Plasma kininogen and kininogen-cleaving proteases in arthritis

关节炎中的血浆激肽原和激肽原裂解蛋白酶

• 批准号: 8622184
• 负责人: Yi Wu
• 金额: $ 7.65万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8622184
• 关键词: Ablation; Address; Adult; Animals; Arthritis; Attenuated; Autoantibodies; Bradykinin; Chronic; Cleaved cell; Clinical Research; Coupling; Development; Disease; Dissection; Etiology; Event; Factor XI; Factor XII; Future; Generations; Genes; Genetic; Goals; High-Molecular-Weight Kininogen; Human; Immune response; Immune system; In Vitro; Indium; Inflammatory; Investigation; Kallikrein-Kinin System; Kininogens; Kinins; Knockout Mice; Knowledge; Mediator of activation protein; Messenger RNA; Modeling; Molecular; Mouse Strains; Mus; Pathogenesis; Patients; Peptide Hydrolases; Plasma; Plasma Kallikrein; Play; Prekallikrein; Production; Prostate-Specific Antigen; Rattus; Reaction; Regulation; Research; Resistance; Rheumatoid Arthritis; Role; Series; Serine Protease; Severities; Surface; System; Testing; Therapeutic; United States; Wild Type Mouse; base; chemokine; chronic pain; cytokine; enzyme substrate; human disease; improved; in vivo; inhibitor/antagonist; innovation; insight; joint injury; monocyte; mouse model; mutant; novel; novel therapeutics; public health relevance; receptor; research study

DESCRIPTION (provided by applicant): Experimental and clinical studies have suggested that the activation of plasma kallikrein-kinin system (KKS) is involved in the pathogenesis f arthritis. The KKS consists of 3 serine proteases, prekallikrein (PK), factor XII (FXII), facto XI (FXI), and a non-enzymatic co-factor, high-molecular-weight kininogen (HK). HK plays an important role in the assembly of this system on activation surfaces. In vitro, the active form of PK, FXII and FXI cleave HK to generate bradykinin and kinin-free HK (called as HKa). Both bradykinin and HKa possess multiple proinflammatory functions. Elevated activities of the KKS components and increased cleavage of HK have been detected in plasma from patients with RA and animals bearing arthritis. In Lewis rat model of arthritis, either HK deficiency or the blockad of HK cleavage inhibits arthritis. Our long term goal is to determine the pathogenic role of the KKS in arthritis and the underlying mechanisms. Understanding the regulation of arthritis by the KKS should provide novel insights into the pathogenesis of arthritis and the systemic complications. In our preliminary studies, we have generated a new mouse strain of HK deficiency (Kng1-/-) by deleting the Kng1 gene. We found that Kng1-/- mice display reduced arthritis. In this proposal, we hypothesize that HK and HK-cleaving proteases play an important role in the pathogenesis of arthritis. Because the KKS of the mouse, compared to the rat, more closely resembles that of the human, our Kng1-/- mouse model becomes a critical approach for evaluating the role of the KKS in arthritis. In this application, our hypothesis will be tested through the following specific aims: In Aim 1, we plan to compare the severity of joint inflammation, the levels of cytokines/chemokines and the activities of the KKS components in plasma, and the expression of cytokine mRNA in monocytes between wild type and Kng1-/- mice. We will examine whether genetic ablation of HK confers protection of arthritis, down regulates cytokines/chemokines production, and decreases the in vivo activation of the KKS. This study will reveal the essential role of HK in the pathogenesis of arthritis. In Aim 2, we will determine which proteases are responsible for HK cleavage in arthritis. Although the active form of plasma kallikrein, FXII and FXI can cleave H in purified systems, no studies have ever been performed to demonstrate which proteases cleave HK in vivo, especially in the setting of arthritis. We have obtained the knockout mice lacking each protease of the KKS, which are in the same genetic background with the Kng1-/- mice. We will determine the requirement of each protease for HK cleavage in vivo. This proposal which utilizes a series of genetically deficient mouse models is highly innovative, because it is the first genetic dissection of the role of HK and HK-cleaving proteases in arthritis. The proposed studies will improve in depth understanding of the contribution of the KKS to the pathogenesis of arthritis, reveal new targets to battle RA, and initiate further studie of the underlying cellular and molecular mechanisms.

描述(申请人提供)：实验和临床研究表明，血浆激肽释放酶-激动素系统(KKS)的激活参与了关节炎的发病。KKS由3种丝氨酸蛋白酶组成，即前激肽释放酶(PK)、凝血因子XII(FXII)、凝血因子XI(FXI)和非酶辅助因子、高分子量激肽原(HK)。HK在该体系在活化表面的组装过程中起着重要的作用。在体外，PK、FXII和FXI的活性形式裂解HK产生缓激肽和不含激动素的HK(称为Hka)。缓激肽和Hka都具有多种促炎作用。在RA患者和关节炎动物的血浆中，检测到KKS组分的活性和HK裂解的增加。在Lewis大鼠关节炎模型中，HK缺乏或HK裂解阻断均可抑制关节炎。我们的长期目标是确定KKS在关节炎中的致病作用和潜在机制。了解KKS对关节炎的调控，将为关节炎的发病机制和全身并发症提供新的见解。在我们的初步研究中，我们通过删除Kng1基因产生了一个新的HK缺乏症小鼠品系(Kng1-/-)。我们发现Kng1-/-小鼠的关节炎症状有所减轻。在这项建议中，我们假设HK和HK裂解酶在关节炎的发病机制中起重要作用。由于与大鼠相比，小鼠的KKS更接近于人类，我们的Kng1/小鼠模型成为评估KKS在关节炎中作用的关键方法。在这一应用中，我们的假设将通过以下具体目标进行验证：在目标1中，我们计划比较野生型和Kng1-/-小鼠关节炎症的严重程度、细胞因子/趋化因子水平和血浆中KKS成分的活性，以及单核细胞中细胞因子mRNA的表达。我们将研究基因消融HK是否可以保护关节炎，下调细胞因子/趋化因子的产生，以及减少KKS在体内的激活。本研究将揭示HK在糖尿病发病机制中的重要作用。 关节炎。在目标2中，我们将确定哪些蛋白水解酶在关节炎中负责HK裂解。虽然活性形式的血浆激肽释放酶、FXII和FXI可以在纯化的系统中裂解H，但还没有研究表明哪些蛋白在体内裂解HK，特别是在关节炎的情况下。我们获得了与Kng1-/-小鼠具有相同遗传背景的KKS各种蛋白缺失的基因敲除小鼠。我们将在体内确定每种蛋白酶对HK切割的需要量。这项建议利用了一系列基因缺陷的小鼠模型，具有很高的创新性，因为这是第一次从基因上剖析HK和HK裂解酶在关节炎中的作用。这些研究将加深对KKS在关节炎发病机制中作用的深入了解，揭示抗击RA的新靶点，并启动对潜在细胞和分子机制的进一步研究。