MLKL Promotes Adhesion Of Monocytes To Endothelial Cells Via Upregulation Of ICAM-1 Expression And Its Correlation With Coronary Heart Disease

BACKGROUNDIn recent years,the incidence and mortality of coronary heart disease(CHD)have been increasing,which has brought great burden to the society.The pathological basis of CHD is inflammation,atherosclerosis(AS)and thrombosis.When the blood vessel is stimulated by some factors,the interaction between endothelial cells and leukocytes leads to inflammatory cascade reaction,promotes the adhesion of monocytes and endothelial cells,and then promotes the formation of AS.Intercellular adhesion molecule-1(ICAM-1)is a member of the immunoglobulin superfamily of adhesion molecules,which can closely bind with vascular endothelial cells and participate in the initial process of adhesion reaction.MLKL,a kinase like protein,has been shown to play an important role in the downstream pathway of programmed necrosis induced by tumor necrosis factor(TNF).Programmed necrosis releases a large number of inflammatory mediators and injury related factors,and participates in the occurrence of ischemic cardiovascular disease.Our previous study found that MLKL can promote the occurrence and development of AS by inhibiting autophagy and aggravating inflammation.However,whether MLKL can promote ICAM-1 expression to participate in the process of endothelial cell adhesion and affect the occurrence and development of AS remains unclear,and the relevant mechanism needs to be further explored.MLKL is widely present in many organs of human body.A large number of studies have shown that MLKL can be used as a biomarker for many kinds of malignant tumors(gastric cancer,pancreatic cancer,etc.),but there are few studies on MLKL and cardiovascular disease.In this study,MLKL level in serum of CHD patients was measured,and the correlation between MLKL level and CHD was analyzed to provide a new laboratory basis for the auxiliary diagnosis and prognosis evaluation of CHD.MATERIALS AND METHODS1.Both human umbilical vein endothelial cells(HUVECs)and human monocytic THP-1 cells were obtained from the American Type Culture Collection(ATCC).(1)MLKL siRNA or recombinant plasmids overexpressing MLKL was used to transfect HUVECs,the expression levels of mRNA and protein for MLKL and ICAM-1 were detected by qRT-PCR and Western blotting.(2)HUVECs were transfected with MLKL siRNA or recombinant plasmids overexpressing MLKL.After 24 hours,HUVECs were incubated with CFSE green fluorescent labeled THP-1 mononuclear cells,and photographed with inverted fluorescence microscope.Image-J software was used to measure the number of fluorescent spots,which was the number of THP-1 mononuclear cells adhered to endothelial cells.2.A total of 100 CHD patients were recruited in this study,including 30 cases of acute myocardial infarction(AMI),35 patients diagnosed with unstable angina pectoris(UA),35 cases of stable angina pectoris(SA).30 healthy people were as normal controls.The control group was consisted of 30 healthy subjects without CHD.According to the results of coronary angiography,patients were divided into 22 with single-vessel disease,25 with double-vessel disease,and 53 with three-vessel disease.The serum MLKL levels of four groups were detected by enzyme-linked immunosorbent assay(ELISA)kit;and the correlation between MLKL and CHD was analyzed.RESULTS1.MLKL overexpression greatly enhanced ICAM-1 expression in human umbilical vein endothelial cells(HUVECs),while pcDNA-MLKL promoted adherence of human monocyte cells(THP-1)to HUVECs.MLKL siRNA dramatically inhibited expression of ICAM-1 and made THP-1 cells less adherent to HUVECs2.MLKL level of CHD group was higher than that of normal control group;MLKL content was the highest in AMI group,and the highest in patients with three branches of coronary artery disease;MLKL was positively correlated with NT-proBNP and CRP levels;serum MLKL levels of AMI group and UA group were positively correlated with Gensini score.CONCLUSION1.MLKL promoted THP-1 cell adhesion to HUVECs via upregulation of ICAM-1 expression,thereby promoting development of atherosclerosis.2.MLKL level is positively correlated with the incidence and degree of CHD,which can be used for the diagnosis and evaluation of CHD.