Orai-IGFBP3 Signaling Complex Regulates High-glucose Exposure-induced Effects Of Human Coronary Artery Endothelial Cells And Its Mechanism

Background: Diabetes can lead to microvascular and macrovascular complications that are characteristic of diabetes.Therefore,improving endothelial function in diabetic patients is an effective way to prevent severe cardiovascular injury.Store Operated Calcium Entry(SOCE)is controlled by an Orai Transient receptor protein Potentical(TRP)and transmembrane Ca2+ sensor Stromal interaction molecules(STIM)are mediated and regulated by endoplasmic reticulum Ca2+ depletion,which is the main mechanism of cellular Ca2+ influx.According to reports in the literature,the SOCE pathway promotes endothelial cell proliferation and IGFBP-3(insulin-like growth factor binding protein-3)has been shown to have anti-apoptotic effects.Objective:Diabetes-related endothelial dysfunction may be related to Ca2+homeostasis disorder.Our main goal was to uncover the underlying mechanism by which high glucose(HG)promotes the proliferation of cultured human coronary endothelial cells(HCAECs)and by which the Orais and IGFBP-3 complex promotes this proliferation.Methods: HCAECs were cultured in high glucose medium and normal glucose medium.After 13,7 and 14 days,the expression of Orais IGFBP3 PCNA was detected by Western blotting;in addition,the expression changes of PCNA and IGFBP3 were detected by Western blotting after treating HCAECs with SOCE agonists or inhibitors under the condition of high glucose and normal glucose;CCK-8method was used to detect the effects of high glucose culture,SOCE agonist and inhibitor treatment on HCAECs proliferation activity;the interaction between Orais and IGFBP3 in HCAECs under high glucose condition was detected byimmunofluorescence and immunoprecipitation;the expression levels of Orais IGFBP3 and PCNA in coronary artery endothelium of diabetic mice induced by STZ were detected by immunohistochemical assay;After IGFBP3 protein expression was knocked out by transfection specific small interfering RNA,SOCE activity was detected by calcium imaging and Orais expression was detected by Western blot.Results:(1)The proliferation and viability of coronary artery endothelial cells cultured in high glucose for 3,7 and 14 days were significantly enhanced,and the proliferation of STZ-induced coronary artery endothelial cells in diabetic mice was also enhanced.(2)After HCAECs were cultured in high glucose medium for 7 days,the protein expression levels of Orai 1,Orai 2 and Orai 3 were increased,and SOCE was significantly activated.The expression of Orais was also increased in STZ-induced coronary endothelial cells of diabetic mice.In addition,SOCE can regulate the proliferation of HCAECs.(3)The expression of IGFBP3 protein in the coronary endothelium of diabetic mice induced by high glucose culture of HCAECs and STZ was significantly increased;By transfecting IGFBP3 specific si RNA,PCNA protein level and proliferation activity of HCAECs can be reduced.(4)Orais interacts with IGFBP3 in HCAECs,and high glucose can enhance the interaction between them.(5)In HCAECs cultured with high glucose,the SOCE agonist ATP enhanced the expression of IGFBP3 protein,while the SOCE inhibitor BTP2 decreased the expression of IGFBP3 protein In addition,down-regulation of IGFBP3 significantly reduced the expression levels of Orai 1,Orai 2 and Orai 3 proteins,and inhibited ATP or TG-induced SOCE in HCAECs under high glucose conditions.The down-regulation of IGFBP3 reduced the increase of cell activity and proliferation induced by ATP or HG,and also blocked the decrease of HCAECs activity and proliferation induced by BTP2.Conclusion:(1)High glucose culture significantly enhanced the proliferation of HCAECs.(2)After 7 days of high glucose culture,SOCE in HCAECs was significantly activated,and SOCE regulated the proliferation of HCAECs.(3)After 7 days of high glucose culture,the expression of IGFBP3 increased significantly,and IGFBP3 regulated the proliferation of HCAECs.(4)IGFBP3 interacts with Orais in HCAECs,and the interaction is enhanced by high glucose culture.(5)IGFBP3-Orais signaling complex regulates HCAECs proliferation and cell activity enhancement induced by high glucose.