Sphingomyelin Synthase 2 Promotes Endothelial Dysfunction By Inducing Endoplasmic Reticulum Stress

Objective:Endothelial dysfunction is involved in the initiation of atherosclerosis,which is the pathological basis of cardiovascular disease.Previous studies in our laboratory have shown that sphingomyelin synthase 2(SMS2)can promote endothelial cell dysfunction by activating Wnt/β-catenin signaling pathways.Meanwhile,endoplasmic reticulum stress can lead to endothelial cell dysfunction,but whether there is an association between SMS2 and endoplasmic reticulum stress remains unclear.To explore their relevance and possible molecular mechanisms,in this paper,we constructed a model of SMS2 overexpression of human umbilical vein endothelial cells(HUVECs)and simultaneously treated cells using 4-phenylbutyric acid,Simvastatin,Lithium chloride,and Salinomycin,respectively,to detect related molecular markers and biological behaviors.Method:1.To explore the possible relationship between SMS2 and endoplasmic reticulum stress,three different treatments were performed in HUVECs:(1)To explore the relevance between SMS2 and endoplasmic reticulum stress,HUVECs were divided into two groups: C(negative control group)and S(SMS2 overexpression group);(2)To explore the relationship between endoplasmic reticulum stress and SMS2,HVUECs were divided into two groups: Control and Tunicamycin(endoplasmic reticulum stress activator);(3)To confirm that SMS2 can lead to endoplasmic reticulum stress,rather than protein overload-induced endoplasmic reticulum stress,HUVECs were divided into two groups: C and Dy105(SMS2inhibitors).We established the cell models according to above groups and detected the expression of GRP78 and SMS2 by WB;In addition,we detected the activity of SMS2 by thin-layer chromatography.2.To explore the possible relationship between Wnt/β-catenin signaling pathway and endoplasmic reticulum stress,HUVECs were divided into three groups: C,Li(LiCl,lithium chloride,activation of Wnt/β-catenin signaling pathway)and Sal(Salinomycin,inhibition of Wnt/β-catenin signaling pathway);To explore the possible mechanism of SMS2 activation of Wnt/β-catenin signaling pathways,HUVECs cells were divided into C and S groups.The expression of endoplasmic reticulum stress-related molecules,β-catenin,P-β-catenin,LRP6 and P-LRP6 were detected by WB.3.To explore the possible relationship between endoplasmic reticulum stress and endothelial cell dysfunction,HUVECs were divided into four groups: C,S,4-PBA(inhibition of endoplasmic reticulum stress)and S+4-PBA.The expression of related molecules were identificated by WB,and endothelial dysfunction correlation index could be tested through cell adhesion experiments.4.To explore the relationship between intracellular cholesterol and endothelial dysfunction,HUVECs were divided into four groups: C,S,Sim(suppression of cholesterol synthesis)and S+Sim.The content of cholesterol was vertified through Filipin assay;WB was used to test the expression of related molecules;the related endothelial dysfunction indexs were detected by LDH,NOS,SOD assay kit and cell adhesion experiment.Results:1.The protein expression levels of GRP78 and SMS2 in the SMS2 overexpression group were significantly increased in constrasted with the blank group,and the protein expression levels of GRP78 decreased after inhibition of SMS2.However,tunicamycin induced endoplasmic reticulum stress that could dramatically enhance the expression of SMS22.Relative to the blank group,activation of Wnt/β-catenin by LiCl increased the protein levels of the endoplasmic reticulum stress-related molecules GRP78,CHOP,ATF6,and β-catenin,but inhibition of Wnt/β-catenin by salinomycin led to opposite results.Overexpression of SMS2 could increase the protein levels ofβ-catenin,P-LRP6 and LRP6 but decrease the content of P-β-catenin.3.Compared with the blank group,the endothelial dysfunction was weakened by endoplasmic reticulum stress inhibitor,the endothelial dysfunction was aggravated after SMS2 overexpression treatment,and the endothelial dysfunction was weakened in constrasted with SMS2 overexpression after combined treatment.4.The intracellular cholesterol and the protein expression levels of the endoplasmic reticulum stress-related molecules GRP78,CHOP and ATF6 decreased compared to the blank group after Simvastatin treatment,and the protein levels of the endoplasmic reticulum stress-related molecules decreased after treatment with the combination of Simvastatin and SMS2 overexpression compared to SMS2 overexpression.In addition,after treating the cells with overexpression of SMS2,the degree of endothelial dysfunction was aggravated,while the damage index was significantly decreased in constrasted with SMS2 overexpression after simvastatin and SMS2 overexpression combined treatment.Conclusion:1.HUVECs can induce endoplasmic reticulum stress after transfection with SMS2overexpression2.SMS2 triggers the Wnt/β-catenin signaling pathway by incresing the phosphorylation of LRP6,which can induce endoplasmic reticulum and ultimately promoting endothelial dysfunction.3.SMS2 can lead to intracell ularcholesterol accumulation,which striggers endoplasmic reticulum and finally contribute to endothelial dysfunction.