Role of MMPs in TGFbeta-induced Cataract Formation

描述(申请人提供)：上皮-间充质转化(EMT)已被证明在包括眼晶状体在内的多个器官和组织的纤维组织中起着重要作用，它导致前囊下白内障(ASC)和后囊混浊(PCO)，也称为继发性白内障。晶状体上皮细胞(LECs)增殖增加，LECs向肌成纤维细胞转化，涉及细胞-细胞黏附分子E-cadherin的丢失和β-SMA的表达，是ASC和PCO的早期事件。转化生长因子β(TGF？)是一种多效性的形态原，已被证明可以诱导晶状体上皮细胞的EMT和随后ASC以及PCO的形成。利用先前建立的大鼠晶状体培养模型，外源性转化生长因子？诱导ASC我们已经证明，用基质金属蛋白酶(MMPs)的抑制剂治疗，尤其是MMP2和MMP9，可以抑制转化生长因子β诱导的白内障改变，包括EMT。上一次赠款期间的研究进一步表明，这两种基质金属蛋白酶可能在这些白内障的发生中协同和/或冗余地发挥作用。例如，使用涉及Ad TGF？的ASC的模型。对于眼睛，我们已经表明，尽管与野生型小鼠相比，MMP-9KO小鼠患上白内障的时间要晚一些。因此，抑制这两种MMP可能是预防EMT和随后的白内障发生所必需的。这些MMP介导EMT和白内障形成的潜在机制是在之前的资金支持期间确定的，涉及E-钙粘附素的破坏。初步数据表明，E-钙粘蛋白的破坏和脱落导致了与EMT相关的下游信号事件，包括β-连环蛋白和肌钙粘蛋白相关转录因子(MRTF-A)的核转位。然而，ASC和PCO对这些信号中间体的需求以及MMP是如何参与的还不清楚。在目前的应用中，我们使用多种ASC和PCO的体外和体内模型来研究这些由转化生长因子？介导的信号通路。此外，我们还概述了将直接确定基质金属蛋白酶-2和基质金属蛋白酶-9在ASC形成中的独特和/或协同作用的实验。最终，我们的目标是确定在ASC和PCO中控制EMT和纤维化的由转化生长因子？介导的通路，以便设计减轻这些疾病的治疗方法。 与公共卫生相关：尽管发达国家有有效的手术可用，但晶状体或白内障的透明度丧失是世界范围内致盲的主要原因。白内障囊外摘除术是北美最常见的外科手术，每年花费超过35亿美元，并经常导致继发性白内障(后囊混浊(PCO))等并发症。因此，为了制定治疗和/或预防白内障的治疗策略，有必要了解调节晶状体正常和病理分化的细胞和分子机制。在这项拟议的研究中，我们将研究两种纤维性白内障，前囊下白内障(ASC)和后发性白内障(PCO)的细胞信号机制，并进一步确定如何抑制这些机制。具体地说，我们将重点放在那些由转化生长因子β(TGF？)和基质金属蛋白酶(MMPs)，因为两者都与这些白内障有关。最终，希望从拟议的研究中获得的数据将导致减轻白内障形成的治疗策略。

DESCRIPTION (provided by applicant): Epithelial-mesenchymal transition (EMT) has been shown to play an important role in the fibroses of multiple organs and tissues, including the ocular lens, where it contributes to both anterior subcapsular cataracts (ASC) and posterior capsular opacification (PCO), also known as secondary cataract. Increased proliferation of lens epithelial cells (LECs), and EMT of LECs into myofibroblasts, involving a loss of the cell-cell adhesion molecule E-cadherin and an induction in ?-smooth muscle actin (?SMA) expression are early events in both ASC and PCO. Transforming growth factor beta (TGF?) is a pleotropic morphogen that has been shown to induce the EMT of LECs and subsequent formation of ASC, as well as, PCO. Using a previously developed rat lens culture model in which exogenous TGF? induces ASC we have shown that treatment with inhibitors to the matrix metalloproteinases (MMP), specifically MMP-2 and MMP-9, suppresses TGF? -induced cataractous changes, including EMT. Studies from the previous grant period further show that these two MMPs likely work cooperatively and/or redundantly in the development of these cataracts. For example, using a model of ASC involving the delivery of Ad TGF? to the eye we have shown that MMP-9 KO mice develop cataracts, albeit they are delayed compared to wild-type mice. Thus, inhibiting both MMPs may be required to prevent EMT and subsequent cataractogenesis. The potential mechanism by which these MMPs mediate EMT and cataract formation was identified during the previous funding period and involves disruption of E-cadherin. Preliminary data suggests that disruption and shedding of E-cadherin results in downstream signaling events linked to EMT including nuclear translocation of ?-catenin and the myocardin-related transcription factor (MRTF-A). However, the requirement for these signaling intermediates in ASC and PCO and how MMPs are involved is not known. In the current application we investigate these TGF? -mediated signaling pathways using multiple ex vivo and in vivo models of ASC and PCO. In addition, we outline experiments that will directly determine the unique and/or cooperative roles of MMP-2 and MMP-9 in ASC formation. Ultimately, our goal is to define the TGF? - mediated pathways controlling EMT and fibrosis in ASC and PCO in order to design therapeutics for mitigating these diseases. PUBLIC HEALTH RELEVANCE: Loss of transparency of the lens, or cataract, is the leading cause of blindness worldwide despite the availability of effective surgery in developed countries. Extracapsular cataract extraction is the most frequently performed surgical procedure in North America, costing over 3.5 billion dollars each year, and can frequently lead to complications such as the development of secondary cataract (posterior capsular opacification (PCO). Thus, an understanding of the cellular and molecular mechanisms regulating the normal and pathological differentiation of the lens is necessary in order to develop therapeutic strategies for the treatment and/or prevention of cataracts. In the proposed research we will investigate the cell signaling mechanisms responsible for two fibrotic cataracts, anterior subcapsular cataracts (ASC) and PCO and further determine how these mechanisms can be inhibited. Specifically we will focus on those pathways mediated by transforming growth factor beta (TGF?) and the matrix metalloproteinases (MMPs) since both of have been implicated in these cataracts. Ultimately, it is hoped that the data obtained from the proposed studies will lead to therapeutic strategies for mitigating cataract formation.