Chaoyang Zhang1,2,3, Hai Xie1,2,3, Qian Yang1,2,3, Yiting Yang1,2,3, Weiye Li1,2,3,4, Haibin Tian1,2,3, Lixia Lu1,2,3, Fang Wang1,2,3, Jing-Ying Xu1,2,3, Furong Gao1,2,3, Juan Wang1,2,3, Caixia Jin1,2,3, Guoxu Xu5, Guo-Tong Xu1,2,3, Jingfa Zhang1,2,3,6,7,8. 1. Department of Ophthalmology of Shanghai Tenth People's Hospital, Tongji Eye Institute, Tongji University School of Medicine, Shanghai, China. 2. Department of Regenerative Medicine, Tongji University School of Medicine, Shanghai, China. 3. Department of Pharmacology, Tongji University School of Medicine, Shanghai, China. 4. Department of Ophthalmology, Drexel University College of Medicine, Philadelphia, Pennsylvania. 5. Department of Ophthalmology, The Second Affiliated Hospital of Soochow University, Suzhou, China. 6. Department of Ophthalmology, Shanghai General Hospital (Shanghai First People's Hospital), Shanghai Jiao Tong University, School of Medicine, Shanghai, China. 7. Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai, China. 8. National Center for Clinical Research of Ophthalmology, Shanghai, China.
Abstract
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), Gö6976, or digoxin, were studied for cell viability and barrier function. The expressions of ZO-1, occludin, VEGFR2, HIF-1α, 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-1α 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. Gö6976 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-1α and JNK signallings, and thus up-regulating ZO-1 and occludin expressions in RPE cells.
PURPOSE: To explore the mechanisms of erythropoietin (EPO) in maintaining outer blood-retinal barrier (BRB) in diabeticrats. 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), Gö6976, or digoxin, were studied for cell viability and barrier function. The expressions of ZO-1, occludin, VEGFR2, HIF-1α, 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 diabeticrats. The leakage was largely ameliorated in EPO-treated diabeticrats. The protein expressions of ZO-1 and occludin in the RPE-Bruch's membrane choriocapillaris complex were significantly decreased, whereas HIF-1α and JNK pathways were activated, in 4-week diabeticrats. 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. Gö6976 and digoxin rescued the tight junction and barrier function in glyoxal-treated ARPE-19 cells. CONCLUSIONS: In early diabeticrats, 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-1α and JNK signallings, and thus up-regulating ZO-1 and occludin expressions in RPE cells.
Authors: Manas R Biswal; Zhaoyao Wang; Ryan J Paulson; Rukshana R Uddin; Yao Tong; Ping Zhu; Hong Li; Alfred S Lewin Journal: Antioxidants (Basel) Date: 2021-05-25