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，是组织炎症、疼痛和修复的有效介质。PARs被丝氨酸蛋白酶激活，如凝血酶、胰蛋白酶和胰蛋白酶，这些蛋白酶在受伤或炎症组织中释放。我们最近报道了角膜表达两种主要的蛋白酶激活受体：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伤口愈合和炎症反应中的作用。该应用将验证丝氨酸蛋白酶（如凝血酶和胰蛋白酶）是角膜上皮细胞炎症和伤口愈合反应的有效调节剂的假设，这些反应是由特定角膜上皮细胞par的激活介导的。在本提案中，我们将承担以下具体目标：具体目标#1：检查seine蛋白酶对角膜炎症和伤口愈合反应关键的人类角膜上皮细胞生物活性的影响；特异性目的2：确定角膜上皮细胞对丝氨酸蛋白酶的反应是否由特异性功能性蛋白酶激活受体（PARs）的激活介导；特定目的#3：确定PARs的丝氨酸蛋白酶激活在介导体内角膜炎症反应中的作用。为了进行拟议的研究，人类角膜上皮细胞和人类角膜组织将用于体外研究，拟议的体内研究将使用正常小鼠以及PAR-1和PAR-2敲除小鼠。丝氨酸蛋白酶影响人角膜上皮细胞增殖、分化、迁移、MMP/TIMP表达和促炎蛋白产生的能力将在体外确定。使用丝氨酸蛋白酶和I特异性天然肽衍生的PAR-1和PAR-2激动剂和拮抗剂，将评估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特定作用。此外，将进行初步的体内“原理证明”研究，以确定丝氨酸蛋白酶和特异性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-1（-/-）和PAR-2（-/-）动物进行比较。蛋白酶和PARs可能是角膜炎症和伤口愈合的重要介质，因此可能被证明是多种眼部疾病的新治疗靶点。

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.