Role of flow-sensitive KLK10 in endothelial dysfunction and atherosclerosis

项目总结

Project Summary Atherosclerosis is an inflammatory disease that underlies heart attacks and stroke, a leading cause of death in the world. Disturbed flow (d-flow or OS) promotes, while stable flow (s-flow or LS) inhibits atherosclerosis by differentially regulating endothelial genes, which in turn regulate endothelial function by the mechanisms that are still not fully understood. We previously reported that kallikrein-related peptidase-10 (KLK10) is the most flow- sensitive gene based on a microarray study using endothelial RNA isolated from mouse arteries. However, it is unknown whether it plays any role in EC biology and atherosclerosis. Our preliminary studies indicate that KLK10 produced by s-flow in ECs appears to regulate several anti-atherogenic responses including EC inflammation, and barrier function, potentially in a protease activated receptors (PAR1 and PAR2)-dependent manner. Here, we propose to test the overall hypothesis that s-flow stimulates endothelial KLK10 production, which provides anti-inflammatory and barrier protective function, via PAR1- and PAR2-mediated pathways, leading to atheroprotection. In contrast, Klk10 expression in EC is reduced by d-flow, resulting in induction of EC inflammation, barrier disruption and atherogenesis. We will test this hypothesis in three Aims. Aim 1 will determine the role of KLK10 in flow-dependent EC function. First, flow-dependent KLK10 expression in cultured human and mouse ECs (HAEC, HUVEC, MAEC) under LS vs. OS and in mice will be determined. Effect of KLK10 on flow-dependent EC function (EC inflammation and permeability) will be determined using rKLK10, KLK10 expression vectors, or siRNA. Next, the role of PAR1/2 in these KLK10- dependent EC functions will be determined using specific PAR1 and PAR2 agonists or antagonists, siRNAs and overexpression vectors. Aim 2 will determine the role of PARs in mediating the role of KLK10 in ECs. We will test whether PAR1/2 mediate the anti-inflammatory and barrier protection function of KLK10 in ECs via PAR1/2-biased agonisms by using pharmacological inhibitors and gene-manipulation approaches. BRET and TANGO assays will determine interaction between PAR1/2 and β-arrestin vs G-proteins. Aim 3 will determine the role of Klk10 in atherosclerosis by the PAR1/2-dependent mechanisms in mouse. ApoE-/- mice will be treated with rKLK10 protein or AAV-KLK10, or KLK10 siRNA in 7C1 EC-targeting nanoparticles. The partial carotid ligation model of atherosclerosis (2 weeks) will be used first, and confirmed in a standard high-fat diet model (3 months). Both pharmacological agents, PAR1-/- and PAR2-/- mice will also be used. Successful completion of these studies would identify KLK10 as a flow-sensitive protein produced and secreted into the circulation, whereby it serves as an autocrine and systemic anti-atherogenic mediator and therapeutic target of atherosclerosis.