Partial characterization of the human retinal endothelial cell tight and adherens junction complexes.

PURPOSE: In diabetic retinopathy and macular edema, the blood-retinal barrier fails to function properly, and there is transvascular leakage of proteins and solutes. The tight junction protein occludin and the adherens junction protein cadherin-5 have been shown to be critical to maintaining the endothelial barrier and regulating paracellular transport of large vessel endothelia. However, the expression and distribution of these junction proteins in the retinal endothelium is not well characterized. METHODS: Human and bovine retinal endothelial cells were isolated as described previously. Western blot analysis and flow cytometry techniques were used to assay for the presence of occludin, zonula occludens-1 (ZO-1), cadherin-5, and beta-catenin. The subcellular localization of the proteins was visualized by immunohistochemistry performed on cultured human retinal endothelial cells and cryosections of bovine retina. RESULTS: Western blot analysis and flow cytometry techniques found occludin, ZO-1, cadherin-5, and beta-catenin in cultured human retinal endothelial cells. Immunofluorescence staining of cultured retinal endothelial cells and cryosections of bovine retina showed junctional localization of occludin, ZO-1, cadherin-5, and beta-catenin. CONCLUSIONS: This report demonstrates the expression of occludin and cadherin-5 in retinal endothelial cells and their localization to sites of cell-cell contact. Expression of their respective regulatory proteins, ZO-1 and beta-catenin, at sites of cell-cell contact suggests that occludin and cadherin-5 play a role in maintaining the retinal endothelial barrier.

Reduced expression of the adherens junction protein cadherin-5 in a diabetic retina.

PURPOSE: Transvascular leakage occurs in diabetic retinopathy. The tight junction proteins occludin and zonula occludens-1 (ZO-1) and adherens junction protein cadherin-5 are critical to the maintenance of endothelial barrier. We report a comparison of junction protein expression in the normal and diabetic retina. METHOD: Case report. Postmortem retinal cryosections were prepared from the left eye of a 73-year-old woman with diabetic retinopathy. Cryosections were immunostained for cadherin-5, occludin, and ZO-1 and compared with retinal cryosections from the right eye of a 72-year-old man with no progression of retinal disease. RESULTS: Immunofluorescence showed positive retinal vessel staining for occludin and ZO-1 in both eyes and cadherin-5 in the normal eye but reduced cadherin-5 staining in the retinal vessels of the diabetic eye. CONCLUSION: Increases in transvascular leakage observed in diabetic retinal vasculature may be associated with reduction in the expression of the critical adherens junction protein, cadherin-5.

Retinal vascular permeability determined by dual-tracer fluorescence angiography.

Diabetic retinopathy is the leading cause of blindness in working age individuals in the United States. Breakdown of the blood-retinal barrier is one of the earliest events in the progression of diabetic retinopathy. Ideally, therapeutic measures would be directed at this early stage, but there are few sensitive, quantitative methods to assess the retinal vascular barrier in vivo. We present here a method that combines fluorescence microangiography and the simultaneous use of two fluorescent tracers to quantitatively assess the retinal vascular barrier. PS/F (permeability x surface area/flow) describing the retinal vasculature of Long Evans rats was found to be 0.086+/-0.031 (n=13, avg.+/-s.d.). Based on estimates of flow and surface area, we estimate the permeability of sodium fluorescein to be approximately 1.2 x 10(-5) cm/s. Infusion of a hyperosmolar solution of 1.6 M mannitol for 5 min significantly increased PS/F in individual veins and significantly increased a flow weighted PS/F from 0.073+/-0.028 to 0.16+/-0.034 (n=3). In conclusion, we have adapted indicator dilution techniques to quantitatively assess the retinal vasculature in vivo. We have found dual-tracer fluorescence angiography to be a sensitive indicator of increases in the blood-retinal barrier produced by hyperosmolar mannitol. This methodology is a promising new minimally invasive strategy which may be adapted to quantitatively track retinal vascular permeability.