Matrix Metalloproteinases-Subcapsular Cataract Formation

描述(申请人提供)：晶状体失去透明度，或白内障，是世界上导致失明的主要原因。两种相关的白内障类型，包膜下白内障和继发性白内障，涉及晶状体上皮细胞(LECs)的增殖增加和转化为大的“纺锤形”肌成纤维细胞斑块，表达收缩细丝-α-SMA(α-SMA)。该项目的长期目标是确定在白内障形成过程中改变晶状体上皮细胞遗传组成和表型的细胞外信号。具体地说，基质金属蛋白酶(MMPs)在晶状体上皮细胞向间充质细胞或肌成纤维细胞(EMT)的转化中起功能作用的假说将被研究，这是包膜下白内障和继发性白内障的特征。本实验室的初步发现表明，在转化生长因子-β诱导的大鼠白内障模型中，伴随着EMT的是明胶酶A(MMP2)的表达诱导。这可能反映了MMP的下游参与，而不是最初的原因。然而，同样可能的是，MMPs在启动导致白内障形成的事件中发挥了更积极的作用。例如，MMPs已被证明可以直接影响许多细胞类型的EMT，并激活潜在的转化生长因子-β。拟议的研究将区分这两种不同的假设。利用转化生长因子-β诱导的大鼠模型，通过免疫组织化学、酶谱分析和免疫印迹分析，检测基质金属蛋白酶-2和其他候选基质金属蛋白酶的诱导时间和水平。这些MMPs的表达将与表达α-SMA的肌成纤维细胞的出现相关。在培养模型中将使用MMPs的合成抑制剂来确定是否需要一个或多个MMPs来介导转化生长因子-β诱导的白内障改变。如果MMPs直接参与白内障的发生，其阻断基质金属蛋白酶表达和活性的能力可能对晶状体和白内障的研究有很大的影响，因为这将为研究预防白内障的新的治疗方法提供基础。最后，将大鼠的转化生长因子-β模型适应于小鼠的晶状体，以便使用基质金属蛋白酶基因敲除小鼠来确定候选基质金属蛋白酶在囊下白内障形成中的单独作用(S)。

DESCRIPTION (provided by applicant): Loss of transparency of the lens, or cataract, is the leading cause of blindness in the world. Two related forms of cataract, subcapsular cataract and secondary cataract, involve the increased proliferation and transformation of lens epithelial cells (LECs) into plaques of large "spindle shaped" myofibroblasts which express the contractile filament, alpha-smooth muscle actin (alpha-SMA). The long-term goal of this project is to determine the extracellular signals, which alter the genetic makeup and phenotype of lens epithelial cells during subcapsular cataract formation. Specifically, the hypothesis that Matrix Metalloproteinases (MMPs), play a functional role in the transition of lens epithelial cells into mesenchymal or myofibroblast cells (EMT), a hallmark feature of subcapsular cataracts and secondary cataract will be investigated. Preliminary findings from this laboratory show that accompanying the EMT in the TGF-beta-induced cataract model in rats is the induction in expression of gelatinase A (MMP-2). This may reflect a downstream involvement of MMPs, secondary to the initial cause. However, it is equally probable that MMPs play a more active role in initiating the events leading up to cataract formation. For example, MMPs have been shown to directly influence the EMT of many cell types and to activate latent TGF-beta. The proposed studies will distinguish between these two alternate hypotheses. Using the TGF-beta induced model in rats, the timing and level of induction of MMP-2 and additional candidate MMPs, including MMP-3 and MMP-9 will be examined with immunohistochemistry, zymography and western blot analysis. Expression of these MMPs will be correlated with the appearance of myofibroblasts expressing alpha-SMA. Synthetic inhibitors of MMPs will be used in the culture model to determine if one or more of the MMPs are required for mediating the TGF-beta induced cataractous changes. If MMPs directly participate in cataractogenesis, the ability to block MMP expression and activity may be of high impact to lens and cataract research since it would provide the basis for investigation of a novel therapy for the prevention of cataracts. Finally, the TGF-beta model in rats will be adapted to the mouse lens in order to use MMP knockout mice for determining the individual role(s) of candidate MMPs in subcapsular cataract formation.