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。最近的临床试验表明，靶向血管内皮生长因子(VEGF)可以有效地防止视力丧失的进展，并对一些患者恢复视力。这些研究表明，视网膜的药物疗法可以有效地治疗糖尿病视网膜病变。然而，并不是所有的患者都对抗血管内皮生长因子疗法有反应，这种疗法需要重复眼内注射，有患眼内炎的风险。除了血管内皮生长因子， 糖尿病视网膜病变患者体内许多炎症因子水平升高，包括肿瘤坏死因子、趋化因子(C-C基序)配体2(CCL2)、白介素6和8。因此，了解生长因子和炎性细胞因子改变视网膜血管内皮细胞导致血管通透性和血管生成的机制具有重要意义。上一次资助时期的研究已经确定了两条重要的信号通路，它们控制着对血管内皮生长因子的通透性。第一个途径涉及传统的蛋白激酶C(PKC)激活和紧密连接蛋白阻滞素的磷酸化，是血管内皮生长因子诱导的血管通透性所必需的。已发表的和初步的数据也表明，封闭蛋白磷酸化有助于生长控制和血管生成。因此，血管内皮生长因子激活下游的阻断素磷酸化有助于血管通透性和血管生成，提示在生长和血-视网膜屏障分化中起重要作用。此外，在前一个资金时期的研究已经发现了第二个信号通路，涉及非典型的PKC激活，这是对血管内皮生长因子和炎性细胞因子如肿瘤坏死因子和CCL2的渗透性反应所必需的。利用质谱磷蛋白组学分析，我们已经确定了将这一途径与小G蛋白调节和通透性控制联系起来的下游靶点。在此，我们建议确定阻滞素磷酸化在体内通透性和血管生成中的作用，同时也阐明了aPKC激活途径对血管通透性的控制。总之，这些研究将对血管内皮生长因子诱导的通透性和血管生成的机制提供新的见解，特别是通过控制紧密连接的复杂和小G蛋白对细胞骨架的调节。这项研究有望为血管生长和成熟的本质提供新的见解，并确定有效对抗生长因子和炎性细胞因子的治疗干预的新靶点。

DESCRIPTION (provided by applicant): Diabetic retinopathy remains a leading cause of blindness in the United States. Recent clinical trials have demonstrated that targeting vascular endothelial growth factor (VEGF) can effectively prevent progression of vision loss and for some patients restore visual acuity. These studies demonstrate that medical therapy for the retina can effectively treat diabetic retinopathy. However, not all patients respond to anti-VEGF therapies, which require repeat intra-ocular injections with the risk of endophthalmitis. In addition to VEGF, a number of inflammatory factors are elevated in patients with diabetic retinopathy that are believed to contribute to disease pathology including tumor necrosis factor, the chemokine (C-C motif) ligand 2 (CCL2), interleukin 6 and 8. Therefore, understanding the mechanisms by which growth factors and inflammatory cytokines alter the retinal vascular endothelium leading to vascular permeability and angiogenesis remains of high significance. Research from the previous funding period has identified two important signaling pathways that control permeability in response to VEGF. The first pathway involves conventional protein kinase C (PKC) activation and phosphorylation of the tight junction protein occludin and is required for VEGF induced vascular permeability. Published and preliminary data also reveal that occludin phosphorylation contributes to growth control and angiogenesis. Thus, occludin phosphorylation downstream of VEGF activation contributes to both vascular permeability and angiogenesis suggesting an important role in growth and blood-retinal barrier differentiation. Further, research over the previous funding period has identified a second signaling pathway involving atypical PKC activation as required for permeability response to both VEGF and inflammatory cytokines such as TNF and CCL2. Utilizing mass spectrometry phosphoproteomic analysis we have identified downstream targets linking this pathway to small G-protein regulation and control of permeability. Here we propose to define the role of occludin phosphorylation in permeability and angiogenesis in vivo while also elucidating the aPKC activation pathway to the control of vascular permeability. Collectively, these studies will provide novel insight into the mechanisms of VEGF induced permeability and angiogenesis specifically through the control of the tight junctions' complex and small G-protein regulation of the cytoskeleton. This research is expected to provide new insight into the nature of blood vessel growth and maturation and identify new targets for therapeutic intervention that are effective against both growth factors and inflammatory cytokines.