| BackgroundThe morbidity and mortality of coronary artery disease (CAD) are always higher both in China and abroad, which field is always focused on by both clinical and basic medical research. The pathogenesis research has made great progress in recent years. However, the gene research of CAD including myocardial infarction (MI) obviously lags behind other diseases till now mainly because that CAD is a complex pathophysiological process, multifactorial and influenced by complex interactions of environmental and genetic factors. Previous studies have demonstrated that hypertension, dyslipidemia, obesity, smoking and other risk factors are clearly associated with the onset of coronary heart disease. Some genetic studies and genome-wide linkage analysis have also found some susceptibility loci and candidate genes associated with CAD, and these loci and gene directly involved in the pathogenesis and regulation of risk factors in coronary heart disease. As a multifactorial complex disease, multiple genes associated with CAD have been found with the sequence variation or mutations, while the role is so weak.Some researchers found that members of the family of transcription factors, myocyte enhancer factor-2 A (MEF2A) was involved in autosomal dominant form of CAD. This viewpoint has been paid more attention by the whole world scholars. A genetic analysis for early onset coronary artery disease in a white American family confirmed that the family of CAD/MI fit a pattern of autosomal dominant inheritance disease.15q26 MEF2A candidate genes are checked out by the method of the genome-wide scan.21 bases (21-bp) missing are demonstrated in extra 11, the line of MEF2A gene in all the cases of the families. The occurrence of MEF2A mutations is about 1.93% in all the 207 patients by genetic analysis, which confirmed by coronary angiography with diagnosis of CAD/MI. These preliminary studies suggested the onset of MEF2A variation occured in CAD/MI, although the hypothesis required further support. However, the study by Weng et al. demonstrated that no MEF2A gene polymorphism and susceptibility were related to early onset coronary artery disease of 300 cases of white in MEF2A gene detection. An Irish crowd MEF2A gene, large-scale study showed 21 bases (21-bp) missing in extra 11, the line of MEF2A gene was not associated with ischemic heart disease. Based on the above controversial results, we supposed that the MEF2A mutations may be a rare, only a few lines of leading to coronary heart disease genes in our country, or not associated with the pathogenesis of genealogy. The conclusion required more data about Chinese CAD/MI family genetic studies to be proved.Object1. The suitable cases and pedigree investigation were selected in CAD/MI family according to the diagnostic criteria of coronary heart disease/myocardial infarction, in the early onset coronary artery disease in Chinese population screening.2. The genetic characteristics of CAD family, traditional risk factors, lipid metabolism and coronary artery lesions were analyzed.3. The family genome-wide linkage analysis, screening of CAD/MI candidate genes, gene sequencing and the meaningful genetic mutations were analyzed.4. Compared with the scattered CAD patients and healthy control, the gene mutation in exon 1-11, MEF2A gene in the line of coronary heart disease was examinated and the MEF2A gene mutation in CAD/MI family was analyzed.Materials and methods1. The patient data came from the medical records which retrieved the clinical diagnosis of early onset coronary heart disease and pedigree investigation and screening of the conforming to the dominant inheritance characteristic. At least two factors in the following conditions as the diagnosis standard of CAD or MI:(1)chest pain, (2)ECG and myocardial enzyme or troponin changes refered to acute myocardial infarction (AMI), (3)Coronary CT angiography or vascular cavity diameter of the stenosis above 50%. All family members were required a medical examination, blood analysis test (blood routine, liver and kidney function, blood sugar, lipids and blood ion analysis), coronary angiography, standardized follow-up, including activity endurance, medication history, and personal habits.2. The CAD control group (n= 311) were described as confirmed sporadic patients after the first myocardial infarction admission parallel coronary angiography in vascular lesions. The healthy control group (n=323) were confirmed no coronary leisions after coronary angiography or coronary CT angiography discharged from CAD, without family history of premature CAD.3. Array Comparative Genomic Hybridization:Genomic DNA from all subjects was extracted from the whole blood using the FlexiGENE DNA kit according to the manufactures’instructions. Array Comparative Genomic Hybridization (aCGH) was performed on DNA samples of the patients with CAD in the extended family. Normal human genomic DNA was used as reference. The aCGH protocol followed. We used oligonucleotide microarrays covering the entire genome with an average of 5-kb spacing, which allowed us to delineate copy number with a high precision. To identify chromosomal aberrations associated with gene-expression changes, copy number variation was assessed by equating the log2 ratio of signal intensity between the health controls and patient samples.4. Sequencing of the MEF2A Gene in Extended CAD/MI family:We performed PCR amplification of the region containing both exons and intron-exon boundaries of the MEF2A gene. Genomic DNA obtained from peripheral blood was subsequently subjected to amplification using Taq DNA polymerase and comprising 35 cycles of 30s at 95_℃,30s at 58_℃, and 30s at 72_℃ followed by extension of 6 min. The oligonucleotide primers for the MEF2A gene were designed with Primer 3.0. Single strand conformation polymorphism(SSCP) was used to detect possible gene mutations in the PCR products. And the products were cloned into pGEM(?)-T Easy Vector and sequenced with the T7 or sp6 primer by the BigDye Terminator cycle sequencing reaction and ABI PRISM 3100 Genetic Analyzer.Results1. For the present study, we identified one CAD/MI extended Chinese family with 34 members (5 members died). This family consisted of 20 females and 14 males distribute into four generations. A female,36 years old patient was diagnosed with LAD coronary artery stenosis more than 80% by CAG, who had no cigarette smoking history, hypertension, diabetes and hyperlipids, in accordance with early onset coronary heart disease diagnosis. And her mother, two elder sisters, one elder brother and brother in law were found with symptoms and signs of CAD/MI.2. CAD risk factors Analysis of the family members:total serum cholesterol and triglyceride moderately elevated in only a few members of the family, but serum low-density (LDL-C) and high-density lipoprotein cholesterol (HDL-c) were normal. No smoking, hypertension, diabetes and obesity were investigated in the family members. Multivariate Logistic regression analysis demonstrated that neither was related to CAD/MI including age, body mass index (BMI), fasting blood sugar, cholesterol and triglyceride.3. Family genetic features:four continuous generations were diagnosed with CAD/MI, including the men and women, and the prevalence of the third generation was about 55.6%(5/9), one of the patients’ parents was a patient, the other was healthy. The offspring of the healthy relatives in these patients was healthy, conformed to the characteristics of autosomal dominant genetic disease.4. Micro-array CGH analysis:difference of chromosome copy numbers of 50kb fragments in the line of chromosome 15q26 in seven of CAD/MI famly patients (Iog2 ratio means< 0.3), The regeions contained 11 important genes (POTEB CXADRP2, LOC646214, GOLGA8C, OR4M2, OR4N4, LOC650137, MEF2A, OR4F4, WASH3P and FAM138E). In these genes, only MEF2A gene was associated with the pathogenesis of CAD after literature retrieval and it was chosen as the strongest candidate gene.5. MEF2A gene sequencing:(1) The changes of gene mutation were not found in 1, 2,3,4,5,6,7,8,9,10 exons sequencing of MEF2A gene of all the family members in this study; (2) Six base pairs (CAGCCG) missing was located in MEF2A 11 exon in all 7 CAD patients and 5 non-CAD patients; (3) Lack of the six bases (bp) was located at MEF2A cDNA sequence from 1671 to 1676; (4) A CAG sequence lied in sequence of exon 11 missing CAGCCG and n CAG repeat sequences were before it; (5) The members without lack of 6-bp (normal phenotype) in the family were not suffering from CAD, the other five family members lacked 6-bp but without suffering from CAD were under 40 years old; (6) MEF2A gene with 6-bp or 21-bp missing in exon 11 coding region was not found in the CAD control group (n=311) and the healthy control group (n= 323).Conclusions1. The Chinese CAD family with dominant inheritance characteristics was successfully screened in this study. The traditional risk factors of CAD with family members had no correlation to CAD, including age, blood pressure, smoking, blood lipid, blood glucose and body mass index which proved that family heredity was the important factors to CAD in part of the populations.2. For the first time, we discovered that a Chinese family genetic type of CAD was closely related to the novelty MEF2A gene mutations The mutation was six bases (CAGCCG) missing, located in exon 11, might be one kind of new mutations in human MEF2A gene types.3. In this study, six bases missing in exon 11 of MEF2A and lack of 21-bp and other exons mutations were not found in the scattered CAD patients and the normal control group, which was reported previously abroad. Hence, we suggested that MEF2A gene mutations in exon 11 were not correlated to scattered CAD patients, might be one of the disease-causing genes in the CAD family.IntroductionAtherosclerosis (AS) is one of the major threaten to human life and health.Among numerous mechanism by which the disease occurs, endothelial dysfunction not only contributes to the occurrence of Atherosclerosis, but also lays a foundation for the progression of the disease. Abnormal glucose metabolism is closely associated with atherosclerosis.Currently Diabetes is the leading factors contributing to atherosclerosis (AS) and coronary heart disease (CHD). Compared with patients without diabetes, atherosclerosis progression is more agressive in patients with diabetes, which has nothing to do with the control of blood glucose level. The results from international large-scale randomized clinical trails have revealed that quitting smoking completely and controlling of blood pressure and blood lipid level strictly can significantly reduced the morbidity and mortality of cardiovascular disease in high risk groups with AS. However, large-scale clinical trails such as the ACCORD, ADVANCE and VA Diabetes trials have suggested that mataining a steady level of glycated hemoglobin in population at high risk of AS with diabetes mellitus can reduce the incidence of microvascular lesions; however it can significantly increase mortality of macrovascular lesions. These clinical researches demonstrate that there is a linear correlation of smoking, hypertension and hypercholesterolemia with cardiovascular events. However, the blood glucose level is not linearly associated with cardiovascular events. The contributions of Diabetes to AS may involve other molecular mechanisms and therapeutic targets, which to be elucidated by more profound basic research.Studies have shown that high glucose can damage endothelial cells directly, induce endothelial cells to secrete and release adhesion molecules, chemokines and other inflammatory mediators, and stimulate inflammation. Moreover, by inducing oxidative stress, lipid metabolic disorder and insulin resistance, high glucose can cause vascular endothelial dysfunction. Based on these factors, hyperglycaemia could play a very important role in the formation of atherosclerosis. The mechanisms of vascular endothelial dysfunction caused by high gluose are very complicated, and the roles of many cytokines and signaling pathway invloved in these mechnaisms have not been clearly clarified. Therefore, besides controlling gluose level, seeking other intervention measures is of great significance in prevention and treatment of diabetes atherosclerosis.Kruppel-like factor belongs to a subclass of the zinc-finger family of transcription factors. Research has revealed that KLF2 play a vital part in the protection of endothelial function, which directly or indirectly regulates the expression of a variety of functional genes of endothelial cells and fuctions as a regulator of many key processes such as inflammation, blood clotting, angiokinesis, angiogenesis and so on. Furthermore, KLF2 operates as a theraputic target of stain. Consequently, KLF2 is essentially involved in the formation of AS. Currently there are few relevant reports on the effects of KLF2 on sugar and lipid metabolism. The purpose of this study is to explore whether KLF2 plays a vital part in the protection of vascular epithelial function under the condition of high glucose in vitro, and to clarify the possible molecular mechcanisms and intervention measures, both of which can offer an important therapeutic target for the prevention and treatment of atherosclerosis with diabetes mellitus.Materials and Method1.Cell culture and survival analysisHuman umbilical vein endothelial cells (HUVECs) were cultured in vitro, with the intervention of high sugar in different concentration and at different time point. The cell growth curve was produced using an MTT colorimetric assay. We decided to use the HUVECs exposed to the medium containing 25.5 mmol/L D-glucose for 24h as the high glucose induction model (high glucose group, HG).2.Cell interventionWe detected the KLF2 expression in HUVECs with 25.5 mmol/L D-glucose and different concentration of atorvastatin or rosiglitazone. SB203580 is used for inhibiting the activition of p38MAPK. Cells that were intervened with 5.6 mmol/L and 5.6 mmol/L D-glucose pulse 19.9 mmol/L mannitol respectively were regarded as controls.3.RT-PCRCells of each group were collected to extract RNA which was applied to the reverse transcription and RT-PCR, and then we could estimate the mRNA expression of KLF2, PPARy, VCAM-1, ICAM-1 within each group.4. Western blotting:Cells of each group were collected to extract protein, in order to estimate the protein expression of KLF2, VCAM-1, ICAM-1, MAPKAPK-2 p38MAPK, p-p38MAPK and PPARy respectively.5. Assay of NOThe medium of each group of cells was collected and the amount of NO released by HUVECs was determined using a NO assay kit according to the manufacturer’s protocol.Results1.High glucose (25.5 mmol/L) significantly increased VCAM-1 and ICAM-1 mRNA and protein expression in HUVECs compared to 5.6 mmol/L glucose and 5.6 mmol/L D-glucose pulse 19.9 mmol/L mannitol, while simultaneously decreased KLF2 mRNA and protein expression and the release of NO. Moreover, there is a strong link between VCAM-1, ICAM-1 expression, the amount of NO and KLF2.2.Atorvastatin (0.1 μM,1 μM and 10 μM) or rosiglitazone (0.1 μM,1 μM and 10 μM) significantly suppressed VCAM-1 and ICAM-1 expression and increased nitric oxide release in HUVECs induced by high glucose. These effects of atorvastatin were dose-dependent. The effect of higher concentration could be more obvious.3.Compared to the controls, the phosphorylation of p38 MAPK was enhanced in HUVECs induced by high glucose, and the expression of KLF2 was increased with the SB203580 inhibitor to p38 MAPK. Medium concentration (1 μM) of atorvastatin or rosiglitazone can significantly inhibit the phosphorylation of p38 MAPK within HUVECs induced by high glucose.Conclusion1.High glucose increased VCAM-1 and ICAM-1 mRNA expression in HUVECs, and decreased the release of NO, leading to endothelial dysfunction. Effects of high glucose on VCAM-1, ICAM-1 expression and the release of NO were closely associated with the decreasement in transcription factor KLF2 expression by high glucose in HUVECs.2.HUVECs incubated with high glucose resulted in a significant decrease of KLF2 expression, which was mediated by p38 MAPK activation.3.High glucose(25.5 mmol/L) decreased KLF2 production, atorvastatin or rosiglitazone can inhibit this kind of activation mediated by high glucose, and suppressed VCAM-1 and ICAM-1 expression and increased nitric oxide release induced by high glucose in HUVECs. |
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