Diabetes and Vascular Endothelial Growth Factor Induce Paracellular Vascular Permeability at Regions of Occludin Redistribution

Abstract

Abstract: : Purpose: Previous data show that the vasculature of diabetic rat retinas contain regions where the tight junction protein occludin is redistributed (IOVS, 41:3561-3568). The aim of this study was to determine if these are also regions of paracellular permeability. In addition the time course of increased paracellular permeability in response to vascular endothelial growth factor (VEGF) and streptozotocin (STZ)-diabetes was determined. Methods: STZ-diabetic rats and control rats injected intravitrealy with VEGF (2ng/eye) were perfused with 2% paraformaldehyde. Then the rats were perfused with the Alexafluor-conjugated lectin concanavalin-A (ConA). ConA has been shown previously to bind specifically to basement membrane but not the luminal surface of endothelial cells. When perfused through fixed vasculature the ConA only binds to regions of basement membrane that were exposed directly to the contents of the vascular lumen prior to paraformaldehyde fixation. Therefore, specific ConA binding identifies regions of paracellular permeability. The excess unbound ConA was flushed out with saline and the whole retinas were post-fixed and mounted for confocal microscopy. Some retinas were also labeled by immunofluorescence for occludin. Results: In control retinas ConA bound only to the venules. 15 minutes after VEGF injection ConA also bound to the post-capillary venules. 30 minutes after VEGF, ConA binding extended to the arterioles. ConA binding was accompanied by decreased occludin immunoreactivity at the cell border. Similarly, after 2 weeks of STZ-diabetes ConA bound to the post-capillary venules. Punctate ConA binding occurred in large caliber vessels and colocalized with interrupted occludin immunoreactivity. After 1 and 2 months of STZ-diabetes, ConA binding extended to the arterioles and other vessels including the capillaries. In both STZ-diabetic and VEGF-injected rats there was an inverse relationship between occludin immunoreactivity and ConA binding. Conclusion: Diabetes and VEGF increase paracellular vascular permeability in the retina. The post-capillary venules are changed first, followed by the arterioles and capillaries of the outer plexiform layer. Paracellular permeability occurs at regions containing reduced occludin immunoreactivity at the cell border. These data suggest a mechanistic link between occludin redistribution and increased paracellular permeability.

Keywords: 388 diabetic retinopathy • 339 cell adhesions/cell junctions • 614 vascular cells