The Effects Of Sestrin 2 On Angiogennic Ability Of Human Umbilical Vein Endothelial Cells Under Hypoxic Condition

Objective:In this study,human umbilical vein endothelial cells(HUVECs)were used as a model,lentivirus transfection was applied to regulate endogenous Sestrin2(SESN2)gene expression.To investigate the influence of SESN2 overexpression on the angiogenesis of HUVECs.Ultimately,our objective was to investigate the possibility of promoting angiogenesis and wound healing through adjustment of SESN2 gene,furthermore,to provide a strategy for vascular tissue engineering.Methods:Using HUVECs as cell model,and lentiviral vector carrying either green fluorescent protein(GFP)only(LV-GFP)or GFP and SESN2 together(LV-SESN2-GFP)were transfected into HUVECs.Following puromycin selection,a stable transfected HUVECs line was generated.The transfection efficiency was detected using fluorescence microscopy,Hoechst 33258 staining along with flow cytometry.Simultaneously,using quantitative real-time PCR(q RT-PCR)and western blotting(WB)to measure the m RNA and protein expression levels of SESN2 in HUVECs.Ultimately,HUVECs line that steadily overexpressed SESN2 protein were obtained.The HUVECs were grouped as follows:Non-infected cells were blank group(WT group),cells infected with LV-GFP were served as negative control(NC group)and cells overexpressed SESN2 protein were in SESN2 group.Successively,all groups of HUVECs were respectively cultured under hypoxic(37?,5%CO₂,1%O₂,94%N₂)and normoxic conditions(37?,5%CO₂).The inverted fluorescence microscope and Cell Counting Kit-8(CCK-8)assay was used to detecte the effects of SESN2 overexpression on the proliferation of HUVECs for 24,48 and 72?h.The wound-healing migration assays,Transwell migration assay and Matrigel tube formation assay were exploited to estimate the effects of SESN2 overexpression on the migration and vascular lumen formation abilities of HUVECs.And then,the expression levels of hypoxia-inducible factor-1?(HIF-1?)and vascular endothelial growth factor(VEGF)protein were assessed by western blotting to explore the effects of SESN2 overexpression on the capacity to secrete pro-angiogenic factors of HUVECs.Results:(1)Using lentiviral vector carrying either GFP only(LV-GFP)or GFP and SESN2 together(LV-SESN2-GFP),we established HUVECs line that steadily overexpressed SESN2 protein.Notably,transfection efficiency detected by fluorescence microscopy,Hoechst 33258 staining and flow cytometry was higher than95%.According to the experimental results of q RT-PCR and western blotting,the expression of SESN2 m RNA and protein levels were much higher in the SESN2group compared with the WT and NC groups(P<0.001).(2)The results of inverted fluorescence microscope and CCK-8 assay suggested that prolonged hypoxia inhibited the proliferation of HUVECs,however,SESN2 overexpression promoted cell proliferation in the HUVECs under normoxic or hypoxic conditions and exerted a protective effect on the HUVECs under hypoxic condition.(3)The wound-healing migration assays and Transwell migration assay confirmed that SESN2overexpression could enhance the cell crawling velocity and migration ability of HUVECs under normoxic or hypoxic conditions,which would contribute to angiogenesis,especially in hypoxic microenvironment.(4)Matrigel tube formation assay shows that representative images were observed after a 7 hours incubation,and the convoluted structure of the tube deteriorated over time in all groups.Meanwhile,the assay has confirmed that SESN2 overexpression significantly improved the ability of HUVECs to form tube networks on Matrigel under normoxic or hypoxic conditions.(4)The WB analysis showing that the overexpression of SESN2 increased the expression of VEGF protein,and had no effect on HIF-1?protein of HUVECs.Conclusion:Our work showed for the first time that the modulation of SESN2overexpression can help the HUVECs adapt to the hypoxic microenvironment and improve the ability of HUVECs to form blood vessel-like structures,which will promote angiogenesis in wound healing and tissue engineering applications.In summary,the findings of this study have provided a new strategie on the vascularization of engineered tissues,and,these findings have significant implications for the tissue-engineered grafts survival and serve physiological roles.