Erythropoietin protects outer blood-retinal barrier in experimental diabetic retinopathy by up-regulating ZO-1 and occludin

Purpose To explore the mechanisms of erythropoietin (EPO) in maintaining outer blood-retinal barrier (BRB) in diabetic rats. Methods Sprague-Dawley rats were rendered diabetic with intraperitoneal injection of streptozotocin, and then followed by intravitreal injection of EPO. Two and four weeks later, the permeability of outer BRB was examined with FITC-dextran leakage assay, following a method to demarcate the inner and outer retina based on retinal blood supply. The glyoxal-treated ARPE-19 cells, incubated with EPO, soluble EPO receptor (sEPOR), Go6976, or digoxin, were studied for cell viability and barrier function. The expressions of ZO-1, occludin, VEGFR2, HIF-1 alpha, MAPKs, and AKT were examined with Western blot and immunofluorescence. Results The major Leakage of FITC-dextran was detected in the outer nuclear layer in both 2- and 4-week diabetic rats. The leakage was largely ameliorated in EPO-treated diabetic rats. The protein expressions of ZO-1 and occludin in the RPE-Bruch's membrane choriocapillaris complex were significantly decreased, whereas HIF-1 alpha and JNK pathways were activated, in 4-week diabetic rats. These changes were prevented by EPO treatment. The in vitro study with ARPE-19 cells confirmed these changes, and the protective effect of EPO was abolished by sEPOR. Go6976 and digoxin rescued the tight junction and barrier function in glyoxal-treated ARPE-19 cells. Conclusions In early diabetic rats, the outer BRB might be more severely damaged and its breakdown is the major factor for retinal oedema. EPO maintains the outer BRB integrity through down-regulation of HIF-1 alpha and JNK signallings, and thus up-regulating ZO-1 and occludin expressions in RPE cells.