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P. Turowski, Z.K. Ockrim, P. Adamson, P. Hykin, J. Greenwood; Mechanisms controlling permeability and junctional architecture of primary retinal endothelial cells in vitro . Invest. Ophthalmol. Vis. Sci. 2004;45(13):451.
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Purpose:Elevated levels of vascular endothelial growth factor (VEGF) can be detected in the vitreous of patients with diabetic macular oedema. VEGF is an angiogenic growth factor with a strong vasoactive potential and thought to be central in the induction of diabetic macular oedema. Indeed, exposure of vascular endothelial cells in vitro to VEGF has been shown to increase their permeability but little is known about the underlying mechanisms. Inter–cellular adherens and tight junctions are key components controlling paracellular permeability of endothelial beds. In the present study we have investigated how VEGF treatment affected the temporal and spatial organisation of inter–cellular junctions. Methods:Primary microvascular endothelial cell cultures were established from rat retinae (RMEC) and subcellular localisation of junctional proteins determined using indirect immunofluorescence. Their subcellular localisation was also determined following treatment with VEGF, thrombin or histamine (two other well–established permeability factors), in combination with various drugs specifically interfering with intra–cellular signalling pathways. Results:RMECs were established and maintained up to three weeks in culture. Cadherin and catenins, components of adherens junctions, as well as the tight junctional proteins occludin, claudin–5 and ZO–1 were all found predominantly at the inter–cellular junctions. VEGF treatment affected the subcellular distribution of many junctional proteins: It induced either their disappearance (for occludin and claudin–5) or their redistribution to the cytoplasm (for catenins and ZO–1). Thrombin and histamine induced similar but not identical rearrangements of junctional proteins. Further data will be presented from our ongoing work testing the involvement of small GTPases, phosphatidylinsositide 3–kinases, and various protein kinases in the signalling pathways modulating permeability in RMECs. Conclusions:Our work suggests that VEGF might contribute to the pathogenesis of diabetic macular oedema by negatively regulating the intracellular junctions of the retinal endothelium. Understanding underlying signalling mechanisms may open novel therapeutic avenues.
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