The Effect Of Insulin Modulating ER-α Methylation On Atherosclerosis

Part I.The effect of insulin modulating ER-α methylation on VSMCs proliferationBackgroundCoronary atherosclerotic heart disease(CAD)and type 2 diabetes mellitus are both high morbidity in the world,and are two main causes of death.Type 2 diatetes mellitus usually accompanied with insulin resistance(IR)which mean the efficiency that insulin promotes taking and utilizing glucose decreased,and consequently increasing endogenous insulin secretion.Lots of studies found that insulin resistance and consequent hyperinsulinemia are independent risk factors of the formation and development of atherosclerosis,and at the same time promote restenosis after vascular injury.One large sample clinical research of diabetes mellitus in British(ACCORD)indicated that controlling of blood glucose and blood pressure effectively reduced microangiopathy,but had not effect on the macroangiopathy,as CAD etc,that is relates to diabetes mellitus accompany with insulin resistance.DCCT/EDIT research group found that diabetic are not sensitive to insulin,which results in plasma insulin level increased,and increases atherosclerosis compare with diabetic who are sensitive to insulin.Vascular smooth muscle cells locate in the media of vascular,regulate arterious systolic and diastolic function,and have some excretion function.The excretive function is strengthened under the effect of pathogenic factor,and excrete and release some inflammatory factors,that mean the cell phenotype translated to excrete phenotype from systolic phenotype.Some researches have illustrated that hyperinsulinemia promote VSMCs proliferation,migration and phonotype translation.With the development of epigenetics,it was found that there is DNA hypermethylation phenomenon in the process of atherosclerosis.Epigenetics means that without modifying the genetic code,gene expression altered heritably,that is genotype is unchanged bue phenotype changed.This kind of change can hand on stably in development and cell proliferation,including DNA methylation and histone acetylation.Many of epigenetic change are reversible,different with DNA sequence change,that make epigenetic disease can be cured.It is found that there are some genes aberrant methylation in the process of VSMCs phenotype translation.Seppo et al found that gene 5-methylcystein was reduced obviously in the atherosclerotic plaque of atherosclerotic patients and ApoE-/-mice,and VSMCs translated to secrete phenotype from systolic phenotype with hypomethylation,which was related to the transcriptional activity increasing,and found that transmethylase expression increased.So we suspect that aberrant methylation and demethylation of special locus may be concurrence at the process of VSMCs phenotype translation promotes atherosclerosis.It is demonstrated that insulin resistance cause to VSMCs phenotype translation.The effect of hyperinsulinemia cause gene methylation has been verified,as Kim et al found that,insulin induce corpus cavernosum smooth muscle cells androgen receptor promoter methylation,and inhibited androgen receptor expression.So we propose the hypothesis that insulin resistance lead to VSMCs phenotype translation through inducing cells epigenetic changes.Estrogen is an important hormone,it can protect cardiovascular system,except for the effect on reproductive system.Estrogen binds to estrogen receptor to have the various effects.Normal ER-α can inhibit growth,and producing protective NO after binding with estrogen,and inhibit VSMCs and platelets activation.ER-α methylation level is increase in atherosclerosis,causing the effect of inhibiting growth reduced,and promote VSMCs proliferation.So we propose the hypothesis that insulin can promote VSMCs proliferation and atherosclerosis through affecting ER-α methylation.We researched the effect of insulin to ER-α expression in the ApoE/Lepr-/-mice and rat VSMCs.Solve the problem will contribute to explain the new mechanism of insulin promote VSMCs proliferation and atherosclerosis,and find the new target for therapy.ObjectiveTo investigate the effect of ER-α methylation in insulin promoting VSMCs proliferation and atherosclerosis.Methods1.Observe the effect of insulin to atherosclerosis formation in Apo E/Lepr-/-mice aorta.ApoE/Lepr-/-mice were intraperitoneal injected insulin or normal saline,and measured the bodyweight and fasting blood glucose every week,and HE dye and ER-α immunohistochemical test were done in mouse aorta paraffin section.2.Research the effect of insulin on rat VSMCs ER-α,DNMT1,DNMT3 a expression.We tested the rat VSMCs ER-α,DNMT1,DNMT3 a protein and mRNA expression using Western Blot and qPCR.3.Research the effect of transmethylase inhibitor 5-Aza on insulin reducing rat VSMCs ER-α expression.We gave rat VSMCs 5-Aza alone or with insulin,and tested the rat VSMCs ER-α protein and m RNA expression using Western Blot and qPCR.4.Research the effect of insulin on rat VSMCs ER-α methylation.We gave rat VSMCs insulin alone or with 5-Aza,and tested VSMCs ER-α methylation level using BSP.5.Research rat VSMCs proliferation and migration after upregulated ER-α expression.We built ER-α high expression lentiviral vector,and transfected to rat VSMCs.We tested the ER-α high expression VSMCs cell cycle by flow cytometry,and tested cell proliferation by CCK-8 and Ed U,and tested cell migration by scratch experiment.6.Research the effect of rat VSMCs ER-α second exon regulatory sequence methylation to gene transcriptional activity.At first,we built the rat ER-α second exon regulation sequence p GL3-promoter luciferin reporter vector;second,rat ER-α second exon regulation sequence article methylation in vitro,and digestion authenticate by methylation sensitive restriction enzymes(Aci I);at last,methylated and unmethylated ER-α second exon regulatory sequence p GL3-promoter luciferin reporter vectors transfected to 293 FT cells,and tested gene transcriptional activity by luciferase reporter gene.Results1.There were no differences in mean body weight or blood glucose between the experimental group and control group,after the 12-week treatment of intraperitoneal insulin injections compared with the control group.The insulin level and HOMA-IR of the experimental group were higher significantly compare with the control group.2.The HE dye result of ApoE/Lepr-/-mice aorta present that mice in the experimental group showed significant atherosclerotic plaques,thickened walls,and d disordered arrangement of nuclei after the 12-week treatment of intraperitoneal insulin injections compared with the control group.Immunohistochemistry revealed that the expression of ER-α protein in the vascular smooth muscle nuclei was significantly decreased in the experimental group compared with the control group.3.The mRNA and protein level of ER-α were decreased significantly after treating VSMCs with insulin for 24,48 and 72 h,and were more decreased with the treat time prolong.While the m RNA and protein level of DNMT1 and DNMT3 a were increased significantly,and were more increased with the treat time prolong also.4.We treated rat VSMCs with insulin and 5-Aza,and we found that the protein level of ER-α was increased,and the mRNA level of ER-a was not changed compared with control group.5.The methylation level of ER-α second exon was increased after treated rat VSMCs with insulin.While the methylation level of ER-α second exon decreased significantly after treated rat VSMCs with insulin and 5-Aza compare with control group and insulin groups.6.We transfected rat VSMCs with ER-α high expression lentiviral vector,upregulated ER-a expression,flow cytometry,CCK-8 and EdU results presented that VSMCs proliferation were inhibited,and scrath experiment results presented that VSMCs migration were inhibited neither.7.Rat VSMCs ER-α second exon regulatory sequence can active gene transcription,while inhibit gene transcription after its methylation.ConclusionsInsulin can promote Apo E/Lepr-/-mice atherosclerotic plaque formation and development,and decrease aorta smooth muscle cells ER-α expression.The mechanism is insulin induce VSMCs ER-α methylation decreased ER-α expression,and promote VSMCs proliferation,migration and atherosclerosis.Our research illustrated the new mechanism of insulin promote VSMCs proliferation and atherosclerosis,and find the new target for therapy of atherosclerosis,and provide new theoretical foundation for whether us ing insulin.Part II.The relationship between insulin resistance and ER-α methylation in CAD patientsBackgroundCoronary atherosclerotic heart disease(CAD)and diabete mellitus are two common and frequently-occurring diseases.The morbidity in our country increased rapidly with the growth of living standard,aging of polulation and westernization of life-style.Diabete is relate to CAD very well,CAD is a main complication and causing death factor of diabete mellitus.The morbidity of CAD in patients of diabete mellitus is twofold to fourfold compare to the people without diabete mellitus.And the patients are younger,and more serious,and 65% patients of diabete mellitus dead because of the CAD.High blood glucose,high plasma insulin level,vascular endothelium dysfunction and vascular smooth muscle cells dysfunction directly of indirectly promote CAD occurrence and development.Research about people in Finland and Quebec region Canada showed that the morbidity of CAD in patients of diabete mellitus with hyperinsulinemia is higher and more serious.Insulin is a hormone that modulate the balance of blood glucose.Abundant studies demonstrated that insulin can keep endovascular balance as an angiectatic hormone,and insulin resistance usually accompanied with vascular diseases.VSMCs play an important role in atherosclerosis formation and development.VSMCs proliferation,migration and produced extracellular matrix involved in the atherosclerotic plaque early formation,and contributed to keeping the stability of plaque.Insulin promotes VSMCs proliferation,migration and taking in glucose,and to promote development of atherosclerosis.But how insulin modulates VSMCs function is still not very clearly.With the development of epigenetics,DNA methylation indicate a new direction for us to study the mechanism of insulin promote VSMCs proliferation.Epigenetics is that DNA sequence unchanged,gene expression occurring hereditary function variation,including DNA methylation,histone acetylation and mi RNA.And DNA methylation is studied extensively.DNA methylation is under the eff ect of transmethylase,genome cytosine 5’-C methylation in Cp G islands.The change is reversible,and can delivery to next generation.It was found that some VSMCs genes were modulated by DNA methylation.These genes of VSMCs involved in VSMCs proliferation,migration and phenotype translation,as coding platelet derived growth factor(PDGF),serum response factor(SRF),and SMC special SM-a,ER-α and ER-β genes.DNA methylation of VSMCs plays a very important role in the occurring and development of atherosclerosis.Estrogen knows as female hormone,it promotes women sexual organs maturing and second sex characters occurring.There is estrogen in men also.Estrogen has important protective effect on our caidiovascular system.Some researches had found that administration estrogen to female or male patients promoted vasodilatation and inproved myocardial blood supply.Estrogen increases the release of vasodilate substrates,and decreases the production of vasoconstriction substrates,and inhibits the effect o f them.Estrogen plays its function after binding to the estrogen receptor.Estrogen receptor is a potential growth inhibition gene,and plays important antiatherosclerotic effect.Jana Ortmann et al found that ER-α inhibited high blood glucose induced human VSMCs proliferation,but ER-b and G-protein-coupled estrogen receptor(GPER)did not.ER-α increases myocardial glucose utilization,and promotes cardiac function recovery after ischemia reperfusion,and reduces infarct.ER-α methylation is one of the important mechanisms about ER-α expression decreased.It had been found that human genome ER-α methylation level increased significantly in atherosclerotic patients.We have found that insulin reduced rat VSMCs ER-α expression by induced ER-α methylation,and that promoted VSMCs proliferation,migration and phenotype switch.To further affirm the relationship between insulin resistance and ER-α methylation in coronary atherosclerotic disease,we analyzed the peripheral blood genome ER-α methylation level in CAD patients with or without insulin resistance.ObjectiveTo investigate the relationship between insulin resistance and ER-α methylation in CAD patients.Methods1.Select 96 CAD patients diagnosis by coronary angiography in the third military medical university during December 2014 to June 2016 who accompany with or without insulin resistance.We excluded the patients with serious cerebrum,liver,kidney and other important organ diseases.We collected the clinical data about age,gender,long-term smoking history,main complication and so on.And we also tested some biochemical criterion,as blood glucose,blood lipid,liver and kidney function,plasma insulin level etc.The patients were divided to two groups,IR group and non-IR group,depend on their plasma insulin level.2.We gathered their elbow venous blood,and extracted the whole blood genome DNA,and detected the genome ER-α methylation level by methylation mass spectrometric detection technology.3.Statistic analysis the difference of genome ER-α methylation level of CAD patients between IR group and non-IR group,and analysis the influence of HOMA-IR,age and gender on ER-α methylation level.Results1.There are not significant difference about common clinical cata,as age,gender,smoking history,fasting blood glucose,blood lipid,liver and kidney function and main complication.The plasma insuli levels of patients in IR group are much higher than that of patients in non-IR group.2.The results of methylation mass spectrometric detection showed that,the genome ER-α first exon methylation level of patients in IR group were much higher compare with non-IR group.3.The genome ER-α first exon methylation levels of patients in IR group were positive correlate to the HOMA-IR.4.The genome ER-α first exon methylation levels of patients in the two groups were positive correlate to age,and the correlation of non-IR group was more significant compare with IR group.5.The gender factor had not influence to the genome ER-α first exon methylation level of patients in both groups.ConclusionsWe compared the of genome ER-α methylation level of patients between IR group and non-IR group,and we found that the genome ER-α first exon methylation level of patients in IR groups was much higher compare with non-IR group,and was positive correlate to HOMA-IR.The genome ER-α methylation levels of patients in both groups were both positive correlate to age,but were not correlated to gender.