Abstract
Abstract: :
Purpose: Vascular endothelial Growth Factor (VEGF) is an angiogenic growth factor with a strong vasoactive potential and is thought to be central to the pathogenesis of diabetic macular edema. Indeed, VEGF treatment of vascular endothelial cells in vitro leads to an increase of permeability but little is known about the underlying mechanisms. Key components controlling paracellular permeability in endothelial beds are thought to be the intracellular adherens and tight junctions. In the present study we have investigated whether VEGF–induced hyperpermeability is mediated through changes in the tight junction protein ZO–1. Methods: Primary microvascular endothelial cell (RMEC) cultures were established from rat retinae. Subcellular localisation of junctional proteins was determined using indirect immunofluorescence. Permeability was assessed by measuring transendothelial resistance or fluorescein–dextran transport. Results: RMECs were established and maintained up to three weeks in culture. VE–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. Long–term treatment induced the disappearance of transmembrane proteins such as VE–cadherin, occludin and claudin–5. Treatment as short as one hour affected the junctional localisation of catenins and of ZO–1. VEGF–induced reduction of junctional ZO–1 depended on signaling via src, Ca/PKC, PI3K, small GTPases and MAP kinases, but not the cytoskeleton. Significantly, this signaling pattern overlapped only partially with that of VEGF–induced hyperpermeability. Conclusions: From these studies ZO–1 appeared to be an unlikely candidate for mediating VEGF–induced hyperpermeability. Nevertheless, our work suggests that VEGF might contribute to the pathogenesis of diabetic macular oedema by negatively regulating the intracellular junctions of the retinal endothelium.
Keywords: growth factors/growth factor receptors • signal transduction • diabetic retinopathy