PROTEINASE ACTIVATED RECEPTORS IN CORNEAL INFLAMMATION

描述(由申请人提供)：蛋白酶激活受体(PARs)包括一个新的细胞膜蛋白家族，它是组织炎症、疼痛和修复的有效介质。PARs由丝氨酸蛋白酶激活，如凝血酶、胰酶和胰蛋白酶，这些酶在损伤或发炎的组织中释放。我们最近报道了角膜表达两种主要的蛋白水解酶激活受体：PAR-1和PAR-2。该项目的长期目标是确定丝氨酸蛋白酶和PARs在介导各种角膜上皮细胞创伤愈合和炎症反应中的作用。这一应用将检验一种假设，即凝血酶和类胰蛋白酶等丝氨酸蛋白酶是通过激活特定的角膜上皮细胞PARs而介导的角膜上皮细胞炎症和伤口愈合反应的有效调节剂。在这项建议中，我们将承担以下特定目标：特定目的#1：研究丝氨酸蛋白酶对人角膜上皮细胞生物学活性的影响，这些细胞对角膜炎症和创伤愈合反应至关重要；特定目的#2：确定角膜上皮细胞对丝氨酸蛋白酶的反应是否通过激活特定的功能性蛋白水解酶激活受体(PARs)来介导；特定目的#3：确定PARs的丝氨酸蛋白酶激活在体内介导角膜炎症反应中的作用。为了进行拟议的研究，将利用人角膜上皮细胞和人角膜组织进行体外研究，拟议的体内研究将利用正常小鼠以及PAR-1和PAR-2基因敲除小鼠。丝氨酸蛋白酶对人角膜上皮细胞增殖、分化、迁移、基质金属蛋白酶/TIMP表达和促炎蛋白产生的影响将在体外确定。PAR-1和PAR-2在这些丝氨酸蛋白酶诱导的人角膜上皮细胞反应中的具体作用将使用丝氨酸蛋白酶和I特定的天然多肽衍生的PAR-1和PAR-2激动剂和拮抗剂来评估。此外，还将进行初步的体内“原则证明”研究，以确定丝氨酸蛋白酶和特定的PAR-1和PAR-2激动剂在野生型小鼠中诱导角膜炎症反应的能力，并与PAR-I(-/-)和I PAR-2(-/-)动物进行比较。蛋白水解酶和PARs可能是角膜炎症和伤口愈合的重要介质，因此可能被证明是一系列眼科疾病的新治疗靶点。

DESCRIPTION (provided by applicant): Proteinase activated receptors (PARs) comprise a novel family of cell membrane proteins that are potent mediators of tissue inflammation, pain, and repair. PARs are activated by serine proteases such as thrombin, trypsin, and tryptase that are released in injured or inflamed tissues. We have recently reported that the cornea expresses two of the principal proteinase activated receptors: PAR-1 and PAR-2. The long-term goal of this project is to determine the role of serine proteases and PARs to mediate a variety of corneal epithelial cell wound healing and inflammatory responses. This application will test the hypothesis that serine proteases such as thrombin and tryptase are potent modulators of corneal epithelial cell inflammatory and wound healing responses that are mediated by the activation of specific corneal epithelial cell PARs. In this proposal, we will undertake the following specific Aims: SPECIFIC AIM #1: To examine the effect of seine proteases on human corneal epithelial cell biological activities critical for corneal inflammatory and wound healing responses; SPECIFIC AIM #2: To determine if corneal epithelial cell responses to serine proteases are mediated by the activation of specific functional protease activated receptors (PARs); SPECIFIC AIM #3: To determine the role of serine protease activation of PARs in mediating in vivo corneal inflammatory responses. To conduct the proposed studies, human corneal epithelial cells and human corneal tissue will be utilized for in vitro studies and the proposed in vivo studies will utilize both normal mice as well as PAR-1 and PAR-2 knockout mice. The ability of serine proteases to influence human corneal epithelial cell proliferation, differentiation, migration, MMP/TIMP expression, and proinflammatory protein production will be determined in vitro. The specific role of PAR-1 and PAR-2 in these serine protease induced human corneal epithelial cell responses will be assessed using serine proteases and I specific native peptide derived PAR-1 and PAR-2 agonists and antagonists. Additionally, preliminary "proof of principle" in vivo studies will be undertaken to determine the capacity of serine proteases and specific PAR-1 and PAR-2 agonists to induce corneal inflammatory responses in wild type mice in comparison to PAR-I(-/-) and I PAR-2 (-/-) animals. Proteases and PARs may be important mediators of corneal inflammation and wound healing and therefore could prove to be novel therapeutic targets for a wide range of ocular diseases.