Long noncoding RNAs (lncRNAs) have emerged as biomarkers and regulators of cardiovascular diseases (CVDs), including, coronary artery disease (CAD), and aortic valve stenosis (AVS). lncRNA HIF1a-AS1 is significantly dysregulated in endothelial cells and CVDs which is located on the antisense strand of the hypoxia-inducible factor 1a gene (HIF1a). However, the specific function and the mode of action of the HIF1a-AS1 is still poorly investigated in CVD. Here, we sought to explore the specific role of HIF1a-AS1 in endothelial cell biology (EC) and CVD, in particular, the role of HIF1a in AVS.
Methods and Results: Deep sequencing of aortic valve tissue samples, explanted from patients with AVS with CAD revealed that HIF1a-AS1 was highly upregulated in comparison to healthy controls. HIF1a was increased in ECs under hypoxia (1% O2) induced by Isoproterenol hydroschloride (IPH), Deferoxamine (DFO), and in hypoxia chambers. siRNA-mediated knockdown of HIF1a-AS1 in ECs increased their effects on cellular function (proliferation, tube formation, angiogenesis). Mechanistically, the RT2 angiogenesis array contains 84-angiogenesis-related genes revealed that some genes, for example, SERPINE1 (Serpin Family E Member 1), eNOS (Nitric Oxide Synthase 3 ), and THBS1 ( thrombospondin 1) were dysregulated, Single real-time PCR experiments showed that the expression of THBS1, an inhibitor of angiogenesis gene, was significantly upregulated. Loss of HIF1a-AS1 promotes THBS1 mRNA and protein expression. Furthermore, to study Endothelial-to-mesenchymal transition (EndMT) in vitro, ECs were treated with transforming growth factor-β2 (TGFβ2) and interleukin-1β (IL-1β), and tumor necrosis factor (TNFa) for 5 days. RT-qPCR results revealed that endothelial markers (von Willebrand factor [vWF], endothelial nitric oxide synthase [eNOS]) were downregulated, in contrast, mesenchymal markers (a-smooth muscle actin [aSMA], SM22) were upregulated, indicating that a successful induction of EndMT. Immunoblotting experiments and immunofluorescence confirmed an upregulated protein level of THBS1 after HIF1a-AS1 knockdown compared with the control group undergoing EndMT. Knockdown of THBS1 by siRNA significantly increased angiogenic capacity (tube formation, proliferation, angiogensis) in endothelial cells. Mechanistically, HIF1A-AS1 binds to the RNA-DNA-RNA triplet complex, therefore, we aim to perform ChiP-seq analysis by using valvular cells followed by validation of the target and regulatory mechanism. We are further discovering molecular mechanisms of EV-lncRNA transfer to regulate recipient cell biology and the specific role of HIF1a-AS1 in AVS by using the murine model for exploring translation relevance in vivo.
Conclusion: These data demonstrate that HIF1a-AS1 is hypoxia–regulated lncRNA regulates EC function, specifically, angiogenic phenotype via a THBS1-dependent mechanism in endothelial cells. The underlying mechanism of HIF1a-AS1-mediated intercellular communication and the regulation of the angiogenic gene network in AVS will be further investigated which will add novel insights into lncRNA-regulation in AVS. via a THBS1-dependent mechanism in endothelial cells. The underlying mechanism of HIF1a-AS1-mediated intercellular communication and the regulation of the angiogenic gene network in AVS will be further investigated which will add novel insights into lncRNA-regulation in AVS.
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