Mechanisms of Retinal Vascular Permeability in Diabetes

描述(申请人提供)：糖尿病视网膜病变在美国仍然是导致失明的主要原因，没有FDA批准的药物治疗。该建议的目的是了解导致血管通透性增加和黄斑水肿的分子机制，并阐明血管通透性与血管生成的关系，从而开发治疗或预防糖尿病视网膜病变的新疗法。糖尿病视网膜病变的血管功能障碍既可能是高血糖、晚期糖基化终末产物和高脂血症对血管内皮细胞的直接作用，也可能是这些代谢产物通过诱导生长因子(血管内皮生长因子)和炎性细胞因子(如肿瘤坏死因子)的间接作用所致。在目前的方案中，假设血管内皮生长因子和肿瘤坏死因子改变紧密连接复合体，导致内皮通透性增加。上一次资助期间的研究已经证明，血管内皮生长因子诱导紧密连接蛋白阻滞素的磷酸化，这种方式依赖于蛋白激酶C，与血管通透性有关。此外，血管内皮细胞生长因子诱导封闭蛋白和其他紧密连接蛋白从细胞膜重新分布到细胞质，随着时间的推移，导致封闭蛋白的降解。通过质谱学分析阻塞素的磷酸化位点，已经确定了血管内皮生长因子响应的磷酸化位点。在这项建议中，提供的数据表明，突变的occludin可以防止磷酸化，阻断血管内皮生长因子诱导的通透性和阻塞素的内吞作用。此外，初步数据表明，肿瘤坏死因子通过减少紧密连接蛋白claudin 5和zonula occludens 1而改变紧密连接复合体，但不减少封闭蛋白的含量，这表明至少存在部分发散机制。封闭蛋白的含量与紧密连接的细胞的增殖密切相关。在视网膜色素上皮细胞中沉默occludin的表达可以诱导细胞增殖增加两倍。因此，我们将探讨血管内皮生长因子和肿瘤坏死因子改变紧密连接复合体诱导内皮细胞通透性的机制，以及血管内皮生长因子诱导的封闭蛋白磷酸化和降解与血管生成的关系。虽然血管内皮生长因子和肿瘤坏死因子在控制阻塞素方面存在分歧，但初步数据表明，这两种因子都利用非典型的PKC途径改变连接复合体，并诱导内皮通透性。针对非典型PKC通路的单独或联合PKC抑制剂的治疗可能为控制糖尿病视网膜病变和其他涉及血管内皮生长因子和炎性细胞因子的视网膜疾病的血管通透性提供有效的手段。与公共卫生相关：本提案中的实验将阐明生长因子和炎性细胞因子改变血-视网膜屏障紧密连接复合体导致糖尿病视网膜病变黄斑水肿的机制。此外，该提案将探索血管通透性和血管生成之间的联系，以及紧密连接蛋白闭合蛋白在内皮细胞生长控制中的作用。

DESCRIPTION (provided by applicant): Diabetic retinopathy remains a leading cause of blindness in the United States with no FDA approved medical therapy. The goal of this proposal is to understand the molecular mechanisms that lead to increased vascular permeability and macular edema and to elucidate the relationship of vascular permeability to angiogenesis so that novel therapies to treat or prevent diabetic retinopathy may be developed. Vascular dysfunction in diabetic retinopathy may result from both the direct effect of hyperglycemia, advanced glycation end products, and hyperlipidemia on vascular endothelial cells and the indirect effect of these metabolites through induction of growth factors such as vascular endothelial growth factor (VEGF) and inflammatory cytokines such as tumor necrosis factor (TNF). In the current proposal, it is hypothesized that VEGF and TNF alter the tight junction complex leading to increased endothelial permeability. Research over the previous funding period has demonstrated that VEGF induces phosphorylation of the tight junction protein occludin in a protein kinase C dependent manner that is associated with vascular permeability. Further, VEGF induces redistribution of occludin and other tight junction proteins from the plasma membrane to the cell cytoplasm and over time, leads to the degradation of occludin. Analysis of occludin phosphorylation sites by mass spectrometry has identified VEGF responsive phospho-sites. In this proposal, data is presented demonstrating that mutation of occludin to prevent phosphorylation, blocks VEGF-induced permeability and occludin endocytosis. Furthermore, preliminary data demonstrate that TNF alters the tight junction complex by reducing the tight junction proteins claudin 5 and zonula occludens 1 but does not decrease occludin content suggesting at least partially divergent mechanisms. Occludin content is closely associated with proliferation of cells that possess tight junctions. Silencing occludin expression in retinal pigment epithelium cells induces a two-fold increase in cell proliferation. Therefore, we will examine the mechanisms by which VEGF and TNF alter the tight junction complex to induce endothelial permeability and the relationship of VEGF-induced occludin phosphorylation and degradation to angiogenesis. While VEGF and TNF diverge in control of occludin, preliminary data demonstrates both factors utilize the atypical PKC pathway to alter the junctional complex and induce endothelial permeability. Therapies targeting atypical PKC pathway alone or in conjunction with PKC inhibitors may provide an effective means to control vascular permeability in diabetic retinopathy and other retinal diseases involving VEGF and inflammatory cytokines. PUBLIC HEALTH RELEVANCE: The experiments in this proposal will elucidate the mechanisms by which growth factors and inflammatory cytokines alter the tight junction complex of the blood-retinal barrier contributing to macular edema in diabetic retinopathy. Further, the proposal will explore the link between vascular permeability and angiogenesis and the role of the tight junction protein occludin in endothelial cell growth control.