Effect Of MEF2A Gene On Vascular Endothelial Cells And The Possible Mechanisms

BackgroundCoronary heart disease (CHD) is one of cardiovascular diseases harmful to human health. But its pathogenesis is still unknown. At present, it is obviously certain that hypertension, hypercholesterolemia, obesity, smoking and diabetes mellitus are risk factors inducing CHD. Coronary heart disease is a polygenic inherited disease of genetic predisposition affected by environment factors. However, its etiology in heredity can't be found all the time. Thus, looking for CHD and atherosclerosis susceptibility gene is the key point to research cardiovascular disease. With the development of molecular genetics, the relationship between genetic mutation and polymorphism and early CHD was paid more attention in recent years.Myocyte enhancer factor-2 (MEF2) transcription factors are the members of MADS-box transcription factors family. There are four mammalian MEF2 genes, MEF2A,-B,-C, and -D, which are originally described as muscle-specific DNA binding proteins and their outstanding function is to control gene transcription during myocyte differentiation. Several studies have indicated that MEF2 proteins may play a pivotal role in the transmission of extracellular signals to the genome and can activate the genetic programs, control cell differentiation, proliferation, morphogenesis, survival and apoptosis in a wide range of cell types. MEF2 transcription factors work in the form of heterodimer or homodimer, its most protein is bound during the control of transcription in the myocyte specific gene regulatory region.In 2003, Wang and coworkers reported that the mutation of MEF2A was a potential genetic cause for familial CHD and researched the CHD pathogenesis at the level of gene and proposed a new signal pathway of CHD pathogenesis. MEF2A gene locates on chromosome 15q26.3 region and its protein is highly expressed in vascular endothelium. Their research showed that maybe its pathogenesis is that genetic mutation changes the transcription process of gene expression, as a result, endothelia develops abnormally to be abnormal or function-deficit vascular endothelium, which will enhance exudation of monocyte and exposure of sub-endothelial matrix layer to make atherosclerosis plague and thrombosis inducing coronary atherosclerosis. The result was awarded "one of top ten achievements" by American Heart Association. However, the genetic variants within MEF2A gene relating to MI/CHD has been challenged by a series of inconsistent results which showed MEF2A variant was not associated with MI in the familial or the sporadic cases.The endothelium is the thin layer of cells that line the interior surface of blood vessels, which is a large protective screening system. The endothelial cells can secrete a series of vascular active substances and cytokines which can maintain basal vascular tone, prevent platelet adhesive, anti-thrombosis, maintain blood flow, inhibit vascular smooth muscle cell proliferation and inflammation, prevent inflammation cell infiltrate and harmful substances in the blood, to maintain the balance inside. Nitric oxide (NO) called endothelial diastolic factor (EDRF) is an important factor of these functions. Dyslipidemia (such as increased oxidative modification LDL), smoking, obesity, diabetes, hypertension, and other factors can stimulate vascular endothelial cells secret inflammatory factors that lead to inflammation, promote inflammatory cells adhesion to endothelial cell and thrombosis, which induces the formation of atherosclerosis.NF-κB involves in many genes especially the expression and regulation of early response genes related to body defense reaction, which plays an important role in the transcription and regulation of synthesis of many bioactive substances in the body and involves in atherosclerosis formation and development. In mammals, the subfamily of NF-κB proteins(also called Rel family) include:RelA (p65), RelB, c-Rel, NF-κB1/p50 and NF-κB2/p52. Polypeptide chain composed of p65 and p50 protein subunits is the common activated form, which include the p50 homodimer, p65 homodimer and p50-p65 heterodimers. NF-κB activation stimulates the expression of leukocyte adhesion molecules such as VCAM-1 increase. The combination of VCAM-1 and receptorα4β1 integrin can induce signal inside endothelial cell to change endothelial cell transformation and adhesion of mediated mononuclear cell, lymphocyte and endothelial cell or vascular smooth myocyte.Some studies show that ox-LDL can also activate of NF-κB in different levels. Activation of NF-κB involves in the adhesion molecule VCAM-1 regulation, as well as involved in the transcriptional regulation of other cytokines. But which functions of endothelium affected by mutant MEF2A and specific action mechanism are unknown. It is possible that MEF2A gene involves in the regulation of endothelial function by the way of affection of vascular endothelial cell NF-κB regulation, but the specific signaling pathways need to be further studied. ObjectiveIn our study, coronary heart disease-related gene MEF2A wild-type and 21 base mutant plasmid was transfected into human umbilical vein endothelial cells lines to explore the possible mechanism of MEF2A on endothelial cell function changes by detecting the proliferation of endothelial cells, the levels of NO, adhesion molecule VCAM-1, NF-κB and so on. Adding ox-LDL to stimulate the normal vascular endothelial cells at different times, we observed the expression of MEF2A protein and functional changes of adhesion in endothelial cells to clarify the common roles of genes and environment impact of the CHD. The research is to investigate the relationship between the MEF2A gene and atherosclerosis by studying the mechanisms of the influences of MEF2A gene on endothelial function.Methods1. The experimental plasmids were a gift from the United States Professor Wang Qing had been confirmed by gene sequencing. These plasmids were transformed into E. coli DH5a sensitive cells to have extracorporeal plasmid amplification. Three groups of human umbilical vein endothelial cells (HUVECs) were studied including the cells transfected with pcDNA3.0-GFP plasmid (the control group), with MEF2A wild-type plasmid (WT group), and with MEF2A 21-base pair deletion mutantion plasmid (Δ21 group). We used liposome Lipofectime2000 to transfect human umbilical vein endothelial cells and Hela cells. MEF2A plasmids contained the Flag tags. Flag protein was detected to determine the success of transfection.2. Flow cytometry was used to detect the GFP expression to choose best and stable transfection methods. Western-blot used to detect the expression of MEF2A protein.3. Luciferase detection system for detecting the transcriptional activity of plasmids.4. Immunofluorescence used to detect the localization of MEF2A protein. 5. Four groups of human umbilical vein endothelial cells (HUVECs) were studied including the cells transfected with pcDNA3.0-GFP plasmid (the control group), MEF2A wild-type plasmid (WT group), MEF2A 21-base pair deletion mutantion plasmid (Δ21 group) and siRNA group. We detected endothelial cell proliferation and NO level of every group after transfection.6. We detected the adhesion molecule and NF-κB levels of each group by extraction of total protein or nuclear protein of above cells with western-blot analytical technique.7. Fluorescent dye pretreated Thp-1 cells were cultured with endothelial cells. Monocyte adhesion test were carried out.8. Using ox-LDL (50μg/ml) to stimulate normal endothelial cells: ox-LDL was stimulated endothelial cells at 0,6,12,24 and 36 hours. Cells were collected for extraction of total protein and nuclear protein. We measured the protein levels of MEF2A, adhesion molecule VCAM-1 as well as the nucleus of NF-κB.Results1. We transfered genes into human umbilical vein endothelial cells by Lipofectime2000 at first than detected Flag protein expression successfully. Transfection rate detected with flow cytometry was 70%.2. Endothelial cell by transfected both MEF2A wild-type and mutant-type plasmid and these of control group can detect expression of MEF2A protein. Compared with control group, MEF2A protein of WT group and mutant group were over expressed, but there was no obvious difference between last two groups.3. Luciferase detection showed that plasmid transcription activity in mutant group was lower than that of wild-type group.4. Cell immunofluorescence staining results showed that wild-type MEF2A protein localized in the nucleus. TheΔ21 mutation of MEF2A protein could be detected in the cytoplasm partly.5. Western blotting showed that MEF2A protein expression was reduced to 80% in the siRNA group compared with that in WT group. The expression of MEF2A protein was significantly higher in the WT andΔ21 groups than that in the control but there was no significant difference between theΔ21 group and the WT group.6. Comparison of proliferation:48 hrs after transfection. As compared to the control group the WT group had higher proliferation (0.344±0.022 vs.0.648±0.053, n=5) (p<0.01). HUVEC proliferation rate of theΔ21 group and the siRNA group has dropped to 36.2% and 63.6% when compared with that in WT group.7. In the 24,36 and 48 hrs after transfection, the WT group had higher NO expression than that of the control group. Transfected 36 hours and 48 hours, NO expression of siRNA group and theΔ21 group decreased significantly when compared with that in wild-type group. And siRNA group declined more marked.8. VCAM-1 expression was lower in wide type HUVEC than that in the control group (0.076±0.011 vs.0.223±0.025, n=5). VCMA-1 expression of the siRNA group and theΔ21 Group were higher than that of wild-type group. (0.435±0.037 vs.0.076±0.011, n=5,p<0.01) (0.207±0.018 vs.0.076±0.011, n=5,p<0.05).9. Mononuclear endothelial adhesion results:Cell numbers of Thp-1 adhesion to endothelial cells reduced in WT group compared with that in control group (p< 0.05). Thp-1 cells increased in siRNA group (p< 0.01) andΔ21 group (p< 0.05) when compared with that in wild type group.10. Western-blot results showed that NF-κB expression of WT group was significantly lower than that in the control group. But the NF-κB expression of the siRNA group and theΔ21 group were higher than that of wild-type group, especially the siRNA group.11. With action time of 50μg/ml ox-LDL on HUVEC extension, the MEF2A protein expression of HUVEC reduced gradually which was time-dependent. The expression of NF-κB p65, as well as VCAM-1 protein increased gradually and reached the maximum levels at 24 hrs.Conclusion1. We transfected MEF2A gene with Lipofectime2000 successfully. We got a well transfection rate.2. This kind of 21-base deletion mutation of MEF2A gene do not change MEF2A protein expression but it can affect the transcriptional activity of plasmid. The transcription activity of the plasmid is lower than that of wild-type obviously. The MEF2A protein located in the cell nucleus in WT group while stayed in the cytoplasm in the 21-base deletion mutant group. This kind of mutation was not included in the NSL region, but it changes the localization of protein..3. MEF2A gene involves in the regulation of vascular endothelial cell proliferation.4. MEF2A gene affects the expression of endothelial cell NO.5. MEF2A gene could regulate the expression of adhesion molecule VCAM-1 and NF-κB.6. Ox-LDL reduces the. expression of MEF2A protein in normal endothelial cells in a time-dependent way, while increases the expression of NK-κB and adhesion molecule VCAM-1.