| ObjectivesThe nuclear receptor superfamily of PPARs consists of isoform α, γ, and β/δ. Although these different members are encoded by separate genes, they have a similar protein structures. PPARs regulate gene expression by binding as heterodimers with retinoid X receptors (RXRs) to specific PPAR response elements (PPRE) in the promoter regions of specific target genes. PPARγ is found predominantly in adipose tissue, where it plays a crucial role in adipocyte differentiation, lipid metabolism and insuline resistance. The PPARγ gene, located at 3p25-24, gives rise to three distinct mRNAs, i.e. PPARγ1, PPARγ2 and PPARγ3. All three PPARγ subtypes contain the common exons 1-6, each differs in their 5' ends and each is under control of their own promoter. PPARγ is mainly expressed in the intestine and in the adipose tissue. Furthermore, PPARγ is expressed in vascular cells including endothelial cells, smooth muscle cells, monocyte/macrophage cells and foam cells. It can be detected out in atherosclerosis and tumor tissue. The antidiabetic glitazones are high-affinity agonists of PPARγ.Atherosclerosis is a multifactorial disease which may result in ischemia of the heart and brain, and infarction. The formation of atherosclerotic lesions involves attraction of monocytes/macrophages and T lymphocytes. Intermediate and advanced lesions typically consist of lipid-laden monocytes/ macrophages (foam cells), migrating and proliferating smooth muscle cells, and the accumulation of cell debris and/or the presence of fibrous caps. Recent studies showed that PPARγ expressed in atherosclerotic lesions and macrophage foam cells, and was associated with obesity-related metabolic diseases such as hyperlipidemia, insulin resistance, and coronary artery disease (CAD).Our objective in this study was to explore the relationship between PPARγ and ET-1 in gene level. At first, we investigated the PPARγC161→T substitution in our well-characterized hospital- based patients of coronary artery disease, in order to observe the relation between PPARγ gene C161→T polymorphisms and the severity of coronary lesion, and the relationship of the polymorphisms with lipid and glucose metabolism in Han Race Chinese. Secondly we established a model of myocardial infarction in rat to observe the effect of PPARγ agonist Rosiglitazone maleate on the ventricular haemodynamics, the histological change, the apoptosis level of cardiomyocytes and PPARγ gene expression after myocardial infarction in rats. Thirdly, we studied the effects of Rosiglitazone on the expression of PPARγ and endothelin-1 (ET-1). We wanted to understand the relationship between the two genes, observe the relation between PPARγ and inflammation elements, such as Tumor Necrosis Factor-α (TNF-α), and understand the effects of PPARγ in protecting the function of endothelial cell and reducing the inflammation response. At last, we observed the expression of ET-1 under the condition that the expression of PPARγ was reduced with the siRNA technique, and by this way we reconfirmed the correlation of the two genes from another way.Methods1, 292 subjects were investigated in this study, including 89 healthy persons, 203 cases diagnosed as CAD. PPARγC161→T gene polymorphism was determined by polymerase chain reaction and restriction fragment length polymorphisms, the blood glucose and the blood lipoprotein were detected, and body height and body weight were measured. The coronary artery lesions were detected by coronary angiography and analysed quantitatively by Gensini score method. The risk factors of CAD were estimated, and the frequencies of PPARγC161→T genotypes and the "T" allele in the CAD and healthy groups were observed.2,Acute myocardial infarction (AMI) model of rat was established by ligation of the left coronary artery. AMI rats were divided into two groups: AMIA group (AMI rats without any treatment) and AMIB group (AMI rats were treated with PPARγ agonist thiazolidinedione - Rosiglitazone 5mg.kg-1/day). The rats in sham group underwent the same procedures but without tying the LAD artery. The infarct size and the changes of the cardiac structure after infarction were assessed by the method of hematoxylin-eosin stain. The cardiac function was evaluated by the physiological signal recording system. The apoptosis level was examined with the method of flow cytometry. The expression of PPARγ was examined by RT-PCR.3, At 24 hour after administrated of Rosiglitazone, the mRNA of PPARγ and ET-1 were determined by RT-PCR and quantitative real-time PCR, the protein of PPARγ and ET-1 precursor were detected by western-blot in the normal and the Angiotensin II (AngII) induced human umbilic vein endothelial cell ( HUVEC) in vitro. The secretion of TNF-α in HUVEC was measured with ELISA.4,The HUVEC was intervened with siRNA Expression Cassette segments in vitro, 24 hours later, the mRNA of PPARγ and ET-1 were detected with RT-PCR, quantitative real-time PCR, the protein of PPARγ was measured with western-blot.Results1,Relationship of PPARγC161-T gene polymorphism and coronary artery disease(1) In normal healthy group, "T" allele frequency was 0.213, "C" allele frequency was 0.787, and in CAD group "T" allele frequency was 0.192, "C" allele frequency was 0.808. There was no significant difference between the two groups.(2) There was a significant association between PPARγC161→T genotypes and the number of significantly disease vessels. "T" allele carriers were far more frequent in patients without than those with significantly diseased vessels (P<0.05), and the CAD risk in the "T" allele carries (OR: 0.56, 95%CI: 0.24-0.63) was much lower than that in the CC homozygote (OR: 1.92, 95%CI:1.09 -2.54). The results showed that Gensini score in patients with CC genotype were markedly higher than that in patients with 'T'allele (p<0.05).(3) apoB was obviously higher in patients with CC homozygote than those with "T" allele carriers (1.02±0.22 与 0.94±0.23,P<0.05), and there was no different in HOMA IR.2, The influences of Rosiglitazone on the ventricular haemodynamics, the histological change, the apoptosis level of cardiomyocytes, and PPARγ gene expression in Rats'myocardial infarction models(1) The cardiac function declined after AMI. AMIA group compared with the sham group, MAP, LVPSP and ±dp/dtmax declined (105.60±10.71 VS 124.17±7.18, 130.63±7.24 VS 150.34±6.82, 3805.30±244.86 VS 5940.83±400.77, -2749.00±131.45 VS 4634.75± 333.96,p<0.05, respectively), but LVEDP rised significantly (15.5±2.35 VS 4.52±0.57,P＜0.05).(2) After 14 days treatmented by Rosiglitazone, the LVEDP declined (10.14±2.28 VS 15.5±2.35, p<0.05), and infarct size decreased 33% compared with AMIA group. The change of the cardiac structure after infarction in AMIB group was better than in AMIA group.(3) AMIA rats' apoptosis level was as high as 21.15-fold of the sham group. Compared with AMIA group, after 14 days treatment by Rosiglitazone, the apoptosis level was decreased significantly (16.04±2.26 VS 26.44±3.51,p<0.05).(4) Compared with AMIA group and the sham group, expression of PPARγ was increased obviously in AMIB group (2.352±0.159 VS 1.574±0.196 VS 0.491±0.078, p＜0.001).3, The effect of Rosiglitazone on gene expression of the nomal and Ang II - induced human umbilia vein endothelial cell in vitro(1) The mRNA and protein expression levels of PPARγ were increased significantly by Rosiglitazone, on the contrary, the level of ET-1 mRNA and its precursor pretein expression reduced (p<0.001). There was relationship between two genes expression ( r=o.914, p=0.011; r=0.999, p=0.028).(2) The mRNA expression of PPARγ and ET-1, and the protein expression of PPAR-γ and ET-1 precursor were markedly increased in HUVEC stimulated by Ang II as compared with that in the control group. 24 hours after administration of Rosiglitazone, the mRNA expression and protein expression of PPARγ rised, at the same time the expression of ET-1 reduced (P<0.05).(3) The secretion of TNF-α increased by Ang II in a dose- dependent manner; and restrained by Rosiglitazone (p<0.05).4,siRNA Expression Cassette 1(1326) down-regulated the mRNA and protein espression of PPARγ and up-regulated ET-1 gene mRNA expression at the same time. A link was found between two genes(r=0.995, p=0.042).Conclusion1, In the Han race Chinese, the distributing trend of PPARγC161→T gene polymorphism in the healthy group is as the same as that in the patients with CAD group; The Gensini score of the coronary artery angiography in persons with CC genotype are significantly higher than that in the persons with "T" allele. It means that there is a lower risk of CAD in the patients with "T" allele.2,Rosiglitazone can decrease infarct size and apoptosis level of AMI in rats. The effect of Rosiglitazone may be associated with the increase of PPARγ gene expression, which can protect the function of cardiac muscle cell and vascularity.3, The expression of PPARγ in the mRNA and protein level can be enhanced, the ET-1 gene expression in the mRNA level and ET-1 precursor expression in the protein level, and TNF-α secreted from the vascular wall can be suppressed by Rosiglitazone through the way of increased PPARγ gene expression in HUVEC.4,siRNA Expression Cassette 1(1326) can cause an inhibition of PPARγ expression which lead to an increase of ET-1 expression. Therefore, both of PPARγ and ET-1 play a key role in formation of the Atherosclorsis.
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