| Aim Diabetic retinopathy and diabetic nephropathy witch are the represents of diabetic microvascular complications, are two of the most common and severe microvascular complications of diabetes. There is a high concordance in the development of diabetic retinopathy and diabetic renal disease in diabetic patients, as well as strong familial aggregation of these complications, suggesting a common underlying genetic mechanism. However, the precise gene(s) and genetic variant(s) involved remain largely unknown. The purpose of this study was to uncover the generic susceptibility of diabetic microvascular complication. Methords: 1. 19 SNPs from 11 candidate genes, which involved in EPO and other important cellular factors that maybe increase susceptibility to diabetic microvascular complications in our three diabetes cohorts, 2672 diabetic patients with or without PDR and ESRD were genotyped and tested for allelic associations. 2. To find that the risk allele that may confer an increased the risk of diabetic microvasscular complications by influencing EPO expression level, MatInspector and luciferase reporter expression were performed to scan putative transcription factor binding sites. 3. To further test the affection of different alleles on EPO protein expression, ELISA, RT-PCR and immunohistrochemistory were perfomred on human vitreous and lymphocytes and retinal samples of non-diabetic individuals and human with the TT genotype compared to those with the GG genotype. Moreover, the EPO mRNA level was determined in the kidney of db/db mouse model and in the retina of oxygen-induced retinopathy (OIR) mouse models by real-time RT-PCR. 4. To explore the function of EPO on angiogenisis, EPO corneal intrastroma injection was performed and new vessels on the cornea were abserved. Results: 1. No significant association was found except for SNP rs1617640 (p=1.91×10-3 for an additive allele-dosage model, T allele: 63.10% in cases versus 54.18% in controls). The association was replicated in other two independent replication cohorts, Boston type 1 diabetes (P=2.1×10-2 cohort and GoKind (P=2.66×10-8) cohort. 2. Using Matlnspector to scan putative transcription factor binding sites within this region, we found that the T allele creates two enhancer binding sites and the.T allele enhanced luciferase reporter expression by 25-fold compared to that of the G allele (P=4.7×10-29), suggesting it may confer an increased risk by influencing EPO expression level. 3. EPO protein concentrations were 7.5-fold higher in vitreous samples of non-diabetic individuals with the TT genotype compared to those with the GG genotype. And EPO mRNA level in lymphocytes in diabetic individuals with the TT genotype was significant higher than in those with GG genotyp (P=3.3×10-2) 4.The expression of EPO was increased about three-fold in the kidney of db/db mice (P=0.0022) and in the mouse ischemic retina (P=1.1×10-7).5 Corneal neovascularization was induced by EPO intrastromal injection in 5 out of 6 eyes injected with EPO protein. However, there is no new vessels were observed in the eyes (n=6) injected with vector. EPO and its receptor EPOR was expressed on epithelia cells, endothelial cells and keratocytes in normal and neovascularized cornea, also expressed on new vessels in neovascularized cornea. Conclusions: EPO has a key role in genetic susceptibility to diabetic microvascular compications.The T allele of rs1617640 plays a significant functional role in EPO expression. This studay identified a potentially new pathogenetic mechanism for advanced micrvascular complications. This study also suggested that the risk would be increased when hrEPO was prescribed to those patients with genetic susceptiblility to diabetic microvasxular complications. A potential effects of EPO on corneal neovascularization was also showed in this studay.
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