FUNCTIONAL CHARACTERIZATION OF ISTHMIN, A NOVEL SECRETED PROTEIN IN ANGIOGENESIS XIANG WEI B.Sc, Wuhan University A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2010   Acknowledgement I will like to express my deepest and most sincere gratitude to my supervisor, Associate Professor Ge Ruowen, for her continuous support, invaluable guidance and encouragement throughout the research work and writing of the thesis. Her intellectual contribution and logical thinking process have enhanced my knowledge, which have been of great value to me. I will also like to express my earnest thanks to Professor Kini, for his helpful suggestions to my project, unwavering support as well as expert opinions on protein expression and purification. The gratitude also goes out to the people from Professor Kini’s laboratory, for their help in the protein purification work. I acknowledge with appreciation the work of Dr. Zhang Yong on the identification of ISM receptor. I also owe my gratitude to the following undergraduate students, Grace Ho-Yuet Cheng and Ishak Darryl Irwan for their involvement in the cell assays. Thanks go to all members, both past and present, from my laboratory for their kindness, assistance and freindship. They are: Dr. Soheila, Dr. Soluchana, Dr. Farooq, Dr. Ke Zhiyuan, Nilesh, Li Yan, Tan Lu wee, Jinghui, Jingyu, Huapeng, Sun Wei, Yalu, Saran, Nithya, Winnie, Zhenyun and Chaojin etc. I also truly acknowledge the research scholarship from National University of Singapore (NUS) and research fund from the Biomedical Research Council (BMRC). Finally, I will like to thank my dear husband Tan Swee Jin, for his understanding, support and love throughout my study. To my beloved parents, Xiang Caigao and Li Zhongnian whose boundless care and support enable me to complete this work. I wish that we will share the delight of my accomplishment soon.   i    TABLE OF CONTENTS ACKNOWLEDGEMENT . I  TABLE OF CONTENTS .II  SUMMARY XI  LIST OF PUBLICATIONS RELATED TO THIS STUDY XIII  LIST OF FIGURES XIV  LIST OF TABLES .XVII  ABBREVIATIONS XVIII  CHAPTER ONE: INTRODUCTION .1  1.1 Angiogenesis 1  1.1.1 Angiogenesis in life, diseases and medicine 3  1.1.2 Tumor angiogenesis and tumor development 4  1.1.3 Anti-angiogenic cancer therapy .6  1.2 Angiogenesis regulators .9  1.2.1 Pro-angiogenic factors .9  1.2.1.1 Vascular endothelial growth factor (VEGF) family 10  1.2.1.2 Fibroblast growth factor (FGF) family 15  1.2.2 Endogenous inhibitors of angiogenesis .16  1.2.2.1 Gene Products .17  1.2.2.2 Natural protein fragments 23  1.2.2.3 Others 24  1.3 Angiogenesis and integrins .25  ii    1.4 Angiogenesis and focal adhesions 29  1.5 Tumor angiogenesis and macrophage as well as matrix metalloproteinases (MMPs) .31  1.6 In vitro angiogenesis assays and in vivo models of angiogenesis used in the study 34  1.6.1 In vitro angiogenesis assays .34  1.6.2 The Directed In vivo Angiogenesis Assay (DIVAA) 36  1.6.3 Tumor angiogenesis using syngenic mouse tumor model and stably modified tumor cell lines 37  1.6.4 Embryonic angiogenesis using zebrafish model 38  1.7 Thrombospondin type repeat (TSR) domain .39  1.8 Adhesion-associated domain in MUC-4 and other proteins (AMOP) domain 41  1.9 Isthmin (ISM) .42  1.10 Thrombospondin and AMOP containing isthmin-like (TAIL) .43  1.11 Aim of this study 44  CHAPTER TWO: MATERIALS AND METHODS .45  2.1 Cell Culture .45  2.1.1 Isolation of Human Umbilical Vein Endothelial Cells (HUVECs) .45  2.1.2 Culture of cell lines and primary cell .46  2.1.3 Preservation of HUVECs and tumor cell lines 47  2.1.4 Quantification of cell number 47  2.2 DNA cloning techniques 48  2.2.1 Polymerase chain reaction (PCR) 48  2.2.2 DNA isolation 48  2.2.3 DNA gel electrophoresis 49  2.2.4 DNA ligation 50  2.2.5 Restriction endonuclease digestion of plasmid DNA 50  iii    2.2.6 Transformation .50  2.2.7 DNA sequence analysis .51  2.2.8 Vectors used .52  2.3 RNA isolation .54  2.3.1 RNA extraction from tissues 54  2.3.2 RNA extraction from culture cells .55  2.4 Reverse transcriptase-PCR .56  2.5 Real-time RT-PCR .56  2.6 Whole mount in situ hybridization on zebrafish embryos 57  2.6.1 Linearization of plasmid DNA .57  2.6.2 Probe synthesis and precipitation .58  2.6.3 Quantification of labeled probe .58  2.6.4 Preparation of zebrafish embryos 58  2.6.5 Proteinase K treatment .59  2.6.6 Prehybridization .59  2.6.7 Hybridization .60  2.6.8 Post-hybridization 60  2.6.9 Preparation of pre-absorbed DIG .60  2.6.10 Incubation with pre-absorbed antibodies .61  2.6.11 Color development .61  2.6 12 Mounting and photography 62  2.7 Protein isolation 63  2.7.1 Protein isolation from cell lysate .63  2.7.2 Protein isolation from tumor tissues 64  2.7.3 Collection of conditioned medium .64  2.8 Expression and purification of recombinant ISM proteins .65  iv    2.8.1 IPTG induction and recombinant protein expression 65  2.8.2 Protein purification 65  2.8.3 Determination of protein concentration .66  2.8.4 Detection of protein endotoxin 66  2.9 In vitro cell assays 67  2.9.1 Acute cytotoxicity assay 67  2.9.2 EC In vitro capillary network formation 67  2.9.3 EC migration assay 68  2.9.4 EC attachment and spreading assay .68  2.9.5 EC proliferation assay 69  2.9.6 EC apoptosis assay .70  2.9.7 Binding assay .71  2.10 Western Blotting .71  2.10.1 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 72  2.10.2 Gel transfer .73  2.10.3 Immunoprobing and detection .73  2.10.4 Stripping and re-probe .74  2.11 Immunocytochemistry 74  2.12 Immunoprecipitation(IP) 75  2.12.1 Antibody conjugation .75  2.12.2 Lysate preclear and IP 76  2.13 Transfection 76  2.13.1 Determination of zeocin sensitivity of tumor cells 76  2.13.2 Lipid transfection .77  2.13.3 Selection of stable expression clones .77  2.14 In vivo pathological angiogenesis models 78  v    2.14.1 Animals 78  2.14.2 Directed In Vivo Angiogenesis Assay 78  2.14.3 Subcutaneous tumor model 79  2.15 Immunohistochemistry .79  2.15.1 Fixation 79  2.15.2 Embedding .79  2.15.3 Sectioning 80  2.15.4 Dewax and rehydration 80  2.15.5 Antigen retrieval 81  2.15.6 Immunohistochemistry 81  2.15.7 TUNEL 81  2.15.8 Microvessel density (MVD) 82  2.16 In vivo physiological angiogenesis model 83  2.16.1 Zebrafish maintenance .83  2.16.2 Microinjection of morpholino oligonucleotide (MO) into embryos 83  2.16.3 Design of MO 84  2.17 Statistical analysis .84  2.18 Lists of primers and morpholino oligos 84  CHAPTER THREE: RESULTS PART I .86  3. Characterization of the role of ISM EC cell angiogenesis .86  3.1 Generation of recombinant mouse ISM and its truncated proteins 86  3.1.1 Comparison of ISM Proteins in vertebrates .86  3.1.2 Cloning, expression and purification of recombinant mouse ISM and its truncated fragments in E.coli 87  3.1.3 Determination of endotoxin level in recombinant ISM proteins .89  3.1.4 Determination of acute cytotoxicity of the recombinant ISM proteins .90  vi    3.2 ISM inhibited various aspects of angiogenesis in vitro 92  3.2.1 ISM inhibited in vitro capillary network formation in a time-dependent manner .92  3.2.2 ISM had no effect on VEGF, bFGF or serum stimulated EC migration .95  3.2.3 ISM did not interfere with EC attachment and spreading onto ECM 99  3.2.4 ISM inhibited VEGF, bFGF or serum-stimulated EC proliferation 103  3.2.5 ISM stimulated EC apoptosis in the presence of VEGF, bFGF or serum .105  3.3 Different anti-angiogenic activities of ISM require different functional domain 107   3.3.1 Only ISM-C inhibited in vitro capillary network formation in a timedependent manner .107  3.3.2 Truncated ISM proteins did not influence EC migration .109  3.3.3 ISM truncates had no effect on EC attachment to matrix 110  3.3.4 ISM-N and ISM-C mildly inhibited VEGF-stimulated EC proliferation .111  3.3.5 None of ISM truncates induced EC apoptosis .112  3.4 The effect of ISM on other cell types .114  3.4.1 ISM mildly inhibited serum-stimulated fibroblast proliferation 114  3.4.2 ISM did not influence serum-stimulated tumor cell proliferation .116  3.4.3 ISM marginally induced fibroblast apoptosis 117  3.4.4 ISM did not affect tumor cell apoptosis .119  3.5 ISM but not ISM-C inhibited angiogenesis in vivo 121  CHAPTER FOUR: RESULTS PART II 124  4. ISM inhibited angiogenesis through multiple mechanisms .124  4.1 Interaction between ISM and integrin αvβ5 .125  4.1.1 ECs bind to immobilized ISM and ISM-C but not ISM-N 125  4.1.2 ISM bound to ECs through integrin αvβ5 127  vii    4.2 ISM disrupted EC focal adhesions .130  4.2.1 ISM inhibited VEGF-stimulated FAK phosphorylation 131  4.2.2 ISM inhibited paxillin relocation into EC focal adhesions 132  4.2.3 ISM inhibited VEGF-induced actin stress fiber formation 134  4.3 ISM induced EC apoptosis through Caspase -dependent pathway 136  4.3.1 Caspase inhibitor abolished the ability of ISM in inducing EC apoptosis .136  4.3.2 ISM promoted EC Caspase activation .137  CHAPTER FIVE: RESULTS PART III .139  5. Characterization of the role of ISM in tumor angiogenesis in mouse .139  5.1 ISM expression in human and mouse .139  5.1.1 Expression analyses of ISM in human tissues and tumors 139  5.1.2 Expression analyses of ISM in mouse tissue .141  5.2 Establishment of stable cell lines overexpressing ISM 143  5.3 In vitro characteristics of ISM overexpressing B16 cells .147  5.3.1 Overexpression of ISM did not affect B16 cells proliferation in vitro 147  5.3.2 Overexpression of ISM did not affect apoptosis of B16 cells in vitro .147  5.4 Overexpression of ISM in B16 cells suppressed tumor growth via inhibiting tumor angiogenesis .149  5.4.1Tumor growth was reduced in ISM-overexpressing tumors .149  5.4.2 Microvessel density was reduced in ISM-overexpressing B16 tumors .152  5.5 Investigating the mechanisms of how ISM inhibited B16 tumor growth and angiogenesis 154  5.5.1 Tumor cell proliferation was not altered in ISM-overexpressing B16 tumors .154  5.5.2 Tumor cell apoptosis was increased in ISM-overexpressing B16 tumors .156  viii    5.5.3 Infiltration of tumor associated macrophages (TAMs) was reduced in ISM-overexpressing B16 tumors 158  5.5.4 VEGF expression was not affected in tumors that overexpress ISM 159  CHAPTER SIX: RESULTS PART IV 160  6. Characterization of the role of ISM in embryonic angiogenesis in zebrafish 160  6.1 Bioinformatic analyses of ISM gene(s) in zebrafish 160  6.1.1 Sequence analyses of zebrafish ism, ism2 and LOC100002267 160  6.1.2 Phylogenetic analyses of zebrafish Ism1, Ism2 and LOC100002267 .163  6.1.3 Synteny analyses of zebrafish ism, tail1a and tail1b .164  6.2 Expression analyses of zebrafish ism, tail1a and tail1b .168  6.2.1 Temporal and spatial expression analysis 168  6.2.2 Adult Tissue expression pattern analyses 173  6.3 Functional study of ism, tail1a and tail1b in zebrafish embryonic angiogenesis 175  6.3.1 ism was required for proper embryonic growth, development and survival .175  6.3.2 Knockdown of ism led to angiogenic defects 179  6.3.3 Knockdown of tail1a had no obvious effect on gross embryonic morphology and vascular development 182  6.3.4 Knockdown of tail1b had no obvious effect on gross morphology and vascular development 185  6.3.5 Double knockdown of ism, tail1a or tail1b .188  CHAPTER SEVEN: DISCUSSION .192  7.1 The position of ISM in angiogenesis inhibitors known so far 194  7.2 The role of ISM in physiological angiogenesis 198  7.3 The role of ISM in pathological angiogenesis 203  7.4 Possible mechanisms of action of ISM in angiogenesis .207  ix    Dhanabal M, Ramchandran R, Waterman MJ, Lu H, Knebelmann B, Segal M, Sukhatme VP. 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Crit Care. 2005; 9: 66-75.   240    [...]... conceivable that many genes regulating angiogenesis in vivo have yet to be discovered In this study, we aim to identify a novel endogenous angiogenesis inhibitor and characterize its function in in vivo angiogenesis Isthmin (ISM) is a secreted 60 kDa protein containing a Thrombospondin Type 1 Repeat (TSR) domain and an Adhesion-associated domain in MUC4 and Other Proteins (AMOP) domain with no known... growth in various organs including kidney, bone, heart and retina (Eremina, et al., 2003,Maes, et al., 2002,Stalmans, et al., 2002) VEGF binds to VEGFR1 and VEGFR2, but mediates its biological functions mainly via stimulating VEGFR2 (Shibuya, 2008) As shown in Fig 1.3, VEGF stimulate angiogenesis via activating several important intracellular signaling pathways It induces EC proliferation through activation... activation of Erk pathway, and inhibits EC apoptosis via Akt/PKB pathway The Akt/PKB pathway regulates EC apoptosis by inhibiting pro-apoptotic molecules such as BAD and Caspase 9 The Akt/PKB pathway also activates endothelial nitric oxide synthase (eNOS), leading to the increase in vascular permeability and EC migration Other molecules implicated in VEGF induced EC migration include p38 mitogen-activated protein. .. 2002) In addition, the degree of tumor vascularization is correlated with tumor grade as well as aggressiveness, which serves as a significant clinical prognosis indicator (Sarbia, et al., 1996,Tanigawa, et al., 1996) The angiogenic switch controlled by a net balance of positive and negative regulators, is the initiation of tumor angiogenesis( Hanahan, et al., 1996) The angiogenic cascade includes an activation... the Food and Drug Administration (FDA) of USA for the clinical use for patients with solid tumors 6    because of their capacity to improve survival in Phase III clinical studies There are Avastin (Genentech; bevacizumab), Torisel (Wyeth Corporation; temsirolimus), sunitinib (C.P Pharmaceuticals International) and Sorafenib (Bayer; Nexavar) Avastin, a human recombinant antibody that neutralizes the... relatively larger nuclei than normal ECs, indicating they have more DNA content A certain percentage of tumor ECs are karyotypically aneuploid (e.g 16% of liposarcoma ECs, 34% of melanoma ECs, 54% of renal carcinoma ECs) whereas normal ECs are diploid In addition, there is upregulation of adhesion molecules such as CD31 or ICAM-1 in lung carcinoma ECs compared to normal ECs (Hida, et al., 2008) Morphologically,... insulin-like growth factor-1 interferons interleukins isthmin intersegmental vessels mitogen-activated protein kinase monocyte chemotactic protein- 1 macrophage colony stimulating factor matrix metalloproteinase morpholino oligonucleotide neuropilin posterior cardinal vein pigment epithelium derived factor platelet factor-4 phosphatidylinositol 3 kinase thrombospondin and AMOP containing isthmin-like... like domains, five calcium-binding and a globular carboxyl-terminal end TSP-1 is an inhibitor of angiogenesis in vitro and in vivo and a potent suppressor of malignant growth (Chen, et al., 2000) Experiments in vitro performed by many laboratories have been shown that TSP-1 inhibits EC migration, proliferation and stimulates EC apoptosis (Iruela-Arispe, et al., 1991) Take advantage of the fact that the... protein kinase (MAPK) and focal adhesion kinase (FAK) as well as paxillin (Cross, et al., 2003) Aparting from these, VEGF also induces vasodilation through the release of eNOS and prostaglandins (Ferrara, et al., 2003) Recently, the effects of VEGF on the lymphatic vasculature have been reported The lymphangiogenic activities of VEGF seem to be linked to the recruitment of inflammatory cells, such as macrophages,... MUC-4 and other proteins basic fibroblast growth factor chick chorioallantoic membrane chondromodulin-I dorsal aorta the directed in vivo angiogenesis assay dorsal longitudinal anastamotic vessels endothelial cell extracellular matrix epidermal growth factor engelbreth-holm-swarm endothelial nitric oxide synthase focal adhesion kinase green fluorescent protein human umbilical vascular endothelial cell insulin-like . in in vivo angiogenesis. Isthmin (ISM) is a secreted 60 kDa protein containing a Thrombospondin Type 1 Repeat (TSR) domain and an Adhesion-associated domain in MUC4 and Other Proteins (AMOP). its various domains in in vitro angiogenesis 197 xviii  Abbreviations ADAMTS a disintegrin and metalloproteinases with thrombospondin motifs AMOP adhesion-associated domain in MUC-4 and. Angiogenesis and focal adhesions 29 1.5 Tumor angiogenesis and macrophage as well as matrix metalloproteinases (MMPs) 31 1.6 In vitro angiogenesis assays and in vivo models of angiogenesis used in the