Over-Expression Of SFLT-1in Bone Marrow Mesenchymal Stem Cells Effects Retinal Microvascular Endothelial Cells By H₂O₂ Injury

Background:Retina and choroid neovascular disease is a major factor to visual impairment and blindness,which is associated with oxidative stress and inflammation.The main treatment method is repeated injection of vascular endothelial growth factor?VEGF?through the vitreous chamber,which may result in elevated intraocular pressure,intraocular inflammation,etc,and increase the economic burden of patients.Bone marrow mesenchymal stem cells?BMSCs?derived from SD rats can secrete a variety of neurotrophic factors,which play a role in neurotrophic and antioxidant damage through paracrine.Soluble vascular endothelial growth factor receptor 1?SFLT-1?can effectively inhibit VEGF and thereby block neovascularization.Retinal microvascular endothelial cells?RMECs?are important participants in oxidative stress and inflammatory responses of retinal angiogenesis.In this study,BMSCs were transfected with SFLT-1overexpressed adenovirus to observe its effect on RMECs induced by oxidative damage.Objective:The study aimed to establish co-culture system of BMSCs and RMECs in vitro to evaluate the influence of SFLT-1 gene-carried BMSCs on oxidatively damaged RMECs by H₂O₂.Methods:The BMSCs of SD rats were extracted and cultured in vitro,with fluorescence-activated cell sorting?FACS?assay carried out and the differentiation ability into osteogenesis,lipid and endothelial cells investigated to confirm the cell line.Adenovirus vector was applied to transfect SFLT1 gene into BMSCs,with RT-qPCR and ELISA methods used to detect the expression levels of SFLT1 mRNA and protein.Human RMECs were cultured in vitro,followed by vWF identification.Cell Counting Kit-8?CCK8?and Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling?TUNEL?assays were used to identify the optimal oxidative damage concentration of H₂O₂ in increasing concentrations.The experiment included 4 groups:normal endothelial cell group?Normal?,H₂O₂ injured endothelial cell group?H₂O₂?,co-culture group of H₂O₂ injured endothelial cell with green fluorescent protein-transfected BMSCs?GFP-BMSCs?,co-culture group of H2O2 injured endothelial cell with SFLT1 gene-transfected BMSCs?SFLT1-BMSCs?.After incubation in the optimal concentration of H₂O₂ for 24h and 48h,the oxidative damage of cells in each group was observed,with enzyme-linked immuno sorbent?ELISA?assay applied to investigate the different levels of VEGF,MDA and SOD,inflammatory factors levels of TNF-?and IL-1?,and neurotrophic factors levels of BDNF and CNTF among the four groups,to evaluate the impact of BMSCs and SFLT1 on RMECs.Results:The FACS results of rat-derived BMSCs demonstrated positive expression of CD44,CD90,CD29 and CD73,and low expression of CD34,CD11b/c and CD45,which were in accordance with the criteria of MSCs.In addition,the BMSCs were induced into adipocytes,osteoblasts and endothelial cells.The adenovirus vector with over-expressed SFLT1 gene was transfected into the BMSCs with the optimal multiplicity of infection?MOI?value of 400.The mRNA and protein expression of SFLT1 were detected successfully.Human RMECs cultured in vitro demonstrated typical"paving stone"structure,with vWF identified positive.CCK8 and TUNEL assays observed H₂O₂induced hypoxia injury of RMECS with drug concentration dependence,demonstrating decreased cell vitality,and apoptosis.An optimal concentration of 400 mol/L was identified for subsequent experiments.After co-culture of 24h and 48h,compared with that in H2O2 group,RMECs in GFP-BMSCs and SFLT1-BMSCs groups showed decreased VEGF and MDA expression,and increased SOD level,with a greater change of the indexes in GFP-BMSCs group than SFLT1-BMSCs group.In regard to inflammatory factors,RMECs in GFP-BMSCs and SFLT1-BMSCs groups showed increased TNF-?and IL-1?levels,compared with that in H₂O₂ group,with no difference between the two groups.In addition,compared with H2O2 group,the levels of inflammatory factor decreased in RMECs supernatant in GFP-BMSCs group and SFLT1-BMSCs group,but there was no difference in the decline level of SFLT1-BMSCS group.In regard to neurotrophic factors,RMECs in GFP-BMSCs and SFLT1-BMSCs groups showed increased CNTF and BDNF levels,compared with that in H₂O₂ group,with a greater change of the indexes in GFP-BMSCs group than SFLT1-BMSCs group.Conclusion:The SFLT1 gene modified BMSCs may effectively relieve oxidative damage and inflammatory response of RMECs induced by H₂O₂,with reduced levels of VEGF,inflammatory oxidative factors,and increased levels of neurotrophic antioxidant factors.The synergistic effect of SFLT1 and BMSCs is better than that of BMSCs alone,both of which can effectively protect retinal microvascular endothelial cells with oxidative damage.