Molecular Signaling in Cataracts

项目摘要 白内障是世界范围内致盲的主要原因之一。在手术摘除白内障晶状体后， 约20%的白内障患者发展为后囊混浊(PCO)，并显著 降低了生活质量。鉴于白内障的患病率和后发性白内障的发生率相对较高 白内障手术，特别是年轻患者，他们患有更具侵袭性的后囊混浊以及 越来越多的糖尿病患者有更高的风险患上PCO，迫切需要治疗来预防 导致这种致盲情况的细胞变化。目前还没有药物疗法可以有效地预防 后囊混浊，除囊膜切开术外无其他治疗方法。因此，这项工作的长期影响将解决 目前未得到满足的临床需求--使用药物抑制生长因子预防后囊混浊 负责PCO发展的信号。转化生长因子-β(转化生长因子-β)是公认的 导致PCO发病的主要生长因子。转化生长因子-β诱导Smad蛋白激活，进而 导致一系列参与上皮细胞向间充质细胞转变的基因转录 表型，称为上皮向间充质转化(EMT)。在初步研究中，我们表明 醛糖还原酶(AR)是一种与糖尿病眼病相关的多元醇途径酶，通过以下方式促进Smad的激活 转化生长因子-β。拟议中的研究试图设计治疗策略，以阻断驱动 PCO工艺。在目标1中，我们将使用转基因小鼠模型作为平台来检验AR的假设 促进EMT在PCO发病中的作用。PCO发展的相对风险将在我们的AR-TG中衡量 小鼠，AR缺失小鼠(ARKO)和野生型C57BL6小鼠，使用免疫染色和定量PCR来 术后不同时间测量晶状体EMT和结构标志物。在目标2中，我们将破译 在PCO的发生中，AR与转化生长因子B介导的信号转导有关的机制。我们将利用突变形式的 AR和转化生长因子受体适配器蛋白来验证AR通过 它与辅助蛋白的相互作用与Smad募集到受体复合体有关。在目标3中，我们 将探索两种不同的治疗策略来阻断PCO动物模型中的EMT信号。首先，我们会 验证药物阻断AR足以防止相关细胞变化的假设 随着PCO的发展。在这项研究的第二个手臂中，我们将测试一种膜渗透形式的 Smad7，Smad信号的抑制剂，以减弱我们的PCO小鼠模型中的EMT。我们还将调查 一种联合使用AR抑制剂和TAT-Smad7的联合疗法。这些研究旨在澄清 导致PCO的分子机制，并导致其预防的治疗策略。

Project Summary Cataract is one of the major causes of blindness worldwide. Following surgical removal of the cataractous lens, approximately 20% of cataract patients develop posterior capsular opacification (PCO) and significantly compromised quality of life. Given the prevalence of cataract and the relatively high rate of PCO following cataract surgery, especially among young patients who develop a more aggressive PCO as well as the increasing number of diabetics at higher risk to develop PCO, there is an urgent need for therapies to prevent cellular changes that lead to this blinding condition. There is currently no medical therapy to effectively prevent PCO and no alternative to capsulotomy for its treatment. Thus, the long-term impact of this work will address a currently unmet clinical need—the prevention of PCO using pharmaceutical agents to suppress growth factor signaling responsible for PCO development. Transforming growth factor-beta (TGF-β) is recognized as one of the major growth factors that drive PCO pathogenesis. TGF-β induces activation of Smad proteins, which then bring about transcription of a battery of genes involved in the shift of epithelial cells to a mesenchymal phenotype, known as epithelial-to-mesenchymal transition (EMT). In preliminary studies we showed that aldose reductase (AR), a polyol pathway enzyme linked to diabetic eye disease, facilitates Smad activation by TGF-β. Proposed studies seek to devise therapeutic strategies to interrupt the pathogenic signals that drive the PCO process. In Aim 1, we will use transgenic mouse models as a platform to test the hypothesis that AR facilitates EMT in PCO pathogensis. Comparative risk for PCO development will be measured in our AR-Tg mice, AR null mice (ARKO), and wild type C57BL6 mice, using immunostaining and quantitative PCR to measure EMT and structural markers in the lens at various times following surgery. In Aim 2, we will decipher the mechanism linking AR to TGFß-mediated signaling in PCO development. We will utilize mutant forms of AR and TGFß-receptor adaptor proteins to test the hypothesis that AR interferes with Smad-activation through its interactions with accessory proteins involved with Smad recruitment to the receptor complex. In Aim 3, we will explore two different therapeutic strategies to block EMT signaling in animal models of PCO. First we will test the hypothesis that pharmacological blockade of AR is sufficient to prevent cellular changes associated with PCO development. In a second arm of this study, we will test the ability of a membrane-permeable form of Smad7, an inhibitor of Smad signaling, to attenuate EMT in our mouse model of PCO. We will also investigate a combination therapy involving combined use of an AR inhibitor and Tat-Smad7. These studies aim to clarify molecular mechanisms leading to PCO and lead to therapeutic strategies for its prevention.