The Role of Mechanosensitive KLK10 in Endothelial Biology and Atherosclerosis Open Access

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

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Physiology Biology, Cell Biology, Molecular
Keyword	Atherosclerosis Mechanobiology Endothelial Inflammation HTRA1 Flow KLK10
Committee Chair / Thesis Advisor	Hanjoong Jo, Emory University
Committee Members	Wilbur Lam, Emory University John Hepler, Emory University Stephen Traynelis, Emory University Morley Hollenberg, University of Calgary, Alberta

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