Mechanisms of Retinal Vascular Permeability in Diabetes

描述（由申请人提供）：在美国，糖尿病视网膜病变仍然是失明的主要原因，没有FDA批准的药物治疗。本研究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，以便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可能是由于高血糖、晚期糖基化终产物和高脂血症对血管内皮细胞的直接作用，以及这些代谢物通过诱导生长因子如血管内皮生长因子（VEGF）和炎症细胞因子如肿瘤坏死因子（TNF）的间接作用。在目前的建议中，假设VEGF和TNF改变紧密连接复合物导致内皮细胞通透性增加。先前资助期的研究表明，VEGF以蛋白激酶C依赖的方式诱导紧密连接蛋白occludin的磷酸化，这与血管通透性有关。此外，VEGF诱导occludin和其他紧密连接蛋白从质膜重新分布到细胞质，并随着时间的推移，导致occludin的降解。质谱法分析occludin磷酸化位点已经确定了VEGF反应磷酸化位点。在这项提议中，数据显示occludin的突变可以阻止磷酸化，阻断vegf诱导的通透性和occludin内吞作用。此外，初步数据表明，TNF通过降低紧密连接蛋白claudin 5和occludens小带1来改变紧密连接复合物，但不降低occludin含量，这表明至少有部分不同的机制。Occludin含量与具有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表达诱导细胞增殖增加两倍。因此，我们将研究VEGF和TNF改变紧密连接复合物诱导内皮通透性的机制，以及VEGF诱导的occludin磷酸化和降解与血管生成的关系。虽然VEGF和TNF在控制occludin方面存在分歧，但初步数据表明，这两个因素都利用非典型PKC途径改变连接复合物并诱导内皮细胞的通透性。针对非典型PKC通路单独或联合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VEGF和炎症细胞因子的视网膜疾病的血管通透性提供有效手段。公共卫生相关性：本提案中的实验将阐明生长因子和炎症细胞因子改变血视网膜屏障紧密连接复合物的机制，从而导致糖尿病视网膜病变中的黄斑水肿。此外，该提案将探讨血管通透性和血管生成之间的联系以及紧密连接蛋白occludin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.