Dynamic Immune Cell Accumulation Corresponds with Vascular Inflammation during Disturbed Flow-Induced Atherogenesis in Mouse Carotid Artery Open Access

Alberts-Grill, Noah Milton (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/3j3332499?locale=en
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Abstract

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the United States. The leading cause of CVD is the progressive growth of lipid-laden plaques within the large arteries of the body, a process known as atherosclerosis. This inflammatory disease is characterized by the infiltration of immune cells into the artery wall at sites that are chronically exposed to disturbed patterns of blood flow (d-flow). Immune cells accumulate within the artery wall where they establish the sustained, local inflammation required to drive plaque development. While the immunogenesis of atherosclerosis has been extensively studied over the past decade or so, the relationship between d-flow and immune cell function in the vasculature remains poorly understood. In order to overcome this knowledge gap, we used a partial carotid ligation model to generate d-flow in the left common carotid artery (LCA), which induces the rapid development of atherosclerosis within 2 weeks along the entire length of LCA in apolipoprotein E-deficient (ApoE-/-) mice fed a high-fat diet (HFD). We developed a 10-fluorochrome, 13-parameter, flow cytometry-based immunophenotyping method to quantify the infiltration of 7 major classes of immune cells into flow-disturbed LCA during rapid atherogenesis. Using this method we provide the first quantitative description of vascular leukocyte number and composition over the entire lifespan of murine atherosclerosis, and showed that d-flow induces rapid and dynamic accumulation of monocyte/macrophages, dendritic cells, granulocytes, NK cells, and T-cells within 4 days. Leukocyte numbers peaked by 7 days, preceding atheroma development in LCA by day 14 days post-ligation. qPCR and ELISA arrays showed that leukocyte infiltration corresponds with the onset of progressive pro-inflammatory cytokine and chemokine expression in the artery wall, demonstrating the close link between d-flow, vascular leukocyte recruitment, and atherosclerosis. Flow cytometry analysis of peripheral blood leukocytes (PBLs) in our model showed that d-flow powerfully inhibits hyperlipidemia-induced leukocytosis in ligated versus sham-operated controls, suggesting that localized d-flow can influence immune function on a systemic level, independent of classical atherosclerosis risk factors such as hyperlipidemia. Together, our studies establish partial carotid ligation as an important experimental model to study the interface between vascular physiology, immunopathology, and atherosclerosis.

Table of Contents

Chapter 1: General Introduction 1

1.1 Atherosclerosis, disturbed flow, and inflammation 2

1.2 Immune cells and atherosclerosis 4

1.3 DC function in atherosclerosis 13

1.4 Characterizing vascular DC subsets 15

1.5 DC function in vascular homeostasis 24

1.6 Summary of vascular DC function 32

1.7 Partial carotid ligation model of atherosclerosis 34

1.8 References 38

Chapter 2: Dynamic immune cell accumulation during flow-induced atherogenesis in mouse carotid artery: An expanded flow 69

2.1 Introduction 70

2.2 Methods 72

2.3 Results 85

2.4 Discussion 105

2.5 References 110

Chapter 3: Disturbed flow induces sustained leucopenia during atherogenesis in hyperlipidemic ApoE-/- mice 118

3.1 Introduction 119

3.2 Methods 121

3.3 Results 124

3.4 Discussion 129

3.5 References 135

Chapter 4: The effect of Cathepsin S deficiency on leukocyte recruitment and atherosclerosis in the flow-disturbed carotid artery 140

4.1 Introduction 141

4.2 Methods 144

4.3 Results 150

4.4 Discussion 162

4.5 References 167

Chapter 5: Fms-like tyrosine kinase 3 ligand treatment fails to control disturbed flow-induced atherosclerosis in ApoE-/- mice 173

5.1 Introduction 174

5.2 Methods 177

5.3 Results 181

5.4 Discussion 186

5.5 References 191

Chapter 6: Discussion 198

6.1 A brief overview of the role of immune responses in atherosclerosis 199

6.2 Summary of experiments and findings 201

6.3 Future directions 207

6.4 Contributions to the field 211

6.5 References 214

INDEX OF FIGURES

Figure 1.1 Advanced atheroma morphology in 3 and 4 week flow-disturbed atherosclerotic lesions 36

Figure 2.1 Partial carotid ligation procedure and arterial leukocyte preparation 74

Figure 2.2 Fluorescence minus one gating controls and single-color stain controls for 13-parameter flow cytometry method 77

Figure 2.3 Development of a thirteen-parameter immunophenotyping study of leukocytes in the murine carotid artery 79

Figure 2.4 Thirteen-parameter, 10-channel flow cytometry staining in splenocytes and peripheral blood leukocytes 86

Figure 2.5 Comparison of carotid and aortic lesions using the 13-parameter flow cytometry method 87

Figure 2.6 Dynamic infiltration of leukocytes into LCA in response to disturbed flow 88

Figure 2.7 Sample quality and leukocyte viability remain fairly consistent between cell preparations from early and developed plaques 90

Figure 2.8 Dynamic infiltration of leukocytes into LCA in response to disturbed flow 91

Figure 2.9 Disturbed flow induces a transient peak accumulation of innate cells, sustained T-cell accumulation, and delayed B-cell entry into LCA 93

Figure 2.10 Disturbed flow induces accumulation of macrophages, DCs, and T-cells in LCA 95

Figure 2.11 Dynamic changes in expression of cytokines and chemokines in LCA by disturbed flow 96

Figure 2.12 Cytokine and chemokine gene expression is upregulated by disturbed flow in partially-ligated LCA 97

Figure 2.13 PLSR modeling distinguishes time sensitive changes in cytokine and chemokine gene expression following partial carotid ligation 103

Figure 2.14 PLSR modeling links IFNg expression with increased leukocyte accumulation in flow-disturbed arterial wall at 7 days, preceding increased inflammation and plaque growth at 14 days post-ligation 104

Figure 3.1 Gating strategy for leukocyte phenotyping panel 124

Figure 3.2 Flow disturbance abrogates hyperlipidemia-induced leukocytosis 125

Figure 3.3 Flow disturbance induces prolonged lymphopenia 126

Figure 3.4 Disturbed flow-induced atherosclerosis development reduces circulating numbers of innate immune cells 128

Figure 4.1 Gating strategy for leukocyte phenotyping panel 147

Figure 4.2 Gating strategy for T-cell phenotyping panel 148

Figure 4.3 CatS deficiency disrupts leukocyte recruitment into flow-disturbed LCA 151

Figure 4.4 CatS deficiency disrupts innate and adaptive immune cell recruitment into flow-disturbed LCA 155

Figure 4.5 Hypercholesterolemia rapidly induces systemic CD4 T-cell responses 157

Figure 4.6 CatS is required for the induction and maintenance of T-cell responses under hypercholesterolemic conditions 159

Figure 4.7 CatS deficiency fails to suppress pro-atherogenic cytokine production capacity in flow-disturbed LCA 160

Figure 4.8 CatS deficiency fails to inhibit atherosclerosis induced by disturbed flow 161

Figure 5.1 Gating strategy for DC flow cytometry panel 179

Figure 5.2 D-flow preferentially recruits CD11b+ Mo-DCs, but not CD103+ cDCs into LCA 182

Figure 5.3 Flt3L treatment increases CD103+ cDC, and overall leukocyte numbers in LCA at 7 days post-ligation 184

Figure 5.4 Flt3L treatment fails to inhibit d-flow-induced atherosclerosis 185

Figure 5.5 Flt3L treatment increases serum HDL levels in hyper-cholesterolemic ApoE-/- mice 186

Figure 6.1 Ligation induces vascular remodeling and leukocyte infiltration in wild-type C57/Bl6 mice, but fails to cause atherosclerosis 209

Figure 6.2 Proposed experimental model for future studies 210

INDEX OF TABLES

Table 1.1 Phenotype and function of vDC subsets 17

Table 1.2 Effect of genetic knockout models on atherosclerosis 31

Table 1.3 Salient features necessary for the proper identification and characterization of vDC subsets 33

Table 2.1 Time course analyses of dynamic leukocyte accumulation in flow-disturbed LCA 89

Table 2.2 PCR array of cytokines and chemokines expressed in LCA and RCA, and their known functions 98

Table 4.1 Summary of two-tailed t-test analyses of leukocyte accumulation in LCA and RCA of ligated ApoE-/- and CatS-/-ApoE-/- mice at 7 days post-ligation 152

Table 4.2 Summary of two-tailed t-test analyses of leukocyte accumulation in LCA and RCA of ligated ApoE-/- and CatS-/-ApoE-/- mice at 14 days post-ligation 153

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