Role of flow-sensitive KLK10 in endothelial dysfunction and atherosclerosis

项目摘要 动脉粥样硬化是一种炎症性疾病，是心脏病发作和中风的基础，是 整个世界。扰动流(d-flow或OS)促进动脉粥样硬化，而稳定流(S-flow或LS)通过以下途径抑制动脉粥样硬化 不同地调节内皮基因，进而通过以下机制调节内皮功能 仍然不能完全理解。我们先前报道，激肽释放酶相关的肽酶-10(KLK10)是最流动的- 基于使用从小鼠动脉中分离的内皮RNA的微阵列研究的敏感基因。然而，它是 尚不清楚它在EC生物学和动脉粥样硬化中是否发挥任何作用。我们的初步研究表明，KLK10 S在内皮细胞中产生的FLOW似乎调节几种抗动脉粥样硬化反应，包括EC炎症， 和屏障功能，可能是以一种蛋白酶激活的受体(PAR1和PAR2)依赖的方式进行的。这里, 我们建议检验S流刺激内皮细胞产生KLK10的总体假设，该假说 通过PAR1和PAR2介导的途径提供抗炎和屏障保护功能， 导致动脉粥样硬化的保护。相反，Klk10在EC中的表达被d-flow减少，导致 诱导内皮细胞炎症、屏障破坏和动脉粥样硬化。我们将在三个月内检验这一假说 目标。目标1将确定KLK10在流依赖的EC功能中的作用。首先，依赖于流的KLK10 人和小鼠内皮细胞(HAEC、HUVEC、MAEC)在LS和OS条件下以及在小鼠体内的表达将 下定决心。KLK10对血流依赖的EC功能(EC炎症和通透性)的影响将是 使用rKLK10、KLK10表达载体或siRNA确定。接下来，PAR1/2在这些KLK10中的作用- 依赖的EC功能将使用特定的PAR1和PAR2激动剂或拮抗剂、siRNA和 过表达载体。目的2确定PARs在调节KLK10在内皮细胞中的作用。我们 将测试PAR1/2是否介导KLK10的抗炎和屏障保护作用 使用药物抑制剂和基因操作方法的PAR1/2偏向的激动剂。Bret和 探戈试验将确定PAR1/2和β-arrestin与G-蛋白之间的相互作用。目标3将决定 Klk10通过PAR1/2依赖机制在小鼠动脉粥样硬化中的作用APOE-/-小鼠将成为 用rKLK10蛋白或AAV-KLK10或KLK10 siRNA在7C1EC靶向纳米粒中处理。不完整的 首先将使用颈动脉结扎的动脉粥样硬化模型(2周)，并在标准高脂饮食中得到确认 模型(3个月)。这两种药物，PAR1-/-和PAR2-/-小鼠也将被使用。成功 这些研究的完成将确定KLK10是一种产生和分泌到细胞内的流动敏感蛋白 循环，从而作为一种自分泌和全身抗动脉粥样硬化的介体和治疗靶点 动脉硬化。

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.