Wednesday October 1, 2003: Poster Session 216 PPARs and other nuclear receptors 3P-0702 Homozygote mutation of C161→T polymorphism in the exon 6 of peroxisome proliferator-activated receptor γ gene is associated with onset of premature myocardial infarction T. Chao 1 , T.-H. Chao 1 , Y.-H. Li 1 , J.-H. Chen 1 , H.-L. Wu 2 , G.-Y. Shi 2 , P.-Y. Liu 1 , P.-S. Chen 2 , W.-C. Tsai 1 . 1 Dept. of Internal Medicine; 2 Dept. of Biochemistry, College of Medicine, National Cheng Kung University, Taiwan Background: Peroxisome proliferator-activated receptor (PPAR) is a nuclear receptor. Activation of PPARγ with ligands could modulate some gene tran- scription, thereby leading to multiple antiatherogenic and fibrinolytic effects. However, the association between effect of C161→T mutation of the exon 6 of PPARγ and the onset of premature myocardial infarction is not clarified. Methods: We recruited 146 patients (pts) (mean age 45 yr, 129 male) with premature myocardial infarction (MI) (onset age < 50 yr) and 146 control subjects (mean age 45.3 yr, 120 male). Polymerase chain reaction and restriction fragment length polymorphism were used to define C161→T polymorphism. Results: The frequency of the PPARγ TT genotype among pts with prema- ture MI was significantly higher than that in control subjects (13% vs 5.5%, odds ratio [OR] 2.6, 95% confidence interval [CI] 1.1 to 6.1, p=0.03). This association was not observed in CC, or CT genotypes. The homozygote TT genotype (OR 3.1, 95% CI 1.2 to 7.9), smoking (OR 3.5, 95% CI 2.1 to 6.0), hypertension (OR 3.6, 95% CI 1.9 to 6.9), and diabetes mellitus (OR 3.5, 95% CI 1.5 to 8.4) were the only independent risk factors for premature MI after adjustment of conventional risk factors. Conclusions: There was a significant association between the PPARγ C161→T homozygote mutation and the onset of premature MI in our pop- ulation. Effect of the TT genotype on the production and activity of PPARγ warrants further investigation. 3P-0703 Influence of polyunsaturated fatty acids on the expression of PPARγ in vascular cells and their effect on gene transcription A.-N. Chiang , T.-C. Kuo, H.-H. Wang. Institute of Biochemistry, National Yang-Ming University, Taiwan Objective: To assess the molecular mechanism of polyunsaturated fatty acids (PUFAs) in atherosclerosis, we investigated the expression and transcrip- tional regulation of peroxisome proliferator-activated receptor γ (PPARγ) in response to PUFA treatment. Methods: ApoE-knockout (E°) mice were assigned randomly to three groups feeding with basal diet, corn oil diet, and fish oil diet for 10 weeks. The areas of atherosclerotic lesions were quantitated and compared among E° mice by histopathological analysis. We also used human macrophages and coronary artery smooth muscle cells as in vitro assay system to determine the expression of PPARγ at mRNA and protein levels by RT-PCR and Western blot analysis, respectively. Transcient transfection experiments with plasmids containing PPARγ-response element (PPRE) cloned in front of the luciferase reporter gene were performed to explore how PUFAs affect transcriptional regulation of gene expression. Results: In animal study, fish oil diet significantly reduced the atheroscle- rotic lesion areas in the aorta of E° mice. In cellular studies, different kinds of PUFAs differentially induced PPARγ gene and protein expression in coronary artery SMCs but not in macrophages. The regulation of PPARγ gene expression seems to be a short-term effect. The results of transfection experiments indicated that the PPRE of LXRα promoter was trans-activated by docosahexaenoic acid (DHA). Conclusion: The present study weights the evidence on regulation of PPARγ by PUFAs with regard to their overall impact on the physiological significance in atherosclerosis. 3P-0704 Oxidized low density lipoprotein-induced macrophage proliferation was inhibited by 15-deoxy prostaglandin J2 via inactivation of NF-kB and induces macrophage apoptosis T. Matsumura , T. Matsuo, T. Senokuchi, T. Nishikawa, E. Araki. Department of Metabolic Medicine, Graduate School of Medical Sciences, Kumamoto University, Japan Peroxisome proliferator-activated receptor-γ (PPARγ) agonists inhibit the de- velopment of atherosclerosis in various animal models. It can be explained by the improvement of glucose and lipid metabolism, but it is also possible that PPARγ agonists could directly affect the cells in atherosclerotic lesions. One of the characteristic features in atherosclerotic lesions is a cluster of macrophage- derived foam cells, which play an important role in the development of atherosclerosis. Here, we show that PPARγ agonists, 15-deoxy-prostaglangin J2 (15d-PGJ2), troglitazone and ciglitazone significantly induced macrophage apoptosis. A pan-caspase inhibitor, z-VAD-fmk, prevented 15d-PGJ2-induced macrophage apoptosis. However, it was failed to prevent 15d-PGJ2-mediated reduction of [ 3 H]thymidine incorporation induced by oxidized low density lipoprotein (Ox-LDL). Moreover, pre-treatment with recombinant granulo- cyte/macrophage colony-stimulating factor (GM-CSF) could not prevent high concentration of 15d-PGJ2 (>10 μM)-mediated reduction of [ 3 H]thymidine incorporation, although it prevented low concentration of 15d-PGJ2 (<5 μM)-mediated reduction of [ 3 H]thymidine incorporation. Ox-LDL induced a significant production of GM-CSF from macrophages, which was effectively inhibited by low concentration of 15d-PGJ2 (<5 μM). 2-Cyclopenten-1-one, which is a cyclopentenone ring of 15d-PGJ2, significantly inhibited Ox- LDL-induced GM-CSF production and macrophage proliferation. However, cyclopentene or cyclopentanone had no effect. Moreover, 2-cyclopenten-1- one as well as 15d-PGJ2 inhibited transactivation of NF-kB. Our results suggest that 15d-PGJ2 possesses two different effects on macrophages, high concentration of 15d-PGJ2 (>10 μM) induces macrophage apoptosis and low concentration of 15d-PGJ2 (<5 μM) mediates reduction of macrophage proliferation via inhibition of GM-CSF production. 3P-0705 Pioglitazone improves conduit artery endothelial function and decreases plasma CRP level in type 2 diabetes F.M.A.C. Martens , F.L.J. Visseren, E.J.P. De Koning, T.J. Rabelink. University Medical Center Utrecht, Internal Medicine, Section of Vascular Medicine and Diabetology, Netherlands Introduction: Large vessel atherosclerotic disease is the major cause of mor- bidity and mortality in patients with type 2 diabetes. Pioglitazone, a PPARγ agonist, not only improves insulin sensitivity, but may also have potential favourable effects on other components of the metabolic syndrome. Being an early event in atherogenesis, we examined the effects of shortterm pioglitazone on endothelial function. Methods and Results: A randomised, cross-over, placebo-controlled, dou- ble blind trial with 30 mg pioglitazone once daily for 4 weeks. Flow mediated dilation (FMD) was used to assess large vessel response of the brachial artery on shear stress. Forearm plethysmography (FBF) was used to evaluate the effect on resistance vessel responses of intra-arterial administration of serotonin, nitroprusside and L-NMMA. Twenty male patients with type 2 dia- betes, treated with oral anti-hyperglycemic drugs, not treated with vaso-active medication and a HbA 1c < 9, were enrolled. Endothelial-dependent FMD was significantly increased by pioglitazone treatment compared to placebo (3.1±0.5% vs. 5.4±0.5, p= 0.001), while the endothelial-independent nitroprusside-induced vasodilation did not change. No significant differences were found in FBF-measurements between pi- oglitazone treatment and placebo. Significant decreases in plasma insulin concentration (18.3±2.4mIU/l vs. 14.8±2.1, p= 0.03), free fatty acid level (641±46mmol/l vs. 542±33, p= 0.04), and C-reactive protein concentration (3.5±0.6mg/l vs. 2.6±0.5, p= 0.01) were observed. Conclusions: Short-term pioglitazone treatment improves endothelial func- tion in conduit arteries and decreases CRP plasma level. The improvement of endothelial function can be attributed to changes in insulin, FFA, direct vascular- and/or anti-inflammatory effects of pioglitazone. 3P-0706 The long-term effects of PPARγ agonist in subjects with type 2 diabetes H. Koshiyama 1 , N. Kuwamura 2 , Y. Nakamura 3 , D. Shimono 2 , Y. Seino 2 . 1 Dept DM & ENDO, Medical Research Institute Kitano Hospital, Osaka; 2 Dept DM & Clin Nutrition, Kyoto Univ Grad Sch Med; 3 Div DM & Endo, Dept Hyogo Pref Amagasaki Hosp, Japan Objective: To clarify the long-term effects of PPARγ agonists on early atherosclerotic process in type diabetes Methods: The intima-media thickness (IMT) of common carotid arter- ies was measured using high-resolution B-mode ultrasonography. The IMT measurements were performed on the three locations of the common carotid arteries, ie at the site of greatest thickness and at two other points, 1-cm up- stream and 1-cm downstream from the site of greatest thickness. All segments were scanned and the localized thickness more than 2.0 mm was excluded as plaque lesion. IMT values were averaged on three determinations for right and left arteries, and the greater value of averaged IMT was adopted as the representative data for each measurement. XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan