Myocardin Inhibits Atherosclerosis By Upregulating Abca1 Expression And The Regulatory Mechanism Of Mir-1192

Atherosclerosis(As)is a multifactorial disease and the underlying mechanisms are extremely complicated.Lipid infiltration-induced foam cell formation,chronic damage in endothelial cells,shearing stress generated by blood flow,oxidative low density lipoprotein(ox-LDL)and other events cause damages in large and medium arterial vascular,leading to a series of clinical events,such as acute coronary events and acute cerebral stroke.Currently,lipid infiltration-induced foam cell formation is considered to be a core link to the occurrence and development of As.Previous studies have shown that more than 50% foam cells in the late As plaques of human body were originated from vascular smooth muscle cells(VSMCs).Therefore,effectively inhibiting of VSMCs-derived foam cells’ formation is very important for prevention and treatment of atherosclerotic cardiovascular disease.As a cell membrane binding protein,ATP binding cassette transporter A1(ABCA1)is to mediate the intracellular free cholesterol(FC)efflux to the lipid-poor apolipoprotein A-1(apo-A1),leading to the synthesis of high density lipoprotein(HDL).It plays a pivotal role in maintaining balance of intracellular cholesterol and inhibiting foam cells formation.Our previous studies have confirmed that apo A-1 binding protein,NF-κB and Apelin-13 and others influence foam cells formation and ameliorate the development of As through regulating ABCA1-mediated intracellular cholesterol efflux.Clinical studies have shown that the degree of As lesion in patients with coronary heartdisease was negatively correlated to ABCA1 expression levels in VSMCs.Besides,many other experiments demonstrated that decreased ABCA1 expression in VSMCs was a key element of intracellular cholesterol accumulation,suggesting that ABCA1 may be a protective factor of VSMCs-derived foam cells formation.Thus,it is of great significance to investigate the underlying mechanisms of how to inhibit VSMCs-derived foam cells formation in the development of As,which is of a great help for proposing some novel anti-As strategies and treatments.Myocardin(MYOCD),a powerful co-transcriptional activator,can promote cardiomyocytes and VSMCs differentiation,and regulate the biological process of cardiomyocytes and VSMCs proliferation,differentiation and phenotypic transformation.Recently,increasing evidence have identified that MYOCD is closely connected to the initiation of atherosclerotic cardiovascular and cerebrovascular disease.Many researches verified that MYOCD expression in coronary artery of patients with coronary heart disease was significantly decrease.Overexpression of MYOCD inhibits vascular inflammation,abnormal proliferation and migration of VSMCs.Moreover,MYOCD promotes formation of caveolin-1,an important protein that mediates intracellular cholesterol migration to cell membrane,indicating that MYOCD is likely to regulate cholesterol metabolism.Therefore,the first question in this research is whether and how MYOCD regulate cholesterol metabolism in VSMCs through increased expression of ABCA1.As a transcription factor,serum response factor(SRF)is widely expressed in myocardium and smooth muscle tissue.It can induce gene expression through binding to CAr G-box in the promoter region of target gene.MYOCD is reported to directly associate with SRF and increase the stability of SRF binding to target gene,implying that MYOCD regulates genes expression through SRF.Therefore,the transcriptional regulatory effects of MYOCD on its target genes expression is closely related to SRF.In this waySRF participates in the process in which MYOCD regulates ABCA1 gene expression in VSMCs.Micro RNA(mi RNAs)is a single chain noncoding RNA with a length of about 22 nucleotides,and plays biological effects by combing with 3’-UTR of target gene m RNA.Our colleagues previously shown that mi R27a/b,mi R-467 b,mi R-186 and other mi RNAs participate in the process of As development.Mi R-1192 was discovered in 2013.Genomic analysis shown that mi R-1192(Gene ID: 100316672)was located in chromosome 19,19 B,and 19 17.91 c M loci.It was reported that mi R-1192 inhibits the repair and differentiation of muscle cells injury.Furthermore,mi R-1192 is also involved in regulating inflammatory responses in the body,suggesting that mi R-1192 may have the biological effects of promoting As development.Bioinformatics analysis indicated that MYOCD 3’-UTR contains binding site of mi R-1192.However,there is no mi R-1192 binding site in SRF and ABCA1 3’-UTR.Taken together,mi R-1192 has a biological basis to associate with MYOCD.Therefore,the second question of this research is to investigate the regulatory mechanism in upstream of MYOCD.We will identify whether mi R-1192 target inhibition of MYOCD and carry out a novel anti-As treatment and strategy based on MYOCD.Accordingly,we proposed our scientific hypothesis: MYOCD is able to upregulate ABCA1 via SRF,promote cholesterol efflux,inhibit VSMCs-derived foam cells formation,and ameliorate the development of As.Mi R-1192 is able to target inhibition of MYOCD,down-regulate the expression of ABCA1,and deteriorate development of As.To confirm our hypothesis we designed 4 parts of experiments.Firstly,we observed the effects of MYOCD on ABCA1 expression and cholesterol efflux in VSMCs,and investigated whether MYOCD promotes abca1 expression and cholesterol efflux via ABCA1 in VSMCs or not.Secondly,we investigated whether MYOCD promotes abca1 expression through bind to SRF or not,andincreased ABCA1-induced cholesterol efflux in VSMCs.Thirdly,we observed the effects of MYOCD on As plaques in apo E-/-mice.We studied a underlying pathway through which MYOCD up-regulated ABCA1 levels,reduced cholesterol loading and ameliorated As,in aorta of apo E-/-mice and in harvested and cultured VSMCs from apo E-/-mice aorta.Finally,we investigated upstream of MYOCD,in which we probed whether mi R-1192 targeted m RNA 3’-UTR of MYOCD and inhibited its expression,leading to a decrease of ABCA1 expression and ABCA1-induced cholesterol efflux in VSMCs.Our research is likely to find a novel anti-As therapeutic target and theoretical basis.Part I: MYOCD inhibited cholesterol accumulation in vascular smooth muscle cells through ABCA1Objective: To observe the effects of MYOCD on the expression of ABCA1 and cholesterol efflux in VSMCs,and explore the underlying mechanisms.Methods: Human aortic smooth muscle cells(HA-VSMCs)were treated with 50μg/ml oxidized low density lipoprotein(ox-LDL)for 96 hours and then HA-VSMCs were turned into VSMCs-derived foam cells,overloaded with lipids.HA-VSMCs-derived foam cells were incubated with different concentration of overexpressed and inhibited vector of MYOCD in the scale of(0,1,10,100 n M),plus 25μg/ml apo A-1 for 24 hours.HA-VSMCs were treated with 100 n M MYOCD2 and 25μg/ml apo A-1 for a series time points as 0,6,12,24 and 48 hours.Next,liquid scintillation counter were carried out to measure the cholesterol efflux content to apo A-1,and high performance liquid chromatography(HPLC)was approached to detect the contents of intracellular total cholesterol(TC),FC and cholesterol ester(CE).Real-time Quantitative PCR(q RT-PCR)and Western blot were selected to examine ABCA1 m RNA and protein level after overexpression or silence MYOCD,respectively.MYOCD and sh ABCA1 were co-transfected into VSMCs.We utilized the liquid scintillation counter to detect cholesterol efflux content to apo A-1,and confirm whether MYOCD-induce cholesterol efflux in VSMCs is dependent on ABCA1 or not.Results: MYOCD accelerated ABCA1-mediated cholesterol efflux from HA-VSMCs-derived foam cells via concentration-and time-dependent manners.Our results showed that MYOCD decreased the contents of intracellular total cholesterol,free cholesterol and cholesterol ester.q RT-PCR and Western blot have demonstrated that overexpression of MYOCD obviously upregulated ABCA1 m RNA and protein level.However,inhibition of MYOCD had opposite effects on ABCA1 expression.MYOCD-induced cholesterol efflux was dependent on ABCA1.Conclusion: MYOCD upregulates ABCA1 m RNA and protein expression,promotes intracellular cholesterol efflux.Part II: MYOCD promoted ABCA1-mediated cholesterol efflux in VSMCs via binding to SRFObjective: To investigate the potential mechanisms of whether MYOCD regulates ABCA1-mediated cholesterol efflux in VSMCs through binding to SRF.Methods: Promo Home Page and other bioinformatics online website prediction analysis system were utilized to found whether the ABCA1 promoter sequence has a binding site of CAr G-box that can combine with SRF.We utilize the chromatinimmunoprecipitation assay(Ch IP assay)to identify the combination between SRF and ABCA1 promotor,and confirm the specific binding site.In addition,the combination of MYOCD,SRF and ABCA1 promoter was examined by double luciferase reporter assay after co-transfecting MYOCD,SRF and wild or mutant ABCA1 promoter region reported gene vectors into HEK 293 T cells.Immunoprecipitation was used totest the effect of MYOCD on the combination of SRF and ABCA1.The overexpressed or vectors or inhibitors,including sh SRF,MYOCD,MYOCD+SRF and MYOCD+sh SRF,respectively transfected into VSMCs,and the q RT-PCR and Western blot were selected to detect ABCA1 m RNA and protein levels.Furthermore,the intracellular cholesterol efflux was performed with liquid scintillation counter.Results: Bioinformatics prediction results suggest that ABCA1 promoter region may have the ability to combine with SRF.Ch IP assay verified that SRF could bind to ABCA1 promoter on the Site 2.Our results showed that MYOCD did not directly bind to ABCA1 promoter,while SRF had the ability to directly combine with ABCA1 promoter region.However,MYOCD could further increase the binding ability of SRF to ABCA1 promoter.Meanwhile,immunoprecipitation assay showed that MYOCD could combined with SRF.Overexpression of MYOCD and silence SRF vectors were constructed.q RT-PCR and Western blot detection found that MYOCD binding to SRF can significantly increase ABCA1 m RNA and protein levels.Furthermore,liquid scintillation counter further verified that MYOCD binding to SRF can obviously promote cholesterol efflux in VSMCs.Conclusion: MYOCD further upregulates ABCA1 expression through combining with SRF.Part III: MYOCD inhibited apo E-/-mice atherosclerotic plaques formationObjective: To investigate the effects of MYOCD on ABCA1 expression in aorta from high-fat diet apo E-/-mice,and explore whether MYOCD affects atherosclerotic lesion formation in high-fat diet apo E-/-miceMethods: The six-weeks-old male mice were randomly divided into control group,MYOCD group and sh MYOCD group with the transfection of recombinant vector including r Ad-MYOCD,r Ad-sh MYOCD and r Ad-separately.The apo E-/-mice fed western diet of HF/HC diet for 12 weeks.After 12 weeks,the blood sample and isolated serum of each group mice were collected,serum levels of alanine transaminase(ALT),alkaline phosphatase(ALP)and aspartate transaminase(AST)were measured by chemiluminescence method.After the aortic samples of apo E-/-mice were harvested,ABCA1 m RNA and protein levels of aorta were detected by q RT-PCR and Western blot respectively.Aorta of apo E-/-mice were longitudinally dissected,including aorta arch to iliac artery branch.The atherosclerotic plaque area in aortic arch and its three main branches(left subclavian artery,left carotid artery and trunk arm artery)were observed under stereoscopic microscope.The percentage of total atherosclerotic lesions of lipid accumulation on aortic surface were shown by Oil red O staining.The aortic sinus sections of each group apo E-/-mice were sectioned by rapid frozen sections technique.The plaque area,lipid accumulation and collagen content in aortic sinus were respectively detected by HE,Oil red O and Masson staining.Immunofluorescence staining detected MYOCD expression in atherosclerotic plaques of aortic valve.Results: The overexpressed or silenced MYOCD expression model were established successfully in apo E-/-mice,and didn’t influence the levels of ALP,ALP and AST,indicating that MYOCD didn’t influence the lipid metabolism in liver.Finally,overexpression of MYOCD reduced the expression levels of ABCA1 in aortic samples from apo E-/-mice.Moreover,overexpression of MYOCD reduced the plaque area in aortic arch and aortic sinus,decreased the contents of lipid accumulation in the aorta and increased collagen contents in the atherosclerotic plaque.However,silenced MYOCD increased the plaque area in aortic arch and aortic sinus,promoted lipid accumulation in the aorta and decreased collagen contents in the atherosclerotic plaque.Conclusion: Overexpressed MYOCD upregulates ABCA1 expression in VSMCs from aorta of apo E-/-mice.MYOCD inhibits the occurrence and development of As in apo E-/-mice.Part IV: mi R-1192 targeted inhibition of MYOCD and reduced cholesterol efflux in VSMCsObjective: To investigate whether mi R-1192 inhibits MYOCD expression by targeting its m RNA 3’-UTR,and reduces ABCA1-mediated VSMCs cholesterol efflux.Methods: Bioinformatics online website were utilized to predicted the combination ability and the free energy scores for the hybridization between mi R-1192 and MYOCD.The binding activity of mi R-1192 to MYOCD3’-UTR was detected by luciferase reporter gene assay after co-transfecting mi R-1192 mimic and wild or mutant MYOCD 3’-UTR reported gene vectors into HEK 293 T cells.Furthermore,p GL3-ABCA1-3’-UTR 、p GL3-SRF-3’-UTR vector were constructed,and luciferase reporter gene assay was used to detect the binding ability of mi R-1192 on ABCA1 or SRF.Vectors including mi R-1192 mimic and inhibitor were transfected into VSMCs,q RT-PCR and Western blot were utilized to test the MYOCD m RNA and protein levels respectively.Similarly,ABCA1 m RNA and protein levels were performed with q RT-PCR and Western blot after transfecting MYOCD,mi R-1192 mimic,mi R-1192 mimic + MYOCD into VSMCs.Liquid scintillation counter examined the levels of intracellular cholesterol efflux.Results: Bioinformatics online website database indicated that mi R-1192 has biological basis for targeting MYOCD.Mi R-1192 and wildtype MYOCD3’-UTR co-transfection obviously inhibited luciferase reporter activity when compared with mimic-neg control group,but mutant MYOCD has no effect on luciferase reporter activity.The two groups co-transfected mi R-1192 mimic with ABCA1 or SRF into HEK 293 T cells had no significant change onluciferase reporter activity compared with control group.q RT-PCR and Western blot detected that mi R-1192 mimic downregulated MYOCD m RNA and protein expression.Compared with MYOCD group,mi R-1192 mimic inhibited ABCA1 m RNA and protein expression through downregulating MYOCD level.Finally,liquid scintillation counter found that mi R-1192 can inhibit cholesterol efflux in VSMCs.Conclusion: Mi R-1192 reduces ABCA1 levels in VSMCs via inhibiting MYOCD m RNA and protein expression,and decreases ABCA1-mediated cholesterol efflux.Conclusions(1)MYOCD upregulates ABCA1 expression,promotes ABCA1-mediated cholesterol efflux in VSMCs and inhibits the TC,FC and CE levels in VSMCs-derived foam cells.(2)MYOCD upregulates ABCA1 expression levels by binding to SRF on ABCA1 gene promotor and promotes cholesterol efflux.(3)MYOCD upregulates ABCA1 Mrna and protein expression levels in the aortic vessle wall of apo E-/-mice and decreases the As plaques formation.(4)Mi R-1192 targets the inhibition of MYOCD expression leading to downregulated ABCA1 expression and reduction of ABCA1-mediated cholesterol efflux from VSMCs.