Function Of TRPM2 In The Development Of Aortic Atherosclerosis And Hyperreactivity To 5-HT In Mouse

Atherosclerosis（AS）is a chronic inflammatory disease,characterized by proliferation and migration of VSMCs.VSMCs are the major component of AS neointima.Alterations in atherosclerotic vascular smooth muscle cells are closely related to the increase of intracellular Ca²⁺concentration and over-production of reactive oxygen species（ROS）.Transient receptor potential melastatin（TRPM）family are vital Ca²⁺channel widly epressed in VSMC membrane.TRPM2 is suggested to be a cellular sensitive sensor for oxidative stress and ROS,mediating excessive Ca²⁺entry and resuling in Ca²⁺overload and consequent cell death.In addition,It has been reported that 5-HT promotes vascular smooth muscle cell proliferation and migration and participates in the development of AS.Based on this,we hypothesized that TRPM2 plays a main role in the pathogenesis of mice AS.In the present study,AS animal model was induced by APOE^-/-mice on 16 weeks’high fat diet.Aortas from AS mice were used to detect TRPM mRNA and protein expression by real time RT-PCR and western blot,as well as to exam 5-HT induced contraction and correspondant inhibition by TRPM2 nonspecific inhibitors ACA and 2-APB.Ox-LDL was introduced to incubate aortic smooth muscle cells to establish in vitro AS cell model.After 24 hours treatment of ACA and 2-APB,the proliferation and migration of ASMCs was detected by MTS assay and cell scratch test respectively.The results suggest a pivotal function role of TRPM2 in the progression of AS and5-HT hyperresponsiveness of atherosclerotic aorta contraction.The study also highlights the possibility of targeting TRPM2 as the potential therapeutic candidate for the treatment of AS.Objective:To investigate the function and the pathologic physiology significance of TRPM2 in the development of AS and 5-HT hyperresponsiveness of atherosclerotic aorta contraction.Methods:The AS mouse model was developed by feeding APOE gene knock out mice(APOE^-/-)on 16 weeks’high-fat diet.The body weight,blood sugar,blood pressure,blood lipid were detected,and aorta was stained with Oil Red O and Hematoxylin to evaluate the atherosclerotic plaques.（2）RT-PCR and Western blot were used to detect TRPM2 mRNA and protein expression of aortas.（3）PCNA protein of aortas were detected by Western blot,MTS assay and cell scratch test were applied to evaluate the proliferation and migration of ASMCs treated by ox-LDL,separately.（4）5-HT induced vasoconstriction and its inhibition by ACA and 2-APB were examed in four groups aortas.Results:（1）Compared with the control groups(C57BL/6J and ApoE^-/-mouse fed on normal diet,C57BL/6J mouse fed on high-fat diet),the body weight,blood glucose and blood lipids of the AS model mice(APOE^-/-mouse were fed on high-fat diet)were significantly increased and typical atherosclerotic plaques were observed in aortas of APOE^-/-+hfd mice by HE staining and Oil Red O staining.This results show that high fat diet on APOE^-/-mice successfully developed atherosclerosis.（2）The expression of TRPM2,TRPM5 and TRPM7 mRNA in atherosclerotic arotas were significantly increased.The expression of TRPM2 protein in atherosclerotic arotas were criticaly increased,suggesting the expression of TRPM2 gene were increased in atherosclerotic arotas.（3）The expression of PCNA protein in atherosclerotic arotas were notely improved,suggesting the proliferation of VSMCs in AS.Compared to the control group,the proliferation and migration of ASMCs treated by ox-LDL were inhibited by ACA（1μM）and 2-APB（10μM）.（4）5-HT induced maximal vasoconstriction in atherosclerotic arotas is higher than the control groups.In addition to,EC50 of AS model mice were notely decreaseed.（5）5-HT induced vasoconstriction was not inhibited by ACA（0.1μmol/L）in four groups aortas.In AS model mice,the aortoas contraction were curbed by ACA（1μM）,whereas the inhibition was not significant in the control groups.（6）The inhibition of 5-HT induced vasoconstriction by 2-APB（10μM）in AS model group(APOE^-/-+hfd)were significantly greater than that in the control group.Conclusion:The treatment of APOE gene knockout and high-fat diet:（1）AS model mice were induced successfully.（2）The expression of TRPM2,TRPM5,TRPM7mRNA and PCNA,TRPM2 protein were up-regulated.（3）The responsiveness to 5-HT of aortas were enhanced.（4）5-HT hyperresponsiveness of atherosclerotic aorta contraction were restrained by inhibitors of TRPM2（ACA and 2-APB）.In addition,the proliferation and migration of ASMCs treated by ox-LDL were inhibited by ACA and 2-APB.These results suggested that treatment of APOE gene knockout and high-fat diet may enhance the function of TRPM2 in 5-HT induced vasoconstriction by up-regulating TRPM2 gene expression.Promoting the proliferation and migration of aortic smooth muscle cells to induce the formation of AS model mice.