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.
This proposal provides innovative research to determine the mechanisms by which endothelial cells proliferate and mature contributing to the blood-retinal barrier and how diabetes alters these vessels leading to macular edema and pathologic angiogenesis. Importantly, this research explores required pathways that regulate vascular permeability and may lead to new therapies for macular edema.
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