The role of flow-sensitive MiRNAs and UBE2C-dependent HIF1α pathway in calcific aortic valve disease

Abstract

Calcific Aortic Valve Disease (CAVD), characterized by aortic valve (AV) stenosis and insufficiency (regurgitation), is a major cause of cardiac-related deaths worldwide, especially in the aging population in advanced countries. Once developed, it is treated mainly with AV repair or replacement by surgical or transcathether methods; however, there are currently no pharmacological treatment options for these patients. This is largely due to a relative paucity in molecular mechanistic understanding of the disease. CAVD was once thought to be a passive degenerative disease, but overwhelming evidence demonstrates that it is actively regulated by cellular and molecular pathways that lead to AV inflammation, sclerosis (thickening and fibrosis), and calcific lesions. MicroRNAs (miRNAs) are a large class of evolutionarily conserved, noncoding RNAs which function as post-transcriptional regulators by interacting with the 3’ untranslated region (3’UTR) of specific target mRNAs in a sequence-specific manner. A single miRNA can typically target hundreds of mRNAs. These miRNAs negatively regulate gene expression through translational repression or mRNA cleavage, depending on the degree of complementarity. Flow-sensitive miRNAs have been mostly characterized in vitro however, their role in human disease has not been fully studied. Previous work in our laboratory has focused on identifying shear-sensitive and side-specific (ventricularis compared to fibrosa layers of the AV) miRNAs relevant to CAVD. To this end, we conducted two independent microRNA array studies. First, we isolated human aortic valve endothelial cells (HAVECs) from each side of the leaflet and exposed them to high-magnitude unidirectional laminar shear stress (LS) or low-magnitude oscillatory shear stress (OS) conditions for 24 hours to discover shear-sensitive miRNAs. Second, we isolated endothelial-enriched total RNAs from each side of the leaflet from porcine AVs to discover side-specific miRNAs. These studies allowed us to identify miR-181b and miR-483 as potential miRNAs for further studies. In Aim 1, we focused on studying shear-sensitive miR-181b. We showed that miR-181b was upregulated in OS conditions and that it regulates matrix metalloproteinases (MMP) activity in valvular endothelium. We conducted an in silico analysis combining predicted gene targets of miR-181b and shear-sensitive target genes from our in vitro HAVEC array and identified tissue inhibitor of metalloproteinases 3 (TIMP3) as a shear-sensitive target of miR-181b responsible for the role of miR-181b in extracellular matrix (ECM) degradation. Therefore, we showed that ECM degradation, a critical step in CAVD, might be mediated by the miR-181b/TIMP3 pathway. In Aim 2, we focused on studying the novel shear-sensitive miR-483-3p. We discovered that it regulated inflammation and endothelial-to-mesenchymal transition (EndMT) in HAVECs. In HAVECs we identified UBE2C as a novel shear-sensitive gene targeted by miR-483; which regulates endothelial inflammation and EndMT. Additionally, UBE2C exerts its function by silencing pVHL, which allows the upregulation and stabilization of HIF1α. Therapeutic studies were conducted in porcine AVs, and we showed that the miR-483 mimic as well as PX478, a HIF1α inhibitor, can inhibit AV calcification. These studies identified two potential therapeutic targets for CAVD and identified a novel mechanistic pathway for CAVD involving the miR-483/UBE2C/pVHL/HIF1α pathway. In Aim 3, we are developing a novel accelerated in vivo model for CAVD by combining hypercholesterolemia (via AAV-PCSK9) and mice with bicuspid AV phenotype (via GATA5 knockout mice). This animal model appears to generate severe sclerosis and microcalcifications in AVs in just four months after PCSK9 injection, allowing for accelerated testing of therapeutics for CAVD in an in vivo setting.