Study Of Coronary Heart Disease-related Genes In Vitro Activity MEF2A Its Expression In Human Cardiac And Vascular Tissue

BackgroundCoronary artery disease (CAD) is one of the major diseases that threaten human life and health, the diagnosis and therapy methods of this disease are updating everyday. The prevention and cure of CAD has been converting from secondary prevention to the leading edge of primary prevention. The mothods of diagnosis and treatment are converting to molecular genetic level from the former evaluation and control of the risks. Following the rapid progression of the molecular genetic techniques, a lot of sensitive or causative genes related with CAD were found by linkage or association study. Myocyte enhancer factor-2A (MEF2A) is by far the most important CAD-related gene. A variety of intracellular signaling pathways can regulate its activation. It plays a central role in linking multiple signaling pathways to the genes responsible for cell division, differentiation and death. It is an important transcription factor.that is essential for myogenesis and vessel development. The study made by Wang et al. in2003first suggested that the certain mutation of the exon11performed as autosomal dominant inheritance style of MEF2A and it might cause the dysfunction of blood vessel endothelium cells, smooth muscle cells and finally trigger the onset and progression of CAD. Following studies by Li J et al. all identified the exon11of MEF2A had other mutation regions, for example, CAG repeating sequence, and they might have some associations with CAD. So it is very important to study the dysfunction of cells which caused by the mutation of MEF2A gene, and its further relationship with the pathological basis of CAD.At present, studies were focusing on the association between the mutation of MEF2A and CAD. If we can illuminate the effects of major CAD risk factors and their corresponding medical therapy to the activity of MEF2A transcriptional level, and the effects of the mutational MEF2A protein in cultured HUVEC and VSMC, we will understand the biologic activity of MEF2A much better and may propose additional or even new pathogenetic mechanism for CAD. Finally, the study will help screening and diagnosing individuals with high risk of hereditary factors early.Objectives1. To study the efficiency and toxicity of transfecting with the constructed pEGFP-N1/MEF2A plasmid, evaluate the possibility of establish the transient and stable transfection cell line.2. To construct the Tet-regulatable eukaryotic expression recombinant plasmid of wild type and mutational MEF2A, and study the function of the mutational MEF2A protein in cultured HUVEC and VSMC for further understanding of its relationship with the pathogenetic mechanism of CAD.3. To study the efficiency and toxicity of transfecting with the pLenti6.3-EGFP vector in cultured HUVEC and VSMC, and evaluate the effect of viral transfection to the migration of VSMC. To construct the regulatable eukaryotic expression recombinant plasmid of mutational MEF2A and transfect target cells. To study the structure and functional changes of the transfected cells.4. To analyze the changes of the transcription level of MEF2A after stimulate in vitro with ox-LDL and Stafine in cultured HUVEC.5. To study the distribution and expression of MEF2A in human cardiac muscular tissue and great/median vascular tissue by analyzing the IHC and IP stain results of the tissue samples from the patient and the donor.Methods1. HUVEC and VSMC cell lines were cultured in vitro and liposome method was used to identify the optimal transfection conditions that are suitable for pEGFP-N1/MEF2A eukaryotic expression reconstruction plasmid. We observed the transfection rate and toxicity effects on different time points after the transfection by reverse fluorescent microscopy, and identified the distribution and nuclear localization of the MEF2A protein expressed by cellular immunochemistry staining.2. We transformed the susceptible cells with inducible tetracycline eukaryotic expression vehicle system, pcDNA4.0/TO and pcDNA6.0/TO, to amplify the plasmids. pEGFP-Nl-MEF2A was bi-enzyme cut by Xho I and Xba I, and the cut-off MEF2A-EGFP fusion gene fragments were inserted into the multi-clone site on pcDNA4.0/TO. In this way we got pcDNA4.0/TO-MEF2A, the reconstructed inducible tetracycline eukaryotic expression vehicle.3. HUVEC and VSMC cells were infected with pLenti6.3-EGFP reconstructed lentiviral vehicle, and viral infection rate and toxicity effects were evaluated with reverse fluorescent microscopy. Optimal selection concentration was identified and stably transfected strains were selected with Blasticidin. Tranwell method was used to observe the effects of viral infection on the migration ability of VSMC cells.4. HUVEC cells were isolated and cultured, and oxLDL alone or combined with Atorvastatin was used to stimulate the cells. MEF2A transcription levels of different stimulation concentrations and time points after stimulation were tested with Real-time PCR.5. Specimens of the aorta and coronary arteries with atherosclerotic plaques, myocardium in the ventricular aneurysms, and the relatively normal auricles were acquired from hearts of the early-onset coronary disease patients who underwent heart transplantation. Control specimens of aorta were fetched from the heart donors during the operation. Hematoxylin&Eosin, MEF2A specific immunofluorescent histochemistry and immuno-enzymatic histochemistry stains were applied to observe the pathological changes.Results1. Liposomal method can be used to transfect HUVEC and VSMC cells in vitro, but the transfection rate is relatively low.2. pEGFP-N1/MEF2A reconstructed eukaryotic expression plasmids could not be highly expressed in HUVEC and VSMC cells, and further improvement is needed to introduce inducible promoters into this vehicle system.3. MEF2A was universally expressed in HUVEC and VSMC in vitro cultures, but mainly located in nucleus and the surrounding cytoplasmic region.4. Our attempt to construct pcDNA4.0/TO-MEF2A inducible tetracycline eukaryotic expression vehicle was not successful, because the pEGFP-N1-MEF2A bi-enzyme cutting with Xho I and Xba I was too slow in efficiency.5. The infection rates of the reconstructed lentiviral vehicle, pLenti6.3-EGFP were70～80%and50～60%in HUVEC and VSMC respectively. Green fluorescent protein, which can still be stably expressed after serial passage, and could aid in indentifying the expression of the target gene.6. We got stably transfected strains of pLenti6.3-EGFP by Blasticidin selection. The optimal selection concentration of Blasticidin was8mg/ml.7. Viral vehicle infection did not show obvious toxicity towards the HUVEC and VSMC cell lines. Neither did it significantly influence the migration ability of VSMC cells.8. Ox-LDL down regulated MEF2A mRNA expression in a dose and time dependent manner.9. Under a given ox-LDL stimulation concentration of100ug/mL, the MEF2A expression at any time points were all reversely correlated with Atorvastatin concentrations.10. MEF2A expression was not time dependently changing when Atorvastatin concentration was1μmol/L; but when it’s10or30μmol/L, MEF2A expression was increased higher with longer time after stimulation.11. Aorta and atherosclerotic coronary arteries of early-onset coronary disease patients demonstrate lower levels of MEF2A expression compared with normal controls. Meanwhile, myocardium in ventricular aneurysms showed lower expression of MEF2A than near normal auricles of the same patients.12. Immunofluorescence histochemistry and immuno-enzymatic histochemistry stains generated consistent results.Conclusions1. MEF2A is universally expressed in HUVEC and VSMC in vitro cultures, but mainly located in nucleus and the surrounding cytoplasmic region. Transfection of pEGFP-N1/MEF2A eukaryotic expression plasmids can not establish high expression of MEF2A in HUVEC and VSMC cell lines.2. We can continue our trial in constructing the MEF2A inducible tetracycline eukaryotic expression vehicle, to let inserted foreign MEF2A gene express in HUVEC and VSMC smoothly, efficiently and under the control of tetracycline induction.3. The lentiviral vehicle infection of HUVEC and VSMC cells shows high infection rates, relatively no toxicity effects to the two cell types and no significant impact on VSMC migration ability. These promising results show that MEF2A-EGFP reconstruct lentiviral vehicles can be used with the HUVEC and VSMC cell models to study the relationship between various MEF2A mutation types and coronary disease.4. Through in vitro cell experiments, we demonstrate that oxLDL, a coronary disease risk factor, down regulates MEF2A mRNA in a dose and time dependent manner, and Atorvastatin intervention further strengthens this relationship.5. By analyzing the IHC and IP stain results of the cardiac muscle and great/median vascular tissues of the premature CAD patient and the dornor, we found that the expression level of MEF2A in ischemic cardiomyocytes and in great/median arteries with athrosclerosis was lower than the normal ones. This result was consistent with the result of the ox LDL-stimulated cultured HUVEC.