Title Page
Contents
ABSTRACT - Korean 11
ABSTRACT - English 12
CHAPTER 1. INTRODUCTION 13
1.1. Vascular endothelium 13
1.2. Endothelial dysfunction 13
1.3. Vascular inflammatory diseases and endothelial phenotypes 14
1.3.1. Endothelial pro-inflammatory phenotype 14
1.3.2. EC-proinflammatory stimuli 17
1.4. Endothelial cell response to shear stress 20
CHAPTER 2. SHEAR STRESS AND VASCULAR ENDOTHELIUM 22
2.1. Shear stress on vascular endothelium 22
2.1.1. Effects of shear stress on leukocyte adhesion 22
2.1.2. Effects of shear stress on vascular permeability 23
2.1.3. Effects of disturbed flow on EndMT 24
2.1.4. Effects of disturbed flow on glucose metabolism 24
2.2. Vascular inflammatory diseases 27
2.2.1. Atherosclerosis 27
2.2.2. Pulmonary Arterial Hypertension 30
2.2.3. Sepsis 32
2.2.5. References 35
CHAPTER 3. HYPOTHESIS AND AIMS 46
3.1. Fibronectin and vascular remodeling 46
3.1.1. PFKFB3 and vascular inflammation 48
3.1.2. JAM-A and vascular inflammation 48
3.1.3. References 50
CHAPTER 4. MATERIALS AND METHODS 52
4.1. Cell culture 52
4.2. Application of flow 52
4.3. Monocyte adhesion assay 53
4.4. Western blotting 54
4.5. Immunostaining 54
4.6. Antibody 54
4.7. Plasmids. 55
4.8. RNA interference 55
4.9. Generation of knockout cells using CRISPR/Cas9 genome editing 56
4.10. Nuclear fractionation 57
4.11. Statistics 58
CHAPTER 5. PFKFB3 ROLE ON ECM-DEPENDENT VASCULAR INFLAMMATION 59
5.1. ABSTRACT 59
5.2. Nonstandard Abbreviations and Acronyms 60
5.3. Novelty and Significance 61
5.3.1. What Is Known? 61
5.3.2. What New Information Does This Article Contribute? 61
5.4. INTRODUCTION 62
5.5. RESULTS 63
5.5.1. Disturbed flow induces upregulation of PFKFB3, which promotes endothelial inflammation 63
5.5.2. ECM regulates PFKFB3 phosphorylation and localization 66
5.5.3. Fibronectin mediated dephosphorylation of PFKFB3 induces inflammatory responses 69
5.5.4. Flow induced inflammation was inhibited by cytoplasmic mutant or kinase inactive mutant of PFKFB 73
5.6. Discussion 75
5.7. References 78
CHAPTER 6. ROLE OF JAM-A IN VASCULAR INFLAMMATION 82
6.1. Abstract 82
6.1.2. Nonstandard Abbreviations and Acronyms 83
6.2. Introduction 84
6.3. Results 85
6.3.1. Fibronectin promotes flow induced dephosphorylation of JAM-A at S285 85
6.3.2. Inhibition of JAM-A reduced the disturbed flow induced vascular inflammation 88
6.3.3. JAM-A mediated monocyte adhesion was independent of NFKB and YAP 90
6.3.4. Disturbed flow induces vascular inflammation via changes in JAM-A phosphorylation 91
6.4. Discussion 93
6.5. References 95
CURRICULUM VITAE 98
Figure 2.1. Effects of oscillatory shear stress on vascular endothelium 26
Figure 2.2. EC dysfunction and the progression of vascular diseases 34
Figure 4.1. Experimental setup of oscillatory shear stress 53
Figure 5.1. ECM dependent changes on PFKFB3 localization and vascular inflammation. 60
Figure 5.2. Disturbed flow induced NFKB activation was reduced by PFKFB3 knock-down. 65
Figure 5.3. Fibronectin promotes dephosphorylation of PFKFB3 at S461 and induces nuclear localization. 68
Figure 5.4. Fibronectin mediated dephosphorylation of PFKFB3 induced inflammatory responses. 72
Figure 5.5. Flow induced inflammation was inhibited by cytoplasmic mutant or kinase inactive mutant of PFKFB3. 75
Figure 6.1. The role of JAM-A in vascular inflammation 83
Figure 6.2. Fibronectin promotes dephosphorylation of JAM-A at S285 87
Figure 6.3. Inhibition of JAM-A reduced the disturbed flow induced vascular inflammation 89
Figure 6.4. JAM-A mediated monocyte adhesion was independent of NF-κB and YAP. 90
Figure 6.5. Disturbed flow induces vascular inflammation via changes in JAM-A phosphorylation. 92