Experimental Study On Endothelial Progenitor Cells In The Pathogenesis And Treatment Of Diabetic Retinopathy

Objective:In this study, we established streptozotocin (STZ)-induced type1diabetes mellitus (T1DM) rats, and investigate the role of EPC in the pathogenesis of DR; we observed the effects of simvastatin on EPC mobilization, which could alter the rate of diabetic retinopathy (DR) formation and progression.Methods:(1) Fifty male Wistar rats were divided into control(CON) group(14rats) and diabetes(DM) group(36rats). Wistar rats were induced with streptozotocin (STZ) injection for diabetic retinopathy model. The rats in the DM were further divided into3groups based on the time of observation,1month(DM1)、3month(DM3) and6month(DM6) groups, with12rats in each group. All eyeballs were examined hematoxylin and eosin(HE) staining and VEGF by immunity set expression.(2) Mononuclear cells were collected by density gradient centrifugation from the bone marrow of rats. The isolated cells were cultivated in dishes coated with fibronectin. Immuno-fluorescence staining and flow cytometry were used to indentify EPC. The colony number of EPC was assayed by CFU counting; proliferation, migration and adhesion function of EPC were assayed by MTT chromatometry, modified Boyden chamber assay and adhesion activity assay.(3) Eighty male Wistar rats were divided into normal control group, DR model group, DR with placebo group, DR with simvastatin group with twenty rats in each group. Control group and DR model group were not given any intervention; simvastatin group was given simvastatin20mg/kg orally. Flow cytometry was used to identify and count the number of EPC from peripheral blood. Vascular permeability was quantified by analyzing albumin leakage using Evans blue method. All eyeballs were examined by HE staining and immunohistochemical analysis of CD31expression in the retina of rats. The expression of eNOS, iNOS and Ang-1mRNA in retina was measured by RT-PCR.Results:(1) Blood glucose of DR rats was significantly increased. Endothelial cells were edema in DM6group. The expression of VEGF in retina was significantly increased in DM1, DM3, DM6groups compared with CON group. The significantly reduced thickness and obvious disorganized retinal cell layers were seen in DR rats. In the diabetic groups, we also found that T1DM rats developed telangiectatic vessels, vacuolar degeneration of ganglion cells and vertically formed neovascularizations in the retina.(2) The cell clusters number, proliferation ability, migration ability and adhesion ability of bone marrow-derived EPC was significantly reduced in DM1, DM3, DM6groups compared with CON group. The thickness of retina was reduced and retinal cells becomed disorganized in DM1and DM3group.(3) The counts of EPC from peripheral blood in DR model group and placebo group were lower; the counts of EPC from peripheral blood count in simvastatin group were higher than DR model group and placebo group. The layers tissue of retina was edema and the retina developed telangiectatic vessels and many ganglion cells developed vacuolar degeneration in DR model group and placebo group. The layers tissue of retina was less edema and gradually arranged in simvastatin group. The osmolality of EB in simvastatin group was significantly lower than DR model group. Retina tissue layers in simvastatin treated rats exhibited decreased edema and became organized when compared to non-simvastatin treated rats. CD31expression in control group was weakly positive, its expression increased in simvastatin group compared with DR model group. The expression of eNOS was down-regulated in DR model group compared with control group, its expression increased in simvastatin group compared with DR model group; but the expression of iNOS and Ang-1was contrary to eNOS.Conclusions:(1) The number and biological dysfunction of EPC from bone marrow reduced in diabetic rats. With the development of DR, the number of EPC increased gradually.(2) EPC from peripheral blood in DR rats could be mobilized by simvastatin, which differentiate into retinal vascular endothelial cells and regulate the expression of eNOS and iNOS, in order to slow down the progress of DR.