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.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Study Section
Biology and Diseases of the Posterior Eye Study Section (BDPE)
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Shen, Grace L
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
United States
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Keep, Richard F; Andjelkovic, Anuska V; Xiang, Jianming et al. (2018) Brain endothelial cell junctions after cerebral hemorrhage: Changes, mechanisms and therapeutic targets. J Cereb Blood Flow Metab 38:1255-1275
Dreffs, Alyssa; Henderson, Desmond; Dmitriev, Andrey V et al. (2018) Retinal pH and Acid Regulation During Metabolic Acidosis. Curr Eye Res 43:902-912
Lin, Cheng-Mao; Titchenell, Paul M; Keil, Jason M et al. (2018) Inhibition of Atypical Protein Kinase C Reduces Inflammation-Induced Retinal Vascular Permeability. Am J Pathol 188:2392-2405
Kady, Nermin M; Liu, Xuwen; Lydic, Todd A et al. (2018) ELOVL4-Mediated Production of Very Long-Chain Ceramides Stabilizes Tight Junctions and Prevents Diabetes-Induced Retinal Vascular Permeability. Diabetes 67:769-781
Ramos, Carla J; Lin, Chengmao; Liu, Xuwen et al. (2018) The EPAC-Rap1 pathway prevents and reverses cytokine-induced retinal vascular permeability. J Biol Chem 293:717-730
Díaz-Coránguez, Mónica; Ramos, Carla; Antonetti, David A (2017) The inner blood-retinal barrier: Cellular basis and development. Vision Res 139:123-137
Ramos, Carla J; Antonetti, David A (2017) The role of small GTPases and EPAC-Rap signaling in the regulation of the blood-brain and blood-retinal barriers. Tissue Barriers 5:e1339768
Díaz-Coránguez, Mónica; Chao, Daniel L; Salero, Enrique L et al. (2017) Cell autonomous sonic hedgehog signaling contributes to maintenance of retinal endothelial tight junctions. Exp Eye Res 164:82-89
Liu, Xuwen; Dreffs, Alyssa; Díaz-Coránguez, Monica et al. (2016) Occludin S490 Phosphorylation Regulates Vascular Endothelial Growth Factor-Induced Retinal Neovascularization. Am J Pathol 186:2486-99
Gonçalves, Andreia; Lin, Cheng-Mao; Muthusamy, Arivalagan et al. (2016) Protective Effect of a GLP-1 Analog on Ischemia-Reperfusion Induced Blood-Retinal Barrier Breakdown and Inflammation. Invest Ophthalmol Vis Sci 57:2584-92

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