The Role of Extracelluar Enzymes in Regulating Corneal Repair

描述(申请人提供)：角膜混浊影响数以百万计的人，是世界上第二大致盲原因。显然需要开发非手术策略来预防和治疗角膜纤维化和随后发展的角膜混浊。以往的研究和初步研究表明，细胞外膜蛋白包括6-O-硫酸酯酶Sulf-1和基质金属蛋白酶(MMPs)包括基质金属蛋白酶-9和基质金属蛋白酶-12只有在角膜损伤后才有表达，提示它们可能在角膜修复中发挥作用。这些酶是由上皮细胞、间质细胞和炎症细胞产生的，它们在角膜修复中的作用还没有得到很好的研究。在上皮中，基质金属蛋白酶-9通过对转化生长因子途径的作用而减少再上皮化，而在基质中，基质金属蛋白酶-12已被证明在其他疾病过程中通过转化生长因子-β对纤维化具有保护作用。此外，基质金属蛋白酶-12的S能够比其他基质金属蛋白酶更有效地产生血管抑素，这表明它可能在防止角膜血管生成中发挥作用。已发现Sulf-1在体外可以调节生长因子和趋化因子的结合。各种生长因子的刺激，尤其是转化生长因子-β2和成纤维细胞生长因子，对角膜纤维化表型和角膜新生血管的形成至关重要。综上所述，这些胞外酶可以调节修复过程的各个方面，既有积极的，也有消极的。在这一应用中，我们建议研究细胞外酶MMP9、MMP12和Sulf-1在角膜上皮、间质和炎症反应中的作用。特异靶1将确定基质金属蛋白酶-9或Sulf-1活性对损伤后上皮细胞增殖反应的影响。特定目的2将使用基质金属蛋白酶-12基因敲除小鼠来检测基质金属蛋白酶-12在角膜基质损伤反应过程中的纤维化和血管生成中的作用。具体目标3将检测这些细胞外酶向角膜募集炎症细胞的能力，并将使用一种基于旋转圆盘共聚焦显微镜的新成像技术来研究体内和实时的角膜炎症细胞动力学。了解细胞外酶在损伤角膜中的作用将有助于我们进一步了解维持角膜透明度的重要机制，并提供基本的见解，从长远来看，这将有助于开发更好的角膜混浊治疗方法。

DESCRIPTION (provided by applicant): Corneal opacification affects millions of people and is the second leading cause of blindness in the world. A clear need exists for the development of non-surgical strategies to prevent and treat corneal fibrosis and the subsequent development of corneal opacification. Previous and preliminary studies show that extracellular enymes including the 6-O-sulfatase Sulf-1 and matrix metalloproteinases (MMPs) including MMP-9 and MMP-12 are expressed in the cornea only upon injury, implicating their possible roles in corneal repair. These enzymes are produced by epithelial, stromal and inflammatory cells, and their roles in corneal repair c have not been well-studied. In the epithelium, MMP-9 appears to decrease re-epithelialization through its effects on the TGF pathway, while in the stroma MMP-12 has been shown to be protective against fibrosis via TGF-p in various other disease processes. In addition, MMP-12's ability to produce angiostatin more efficiently than other MMPs suggests that it might play a role in preventing corneal angiogenesis. Sulf-1 has been found to modulate the binding of growth factors and chemokines in vitro. Stimulation of various growth factors, particularly TGF-p2 and FGF, are critical to the corneal fibrotic phenotype and corneal neovascularization. Taken together, these extracellular enzymes may regulate various aspects of the repair process, both positively and negatively. In this application, we propose to investigate the roles of the extracellular enzymes MMP-9, MMP-12, and Sulf-1 in the epithelial, stromal, and inflammatory corneal response to injury. Specific Aim 1 will determine the effects of MMP-9 or Sulf-1 activity on the epithelial cell proliferation response to injury. Specific Aim 2 will use MMP-12 knockout mice to examine the role of MMP- 12 in fibrosis and angiogenesis during the corneal stromal response to injury. Specific Aim 3 will examine the ability of these extracellular enzymes to recruit inflammatory cells to the cornea and will use a novel imaging technique based on spinning disk confocal microscopy to study inflammatory cell dynamics in the cornea in vivo and in real-time. Understanding the roles of extracellular enzymes in wounded corneas should further our understanding of the mechanisms important to the maintenance of corneal clarity and provide basic insights that will, in the long term, allow for the development of better treatment of corneal opacification.