The Role of Shear-Sensitive Protein Heart of Glass Homolog 1 (HEG1) in Endothelial Biology and Atherosclerosis Open Access

Abstract

Atherosclerotic diseases such as myocardial infarction, ischemic stroke, and peripheral arterial disease continue to be leading causes of death worldwide despite the success of cholesterol-lowering drugs and drug-eluting stents, raising the dire need to identify additional therapeutic targets. Interestingly, atherosclerosis preferentially develops in curved and branching arterial regions, where endothelial cells are exposed to disturbed blood flow with characteristic low-magnitude and oscillatory shear stress. In contrast, straight arterial regions exposed to stable flow, associated with high-magnitude, unidirectional shear stress, are relatively well protected from the disease through shear-dependent, atheroprotective endothelial responses, such as induction of atheroprotective protein expression. In the current study, heart of glass homolog 1 (HEG1) is identified as a stable flow-induced endothelial protein. HEG1 is an endothelial-enriched single-pass transmembrane glycoprotein that is essential for vascular development and integrity. Due to its demonstrated importance in vascular biology, and its induction by stable flow, HEG1 was hypothesized to mediate atheroprotective endothelial responses to stable flow and protect against atherosclerotic plaque formation. To test this hypothesis, mechanistic and functional responses to stable flow in endothelial cells with and without HEG1 expression were compared using an in vitro cone and plate shear system. HEG1 expression was found to be necessary for stable flow-induced Krüppel-like factor 2 and 4 (KLF2/4) expression, two critical transcription factors that control the majority of flow-sensitive endothelial genes. HEG1 expression was also required to mediate the pro-alignment, anti-inflammatory, anti-permeability, anti-migratory and anti-angiogenic effects of stable flow. Using the in vivo partial carotid ligation model of acute atherosclerosis, inducible endothelial cell HEG1 knockout mice (HEG1iECKO) were found to have decreased endothelial KLF2/4 expression, and to have more extensive atherosclerotic plaques as compared to controls. Together, these data indicate that HEG1 protein is required to mediate many atheroprotective effects of stable flow via control of KLF2/4 expression, and that HEG1 is a promising new therapeutic target for atherosclerosis.

Table of Contents

Acknowledgements ..................................................................................................................... VI

Table of Contents ....................................................................................................................... VII

List of Figures .............................................................................................................................. IX

List of Tables ................................................................................................................................. X

List of Abbreviations ................................................................................................................... XI

1 Introduction ........................................................................................................................... 1

1.1 Background ..................................................................................................................................... 1

1.1.1 Atherosclerosis ........................................................................................................................................ 1

1.1.2 Disturbed blood flow induces atherosclerosis .......................................................................................... 3

1.1.3 Flow potently regulates EC function ....................................................................................................... 7

1.1.4 Mechanosensors and mechanosignal transduction in endothelial cells ..................................................... 9

1.1.5 OMICs approaches to determine flow-sensitive endothelial genes, proteins, and pathways ................... 15

1.1.6 Therapeutic implications of flow-sensitive genes, proteins, and pathways in atherosclerosis .................. 17

1.2 Hypothesis and Specific Aims ................................................................................................. 20

1.2.1 Hypothesis – HEG1 is a flow-sensitive, atheroprotective protein in endothelial cells ............................ 20

1.2.2 Aim 1 – Determine whether endothelial HEG1 expression is flow-sensitive. ........................................ 22

1.2.3 Aim 2 – Determine the role of HEG1 in endothelial function and dysfunction. .................................... 22

1.2.4 Aim 3 – Determine the role of HEG1 in the pathogenesis of atherosclerosis. ....................................... 24

2 Materials and Methods ....................................................................................................... 25

2.1 PCL surgery ............................................................................................................................... 25

2.2 scRNA-seq and Gene Array ..................................................................................................... 25

2.3 Hybridization chain reaction (HCR) ...................................................................................... 25

2.4 Cell culture ................................................................................................................................ 26

2.5 In vitro shear stress experiments ............................................................................................. 27

2.6 qPCR .......................................................................................................................................... 27

2.7 Western blot .............................................................................................................................. 28

2.8 Immunocytochemistry ............................................................................................................. 30

2.9 siRNA knockdown.................................................................................................................... 31

2.10 Endothelial function studies .................................................................................................... 31

2.11 Plasmid overexpression and viral particle transduction ........................................................ 33

2.12 HEG1iECKO mice ........................................................................................................................ 33

2.13 Mouse atherosclerosis studies ................................................................................................. 34

2.14 Human coronary artery studies ............................................................................................... 35

2.15 Statistical analyses .................................................................................................................... 37

3 Results .................................................................................................................................. 38

3.1 Aim 1 - Determine whether endothelial HEG1 expression is flow-sensitive. ...................... 38

3.1.1 Flow controls HEG1 expression and subcellular translocation in endothelial cells ................................ 38

3.1.2 Flow-induced HEG1 transcription is promoted by increased chromatin availability and performed by transcription factors KLF2 and KLF4 ................................................................................................................. 42

3.2 Aim 2 - Determine the role of HEG1 in endothelial function and dysfunction. .................. 44

3.2.1 HEG1 mediates multiple ULS-induced functional changes in HAECs .................................................. 44

3.2.2 HEG1 mediates flow-induced KLF2/4 expression by the MEKK3-MEK5-ERK5-MEF2 pathway ...... 44

3.2.3 HEG1 controls protein level of MEKK3-inhibitor KRIT1 by co-secretion in response to flow ............ 50

3.3 Aim 3 - Determine the role of HEG1 in the pathogenesis of atherosclerosis. ..................... 53

3.3.1 HEG1iECKO mice have reduced endothelial KLF2/4 and eNOS expression and increased levels of atherosclerosis ..................................................................................................................................................... 53

3.3.2 Endothelial HEG1 expression is negatively correlated with plaque severity and hypertension in human coronary arteries .................................................................................................................................................. 58

4 Discussion ............................................................................................................................ 61

4.1 Summary .................................................................................................................................... 61

4.2 Conclusion ................................................................................................................................. 62

4.3 Future Directions ...................................................................................................................... 63

5 References ............................................................................................................................ 66

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	Health Sciences, Medicine and Surgery Biology, Molecular Engineering, Biomedical
Keyword	Mechanobiology Endothelial cells Atherosclerosis Vascular Biology
Committee Chair / Thesis Advisor	Hanjoong Jo, Emory University
Committee Members	W. Robert Taylor, Emory University Khalid Salaita, Emory University Roy Sutliff, NHLBI Kathy Griendling, Emory University

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