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.

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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 1 INTRODUCTION

1

1.1 Introduction

1

1.2 General introduction of Aniogenesis and wound healing

2

1.2.1 Angiogenesis

2
I. Angiogenic process

3

II. Angiogenesis and inflammatory diseases

5

1.2.2 Wound healing

6
I. Wound healing process

7

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

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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

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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

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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.

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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

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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 1

xvi


a-SMA

a-Smooth muscle actin

TGF-β1

transforming growth factor-beta 1

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

1. 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.
2. Gong Y, Koh DR. Role of neurophil in skin wound healing (manuscript under
submission)
CONFERENCE PAPERS
1. Gong Yue, Koh Dow Rhoon. Wound Healing is Impaired without Fas. 12th
International Congress of Immunology and 4th Annual Conference of FOCIS. Canada
2004
2. Gong Yue, Koh Dow Rhoon. Mouse Model of Inflammation-induced Angiogenesis. 6th

NUS-NUH Annual Scientific Meeting. Singapore 2002

xviii