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，是心脏病患者死亡的主要原因。 世界扰动血流（d-血流或OS）促进动脉粥样硬化，而稳定血流（s-血流或LS）抑制动脉粥样硬化， 差异调节内皮细胞基因，这些基因又通过以下机制调节内皮细胞功能： 仍然没有完全理解。我们以前报道过，激肽释放酶相关肽酶-10（KLK 10）是最流动的， 敏感基因的基础上，使用从小鼠动脉内皮RNA分离的微阵列研究。但据 尚不清楚它是否在EC生物学和动脉粥样硬化中起任何作用。我们的初步研究表明，KLK 10 由内皮细胞中的s-流产生的β-CD似乎调节几种抗动脉粥样硬化反应， 和屏障功能，潜在地以蛋白酶激活受体（PAR1和PAR2）依赖性方式。在这里， 我们建议检验s流刺激内皮细胞KLK 10产生的总体假设， 通过PAR 1和PAR 2介导的途径提供抗炎和屏障保护功能， 导致动脉粥样硬化保护。相比之下，Klk10在EC中的表达被d-flow降低，导致 诱导EC炎症、屏障破坏和动脉粥样硬化形成。我们将在三个方面来检验这个假设。 目标。目的1将确定KLK 10在流量依赖性EC功能中的作用。第一，流量依赖型KLK 10 在LS与OS下培养的人和小鼠EC（HAEC、HUVEC、MAEC）中以及在小鼠中的表达将是 测定KLK10对流动依赖性EC功能（EC炎症和渗透性）的影响将在下文描述。 使用rKLK10、KLK10表达载体或siRNA确定。接下来，PAR1/2在这些KLK 10中的作用- 依赖性EC功能将使用特异性PAR1和PAR2激动剂或拮抗剂、siRNA和 过表达载体。目的2将确定PAR在介导KLK 10在EC中的作用中的作用。我们 将测试PAR1/2是否介导KLK 10在EC中的抗炎和屏障保护功能， 通过使用药理学抑制剂和基因操作方法的PAR 1/2偏向激动。bret和 TANGO测定将确定PAR 1/2和β-抑制蛋白与G蛋白之间的相互作用。目标3将决定 通过PAR 1/2依赖机制探讨Klk 10在小鼠动脉粥样硬化中的作用。ApoE-/-小鼠将 用7C1 EC靶向纳米颗粒中的rKLK10蛋白或AAV-KLK10或KLK10 siRNA处理。部分 首先使用动脉粥样硬化的颈动脉结扎模型（2周），并在标准高脂饮食中确认 模型（3个月）。还将使用两种药理学试剂，PAR1-/-和PAR2-/-小鼠。成功 这些研究的完成将鉴定KLK 10为产生并分泌到细胞中的流动敏感蛋白。 循环，从而它作为一种自分泌和全身抗动脉粥样硬化介质和治疗靶点， 动脉粥样硬化

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.