Diabetes mellitus is a common disease where blood glucose levels are elevated due to a lack of insulin action. Diabetic retinopathy is a complication of diabetes where the retina is damaged causing vision loss that can lead to blindness. The tight junctions in blood vessels of those with diabetic retinopathy are disrupted and allow fluid and blood to leak into the retina. Occludin and zonula occludin 1(ZO-1) are two critical proteins involved in the formation of these tight junctions. When these proteins are phosphorylated, they disassemble from the plasma membrane and are trapped in vesicles within cells. This leads to an increase in the permeability of blood vessels. Vascular endothelial growth factor (VEGF) is elevated in the vitreous humor of those with diabetes and has been shown to increase the phosphorylation of occludin and ZO-1. We hypothesize that VEGF phosphorylates these proteins through the mitogen-activated protein kinase (MAPK) signaling pathway. In this experiment, we use an accepted cell model in Madin-Darby canine kidney (MDCK) cells to analyze the signaling cascade that leads to the disassembly of tight junctions. We treat cells with platelet-derived growth factor (PDGF) because PDGF activates the same molecular pathway that VEGF activates. We add U0126, a MAPK pathway inhibitor, to cells treated with PDGF. We expect that the phosphorylation that would occur from the treatment with PDGF will not be present if the MAPK signaling pathway is inhibited. Following treatment with growth factor and inhibitor, ZO-1 is isolated by immunoprecipitation. The proteins are then run on an SDS PAGE to separate the mixture, and western blot analysis determines the phosphorylation state of ZO-1. This project will provide data that can be used to determine the pathway that is activated in diabetic retinopathy. Understanding the molecular mechanisms of tight junction disassembly will aid development of treatments that prevent the phosphorylation of ZO-1 and consequent disassembly of tight junctions in diabetic patients’ eyes.

Determination of the molecular pathway causing phosphorylation of zonula occludin 1 and disassembly of tight junctions in diabetes

253rd National Meeting (2017)

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