Effet Of Myocyte Enhancer Factor 2A Gene By RNA Interference On Atherosclerosis And The Possible Mechanisms

Part I RNA interference of myocyte enhancer factor 2A induces increased expression of inflammatory genes in mouse aortrc: normal aortic endothelial cells BackgroundAtherosclerosis(AS) is a multi-factorial complex process intertwined with inflammation. The damage and dysfunction of vascular endothelium are the intial factors of atherosclerosis which may therefore induce the stenosis of arterial lumen. The arterial lumen stenosis can further cause ischemia and necrosis in the relative organs and tissues. Myocyte enhancer factor 2A(MEF 2A) is aslo named platelet activating factor acetylhydrolase(PAFAH). Increasing sutides have been suggested that MEF 2A ia a novel bioactive substance of atherosclerosis. MEF 2A combines with the specific gene control region containing A/T sequences in the muslcle cells, and regulates the transcription of target genes which take part in the development of cardiac and skeletal muscle, and play an important role in the growth and development of blood vessels. RNAi is a clinically feasible technology to down-regulate the expression of target genes efficiently and selectively. In the present study, RNA interference(RNAi) technique was adopted to reduce the expression of MEF 2A in mouse aortrc: normal aortic endothelial cells. The expression of MEF 2A and the relative function of blood vessels were measured to provide theoretical basis in predicting atherosclerosis. ObjectivesTo identify the effects of RNA interference of MEF 2A on the expressions of MEF 2A and relative inflammatory genes, ICAM-1, VCAM-1 and PAI-1, in mouse aortrc: normal aortic endothelial cells. MethodsMEF 2A RNAi or scrambled negative control(NC) lentivirus viral suspensions were constructed, and the titers averaged 1×105 TU/ml. Mouse aortrc: normal aortic endothelial cells were untransfected or transfected with NC lentivirus or MEF 2A RNAi lentivirus(multiplicity of infection=40) to determine their silencing efficiency. MEF 2A and inflammatory genes expressions were determined by ELLISA, and m RNA level of MEF 2A and inflammatory genes were further examined by real-time PCR. Results1. Gene-silencing analysis revealed that the site B lentivirus blocked MEF 2A m RNA and protein expression most effectively. This effect acted in the concentraction dependent manner and reached the platform stage at MOI = 40.2. Lentivirus-mediated RNA interference of MEF 2A could statistically increase the activies and m RNA expressions of ICAM-1, VCAM-1 and PAI-1 in mouse aortrc: normal aortic endothelial cells.ConclusionsLentivirus-mediated RNA interference of MEF 2A can significantly inhibit the expression of MEF 2A and enhance the expression of inflammatory genes ICAM-1, VCAM-1 and PAI-1.Part ⅡAcceleration of atherosclerosis in apolipoprotein E-deficient mice by lentivirus-mediated RNA interference of myocyte enhancer factor 2A BackgroundAtherosclerosis(AS), one of the major common diseases, is severely threatening the human health and becoming the main cause of death in adult. Despite intensive management of the conventional atherosclerosis risk factors, many patients continue to experience the onset and recurrence of coronary events. Therefore, elucidating new risk factors and potential therapeutic target of atherosclerosis is urgently needed. MEF 2A is the novel important bioactive substance that keep the stability of blood vessel endothelium. Numerous evdiences have been showing that the decreased expression of MEF 2A may be one of the risk factors of atherosclerosis, and MEF 2A has the ability to predict and monitor the progress of a varity of diseases. Moreover, it has been demonstrated that MEF 2A is associated with the integrity of the vascular endothelium. MEF 2A can modulate the expression levels of inflammatory factors which can induce the emergence of atherosclerosis. These fingdings may provide the novel approach to predict the development of atherosclerosis. In the current study, we set up atherosclerosis mouse model by feeding a high-fat diet and palcing constrictive collar in left carotid artery in apolipoprotein E-deficient mice. RNA interference(RNAi) technique was then used to efficiently and selectively reduce the expression of MEF 2A. We next examined the expression levels of relative inflammatory genes and the morphology of plaques to further elucidate the underlying mechanisms involved in this process. ObjectivesTo efficiently and specifically down-regulate the expression level of MEF 2A by lentivirus-mediated RNA interference and determine whether lentivirus-mediated MEF 2A silencing could modulate the expression levels of inflammatory genes and stability of plaques in the apolipoprotein E-deficient mice.MethodsThe apolipoprotein E-deficient mice were fed a high-fat diet and a constrictive collar was placed around the left carotid artery to induce plaque formation. In brief, the common carotid arteries were dissected and a constrictive silastic collar(0.30 mm) was placed on the left common carotid artery by placement of 3 circumferential silk ties in all mice. The mice were randomly divided into control, negative control(NC) and RNA interference(RNAi) groups. MEF 2A RNAi or scrambled NC lentivirus viral suspensions were constructed and transfected into the carotid plaques 5 weeks after surgery; the control group was administered saline. The carotid plaques were assessed seven weeks later using hematoxylin and eosin(H&E), Masson’s trichrome and oil red O staining; plasma and lesion inflammatory gene expression were examined using ELISA and real-time PCR.Results1. Four weeks after transfection, RNAi group showed the statistical lowest serum concentration and plaque m RNA expression of MEF 2A than control and negative control(NC) groups. However, significant increased systemic inflammatory genes expressions were observed in the plaque and serum of MEF 2A RNAi group..2. MEF 2A RNAi also promoted plaque progression, increased the plaque area, fibrous cap thickness and plaque collagen content, whereas on changes on plaque lipid content were presented in MEF 2A RNAi group.3. There were no obvious differences on the total cholesterol and triglyceride levels among control, NC and RNAi groups. Therefore, the effects of MEF 2A RNAi were independent of serum lipoprotein levels. ConclusionsOur findings demonstrate that lentivirus-mediated MEF 2A RNA interference has the ability to promote the emergency of atherosclerosis and decline the plaque stability, without altering the plasma lipoprotein profile in apo E-/- mice.Part ⅢThe expression levels of plasma MEF 2A and relative inflammatory factors in patients with acute coronary syndrome and stable aninapectoris BackgroundCoronary artery heart disease(CHD) is one kind of myocardial damage caused by imbalance between coronary blood flow and myocardial demand because of the functional and/or organic changes of coronary artery. CHD is known as the polygene desease of interaction between heredity and environment, which leads to high morbidity and mortality. Therefores, in oder to improve the prevention strategies of CHD, a large number of researches have been conducted to determine its pathogenesis and risk factors. The basic pathological change of CHD is coronary atherosclerosis. The damage of vascular endothelium is the intial factor of atherosclerosis. The structure and functional disorder of vascular endothelium can bring out the presence of macrophage in subendocardial. Subsquently, a range of vascular inflammatory changes arise, such as lipid deposition, intima thickening, thrombogenesis and plaques, which finally causes atherosclerosis. In this study, the mechanisms involved in this process were elucidated. ObjectivesTo test the expression levels of plasma MEF 2A and relative inflammatory factors, ICAM-1, VCAM-1. PAI-1, MCP-1, MMP-8 and TNF-α, in patients with acute coronary syndrome(SAP) and stable anina pectoris(ACS), and find the underlying mechanisms. MethodsThe plasmas were first collected from patients with SAP, ACS, and normal control. ELTSA were then conducted according to the the manufacturer’s instruction to detect the expression levels of plasma MEF 2A and relative inflammatory factors in 3 groups. Results1. A dramatic decrease of plasma concentration of MEF 2A was displayed in patients with ACS compared to the SAP and normal control group.2. Compared with normal control and SAP patients, the patients with ACS showed considerable higher plasma concentrations of ICAM-1, VCAM-1. PAI-1, MCP-1, MMP-8 and TNF-α.3. No significant differences on the plasma concentrations of MEF 2A, ICAM-1, VCAM-1. PAI-1, MCP-1, MMP-8 and TNF-α were exhibited among patients with single-vessel, double-vessel and triple-vessel diseases.4. The negative correlations between MEF 2A and ICAM-1, VCAM-1. PAI-1, MCP-1, MMP-8 and TNF-α were found in ACS(r =-0.854, P < 0.05) and SAP(r =-0.748, P < 0.05) groups. ConclusionsThe patients with ACS and SAP show significant decrease plasma concentraction of MEF 2A that is negative correlation with ICAM-1, VCAM-1. PAI-1, MCP-1, MMP-8 and TNF-α.. These results suggest that plasma concentractions of MEF 2A, ICAM-1, VCAM-1. PAI-1, MCP-1, MMP-8 and TNF-α hold the potential to predict the inflammatory reaction and plaque stability in patients with ACS and SAP.