Matrix Metalloproteinases-Subcapsular Cataract Formation

描述（由申请人提供）：透镜透明度丧失或白内障是世界上导致失明的主要原因。 两种相关形式的白内障，即囊下白内障和继发性白内障，涉及透镜上皮细胞（LEC）增殖增加和转化为表达收缩丝α-平滑肌肌动蛋白（α-SMA）的大的“纺锤形”肌成纤维细胞的斑块。本项目的长期目标是确定在囊下白内障形成过程中改变透镜上皮细胞遗传组成和表型的细胞外信号。 具体而言，将研究基质金属蛋白酶（MMP）在透镜上皮细胞向间充质或肌成纤维细胞（EMT）的转变中发挥功能作用的假设，这是囊下白内障和继发性白内障的标志性特征。 该实验室的初步发现表明，在大鼠TGF-β诱导的白内障模型中，伴随EMT的是明胶酶A（MMP-2）表达的诱导。这可能反映了MMPs的下游参与，继发于最初的原因。 然而，同样可能的是，MMPs在引发导致白内障形成的事件中发挥更积极的作用。 例如，MMPs已显示直接影响许多细胞类型的EMT并激活潜伏的TGF-β。拟议的研究将区分这两种替代假设。在大鼠中使用TGF-β诱导的模型，将用免疫组织化学、酶谱和蛋白质印迹分析来检查MMP-2和另外的候选MMP（包括MMP-3和MMP-9）的诱导的时间和水平。 这些MMP的表达将与表达α-SMA的肌成纤维细胞的出现相关。MMP的合成抑制剂将用于培养模型中，以确定是否需要一种或多种MMP来介导TGF-β诱导的白内障变化。 如果基质金属蛋白酶直接参与白内障的发生，那么阻断基质金属蛋白酶表达和活性的能力可能对透镜和白内障的研究具有重要影响，因为它将为研究预防白内障的新疗法提供基础。 最后，大鼠中的TGF-β模型将适用于小鼠透镜，以便使用MMP敲除小鼠来确定候选MMP在囊下白内障形成中的个体作用。

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.