The Mechanism Of Enhancer Factor 2A Gene Mutation Regulating Human Vascular Smooth Muscle Cells And Intervention Study

BackgroundThe main pathological process of coronary heart disease (CHD) is atherosclerosis, but the genetic basis of atherosclerosis remains largely unknown. Myocyte enhancer factor 2A (MEF2A) regulates and mediates cell differentiation in spinal animals. Recently, some studies have reported the linkage between CHD and 7-amino acids deletion (21-bp deletion orΔ21) of MEF2A gene, but the biological effect of this gene on atherosclerosis needs to be clarified.Vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of atherosclerosis. In normal condition, VSMCs is located in the media of artery. During the process of atherosclerosis, VSMCs switch from contractile phenotyps to synthetic phenotypes that tend to more facilely proliferate and migrate into the intima of artery, and to be more efficient in lipid uptaking and foam cell formation. Compared to contractile VSMCs, synthetic VSMCs express lower levels of contractile proteins such as smooth muscleα-actin (α-SM-actin) or smooth muscle 22 alpha (SM22α), but higher levels of the proteins like osteopontin (OPN). So these proteins are the markers of phenotype switching of VSMCs. Mitogen-activated Protein Kinase (MAPK) is a member of important signal transduction systems in vivo and p38 and ERK 1/2 belong to MAPK family. After being phosphorylated, p38 and ERK 1/2 are transcription factors, which play an important role in the physiological and pathological signal transduction pathway, such as proliferation, migration and phenotype switching of VSMCs. Some data show that MEF2A gene is highly expressed in VSMCs. In addition, the potential regulatory effect of MEF2A gene has been implicated in VSMCs phenotype switchinging. We try to establish the cell models to observe the effect of MEF2A gene mutation on the biological characteristics of VSMCs and to understand the relationship between of MEF2A gene mutation and atherosclerosis, and to further understand the pathogenesis of CHD.Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors), the most widely prescribed cholesterol-lowering drugs, could effectively reduced the morbility of CHD events. Studies indicate that beyond the lipid-lowering effect, some antiatherosclerosic effects of statins involve anti-inflammation, antioxidation, improving endothelial function, decreasing monocyte adhesion and infiltration vessel wall, and enhancing the stability of atherosclerotic plaques. However, the potential effects of statins on VSMCs with MEF2A gene mutation remain unclear.ObjectiveThe aim of this study was to investigate the effect of MEF2A gene mutation on VSMCs through by established the VSMCs cell models of MEF2A wild type (WT), MEF2AΔ21 dominant negative mutation (Δ21) and MEF2A siRNA, and to observe the effects of atorvastatin on proliferation, migration, phenotype switching and MAPK signal pathway of VSMCs induced by MEF2A gene mutation. Besides, we would demonstrate the association between MEF2A gene mutation and CHD, as well as other atheroprotective effects of atorvastatin independent of its lipid-lowering effects.MethodsⅠ. A cell model of VSMCs with MEF2A gene mutationVSMCs were maintained in D/F medium containing 10% (v/v) fetal bovine Serum(FBS),100 units/ml penicillin and 100 units/ml streptomycin, trypsinized with 0.25% trypsin and then were propagated each 5 days. The cells were characterized by microscopic observation and detecting the expression ofα-SM-actin. According to the cell transfection, we divided VSMCs into four groups:(1)control (blank) group: transfected with pc-DNA3.1(+)-Green fluorescent protein(GFP) plasmid; (2)WT group:transfected with MEF2A pc-DNA3.1(+)-MEF2A(WT) plasmid; (3)Δ21 group:transfected with MEF2A pc-DNA3.1(+)-21bp(Δ21) plasmid; and (4)siRNA group:transfected with MEF2A siRNA. The MEF2A plasmids and siRNA were transiently transfected into VSMCs using EntransterTM-D and EntransterTM-R. MEF2A plasmids were marked with Flag tags. The success of transfection was determine by detecting Flag protein expression using western blotting with Flag antibody, and transfection efficiency was evaluated by counting GFP positive cells under fluorescence microscope after 24 hours of transfection. After 48 hours of transfection, we detected MEF2A protein of VSMCs by western blotting, to make sure the establishing of cell models VSMCs with MEF2A gene mutation.Ⅱ. Effects of MEF2A gene mutation on VSMCsVSMCs were divided into four groups according to different plasmid transfection:(1)control (blank) group:transfected with pc-DNA3.1(+)-GFP plasmid; (2)WT group:with MEF2A pc-DNA3.1(+)-MEF2A(WT) plasmid; (3)Δ21 group:with MEF2A pc-DNA3.1(+)-21bp(Δ21) plasmid; and (4)siRNA group:with MEF2A siRNA. The MEF2A plasmids and siRNA were transiently transfected into VSMCs by means of EntransterTM-D and EntransterTM-R.1. Proliferation analysis of VSMCs:After 24 hours of transfection, cells were maintained in the D/F medium containing 0.1% FBS for another 24 hours. Quiescent VSMCs were then planted 96-well-culture plates with the density of 5×104 cells/well, and maintained in D/F medium containing 10% (v/v) FBS. Each group had three batches, and each batch had five plates. Following the incubation period of 24 hours, 48 hours and 72 hours,20μL of Methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution (5 mg/ml) was added into VSMCs plates of the 1st,2nd and 3rd batch, and the cells were incubated for 4 hours. The medium was removed and 150μL of dimethyl sulfoxide (DMSO) was added into plates to dissolve the MTT crystals. Then we placed the plates on shaking table for low speed oscillation 10 minutes, and the optical density was read using Multiskan Spectrum at 570 nm wave length.2. Migration analysis of VSMCs:500μL D/F medium containing platelet derived growth factor (PGDF)-BB solution (10ng/ml) without FBS was added into the lower chamber. After 24 hours transfection, cells were maintained in the D/F medium containing 0.1% FBS for another 24 hours. Then cell suspension was made by a density of 5×105/ml with medium containing 0.1% FBS. Cell suspension (200μL) was planted into the upper Millicell transwell chamber. After 12 hours of incubation, filters were fixed with methanol and stained with hematoxylin and eosin. The cells on the upper surface of filters were removed by wiping with cotton swabs. The cells that had migrated to various areas of the lower surface were manually counted under a microscope.3. Western blotting analysis of VSMCs phenotype, p38 and ERK 1/2 MAPK signaling pathway:After being transfected for 48 hours, cells were solubilized with protease inhibitors on ice for 30 minutes. Lysates were centrifuged twice at 12000 r/min (4℃) for 10 minutes each to deposit the insoluble materials. The proteins level in soluble fraction were analysed by means of SDS polyacrylamide gel electrophoresis (SDS-PAGE) and followed by Western blotting with antibody againstα-SM-actin, SM22α, OPN, p38 MAPK, phospho-p38 MAPK, ERK 1/2 MAPK and phospho-ERK 1/2 MAPK.Ⅲ. Effects of atorvastatin on VSMCs with MEF2A gene mutationVSMCs were divided into three groups according to different plasmid transfection:(1)WT group:transfected with MEF2A pc-DNA3.1(+)-MEF2A(WT) plasmid; (2)Δ21 group:with MEF2A pc-DNA3.1(+)-21bp(Δ21) plasmid; and (3)statin group:with MEF2A pc-DNA3.1(+)-21bp(Δ21) plasmid. The MEF2A plasmids were transiently transfected into VSMCs by means of EntransterTM-D.1. Proliferation analysis of VSMCs:After being transfected for 24 hours, cells were maintained in the D/F medium containing 0.1% FBS for another 24 hours. Quiescent VSMCs were then planted into 96-well-culture plates with a density of 5×104 cells/well, and maintained in D/F medium containing 10%(v/v) FBS. Each group had three batches, and each batch had five plates, then added atorvastatin solution (final concentration 100μmol/L) into statin group plates. Following the incubation period of 24 hours,48 hours and 72 hours,20μL of MTT solution (5 mg/ml) was added in VSMCs plates of the 1 st,2nd and 3rd batch, and the cells were incubated for 4 hours. The medium was removed and 150μL of dimethyl sulfoxide (DMSO) was added to dissolve the MTT crystals.Then we placed the plates on shaking table for low speed oscillation 10 minutes, and the optical density was read using Multiskan Spectrum at 570 nm wave length.2. Migration analysis of VSMCs:500μL PGDF-BB solution (10ng/ml) without FBS was added into the lower chamber. After 24 hours of transfection, cells were maintained in the D/F medium containing 0.1% FBS for another 24 hours, and cell suspension was made with a density of 5×105 cells/ml medium containing 0.1% FBS.200μL cell suspension was planted into the upper Millicell transwell chamber, and added atorvastatin solution (final concentration 100μmol/L) into statin group chambers. After 12 hours of incubation, the filters were fixed with methanol and stained with hematoxylin and eosin. The cells on the upper surface of the filters were removed by wiping with cotton swabs. The cells that had migrated to various areas of the lower surface were manually counted under a microscope.3. Western blotting analysis of VSMCs phenotype, p38 and ERK 1/2 MAPK signaling pathway:After being transfected for 24 hours, we added atorvastatin solution (final concentration 100μmol/L) into statin group plates, and continued cultivation for 24 hours, cells were solubilized with protease inhibitors on ice for 30 minutes. Lysates were centrifuged twice at 12000 r/min (4℃) for 10 min each to deposit the insoluble materials. The protein level s in soluble fraction were analysed by means of SDS polyacrylamide gel electrophoresis (SDS-PAGE) and followed by Western blotting with antibody againstα-SM-actin, SM22α, OPN, p38 MAPK, phospho-p38 MAPK, ERK 1/2 MAPK and phospho-ERK 1/2 MAPK.ResultsⅠ. A cell model of VSMCs with MEF2A gene mutation1. VSMCs cells appeared spindle-shaped and were arranged in bundles. Multiple layers of cells were observed in several areas, while single layers grew in other areas. The characteristic peak and valley features of VSMCs colonies were also evident. Western blotting analysis using a VSMCs-specificα-SM-actin antibody revealed that the cells expressedα-SM-actin protein. These tests served to confirm the identity of the cultured VSMCs.2. Western blotting analysis with Flag antibody showed that only WT group andΔ21 group had Flag expression. It identified the transfection of MEF2A WT and MEF2AΔ21 plasmid in VSMCs. The transfection efficiency was about 70%, which was estimated by cotransfection of GFP plasmid.3. Compared with control group, MEF2A protein was overexpressed in VSMCs transfected either with MEF2A WT plasmid or MEF2AΔ21 plasmid (P<0.01), while MEF2A siRNA obviously knockdown MEF2A protein in VSMCs (P<0.05). Ⅱ. Effects of MEF2A gene mutation on VSMCs1. Optical density of MTT showed that there was no statistical difference of the VSMCs proliferation among these groups on 24 hours (P>0.05).2. On the incubation period of 48 hours, there was still no statistical difference between control group and WT group (P>0.05). Compared with WT group,Δ21 group and siRNA group VSMCs proliferation had an significant increase by 56.9%and 71.5%, respectively (P<0.01).3. After 72 hours, there was still no statistical difference between control group and WT group (P>0.05). The cell growth rate inΔ21 group and siRNA group rised to 102.6% and 112.2% compared with WT group (both P<0.01).4. After 12 hours of culture, the quantity of the PDGF-BB-directed VSMCs migration in Millicell transwell chamber were similar between the control and WT group (P>0.05). Compared with WT group, more total cell counts migrating to the lower surface were observed inΔ21 group and siRNA group (P<0.01).5. Compared to WT group,α-SM-actin and SM22a proteins in VSMCs were downregulated, while OPN protein increased inΔ21 and siRNA groups (all P<0.01). No significant differences of the three protein expressions in VSMCs were observed between control group and WT group (P>0.05). 6. The protein expression levels of p38 and ERK 1/2 in VSMCs were similar among all groups. No significant differences were observed in the protein expressions of phosphorylated p38 and phosphorylated ERK 1/2 between control group and WT group (P>0.05). But higher expressions of phosphorylated p38 and phosphorylated ERK 1/2 were obtained inΔ21 and siRNA groups than WT group (both P<0.01).Ⅲ. Effects of atorvastatin on VSMCs with MEF2A gene mutation1. Optical density of MTT showed no statistical difference for VSMCs proliferation among all groups at 24 hours (P>0.05).2. After 48 hours of incubation, an increase by 33% of VSMCs proliferation were detected inΔ21 group than WT group (P<0.05), but no significant differences were seen between WT group and statin group.3. At 72 hour, the cell growth rate inΔ21 group increases to 90.2% than WT group (P<0.05). The cell growth rate in statin group was higher by 24.3% than WT group (P<0.05), but lower thanΔ21 group (P<0.05).4. After 12 hours culture, the cell migration rates in Millicell transwell chamber were higher inΔ21 group and statin group than WT group (P<0.05), but the migration rate of statin group was lower than that ofΔ21 group (P<0.05).5. Compared with WT group, the expressions ofα-SM-actin and SM22αin VSMCs ofΔ21 group were downregulated, while OPN expression ofΔ21 group increased (P<0.01). Besides, the expressions of α-SM-actin and SM22αwere similar between WT group and statin group. However, OPN expression of statin group was higher than that of WT group, but lower than that ofΔ21 group (both P<0.05).6. The expressions of p38 and ERK 1/2 were similar in all groups. Similarly, no significant differences in the expressions of phosphorylated p38 and phosphorylated ERK 1/2 were observed between WT group and statin group (P>0.05). However, the expressions of phosphorylated p38 and phosphorylated ERK 1/2 were higher inΔ21 group than in WT group (P<0.01).Conclusions1. Human VSMCs can be successfully cultured in D/F medium containing 10%(v/v) FBS,100 units/ml penicillin and 100 units/ml streptomycin.2. Transient transfection of MEF2A dominant negative mutation plasmid into VSMCs could be a feasible way to build a cell model of VSMCs with MEF2A dominant gene mutation.3. Transient transfection of MEF2A RNA silence in VSMCs can knockdown the expression of MEF2A.4. MEF2A dominant negative mutation and RNA silence can increase the proliferation and migration of VSMCs.5. MEF2A dominant negative mutation and RNA silence can induce the phenotype switching of VSMCs from contractile phenotype to proliferative one.6. Both p38 and ERK 1/2 MAPK signaling pathways are implicated in MEF2A gene mutation of VSMCs.7. Atorvastatin reduces the proliferation and migration of VSMCs induced by MEF2A gene mutation.8. Atorvastatin has an antagonism effect on the phenotype switching of VSMCs induced by MEF2A gene mutation.9. The antagonism effect of atorvastatin on MEF2A gene mutation may be implemented by inhibiting p38 and ERK 1/2 MAPK signaling pathway.