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首页> 外文学位 >The role of alpha v integrins in lens epithelial mesenchymal transition and posterior capsular opacification.
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The role of alpha v integrins in lens epithelial mesenchymal transition and posterior capsular opacification.

机译：αv整合素在晶状体上皮间质转化和后囊膜混浊中的作用。

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摘要

Posterior Capsular Opacification (PCO) is the major complication after cataract surgery. PCO occurs when residual lens cells (LCs) remaining following cataract surgery undergo a wound healing response producing a mixture of alphaSMA expressing myofibroblasts and lens fiber cells which impair vision. Prior investigations have proposed that integrins play a central role in PCO while my data indicated that alphaV integrin and its interacting beta-subunits; beta1, beta5, beta6, beta8 along with alpha-smooth muscle actin (alphaSMA) are upregulated in residual lens epithelial cells in a mouse model of cataract surgery. To test the hypothesis that alphaV integrins are functionally important in PCO pathogenesis, I created mice lacking the alphaV integrin subunit in all lens cells using a conditional knockout approach. Adult lenses lacking alphaV integrins were analyzed for function and cellular organization by grid analysis, conventional light, confocal and scanning electron microscopy. At three months of age, fiber cells were surgically removed from alphaV integrin null and control lenses and the extent of EMT post-surgery was measured by QRT-PCR and immunofluorescent detection of EMT markers. Lenses lacking alphaV integrin subunits are transparent and show no apparent morphological abnormalities when compared to control lenses. When challenged with surgery/injury, control lenses developed LC multilayering along with upregulation of alphaSMA, beta1-integrin, fibronectin, vitronectin, tenascin-C and TGF-beta induced protein within 48hrs. In contrast, LECs lacking alphaV integrins showed no increase in LC multilayering with little to no upregulation of alphaSMA, fibronectin, vitronectin, and TGF-beta induced protein 48hrs post-surgery. In addition, I saw a significant increase in the mRNA expression of alphaSMA, fibronectin, tenascin-C and TGF-beta induced protein 24hrs post-surgery in control lenses with no significant increase in lenses lacking the alphaV integrin subunit. This effect appears to result from the known roles of alphaV integrins in latent TGF- beta activation since alphaV integrin null lenses do not exhibit detectable SMAD-3 phosphorylation after surgery, while this occurs robustly in control lenses, consistent with the known roles for TGF-beta in fibrotic PCO as well as Anterior Subcapsular Cataracts (ASC). To look further into whether alphaV integrins play a role in the TGF-beta associated fibrotic lens EMT, I utilized mice homozygous for a 12 nucleotide deletion in the betaB2-crystallin gene (Crybb2 Phil/Phill), which have been previously demonstrated in our lab to undergo a juvenile lens epithelium EMT as early as four weeks postnatal, leading to ASC which is followed by the development of severe lens abnormalities. At 2 months age, Crybb2 Phil/Phill demonstrates identical EMT characteristics as seen in wildtype mice at 48hrs post-surgery. Their lens epithelium becomes multilayered and upregulates alphaSMA, fibronectin, vitronectin, TGF-beta induced protein and phosphorylated SMAD-3. Moreover, a SuperArray RT-PCR gene expression analysis on mRNA expression of TGF-beta associated genes showed that, while all TGF-beta ligands 1, 2 and 3 and the vast majority of the down-stream canonical TGF-beta signaling associated players, including SMAD, 2, 3 or 4 were not upregulated in Crybb2 Phil/Phill epithelium, TGF-beta receptor II, TGF-betai and Follistatin which are genes known to upregulate in the aftermath of TGF-beta signaling, were significantly upregulated. These findings suggested that the Crybb2 Phil/Phill epithelium was undergoing an extreme unregulation of TGF-beta signaling which seem to be regulated mainly at the TGF-beta activation level point. This was attested by an active TGF-beta assay which confirmed that Crybb2 Phil/Phill lenses had four times the amount of active TGF-beta as compared to wildtype lenses. In addition, alphaV integrins were found to upregulate at both the mRNA and protein level in Crybb2 Phil/Phill lens epithelium EMT similar to that seen in the lens post-surgery suggesting that betaB2-crystallin may have a non-refractive function in maintaining lens epithelial integrity which will be a topic for future investigations. Overall, while alphaV integrin only subtlety regulates the development and function of the lens, it is involved in regulating the phenotypic alterations in lens epithelial cells that occurs following lens injury/cataract surgery leading to PCO. This is the first study to examine the roles of alphaV integrins in lens development, refractive functions and the pathogenesis of PCO/ASC. These data suggest that therapeutics antagonizing alphaV integrin function could be used to prevent TGF-beta fibrotic PCO following cataract surgery as well as ASC development.
机译：后囊膜混浊（PCO）是白内障手术后的主要并发症。白内障手术后残留的残余晶状体细胞（LC）经历伤口愈合反应，产生表达αSMA的成肌纤维细胞和晶状体纤维细胞损害视力的混合物，就会发生PCO。先前的研究表明，整合素在PCO中起着核心作用，而我的数据表明alphaV整合素及其相互作用的β亚基。在白内障手术的小鼠模型中，残留的晶状体上皮细胞中的beta1，beta5，beta6，beta8和alpha平滑肌肌动蛋白（alphaSMA）上调。为了检验αV整合素在PCO发病机理中功能上重要的假设，我使用条件敲除方法创建了在所有晶状体细胞中都缺乏alphaV整合素亚基的小鼠。通过网格分析，常规光，共聚焦和扫描电子显微镜对缺乏alphaV整合素的成人晶状体进行功能和细胞组织分析。在三个月大时，通过手术从alphaV整联蛋白缺失和对照镜中去除纤维细胞，并通过QRT-PCR和EMT标记的免疫荧光检测来测量术后EMT的程度。与对照镜片相比，缺少alphaV整联蛋白亚基的镜片是透明的，没有明显的形态异常。当受到手术/损伤的挑战时，对照镜片会在48小时内开发出LC多层膜，同时上调了αSMA，β1-整合素，纤连蛋白，玻连蛋白，腱生蛋白-C和TGF-β诱导的蛋白质。相反，缺乏alphaV整合素的LEC术后48小时，LC多层膜没有增加，αSMA，纤连蛋白，玻连蛋白和TGF-β诱导蛋白几乎没有上调。此外，我发现术后24小时在对照镜片中，αSMA，纤连蛋白，肌腱蛋白C和TGF-β诱导的蛋白的mRNA表达显着增加，而缺少alphaV整联蛋白亚基的镜片则没有显着增加。这种作用似乎是由于alphaV整联蛋白在潜在的TGF-β激活中的已知作用引起的，因为alphaV整联蛋白无效透镜在手术后未显示出可检测的SMAD-3磷酸化，而这种情况在对照透镜中发生得很强烈，与TGF-β的已知作用一致。 β在纤维化PCO和前囊膜白内障（ASC）中。为了进一步研究alphaV整合素是否在TGF-β相关的纤维化晶状体EMT中发挥作用，我利用纯合的小鼠在betaB2-crystallin基因（Crybb2 Phil / Phill）中删除了12个核苷酸，这在我们的实验室中已经得到证明。最早在产后四周接受青少年晶状体上皮EMT，导致ASC，随后出现严重的晶状体异常。在2个月大时，Crybb2 Phil / Phill表现出与手术后48小时野生型小鼠相同的EMT特征。他们的晶状体上皮变得多层并且上调αSMA，纤连蛋白，玻连蛋白，TGF-β诱导的蛋白和磷酸化的SMAD-3。而且，对TGF-β相关基因的mRNA表达进行的SuperArray RT-PCR基因表达分析表明，尽管所有TGF-β配体1、2和3以及绝大多数下游规范性TGF-β信号传导相关者，包括SMAD，2、3或4的Crybb2 Phil / Phill上皮细胞未上调，TGF-β受体II，TGF-β1和Follistatin是已知的在TGF-β信号转导后会上调的基因，但均被上调。这些发现表明，Crybb2 Phil / Phill上皮细胞正在经历TGF-β信号的极端失调，而TGF-β信号似乎主要在TGF-β激活水平上受到调节。这是通过活性TGF-β测定法证明的，该试验证实Crybb2 Phil / Phill晶状体的活性TGF-β量是野生型晶状体的四倍。此外，发现Crybb2 Phil / Phill晶状体上皮EMT中的αV整合素在mRNA和蛋白水平上调，与晶状体术后的相似，表明βB2-晶状体蛋白在维持晶状体上皮中可能具有非屈光功能。完整性，这将是未来调查的主题。总体而言，尽管αV整联蛋白仅微妙地调节晶状体的发育和功能，但它参与调节晶状体上皮细胞的表型改变，这种改变在晶状体损伤/白内障手术导致PCO之后发生。这是第一项研究αV整合素在晶状体发育，屈光功能和PCO / ASC发病机理中的作用的研究。这些数据表明，拮抗αV整联蛋白功能的治疗剂可用于预防白内障手术以及ASC发生后的TGF-β纤维化PCO。

著录项

  • 作者

    Mamuya, Fahmy A.;

  • 作者单位

    University of Delaware.;

  • 授予单位 University of Delaware.;
  • 学科 Biology.;Medicine.;Physiology.
  • 学位 Ph.D.
  • 年度 2014
  • 页码 219 p.
  • 总页数 219
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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