Corneal Metalloproteinase-Matrix Interactions

描述(申请人提供)：角膜透明度丧失是许多致盲情况的共同终点。角膜混浊是世界范围内致盲的主要原因。我们的实验室正在研究与角膜伤口修复有关的调节圆锥细胞透明度的机制。具体地说，我们已经在角膜中发现了一种内源性基质金属蛋白酶，它有助于防止准分子激光损伤后的角膜混浊。基质溶素(MMP7)缺陷的小鼠在激光损伤后会有大量的疤痕，这对野生型的小鼠是无害的。 在这项应用中，我们将研究基质金属蛋白酶-7与细胞外基质(ECM)成分的相互作用，这些成分定位于天然和损伤的角膜。 特定目标A将结合免疫组织化学、免疫印迹、酶谱和转基因动物来定位损伤和未损伤的角膜中表达的基质金属蛋白酶-7的数量。 特定目的B扩展这些研究以确定体外培养的角膜上皮细胞和角膜基质细胞来源的基质金属蛋白酶-7的特异性。在这个目标中，我们将通过分析角膜上皮在二维基质凝胶上的迁移，以及角膜基质细胞在三维基质凝胶中的增殖来检测角膜细胞与细胞外基质的相互作用。 特定的目标C将把体外结果转化为基质金属蛋白酶-7基因敲除小鼠。采用免疫组织化学、Western blotting和实时定量聚合酶链式反应(Real-time PCR)检测野生型和基质溶素基因敲除小鼠准分子激光损伤后细胞外基质成分的表达。AIM C的第二部分将仔细检查这些小鼠角膜细胞外基质中依赖于基质金属蛋白酶-7的分解产物。最后，利用基质金属蛋白酶-7/细胞外基质双基因敲除小鼠，研究降低角膜透明度对特定细胞外基质成分的需求。 我们对损伤角膜中的蛋白酶-基质相互作用的研究将进一步加深我们对维持角膜透明度的机制的理解。这些在角膜上的研究结果可能最终也有助于理解其他器官的愈合。

DESCRIPTION (provided by applicant): Loss of corneal clarity is a common endpoint of many blinding conditions. Corneal opacification is a major cause of blindness worldwide. Our laboratory is studying the mechanisms associated with corneal wound repair that regulate coneal clarity. Specifically, we have identified an endogenous matrix metalloproteinase within the cornea which helps prevent corneal opcification after an excimer laser wound. Matrilysin (MMP-7) deficient mice have exuberant scarring following laser injuries which are innocuous to their wild-type counterparts. In this application we will investigate the interaction of MMP-7 with extracelluar matrix (ECM) components localized to native and wounded corneas. Specific Aim A will use a combination of immunohistochemistry, western blotting, zymography, and transgenic animals to localize the sites and quantify the amount of MMP-7 expressed in wounded and unwounded corneas. Specific Aim B extends these studies to determine the matrix substrate specificities of corneal epithelium- and keratocyte-derived MMP-7 in vitro. In this aim we will examine corneal celI-ECM interactions by assaying the migration of epithelium on 2-dimensional matrix gels, and the proliferation of keratocytes in 3-dimensional matrix gels. Specific Aim C will translate the in vitro results to MMP-7 knockout mice. The expression of xtracellular matrix components in wild-type and matrilysin knockout mice after excimer laser wounding will be evaluated by immunohistochemistry, western blotting and real-time PCR. The second portion of Aim C will carefully examine the MMP-7 dependent breakdown products of corneal extracellular matrix in these mice. Lastly, using MMP-7/ECM double knockout mice, the requirement of specific extracellular matrix components for reduced corneal clarity will be investigated. Our studies of protease-matrix interactions in wounded corneas will further our understanding of the mechanisms for maintaining corneal clarity. Results of these studies in the cornea may ultimately be helpful in understanding healing in other organs as well.