The Role of Mechanosensitive KLK10 in Endothelial Biology and Atherosclerosis Pubblico
Williams, Darian (Fall 2021)
Abstract
Introduction: Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified Kallikrein-Related Peptidase 10 (KLK10, a secreted serine protease) as a flow-sensitive gene in arterial endothelial cells, but its role in endothelial biology and atherosclerosis was unknown.
Methods and Results: Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo mouse models and in vitro studies with cultured endothelial cells (ECs). Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated KLK10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced inflammation and permeability dysfunction in human artery ECs (HAECs). Further, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of KLK10-expressing plasmids inhibited atherosclerosis in ApoE-/- mice. Studies using pharmacological inhibitors and siRNAs revealed that the anti-inflammatory effects of KLK10 were mediated by a Protease Activated Receptors (PAR1/2)-dependent manner. However, unexpectedly, KLK10 did not cleave the PARs. Through a proteomics study, we identified HTRA1 (High-temperature requirement A serine peptidase 1), which bound and cleaved KLK10. Further, siRNA knockdown of HTRA1 prevented KLK10’s anti-inflammatory and barrier protective function in HAECs, suggesting that HTRA1 regulates KLK10 function. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques.
Conclusion: KLK10 is a flow-sensitive endothelial protein and, in collaboration with HTRA1, serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.
Table of Contents
TABLE OF CONTENTS
ACKNOWLEDGMENTS VI
LIST OF FIGURES IX
LIST OF TABLES X
SUMMARY 1
1. INTRODUCTION 3
1.1 VASCULAR HEMODYNAMICS AND SHEAR STRESS IN ENDOTHELIAL BIOLOGY 7
1.2 MECHANOSENSORS IN THE ENDOTHELIUM 9
1.3 REGULATION OF FLOW-SENSITIVE NON-CODING GENES 14
1.4 IN VIVO MODELS OF ATHEROSCLEROSIS 35
1.5 IN VITRO MODELS OF SHEAR STRESS 37
1.6 KLK10 AND THE KALLIKREINS 39
2. SPECIFIC AIMS AND HYPOTHESES 48
2.1 SIGNIFICANCE AND IMPACT 48
2.2 RATIONALE 49
2.3 INNOVATION 51
2.4 PROJECT OBJECTIVE 52
2.5 OVERALL HYPOTHESIS 52
2.6 SPECIFIC AIM 1 52
2.7 SPECIFIC AIM 2 54
2.8 SPECIFIC AIM 3 56
2.9 POTENTIAL SIGNIFICANCE 57
3. MATERIALS AND METHODS 58
3.1 CELL CULTURE 58
3.2 SHEAR STRESS EXPERIMENTS 59
3.3 PARTIAL CAROTID LIGATION AND ENDOTHELIAL RNA ENRICHMENT 61
3.4 EN FACE PREPARATION AND IMMUNOHISTOCHEMICAL STAINING OF MOUSE ARTERIES 62
3.5 IMMUNOHISTOCHEMICAL STAINING OF HUMAN CORONARY SECTIONS 63
3.6 QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QPCR) 64
3.7 RECOMBINANT KLK10 PRODUCTION IN CHO-K1 CELLS AND TREATMENT OF ENDOTHELIAL CELLS IN VITRO 65
3.8 TRANSFECTION OF NUCLEIC ACIDS IN VITRO 66
3.9 ENDOTHELIAL FUNCTIONAL ASSAYS 66
3.10 PREPARATION OF WHOLE-CELL LYSATE AND IMMUNOBLOTTING 68
3.12 PAR CLEAVAGE ASSAYS 70
3.13 KLK10 AFFINITY PULLDOWN USING TRICEPS 71
3.14 PROXIMITY LIGATION ASSAY 71
3.15 RKLK10:HTRA1 CLEAVAGE ASSAY AND MASS SPECTROMETRY ANALYSIS OF KLK10 CLEAVAGE PRODUCTS 72
3.16 IN-GEL DIGESTION 72
3.17 MASS SPECTROMETRY 73
3.18 PROTEIN IDENTIFICATION 73
3.19 SERUM LIPID ANALYSIS 74
3.20 KLK10 ELISAS 74
3.21 SINGLE-CELL RNASEQ AND ATACSEQ 74
3.22 STATISTICAL ANALYSES 78
4. DETERMING THE ROLE OF KLK10 IN FLOW-MEDIATED EC DYSFUNCTION 80
4.1 INTRODUCTION 80
4.2 RESULTS 82
4.3 SUMMARY 96
4.4 DISCUSSION 97
5. ASSESSING THE THERAPETIC POTENTIAL OF KLK10 IN ATHEROSCLEROSIS 99
5.1 INTRODUCTION 99
5.2 RESULTS 101
5.3 SUMMARY 108
5.4 DISCUSSION 109
6. INVESTIGATING THE KLK10 MECHANISM OF ACTION 111
6.1 INTRODUCTION 111
6.2 RESULTS 113
6.3 SUMMARY 122
6.4 DISCUSSION 124
7. DISCUSSION 127
7.1 SUMMARY 127
7.2 CONCLUSIONS 130
7.3 FUTURE DIRECTIONS 130
APPENDIX 134
REFERENCES 141
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