Highlights d scRNA-seq and scATAC-seq of mouse carotid arteries after PCL surgery are presented d Carotid artery endothelial cells are heterogeneous and plastic in response to flow d Disturbed flow reprograms ECs to proatherogenic EndMT and EndICLT phenotypes d Disturbed flow regulates access to known and unreported TF binding sites

Objective— Calcific aortic valve (AV) disease, characterized by AV sclerosis and calcification, is a major cause of death in the aging population; however, there are no effective medical therapies other than valve replacement. AV calcification preferentially occurs on the fibrosa side, exposed to disturbed flow (d-flow), whereas the ventricularis side exposed to predominantly stable flow remains protected by unclear mechanisms. Here, we tested the role of novel flow-sensitive UBE2C (ubiquitin E2 ligase C) and microRNA-483-3p (miR-483) in flow-dependent AV endothelial function and AV calcification. Approach and Results— Human AV endothelial cells and fresh porcine AV leaflets were exposed to stable flow or d-flow. We found that UBE2C was upregulated by d-flow in human AV endothelial cells in the miR-483–dependent manner. UBE2C mediated OS-induced endothelial inflammation and endothelial-mesenchymal transition by increasing the HIF-1α (hypoxia-inducible factor-1α) level. UBE2C increased HIF-1α by ubiquitinating and degrading its upstream regulator pVHL (von Hippel-Lindau protein). These in vitro findings were corroborated by immunostaining studies using diseased human AV leaflets. In addition, we found that reduction of miR-483 by d-flow led to increased UBE2C expression in human AV endothelial cells. The miR-483 mimic protected against endothelial inflammation and endothelial-mesenchymal transition in human AV endothelial cells and calcification of porcine AV leaflets by downregulating UBE2C. Moreover, treatment with the HIF-1α inhibitor (PX478) significantly reduced porcine AV calcification in static and d-flow conditions. Conclusions— These results suggest that miR-483 and UBE2C and pVHL are novel flow-sensitive anti- and pro-calcific AV disease molecules, respectively, that regulate the HIF-1α pathway in AV. The miR-483 mimic and HIF-1α pathway inhibitors may serve as potential therapeutics of calcific AV disease.

Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified Kallikrein-Related Peptidase 10 (Klk10, a secreted serine protease) as a flow-sensitive gene in mouse arterial endothelial cells, but its role in endothelial biology and atherosclerosis was unknown. Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo& mouse models and in vitro studies with cultured endothelial cells (ECs). Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated Klk10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced permeability dysfunction and inflammation in human artery ECs (HAECs), as determined by NFkB activation, expression of vascular cell adhesion molecule 1 (VCAM1) and intracellular adhesion molecule 1 (ICAM1), and monocyte adhesion. Further, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of Klk10-expressing plasmids inhibited atherosclerosis in Apoe-/- mice. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. KLK10 is a flow-sensitive endothelial protein that serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.