Role of flow-sensitive KLK10 in endothelial dysfunction and atherosclerosis

流量敏感的 KLK10 在内皮功能障碍和动脉粥样硬化中的作用

• 批准号: 10210428
• 负责人: Hanjoong Jo
• 金额: $ 39万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10210428
• 关键词: 1-Phosphatidylinositol 3-Kinase; ARNT gene; Adhesions; Agonist; Amino Acid Sequence; Anti-Inflammatory Agents; Antiatherogenic; Apolipoprotein E; Apoptosis; Arteries; Atherosclerosis; Biological Assay; Blood Circulation; Blood flow; Carotid Arteries; Cause of Death; Cell physiology; Cellular biology; Characteristics; Cleaved cell; Complex; Coronary Arteriosclerosis; Cultured Cells; Development; Dextrans; Disease; Endothelial Cells; Endothelium; Exercise; Exposure to; F2R gene; Functional disorder; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genes; High Fat Diet; Human; In Vitro; Inflammation; Inflammatory; Kininogenase; Left; Ligation; Mediating; Mediator of activation protein; Modeling; Molecular; Mus; Myocardial Infarction; Operative Surgical Procedures; Pathway interactions; Peptide Hydrolases; Permeability; Pharmacology; Play; Prevention; Production; Proteinase-Activated Receptors; Proteins; RNA; Reporting; Role; Small Interfering RNA; Stroke; Testing; Thrombin; Tissues; Tube; Western Blotting; aortic arch; atherogenesis; atheroprotective; autocrine; base; beta-arrestin; endothelial dysfunction; expression vector; genetic manipulation; high standard; in vivo; inflammatory marker; inhibitor/antagonist; migration; monocyte; nanoparticle; new therapeutic target; novel; overexpression; response; rho; shear stress; therapeutic target; validation studies; vector; 1-Phosphatidylinositol 3-Kinase; ARNT gene; Adhesions; Agonist; Amino Acid Sequence; Anti-Inflammatory Agents; Antiatherogenic; Apolipoprotein E; Apoptosis; Arteries; Atherosclerosis; Biological Assay; Blood Circulation; Blood flow; Carotid Arteries; Cause of Death; Cell physiology; Cellular biology; Characteristics; Cleaved cell; Complex; Coronary Arteriosclerosis; Cultured Cells; Development; Dextrans; Disease; Endothelial Cells; Endothelium; Exercise; Exposure to; F2R gene; Functional disorder; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genes; High Fat Diet; Human; In Vitro; Inflammation; Inflammatory; Kininogenase; Left; Ligation; Mediating; Mediator of activation protein; Modeling; Molecular; Mus; Myocardial Infarction; Operative Surgical Procedures; Pathway interactions; Peptide Hydrolases; Permeability; Pharmacology; Play; Prevention; Production; Proteinase-Activated Receptors; Proteins; RNA; Reporting; Role; Small Interfering RNA; Stroke; Testing; Thrombin; Tissues; Tube; Western Blotting; aortic arch; atherogenesis; atheroprotective; autocrine; base; beta-arrestin; endothelial dysfunction; expression vector; genetic manipulation; high standard; in vivo; inflammatory marker; inhibitor/antagonist; migration; monocyte; nanoparticle; new therapeutic target; novel; overexpression; response; rho; shear stress; therapeutic target; validation studies; vector

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.

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，这是死亡的主要原因 世界。流动障碍（D-Flow或OS）促进，而稳定的流动（S-Flow或LS）抑制动脉粥样硬化 差异性内皮基因，进而通过机制调节内皮功能 仍然没有完全理解。我们以前报道说，Kallikrein相关的肽-10（KLK10）是流量最多的 敏感基因基于一项微阵列研究，使用从小鼠动脉中分离的内皮RNA。但是，是 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KLK10 EC中的S-Flow产生的似乎调节了几种抗动脉生就反应，包括EC炎症， 和屏障功能，可能在蛋白酶激活的接收器（PAR1和PAR2）依赖性方式中。这里， 我们建议测试S-Flow刺激内皮KLK10产生的总体假设，该假设 通过PAR1和PAR2介导的途径提供抗炎和屏障保护功能， 导致动脉保护。相比之下，d-flow降低了EC中的KLK10表达，从而导致 诱导EC炎症，屏障破坏和动脉粥样硬化。我们将在三个中检验这一假设 目标。 AIM 1将确定KLK10在流动依赖性EC函数中的作用。首先，依赖流量的klk10 在LS与OS和小鼠下的培养的人和小鼠EC（HAEC，HUVEC，MAEC）中的表达将是 决定。 KLK10对流动依赖性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EC功能将使用特定的PAR1和PAR2激动剂或拮抗剂，siRNA和 过表达向量。 AIM 2将确定PAR在介导KLK10在EC中的作用中的作用。我们 将测试PAR1/2是否介导ECS中KLK10的抗炎和屏障保护功能 通过使用药物抑制剂和基因操纵方法，PAR1/2偏置的激动剂。布雷特和 探戈测定将确定PAR1/2与β-arrestin vs G蛋白之间的相互作用。 AIM 3将确定 KLK10在小鼠中依赖于PAR1/2依赖机制在动脉粥样硬化中的作用。 apoe - / - 小鼠将 用RKLK10蛋白或AAV-KLK10或7C1 EC靶向纳米颗粒中的KLK10 siRNA处理。部分 动脉粥样硬化的颈动脉连接模型（2周）将首先使用，并在标准的高脂饮食中得到证实 型号（3个月）。也将使用PAR1 - / - 和PAR2 - / - 小鼠的药物剂。成功的 这些研究的完成将确定klk10是产生并分泌到的流动敏感蛋白 循环，它是一种自分泌和系统性的抗动脉生育介体和治疗靶标的 动脉粥样硬化。