Study On Mechanism And Intervention Of Receptor For Advanced Glycation End Products (RAGE) On Diabetic Retinopahty

Chronic hyperglycemia is a major initiator of microvascular complications of diabetes, such as retinopathy. In condition of hyperglycemia, reducing sugars such as glucose can react non-enzymically with the amino groups of ciculating and tissue proteins to form, via a cascade of events, irreversible structures known collectively as advanced glycation end-products (AGEs). The accumulation of AGEs in vivo has been found to increase with age, and occurs at an accelerated rate in subjects with diabetes.RAGE is a multi-ligand member of the immunoglobulin superfamily of cell surface molecules, expressing on several cell-surface sites. But there was only a little research done on the expression of RAGE on the retinal microvascular endothelium and pericytes. To study the characteristics of RAGE and find more effective way to block the receptor are helpful to explore the mechanisms of AGEs on the diabetic retinopathy.Previous study in our laboratory have demonstrated that AGEs were present in abundance in diabetec but not in control retina by immunohistochemical staining. And the injection of extrogenous advanced glycation end products to normal rats can induce diabetic-like retinopathy. While, this study indicates the expression of RAGE on the cells, the effect of RAGE-ASODNs, the change of RAGEmRNA in vivo when the duration is longer and the intervention of aminoguanidin.Section 1 Mechanisms of receptors for advanced glycosylation end products on diabetic retinopathyFirstly, by using reverse transcriptase polymerase chain reaction analysis RAGEmRNA expression was detected individually on 2wk, 4wk and 8wk after the onset of diabetes, with aminoguanidine received for 8 weeks. The result showed RAGEmRNA levels on the retina increased when duration of diabetes was prolonged. The level of RAGEmRNA on 4wk (relative IOD =0.3350?.0696) was higher than 2wk (relative IOD =0.2134?.0554) (P<0.01). There was no significant difference of RAGEmRNA between 8wk (relative IOD=0.2956?.0255) and 4wk diabetic groups(P>0.05), for RAGE didn't increase without limit, with the receptor saturated both in tissue and on the cell-surface. The RAGEmRNA levels of control rats did not change significantly with age, but could be detected. The results indicate that RAGEmRNA were distributed in normal tissues. The changes of RAGEmRNA expression in diabetes suggest that hyperglymia may regulate RAGE expression, which would contribute to the development of diabetic retinopathy by enhancing AGE-RAGE interactions. Under continuous administration up to the eighth week, RAGEmRNA in diabetic retina (relative IOD ?0.1237?.0225) was attenuated, compared with no-treatment diabetic group (relative IOD=0.2956?.0255) (PO.01), but no significant difference had been found between treatment control group (relative IOD =0.1055?.0218) and normal control group (relative IOD = 0.1460?.0282) (P>0.05). These data suggest that the decrease of AGEs levels by aminoguanidine therapy attenuated the abnormal RAGE gene expression that could contribute to preventing retinal damage from AGEs accumulation.Secondly, combined with the isolation of active retinal blood vessels, the optimal setting for the selective growth of BREC was a fibronectin coated substrate and Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% human serum and lOOug/ml heparin. On the other hand, homogeneous cultures of retinal pericytes were obtained when isolated microvessels were seeded to uncoated dishes and grown in DMEM supplemented with 20% fetal bovine serum. These cells were identified by morphological criteria, by their differential staining for Von Willebrand factor and the a-isoform of smooth-muscle actin, or by Dil-Ac-LDL incorporation. Selective culture provides a simple method for the reproducible culture of near homogeneous endothelial cells and pericytes. BREC were first seen as small, well-defined colonies in close apposition, sometimes attached to microvessel fragment. BRP initially appeared in primary cultures as large isolated cells or as loose colonies of overlap...