oa Shear- and Side-dependent microRNAs and Messenger RNAs in Aortic Valvular Endothelium

Aortic valve (AV) disease is a major cause of cardiovascular-linked deaths globally. In addition, AV disease is a strong risk factor for additional cardiovascular events; however, the mechanism by which it initiates and progresses is not well-understood. We hypothesize that low and oscillatory flow is present on the fibrosa side of the AV and stimulates ECs to differentially regulate microRNA (miRNA) and mRNAs and influence AV disease progression. This hypothesis was tested employing both in vitro and in vivo approaches, high throughput microarray and pathway analyses, as well as a variety of functional assays. First, we isolated and characterized side-dependent, human aortic valvular endothelial cells (HAVECs) isolated from transplant recipient AVs. We found that HAVECs express both endothelial cell markers (VE-Cadherin, vWF, and PECAM) as well as smooth muscle cell markers (SMA and basic calponin). Further, HAVECs align in the direction of the flow as well as uptake acetylated LDL. Using microarray analysis on sheared, side-specific HAVECs, we identified side- and shear-induced changes in miRNA and mRNA expression profiles. More specifically, we identified over 1000 shear-responsive mRNAs which showed robust validation (93% of those tested). We then used Ingenuity Pathway Analysis to identify key miRNAs, including those with many relationships to other genes (for example, thrombospondin and IκB) and those that are members of over-represented pathways and processes (for example, sulfur metabolism). Furthermore, we validated five shear-sensitive miRNAs: miR-139-3p, miR-148a, miR-187, miR-192, and miR-486-5p and one side-dependent miRNA, miR-370. To prioritize these miRNAs, we performed in silico analysis to group these key miRNAs by cellular functions related to AV disease (including tissue remodeling, inflammation, and calcification). Additional miRNAs of interest (including miR-7 and miR-506) were determined through analysis of overrepresented miRNA binding sequences in the shear-sensitive mRNA array. Next, to compare our in vitro HAVEC results in vivo, we developed a method to isolate endothelial-enriched, side-dependent total RNA and identify and validate side-dependent (fibrosa vs. ventricularis) miRNAs in porcine aortic valvular endothelium. From this analysis, we discovered and validated eight side-dependent miRNAs in porcine endothelial-enriched AV RNA, including one miRNA previously identified in vitro, miR-486-5p, as well as a shear-responsive miRNA cluster, miR-199a/214. Through microarray studies and in silico analysis, we have prioritized key miRNAs which may serve as master regulators of AV disease. Better understanding of AV biology and disease in terms of gene-regulation under different hemodynamic conditions will facilitate the design of a tissue-engineered valve and provide alternative treatment options.