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DA Antonetti, EB Wolpert, TW GardnerPenn State Retina Research Group; Retinal Endothelial Occludin Phosphorylation Increases with Diabetes and VEGF Activation and is Downstream of Protein Kinase C . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2970.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Diabetic retinopathy is characterized by increased vascular permeability causing retinal edema. At least part of the increase in permeability can be attributed to elevated VEGF within the eye. We have previously shown that VEGF induces phosphorylation of the tight junction protein occludin in retinal endothelial cells in vivo and in primary bovine retinal endothelial cell (BREC) culture. This phosphorylation can be detected as a gel shift in the migration of occludin on Western blots. In the present study we determined whether diabetes increases retinal occludin phosphorylation. In addition, mounting evidence indicates both protein kinase C (PKC) and nitric oxide synthase (NOS) contribute to vascular permeability induced by VEGF. Therefore, specific PKC and NOS inhibitors were tested to determine the contribution of these enzymes in VEGF stimulated occludin phosphorylation. Methods: Animals were made diabetic with streptozotocin and diabetes was confirmed by monitoring blood glucose. Retinas were removed and homogenized in an SDS/Triton X-100/deoxycholate extraction buffer. Occludin phosphorylation was quantified by gel shift on Western blots and the ratio of hyperphosphorylated to hypophosphorylated occludin was determined. Results: Occludin phosphorylation increased after 1 month (p<0.005) and 3 months (p<0.05) of diabetes. Furthermore, VEGF induced a similar increase in occludin phosphorylation in BREC. The NOS inhibitor L-NMMA blocked occludin phosphorylation in response to VEGF but had no effect on occludin phosphorylation in control samples. In addition, a PKC inhibitor blocked occludin phosphorylation at both 600 nM (broad spectrum PKC inhibition) and at 30 nM (PKC ß specific inhibition). Conclusion: These studies reveal that diabetes and VEGF induce occludin phosphorylation through an NOS and PKC ß dependent pathway. PKC ß regulation of occludin phosphorylation may induce vascular permeability.
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