Modifiers of inflammatory angiogenesis in a murine model 1

ACKNOWLEDGEMENTS First and foremost, I would like to express my sincere gratitude to Professor Koh Dow Rhoon, for his advice and guidance throughout the course of my study in the lab I am greatly indebted to him for his time in engaging valuable scientific discussion and advices with me I also wish to express my sincere gratitude to Professor Hooi Shing Chuan, for his kind help and support Special thanks belong to my colleagues, especially Dr Tin Kyaw, for his scientific guidance, knowledgeable discussion and constant encouragement I also wish to thank the administrative staffs in the office of the department of Physiology, especially Ms Asha Das, who has given me support throughout the course of my study in the department of Physiology Many thanks to all the people in the Physiology department for helping me out in one way or another , and creating such a wonderful environment to work in I wish to acknowledge my deepest appreciation to my husband, my parents, and my sister who have been my constant source of encouragement and support I specially wish to dedicate the thesis to my little daughter, Sun Yidan, who gives me the strength to fulfill the project I would also like to thank Dr.Thai Tran for her time spent proofreading my thesis i TABLE OF CONTENTS Acknowledgements i Table of Contents ii Summary viii List of Tables xi List of Figures xii List of Abbreviations xv List of Publications xviii CHAPTER INTRODUCTION 1.1 Introduction 1.2 General introduction of Aniogenesis and wound healing 1.2.1 Angiogenesis I Angiogenic process II Angiogenesis and inflammatory diseases 1.2.2 Wound healing I Wound healing process II Scar formation 11 III Abnormal wound healing 12 1.3 Cytokines in angiogenesis and wound healing 1.3.1 Chemokines in angiogenesis and wound healing 14 14 ii 1.3.2 TNF-α: proinflammatory cytokine 18 1.3.3 VEGF: angiogenic factor 18 1.3.4 TGF-β1 22 1.4 Cellular response in angiogenesis and wound healing 25 1.4.1 Roles of neutrophils in angiogenesis and wound healing 25 I Neutrophil biology 25 II Neutrophil derived cytokine 32 III Roles of neutrophils in angiogenesis 35 IV Roles of neutrophils in wound healing 38 1.4.2 Roles of lymphocytes in angiogenesis and wound healing 39 1.4.3 Roles of monocyte/macrophages in angiogenesis and wound healing 40 I Monocyte/macrophage infiltlration 40 II Key role of macrophage in angiogenesis and wound healing41 1.5 Aims of the study 43 Chapter II Material and methods 45 2.1 Regents 45 2.2 Mice 47 2.3 Genotyping of mice 47 2.3.1 DNA extraction from mouse tail 47 2.3.2 PCR genotyping of mice 48 2.4 Corneal injury model 2.4.1 Introduction of corneal injury model 49 49 iii 2.4.2 Experimental design 50 I The corneal injury model in BALB/c male and female mice 51 II The corneal injury model in BALB/c and Rag1KO mice 51 III Effects of RB6-8C5 treatment on angiogenesis in the corneal injury model 51 2.4.3 Neutrophil depletion by RB6-8C5 treatment 52 2.4.4 Assessment of the degree of corneal opaciy 56 2.5 Skin injury model 56 2.5.1 Introduction of murine skin injury model 56 2.5.2 Experimental design 57 I The skin injury model in BALB/c male and female mice 57 II Skin wound healing in control, Rag1KO, RB6-8C5, and RB6-8C5 control mice 2.5.3 Neutrophil depletion in skin injury model 2.6 Immuno-histochemical technique 58 61 61 2.6.1 Immunohistochemistry staining of neutrophil, F4/80, CD3ε and CD31 61 2.6.2 Immunohistochemistry staining of VEGF in paraffin embedded slides 63 2.7 Staining technique 64 2.7.1 Blood smear preparation and evaluation 64 2.7.2 H&E staining 64 2.7.3 Giemsa staining 65 2.7.4 Trichrome staining 65 2.8 Enzye-linked immunosorbent assay (ELISA) 66 iv 2.8.1 Measurement of MCP-1 level 66 2.8.2 Measurement of MIP-1α, MIP-2, VEGF and TNF-α level 67 2.8.3 Measurement of TGF-β1 level 68 2.9 Protein assay 68 2.10 Isolation of murine neutrophils and neutrophil activation study 69 2.10.1 Preparation of murine neutrophils 69 2.10.2 Neutrophil activation study 70 2.11 Statistics 70 2.12 Buffers 70 Chapter III RESULTS 74 3.1 Introduction of corneal and skin injury model 74 3.1.1 Angiogenesis in the corneal injury model 74 3.1.2 Sexual dimorphism in angiogenesis in the corneal injury model 74 3.2 Key roles of neutrophil in angiogenesis in the corneal injury model 79 3.2.1 Lymphocytes have little impacts on angiogenesis in the corneal injury model 79 3.2.2 Key roles of neutrophils in angiogenesis in the corneal injury model 81 I Efficacy of neutrophil depletion by RB6-8C5 treatment 81 II Effects of neutrophil depletion on corneal angiogenesis 82 III Effects of neutrophil depletion on corneal inflammation 83 IV.Effects of neutrophil depletion on neutrophil infiltration 84 V Induction and localization of VEGF in cornea 91 VI Effects of neutrophil depletion Induction of MIP-1α, MIP-2, v and TNF-α in cornea 95 VII PMA-induced VEGF, MIP-1α, and MIP-2 release from murine neutrophils in a in vitro study 3.3 Important roles of neutrophils and lymphocytes in wound healing in the skin injury model 96 104 3.3.1 Important roles of lymphocytes in skin wound healing 104 3.3.2 Key roles of neutrophils in skin wound healing 105 I Efficacy of neutrophil depletion by RB6-8C5 treatment 105 II Effects of neutrophil depletion on skin wound healing 107 III Effects of neutrophil depletion on neutrophil infiltration 110 IV Effects of neutrophil depletion on macrophage infiltration 112 V Effects of neutrophil depletion on T cell infiltration 112 VI Effects of neutrophil depletion on angiogenesis 116 VII Effects of neutrophil depletion on VEGF protein level 117 VIII Effects of neutrophil depletion on the induction of MIP-1α, MIP-2, MCP-1, TNF-α, and TGF-β1 120 IX Effects of neutrophil depletion on scar formation 127 Chapter IV DISCUSSION 129 4.1 Introduction of animal models used in the current study 129 4.1.1 To study angiogenesis in the corneal and skin injury model 129 4.1.2 Sexual dimorphism in angiogenesis in the corneal injury model 130 4.2 Roles of lymphocytes in angiogenesis and wound healing 131 4.2.1 Lymphocytes have little impacts on angiogenesis vi in the corneal and skin injury models 4.2.2 Roles of lymphocytes in wound healing in the skin injury model 4.3 Important oles of neutrophils in angiogenesis and wound healing 4.3.1 The specificity of RB6-8C5 treatment in depleting neutrophils 131 132 133 133 4.3.2 Key rolel of neurophils in angiogenesis in the corneal and skin injury model 135 4.3.3 Important roles of neutrophil in wound healing in a skin injury model 141 4.4 Scar formation and inflammatory cells 148 CHAPTER V CONCLUSION 150 REFERENCES 152 vii SUMMARY Wound healing is a body’s response to injury, in which angiogenesis play a critical part Immune cells play a role in both angiogenesis and wound healing Understanding the mechanisms of wound healing, angiogenesis in the context of the immune response will help lay the foundation for better treatment of pathologies related to aberrant angiogenesis and wound healing The roles of neutrophils in angiogenesis have been implicated by previous studies However, no direct in vivo evidence relates the neutrophil to natural inflammatory angiogenesis Moreover, there are controversial results on the role of neutrophils in wound healing Similarly, although lymphocytes have been shown to produce angiogeneic factors in pathological conditions, the role of lymphocytes in natural inflammatory angiogenesis is still unclear Lymphocytes play an important part in skin wound healing, but the mechanism need further exploration In the present study, we investigated the role of neutrophils in inflammatory angiogenesis and wound healing in the corneal and skin injury model by depleting neutrophil using RB6-8C5, a neutrophil-depleting antibody We also investigated the role of lymphocytes in inflammatory angiogenesis and wound healing by establishing the corneal and skin injury model on Rag1 knock-out mice and the control mice Angiogenesis, inflammatory cell infiltration, protein levels of vascular endothelial growth factor (VEGF) macrophage inflammatory protein-1alpha (MIP-1α), macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor alpha (TNF-α) were viii investigated in the injured cornea and skin of control and RB6-8C5-treated mice An in vitro model of neutrophil activation was also used to examine the ability of neutrophils to produce and release VEGF, MIP-1α, and MIP-2 We found that enhanced protein levels of VEGF, MIP-1α, and MIP-2 correlated with the degree of neutrophil infiltration in the corneal and skin injury model Neutrophil depletion significantly inhibited angiogenesis and reduced the protein levels of VEGF, MIP-1α, and MIP-2 in the injured cornea and skin Upon stimulation, isolated neutrophils released VEGF from preformed stores and MIP-1α and MIP-2 by de novo synthesis The skin injury model was also used to study the role of neutrophils in skin wound healing We observed the wound healing rate, protein levels of monocyte chemotactic protein-1 (MCP-1), and transforming growth factor beta-1(TGF-β1) and scar formation in the skin wound healing model We found that neutrophil depletion severely impaired wound healing rate, and reduced the protein levels of TGF-β1 In the present study we found that there was no difference in angiogenesis in the corneal and skin injury model between Rag1KO and control mice This finding indicates that lymphocytes may not play a role in the inflammatory angiogenesis However, the skin wound healing was delayed in the Rag1KO mice compared with control mice There were no differences in neutrophil and monocyte infiltration, angiogenesis, and the protein levels of VEGF, MIP-1α, MCP-1 and TNF-α between the Rag1KO and control mice In the Rag1KO mice, the protein levels of MIP-2 and TGF-β1 was decreased ix In conclusion, neutrophils play an important role in the natural inflammatory angiogenesis most likely by releasing proangiogenic factors such as VEGF Neutrophils play an important role in wound healing by inducing angiogenesis and the upregulation of TGF-β1 Lymphocytes may not play a significant role in inflammatory angiogenesis They play an important role in skin wound healing, unrelated to angiogenesis x LIST OF TABLES Table 2.1 Antibodies 46 Table 2.2 Drugs used in mouse surgery care 46 Table 3.1 Peripheral blood counts in the control and RB6-8C5 treated mice 85 xi LIST OF FIGURES Fig 2.1 Corneal injury model in BALB/c male and female mice 53 Fig 2.2 Corneal injury model in BALB/c female and Rag1KO mice 54 Fig 2.3 Experiment design to study the effects of RB7-8C5 on angiogenesis 55 in the corneal injury model Fig 2.4 Skin injury model in BALB/c male and female mice 59 Fig 2.5 Skin wound healing model in control, Rag1KO, RB6-control, and 60 RB6-Rag1KO Fig 3.1 Angiogenesis response in the corneal injury model 76 Fig 3.2 Comparision of corneal angiogenesis between BALB/c female and 77 male mice Fig 3.3 Skin wound healing in BALB/c female and male mice 78 Fig 3.4 Comparision of corneal angiogenesis between BALB/c and Rag1KO 80 mice Fig 3.5 Effects of neutrophil depletion on angiogenesis in the corneal injury 86 model Fig 3.6 Effects of neutrophil depletion on microvessel density in the cornea 87 Fig 3.7 Effects of neutrophil depletion on the degree of corneal opacity 88 Fig 3.8 Infiltration of neutrophils in the corneal angiogenesis 89 Fig 3.9 Detection of VEGF in the cornea during angiogenesis 92 Fig 3.10 Time kinetics for protein levels of MIP-1α in the corneal injury 98 model xii Fig 3.11 Time kinetics for protein levels of MIP-2 in the corneal injury model 99 Fig 3.12 Time kinetics for protein levels of TNF-α in the corneal injury model 100 Fig 3.13 Time kinetics for protein levels of MCP-1 in the corneal injury 101 model Fig 3.14 Effects of PMA on the release of VEGF from murine neutrophils 102 Fig 3.15 Effects of PMA on the release of MIP-1α and MIP-2 from murine 103 neutrophils Fig 3.16 Effects of RB6-8C5 on the neutrophil differential count in control, 106 Rag1KO, RB6-control and RB6-Rag1KO mice Fig 3.17 Skin wound healing in control, Rag1KO, RB6-control and 108 RB6-Rag1KO mice Fig 3.18 Neutrophil infiltration in skin wounds in control, Rag1KO, 111 RB6-control and RB6-Rag1KO mice Fig 3.19 Monocyte infiltration in skin wounds in control, Rag1KO, 114 RB6-control and RB6-Rag1KO mice Fig 3.20 T cell infiltration in skin wounds in control and RB6-control mice 115 Fig 3.21 Angiogenesis in skin wounds in control, Rag1KO, RB6-control and 118 RB6-Rag1KO mice Fig 3.22 Time kinetics for protein levels of VEGF in skin wounds of control, 119 Rag1KO, RB6-control and RB6-Rag1KO mice Fig 3.23 Time kinetics for protein levels of MIP-1α in skin wounds of control, 122 Rag1KO, RB6-control and RB6-Rag1KO mice Fig 3.24 Time kinetics for protein levels of MIP-2 in skin wounds of control, 123 xiii Rag1KO, RB6-control and RB6-Rag1KO mice Fig 3.25 Time kinetics for protein levels of MCP-1 in skin wounds of control, 124 Rag1KO, RB6-control and RB6-Rag1KO mice Fig 3.26 Time kinetics for protein levels of TNF-A in skin wounds of control, 125 Rag1KO, RB6-control and RB6-Rag1KO mice Fig 3.27 Time kinetics for protein levels of TGF-β1 in skin wounds of 126 control, Rag1KO, RB6-control and RB6-Rag1KO mice Fig 3.28 Trichrome stain of scar formation in control and RB6-control mice 128 xiv LIST OF ABBREVIATIONS Ab antibody bFGF basic fibroblast growth factor BM basement membrane CCL CC ligand CCR CC receptor CXCL CXC ligand CXCR CXC receptor DAB 30,3-Diaminobenzidine DETC dendritic epidermal T cel DMSO dimethyl sulfoxide EC endothelial cell ECM extracellular matrix EGF epidermal growth factor EGF-R epidermal growth factor receptor FGF fibroblast growth factor FGFR fibroblast growth factor receptor G-CSF granulocyte-colony stimulating factor GM-CSF granulocyte-macrophage-colony stimulating factor HBSS hanks’ balanced salts H&E Hematoxylin & eosin HIF-1a hypoxia-inducible factor-1a HRP horseradish peroxidase xv IGF-I insulin-like growth factor-I IGF-I-R insulin-like growth factor-I receptor IFN interferon KO knock-out IL-8 interlukin-8 KO knock-out LPS Lipopolysaccharide MCP-1 macrophage chemotactic protein-1 MIP-1α macrophage inflammatory protein- 1alpha MIP-2 macrophage inflammatory protein-2 MMP matrix metalloproteinases MVD microvessel Density PA plasminogen activator PBS phosphate buffered saline PD-ECGF platelet-derived endothelial cell growth factor PDGF plateletderived growth factor PECAM-1 platelet/endothelial cell adhesion molecule 1,CD31 PEDF pigment epithelium-derived factor PDGF platelet derived growth factor PMA phorbol-12-myristate 13-acetate PMN polymorphonuclear neutrophils RA rheumatoid arthritis Rag1 recombination activating gene xvi a-SMA a-Smooth muscle actin TGF-β1 transforming growth factor-beta Th T helper lymphocyte TIMP tussue inhibitors of metalloproteinases TNF-α tumour necrosis factor alpha tPA tissue-type plasminogen activator uPA urokinase-type plasminogen activator VEGF vascular endothelial growth factor VEGFR vascular endothelial growth factor receptor VEGF-A vascular endothelial growth factor-A xvii LIST OF PUBLICATIONS Gong Y, Koh DR Neutrophils promote inflammatory angiogenesis via release of preformed VEGF in an in vivo corneal model Cell Tissue Res 2010 Feb;339(2):437-48 Gong Y, Koh DR Role of neurophil in skin wound healing (manuscript under submission) CONFERENCE PAPERS Gong Yue, Koh Dow Rhoon Wound Healing is Impaired without Fas 12th International Congress of Immunology and 4th Annual Conference of FOCIS Canada 2004 Gong Yue, Koh Dow Rhoon Mouse Model of Inflammation-induced Angiogenesis 6th NUS-NUH Annual Scientific Meeting Singapore 2002 xviii ... formation 11 III Abnormal wound healing 12 1. 3 Cytokines in angiogenesis and wound healing 1. 3 .1 Chemokines in angiogenesis and wound healing 14 14 ii 1. 3.2 TNF-α: proinflammatory cytokine 18 1. 3.3... macrophage inflammatory protein-1alpha (MIP -1? ?), macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor alpha (TNF-α) were viii investigated in the injured cornea and skin of control and... protein -1 MIP -1? ? macrophage inflammatory protein- 1alpha MIP-2 macrophage inflammatory protein-2 MMP matrix metalloproteinases MVD microvessel Density PA plasminogen activator PBS phosphate buffered