Plasma kininogen and kininogen-cleaving proteases in arthritis

描述（由申请人提供）：实验和临床研究表明，血浆钾likrein-kinin系统（KKS）的激活与关节炎的发病有关。KKS由3个丝氨酸蛋白酶组成，prekallikrein (PK), factor XII (FXII), facto XI （FXI）和一个非酶促因子，高分子量激肽原（HK）。HK在该系统在活化表面的组装中起着重要作用。在体外，活性形式的PK、FXII和FXI裂解HK生成缓激肽和无激肽HK（称为HKa）。缓激肽和HKa都具有多种促炎功能。在RA患者和患有关节炎的动物的血浆中检测到KKS成分活性升高和HK裂解增加。在Lewis大鼠关节炎模型中，HK缺乏或阻断HK裂解均可抑制关节炎。我们的长期目标是确定KKS在关节炎中的致病作用及其潜在机制。了解KKS对关节炎的调控应该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基因产生了一种新的HK缺乏症小鼠品系（Kng1-/-）。我们发现kn1 -/-小鼠显示关节炎减轻。在本文中，我们假设HK和HK切割蛋白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，因此我们的kn1 -/-小鼠模型成为评估KKS在关节炎中的作用的关键方法。在本申请中，我们的假设将通过以下具体目的进行检验：在Aim 1中，我们计划比较野生型和Kng1-/-小鼠关节炎症的严重程度、血浆中细胞因子/趋化因子水平和KKS组分的活性，以及单核细胞中细胞因子mRNA的表达。我们将研究HK基因消融是否能保护关节炎，下调细胞因子/趋化因子的产生，并降低KKS的体内激活。本研究将揭示HK在肝癌发病机制中的重要作用

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.