagy .................................................................................... 14 1.5.1 Introduction ............................................................................................... 14 1.5.2 Processes of autophagy ............................................................................. 15 1.5.3 Regulation of autophagy ........................................................................... 17 1.5.4 Physiological and pathophysiological roles of autophagy ....................... 18 1.5.5 The balance between autophagy and apoptosis .....................................