Aortic Valve Mechanobiology- Role of Altered Hemodynamics in Mediating Aortic Valve Inflammation and Calcification
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Calcific aortic valve (AV) disease is a strong risk factor for cardiovascular related deaths and is a significant source of mortality worldwide, with the number of patients requiring AV surgery expected to increase from 250,000 to 850,000 by 2050. However, the molecular mechanisms underlying AV disease have not been well studied or understood. Further, identification of biomarkers that can be used to detect early stage AV disease is also understudied but vital to successfully preventing and/or treating AV disease. It was hypothesized that sclerosis, inflammation and calcification preferentially occurs in the fibrosa compared to the ventricularis due to differential gene expression and oscillatory shear stress. Freshly isolated porcine AV leaflets and an ex vivo shear stress bioreactor was used to test this hypothesis. The low magnitude oscillatory shear stress (OS) appeared to predispose fibrosa to side-dependent calcification via increasing collagen turnover (Col1a1), and thickening of the extracellular matrix (ECM) (fibrosis) and decreasing the expression of genes that protect endothelial function (Klf4 and Enos). The unidirectional pulsatile shear, LS, however, preserved the ECM and gene expression in the ventricularis. The involvement of miRNAs in OS mediated AV pathogenesis was also investigated in a shear- and side-dependent manner. The miR-214 was found to play a role in this OS induced pathogenesis in fibrosa but not ventricularis. Using an ex vivo miRNA silencing protocol, anti-miRNA was delivered to both endothelial and interstitial cells of the AV tissue without compromising the cell viability. Silencing of miR-214 showed that the OS induced pathology in the fibrosa is likely to be mediated via miR-214, klf4 and Tgfβ1 dependent pathway that can lead to AV fibrosis, endothelial-to-mesenchymal transition and eventually sclerosis. The miR-214, however, did not play a role in shear-induced inflammation and calcification. The miR-214, as such, is likely to play a key role in the early onset of side- and shear- dependent AV disease and has a potential to serve as a disease biomarker. Further, an ex vivo AV calcification model was also developed to understand the role of endogenous pro- and anti-calcification factors, such as inorganic pyrophosphate, orthophosphate, and alkaline phosphatase. The functional studies carried out in this dissertation aim to link the mechanosensitive miRNAs to the genes involved in inflammation, endothelial-to-mesenchymal transition, and cell apoptosis etc, which eventually causes AV leaflets to calcify. Thus improved understanding of AV disease mechanisms under different hemodynamic conditions will enable us to improve the design of tissue-engineered valves and develop non-surgical treatment options.