ObjectiveTo investigate the inhibitory effects and related mechanism of S100A4 gene silencing on oxygen-induced retinal neovascularization. Methods7-day-old C57BL/6J mice were randomly divided into 5 groups including normal group, normal-S100A4 group, oxygen induced retinopathy (OIR) group, OIR-S100A4 group, OIR-green fluorescent protein (GFP) group. To establish the OIR model, mice from all groups except normal one were exposed to (75±2)% oxygen for 5 days and then to room air. In the OIR-S100A4 group and OIR-GFP group, the OIR mice were given an intravitreal injection of 1μl of 1.0×109 PFU/ml adenovirus of Ad-S100A4-RNAi or Ad-GFP at P12, and then returned to normoxia for the next 5 days. In the OIR group, OIR was induced in C57bl/6J mice from P7 to P17. In the normal-S100A4 group, the normal P12 mice were give an intravitreal injection of 1 μl of Ad-S100A4-RNAi adenovirus, and maintained in room air from P12 to P17. In normal group, newborn mouse litters were maintained in room air from P0 to P17 without any treatment. Mice in all five groups were euthanized at P17, and retinas were collected for biochemical assays and morphological study. Retinal neovascularization (RNV) was evaluated by counting the number of pre-retinal neovascular cells and the whole mount immunofluorescent staining of the mouse retina. Protein and mRNA expression levels of S100A4, cAMP responsive element binding protein (CREB), B cell lymphoma-2 (bcl-2), Caspase-3 were determined with western blot and real-time PCR. ResultsThe number of pre-retinal neovascular cell nuclei in retinas from OIR-S100A4 group were obviously lower than those in the retinas from OIR group and OIR-GFP group (t=13.61, 14.64; P < 0.05). In OIR-S100A4 group, the retinal neovascular tufts area and the vaso-oblitertion area were both significantly smaller than those in OIR group and OIR-GFP group (P < 0.05). Protein level of CREB and bcl-2 were significantly down-regulated in OIR-S100A4 group than those in OIR and OIR-GFP group (P < 0.05).On the contrary, protein levels of Caspase-3 were up-regulated in OIR-S100A4 group than those in OIR and OIR-GFP group (P < 0.05). ConclusionAd-S100A4-RNAi transfer ameliorates RNV in mouse model of OIR maybe through down-regulating the expression of bcl-2 and CREB, and up-regulating the Caspase-3.
ObjectiveTo observe the expression of S100A8 in plasma exosomes, microvesicles (MV), plasma and vitreous in patients with diabetic retinopathy (DR), and verify it in a diabetic rat model, and to preliminarily explore its role in the occurrence and development of DR.MethodsA case-control study. From September 2018 to December 2019, a total of 73 patients with type 2 diabetes, hospitalized patients undergoing vitrectomy, and healthy physical examinations in the Tianjin Medical University Eye Hospital were included in the study. Among them, plasma were collected from 32 patients and vitreous fluid were collected from 41 patients, which were divided into plasma sample research cohort and vitreous sample research cohort. The subjects were divided into simple diabetes group (DM group), non-proliferative DR group (NPDR group) and proliferative DR group (PDR group) without fundus changes; healthy subjects were regarded as normal control group (NC group). In the study cohort of vitreous samples, the control group was the vitreous humor of patients with epimacular membrane or macular hole. Plasma exosomes and microvesicles (MVs) were separated using ultracentrifugation. Transmission electron microscopy, nanometer particle size analyzer and Western blot (WB) were used to characterize exosomes and MVs. The mass concentration of S100A8 was determined by enzyme-linked immunosorbent assay. Eighteen healthy male Brown Norway rats were divided into normal control group and diabetic group with 9 rats in each group by random number table method. The rats of diabetes group were induced by streptozotocin to establish diabetic model. Five months after modeling, immunohistochemical staining and WB were used to detect the expression of S100A8 in the retina of rats in the normal control group and the diabetes group. t test was used for the comparison of measurement data between the two groups. Single-factor analysis of variance were used for the comparison of multiple groups of measurement data.parison of measurement data between the two groups. Single-factor analysis of variance were used for the comparison of multiple groups of measurement data.ResultsExosomes and MVs with their own characteristics were successfully separated from plasma. The concentrations of plasma exosomes and vitreous S100A8 in the PDR group were higher than those in the NPDR group, DM group, NC group, and the difference was statistically significant (P=0.039, 0.020, 0.002, 0.002, P＜0.000,＜0.000). In the plasma sample cohort study, It was not statistically significant that the overall comparison of the S100A8 mass concentrations of plasma and plasma MV in the four groups of subjects (F=0.283, 0.015; P=0.836, 0.996). Immunohistochemical staining showed that retinal ganglion cells, bipolar cells, cone rod cells and vascular endothelial cells in the diabetic group all expressed S100A8 protein. Compared with the normal control group, the expression level of S100A8 in the retina of the diabetic group increased, and the difference was statistically significant (t=8.028, P=0.001).ConclusionsThe level of S100A8 protein in circulating exosomes increases significantly with the severity of DR in patients with type 2 diabetes. S100A8 may be an influential factor in the inflammatory environment of DR and a potential anti-inflammatory therapeutic target.