The Effect And Potential Mechanisms Of TRPM7 On Mice Atherosclerosis

Background:Atherosclerosis（AS）,a chronic vascular inflammatory disease,is the leading cause of death with increasing morbidity and mortality worldwide.The pathophysiology of AS is that the circulating lipids infiltrate into the damaged endothelium and undergo oxidative modification.Monocytes and vascular smooth muscle cells migrate into the intima and take in oxidized LDL to form foam cells.Vascular smooth muscle cells play an important role in the pathogenesis of AS and can influence the development of AS through phenotype switching,autophagy and senescence.Vascular smooth muscle cell phenotypic switching is characterized by decreased expression of contractile markers and increased expression of synthetic markers.Phenotype switching of smooth muscle cells promoted the development of AS.Autophagy is a lysosomal-dependent process degradating intracellular proteins and damaged organelles.Under the stimulation of lipid peroxide,autophagy maintains the survival of vascular smooth muscle cells.When autophagy is inhibited,vascular smooth muscle cells undergo senescence or apoptosis.Therefore,autophagy activation in vascular smooth muscle cells contributes to plaque stabilization and has a protective effect on AS.Senescence is an irreversible loss of cell proliferative ability.Senescence of vascular smooth muscle cells accelerates the development of AS by inducing plaque inflammation,reducing collagen secretion,degrading the extracellular matrix,and thus leading to plaque instability.Transient receptor potential melastatin 7（TRPM7）channel is a membrane protein with a non-selective cation channels structure and a protein kinase structure.TRPM7 participate in the occurrence and development of vascular diseases such as hypertension and pulmonary hypertension through regulating vascular smooth muscle cell function.However,its effect on AS remains unknown.Objectives:1)To investigate the expression of TRPM7 in diet-induced mice AS and 2)to explore the effect and potential mechanisms of vascular smooth muscle cells TRPM7 on mice AS.Method:1.Identifing the changes of the expression of TRPM7 in the aorta of diet-induced AS mice:1)8-week-old male ApoE-deficient(ApoE^-/-)male mice were given a normal chow diet（NCD）or a high fat diet（HFD）for 12 weeks to construct AS model,wild-type C57（WT）mice were given a NCD as control.Body weight,blood lipid profiles,and plaque areas were measured to verify the modeling.2)Using WT mice as a control,the expression of TRPM7 was detected by RT-qPCR,western blot,immunohistochemical staining and immunofluorescence staining.Meanwhile,the co-localization expression of TRPM7 with endothelial cells,macrophages and vascular smooth muscle cells was measured by immunofluorescence staining to clarify the location of TRPM7 in the mice aorta sinus.2.Confirming the effect of vascular smooth muscle cell-TRPM7specific knockout(Trpm7^SMCKO)on AS plaque area in mice:1)Trpm7^SMCKOmice were induced by intraperitoneal injection of tamoxifen for the period of five consecutive days in 6-week-old male mice,whose genotyping result is ApoE^-/-;Trpm7^flox/flox;Myh11-Cre ER^T2.Their littermates were used as control by intraperitoneal injection of corn oil,taken as a Trpm7^fl/flmice.2)Genotyping,RT-qPCR,western blot and immunofluorescence staining were used to verified the modeling of Trpm7^SMCKOmice.3)The diet-induced AS modeling was started at 8-week-old mice.Trpm7^SMCKOmice were given a NCD or a HFD for 12 or 16 weeks to establish AS models,with Trpm7^fl/flmice giving the same interventions as controls.Body weight,blood lipid profiles,and plaque areas were measured to investigate the effect of TRPM7 on mice mensioned above.3.Detecting the possible mechanisms for protective effect of TRPM7 on AS mice:RT-qPCR and western blot were used to detect the changes of markers of phenotype switching,autophagy and cell senescence in aortic tissue in Trpm7^fl/flmice and Trpm7^SMCKOmice under different dietary interventions.Results:1.Identifing the changes of the expression of TRPM7 in the aorta of diet-induced AS mice:1)After 12 weeks of diet intervention,comparing with the NCD-fed ApoE^-/-mice,HFD-fed ApoE^-/-mice had increased body weight,blood lipid levels,and aortic AS plaques（all P<0.05）,indicating that the mice AS model was successfully constructed.2)Comparing with the NCD-fed WT mice,the mRNA expression of TRPM7 in aorta of NCD-fed ApoE^-/-mice decreased by 80%,and the protein level of TRPM7 decreased by 2.2 times（all P<0.05）.While the expression of TRPM7 in the aortic sinuses showed no significant differences.3)Comparing with the NCD-fed ApoE^-/-mice,the mRNA expression of TRPM7 in aorta of HFD-fed ApoE^-/-mice increased by 3.4 times,and the protein level of TRPM7 increased by 1.8 times.4)The fluorescence colocation of TRPM7 with endothelial cells,macrophages,and vascular smooth muscle cells in the aortic sinuses suggests that TRPM7 is predominantly expressed in vascular smooth muscle cells.5)While the expression of TRPM7 in the aortic sinuses and in plaque measured by immunohistochemical staining were elevated by 1.9,2.0 times,respectively（all P<0.05）.2.Vascular smooth muscle cell-TRPM7 specific knockout in mice decreased plaque areas in AS mice:1)Successfully modeling of vascular smooth muscle cells TRPM7-spcific knockout(Trpm7^SMCKO)mice was verified by genotyping,RT-qPCR,western blot,and immunofluorescence staining.The genotyping result is ApoE^-/-;Trpm7^flox/flox;Myh11-Cre ER^T2.2)After 12 or 16 weeks of a NCD or a HFD intervention,Trpm7^SMCKOmice had no significant changes in body weight and blood lipid profiles compared with the control group(Trpm7^fl/fl).However,the oil red O staining and aortic sinus HE staining showed a decreased plaque areas in Trpm7^SMCKOmice（all P<0.05）,suggesting that specific knockout of TRPM7 in vascular smooth muscle cells had a protective effect on mice AS.3.Detecting the possible mechanisms for protective effect of TRPM7 on AS mice:1)In terms of phenotypic switching,compared with Trpm7^fl/flmice,the expression of the systolic marker ACTA2 in Trpm7^SMCKOmice was increased（all P<0.05）,but there was no significant change in CNN-1.On the other hand,the expression of synthetic markers OPN and MMP2 were decreased in Trpm7^SMCKOmice（all P<0.05）,but there was no significant change in MMP9.2)In terms of autophagy,compared with Trpm7^fl/flmice,the expression of aortic LC3-II/I and beclin-1 in Trpm7^SMCKOmice were increased,and the expression of p62 was reduced（all P<0.05）.3)In terms of senescence,compared with Trpm7^fl/flmice,the expression of p21 in Trpm7^SMCKOmice fed by HFD was decreased（P<0.05）,while there were no significant changes in p53 and p16.Conclusions:1.In diet-induced AS mice model,the expression of TRPM7 is increased in aortic tissue and plaque,and TRPM7 is mainly expressed in vascular smooth muscle cells.2.Specific knockout TRPM7 in vascular smooth muscle cells alleviated AS in mice probably by inhibiting phenotypic switching,promoting autophagy,and inhibiting senescence.