Mechanosensitive miRNA targets TIMP3 to increase matrix degradation in the aortic valve endothelium Open Access

Khambouneheuang, Lucky (2016)

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Heart disease, including calcified aortic valve disease (CAVD), remains the leading cause of death worldwide. The role of endothelial cells (ECs) in CAVD remains relatively unknown; however, disease develops in a side-dependent manner. Preferentially, calcification occurs on the fibrosa endothelium, which is subjected to disturbed blood flow, whereas the ventricularis endothelium, which is subjected to stable blood flow, is relatively unaffected. Our research goal is to discover mechanisms, especially a new type of small molecules (known as microRNAs) by which mechanical force regulates endothelial inflammation and the resulting aortic valve (AV) calcification. We hypothesize that miRNA181b is upregulated by disturbed flow at the AV fibrosa endothelium, leading to decreased expression of TIMP3 and increased matrix degradation. Several microRNAs (miRNAs) have been shown to be mechanosensitive (flow-sensitive), and thus may play a role in the development of AV calcification. Using human aortic valve endothelial cells (HAVECs) in our in vitro cone-and-plate system that simulates different blood flow conditions, laminar shear (LS) versus oscillatory shear (OS), we have found several miRNAs that are regulated by flow, including miRNA-181b. Compared to HAVECs subjected to LS, HAVECs in OS demonstrated significant upregulation in miRNA-181a and -181b expression. Using miRTarBase, we further identified and studied miRNA-181b targets, including genes GATA6, SIRT1, and TIMP3. We also found significantly decreased GATA6, SIRT1, and TIMP3 expression in HAVECs subjected to OS stress. Moreover, our gelatinase assay significantly confirmed increased MMP activity and extracellular matrix degradation in HAVECs subjected to OS. We further transfected HAVECs with pre- and anti-miRNA-181b to study the direct relationship between miRNA-181b and TIMP3. For pre-miRNA-181b treated samples, we observed significant decrease in expression for GATA6, SIRT1, and TIMP3 in HAVEC in OS. For anti-miRNA-181b treated HAVECs, we observed significant decreased MMP activity compared to control samples. Our study overall correlatively demonstrates that in ECs subjected to OS, miRNA-181b is upregulated, leading to the decrease expression of TIMP3 and increased ECM degradation. In the future, our ongoing work may provide insight into novel therapeutic treatments targeting specific mechanosensitive miRNAs to treat AV stenosis and disease.

Table of Contents

Introduction. 1

Clinical Significance of Calcific Aortic Valve Disease. 1

Aortic Valve Structure, Cellular Components, and Functions. 2

Aortic Valve Calcification. 5

Biogenesis of microRNAs. 7

Foundational studies. 9

Objective of study. 10

Materials and Methods. 13

Cells and Cell Culture. 13

Shear stress using cone-and-plate system. 14

Isolation of total RNA. 15

cDNA synthesis & quantitative Polymerase Chain Reaction (qPCR). 15

Gelatinase Assay. 17

miRNA-181b Transfection. 18

Statistical Analysis. 18

Results. 19

LS/OS stress modulates miRNAs and target mRNA in HAVECs. 19

Differential MMP activity and ECM degradation in LS/OS HAVECs. 20

Validating TIMP3 as a miRNA-181b target. 20

Discussion. 27

References. 33

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