Anti-VEGF effects of intravitreal erythropoietin in early diabetic retinopathy
Jingfa Zhang1,2, Liu-Mei Hu3, Guoxu Xu4, Yalan Wu2, Jianfeng Shen2, Yan Luo3, Yong Zhong3, Stephen H. Sinclair5, Myron.Yanoff5, Weiye Li1,2,3,5, Guo-Tong Xu1,2
1
Tongji Eye Institute and Department of Regenerative Medicine, Tongji University School of Medicine, Shanghai, China, 2Laboratory of Clinical Visual Sciences, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai JiaoTong University School of Medicine, Shanghai, China, 3Dept. of Ophthalmology, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China, 4 Dept. of Ophthalmology, Second Affiliated Hospital of Soochow University, Suzhou, China, 5Dept. of Ophthalmology, Drexel University College of Medicine, Philadelphia, PA, USA
TABLE OF CONTENTS
- 1. Abstract
- 2. Introduction
- 3. Materials and methods
- 3.1. Reagents
- 3.2. Experimental animals and intravitreal erythropoietin (EPO) treatment
- 3.3. Examination of blood-retinal barrier (BRB) permeability
- 3.4. RNA isolation and gene expression determined by real-time PCR
- 3.5. Western blotting analysis for HIF-1 alpha, VEGF, EPO and EPO receptor (EpoR)
- 3.6. Sample preparation for morphologic studies, and measurement of the retinal thickness and cell counts
- 3.7. In situ detection of cell death in retina by TUNEL assay
- 3.8. Electron microscopy (EM)
- 3.9. Statistics
- 4. Results
- 4.1. EPO protection of retinal neurons from cell death in early diabetes
- 4.2. EM evidence of the protective effect of EPO on retinal cells in diabetes
- 4.3. Retinal VEGF was up-regulated with the progression of diabetes, and down-regulated by time-dependent EPO treatment
- 4.4. Down-regulation of HIF-1 alpha and VEGF expression by intravitreal EPO was correlated with the BRB restoration in a time- and dose-dependent manner
- 4.5. Down-regulation of PHDs and VHL expressions after intravitreal injection of EPO as post-translational regulation to HIF-1 pathway
- 5. Discussion
- 6. Acknowledgement
- 7. References
1. ABSTRACT
In the present study, a single intravitreal erythropoietin (EPO) to diabetic rats produced therapeutic effects on blood-retinal barrier (BRB) function and neuronal survival at different time courses of retinopathy. In parallel, the >hypoxia-inducible factor 1 alpha(HIF-1 alpha) pathway has been quantitatively studied, including VEGF-A, endogenous EPO, EPO receptor (EpoR), prolyl hydroxylases (PHD1-3) and von Hippel-Lindau tumor suppressor (VHL). The mRNA levels of HIF-1 alpha, VEGF-A, endogenous EPO, PHD1-3 and VHL are all up-regulated in the diabetic retina, and suppressed by exogenous EPO. The increased protein levels of HIF-1 alpha, VEGF-A, and endogenous EPO found in diabetic retinas also have been down-regulated by exogenous EPO. The results demonstrate that the HIF-1 pathway is activated in the retina in early diabetes, but is negatively regulated by a feedback loop following the administration of exogenous EPO. Exogenous EPO at pharmacologic levels leads to suppression of VEGF and in turn, restoration of the normal functions of BRB in a time-dependent manner. In the diabetic retina, the same level of exogenous EPO that inhibits VEGF also exerted neuronal protection.
