3P-0883 B- and M-mode ultrasound studies of arterial wall thickness and arterial wall stiffness in patients with Marfan syndrome and unaffected controls

Wednesday October 1, 2003: Poster Session Aortic and peripheral vascular diseases produced a lipoprotein cholesterol profile similar to that of Ldlr–/– mice. Apoeh/hLdr-/- mice will be valuable for examining the role of apoE isoforms in the progression and regression of atherosclerosis and in plaque vulnerability in peripheral vascular disease. 3P-0879

Anti-atherogenic effect of E040, a new synthetic compound, is mediated by inhibition of macrophage infiltration

K.-H. Nam, C.-H. Lee, C.-T. Kim, B.-H. Kim, J.-Y. Kim, G.-T. Oh. Korea Institution of Bioscience and Biotechnology, Republic of Korea Objective: Atherosclerosis is the leading cause of the heart disease and stroke. Here we studied the effect of E040, a derivative of naringenin (one of the citrus flavonoids), on atherogenesis in Ldlr-/- mice and New Zealand White rabbits both of which fed high cholesterol diets and investigated the underlying anti-atherogenic mechanism. Methods and Results: Feeding high cholesterol diets in both animals, induced considerable lipid accumulation in their aortic arterial wall. In contrast to the mice in the high cholesterol diet group, macrophage infiltration into the atherosclerotic lesion was not so prominent in the mice fed the same diet containing 0.025% E040. To investigate the mechanism by which E040 ameliorate atherogenesis, THP-1 cells were treated with E040. The culture supernatant of THP-1 cells activated with PMA and LPS in the presence of E040 showed reduced chemoattractic activity for naive THP-1 cells. The productions of inflammatory cytokines and chemokine by the cells, however, were not affected by E040. Surface molecules including ICAM-1 and CD36 did not changed by the presence of E040 during THP-1 cell activation, indicating no direct effect of E040 on THP-1 cell activation and migration properties. On the other hand, E040 inhibited VCAM-1 expression on HUVEC activated with TNF-α. In addition, electrophoretic mobility shift assay (EMSA) and luciferase reporter gene expression assay showed inhibition of TNF-α induced NF-kB activation in HUVEC by E040 treatment. TNF-α induced IL-8 production by HUVEC was also suppressed by E040 treatment. Conclusions: A new synthetic compound, E040, markedly reduced atherosclerotic lesion size in animal models, at least partially, by reducing infiltration of macrophage. This is mediated by reduced expression of adhesion molecules on endothelial cells and by regulating production of monocyte attracting molecule(s) by macrophages in the lesions. 3P-0880

Alpha-lipoic acid inhibits vascular smooth muscle cell migration by modulating production of MMP-9 and adhesion molecule

H.S. Kim 1 , M.J. Kim 1 , K.G. Park 1 , H.J. Lee 1 , S.Y. Jung 1 , Y.D. Kim 1 , J.H. Seo 1 , K.U. Lee 2 , J.Y. Park 2 , I.K. Lee 1 . 1 Keimyung University School of Medicine; 2 Ulsan University School of Medicine, Republic of Korea

Cyclic AMP inhibited proliferation of human aortic vascular smooth muscle cells by regulating of E2F expression

M.J. Kim, J.D. Ahn, H.J. Kim, S.-I. Yeo, K.M. Lee, K.S. Kim, I.K. Lee. Keimyung University, School of Medicine, Dong-San Medical Center, Republic of Korea Background and Aims: Proliferation of vascular smooth muscle cells (VSMCs) is an important feature of atherosclerosis and restenosis. Cilostazol is a potent antiplatelet agent that increases the concentration of cAMP within platelets by selectively blocking phosphodiesterase III. E2F sites are growth/cell cycle related genes that are involved in control of the cell cycle or DNA synthesis. In the present study, we investigated whether the expression of E2F in cultured human VSMCs is suppressed by cilostazol. Methods: VSMCs were cultured in DMEM contaning 20% FBS. We evaluated the effect of cilostazol in E2F gene expression in human VSMCs under high-D-glucose and angiotensin II using E2F promoter assay. EMSA was performed for evaluation of E2F activity. Western blotting was performed for analysis of PCNA and E2F expression. And we also evaluated neointimal formation in vivo model of rat carotid injury. Results: Cilostazol downregulated the promotor activiy of E2F in VSMCs induced by high glucose and angiotensin II. PCNA and E2F expression was attenuated by cilostazol in dose-dependent manner. Cilostazol significantly reduced high glucose- induced E2F binding activity in dose-dependent manner. Administration cilostazol virtually abolished neointimal formation after balloon injury to the rat carotid artery in vivo. Conclusion: This study shows that cilostazol have inhibitory effects on VSMCs, accompanied by decreased expression of E2F. Cilostazol may have potential to prevent development of atherosclerosis and restenosis. 3P-0882

Inhibition of cytokine induced I-kB kinase activation as a mechanism contributing to the anti-atherogenic activity of tilianin in hyperlipidemic mice

G.T. Oh, K.-W. Nam, J. Kim, J.-J. Hong, J.-H. Choi, K.-H. Nam. Korea Institution of Bioscience and Biotechnology, Taejeon, Republic of Korea Background: In previous study, we demonstrated tilianin inhibits TNF-α induced VCAM-1 expression in HUVEC. In this study, we demonstrate inhibition of the production of atherogenic cytokines and the anti-atherogenic effects of tilianin in Ldlr-/- mice. Methods and Results: Twenty-six male Ldlr -/- mice were divided into three groups. One group of mice received high fat diet (1.25% cholesterol, 15% fat and 0.5% sodium cholate) and two groups of mice received high fat diet plus 1mg/kg lovastatin and 250mg/kg tilianin, respectively. After 10 weeks, mice were sacrificed and plasma lipid level (total cholesterol, HDL cholesterol triglyceride) and atherosclerosis lesion size were determined. Mice fed high fat diet with tilianin had slightly lower levels of total cholesterol than those fed the unsupplemented high fat diet. The atherosclerosis lesion was significantly reduced by 41.9% compared with control group (p<0.01). Tilianin treatment reduced the plasma levels of TNF-α and IL-1β that can upregulate the expression of celluar adhesion molecules through the activation of NF-κB. Macrophage is one of the major producer of the TNF-α and IL-1β. The expression levels of mRNA and productions of TNF-α and IL-1β were decreased in the tilianin treated peritoneal macrophages from Ldlr-/- mice. Tilianin also inhibit the activation of NF-κB that regulates TNF-α and IL-1β gene expression, but not the activation of AP-1. The inhibition of NF-κB activation was due to its blockade of the upstream signals mediated I-κB kinase (IKK) activation, and subsequent phosphorylation and degradation of IκB in RAW 264.7 cells. Conclusions: Tilianin ameliorates atherosclerosis by inhibition of TNF-α and IL-1β gene expression that are regulated by NF-kB. The possible molecular mechanism against atherosclerosis of tilianin is an inhibition of IKK activation, and subsequent phosphorylation and degradation of IkB. 3P-0883

B- and M-mode ultrasound studies of arterial wall thickness and arterial wall stiffness in patients with Marfan syndrome and unaffected controls

F. Zijta. Academic Medical Centre, Amsterdam, Netherlands Objectives: Marfan syndrome is an autosomal dominant inherited disorder of connective tissue with variable manifestations in primarily skeletal, ocular and cardiovascular organ systems. Prognosis is mainly determined by progressive dilation of the aortic root that may lead to aortic dissection and death. Medical and surgical therapy have greatly improved prognosis of Marfan patients.

XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan

WEDNESDAY

Background: Migration and proliferation of vascular smooth muscle cells (VSMCs) is a critical event in the formation of atherosclerosis and restenosis after percutaneous transluminal angioplasty (PTC). Matrix metalloproteinases (MMPs) and cell adhesion molecules such as intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) are important for VSMC migration into the intima. It has been reported that induction of these molecules depends on activation of the transcription factor nuclear factor-kappa B (NF-kappa B) and is associated with oxidative stress. Alphalipoic acid (ALA), the metabolic thiol antioxidant, may be useful in treating pathologies associated with redox imbalances. In this study, we investigated the effects of ALA on MMP-9 and adhesion molecule expression and NFkappa B activity in human VSMC stimulated by high glucose, high glucose with angiotensin II and TNF-α. Methods: Human VSMCs were obtained from the aorta of transplant donor and were cultured in DMEM containing 20% FBS. We used zymography for MMP9 expression and Northern and Western blot analysis for ICAM-1. VSMC migration assay was done by Boyden chamber assay and electrophoretic mobility shift assay (EMSA) was done for evaluation of NF-kappa B activity. Results: ALA inhibited stimulant induced VSMC migration in Boyden chamber assy. Preincubation of VSMCs with ALA downregulated NF-kappa B activity and reduced expression of MMP-9 and ICAM-1 in human VSMCs stimulated by high glucose, high glucose with angiotensin II and TNF-α. Conclusion: Our data show that ALA reduces expression of MMP-9 and ICAM-1 and inhibits VSMC migration through the inhibition of the NF-kappa B signaling pathway and thus provides potential therapeutic effects for development of atherosclerosis or prevention of restenosis after angioplasty in human.

3P-0881

255

Wednesday October 1, 2003: Poster Session Gene therapy

256

Little is known about the in-vivo elastic properties of arterial segments other then the aortic root. Here we describe arterial wall thickness as a morphological parameter and arterial wall stiffness as a functional parameter. The carotid and femoral arteries were taken as representatives of vessels more downstream of the aortic root in patients with Marfan syndrome. Methods: Thirty-nine Marfan patients (age 32(SD5)years) and 40 controls were investigated. B-mode ultrasound imaging was used to assess carotid and femoral intima-media thickness (IMT). M-mode ultrasound was used to assess distal carotid distensibility. Results: IMT (0.58(0.12) and 0.57(0.06)mm) and distal common carotid lumen diameter (6.15(0.54) and 6.19(0.65) mm) were similar in patients and controls. Carotid artery distensibility was significantly decreased in Marfan patients (42.7(12.8) vs. 47.9(10.7)kPa in controls). Pulse pressure in patients was increased due to a lower diastolic blood pressure (SBP was 121(11) and 120 (10) mmHg (p=0.3) and DBP was 70(8) and 75(9) mmHg (p=0.01) in patients and controls respectively). Conclusion: Marfan patients showed increased carotid arterial wall stiffness independent of increase in arterial wall thickness and arterial diameter. This is most likely caused by the increased pulse pressure in patients. The findings indicate that the impairment of elastic properties of the arterial media in Marfan patients is not confined to the aortic root and aorta, but also to vessels more downstream of the aortic root. 3P-0884

Distinct findings in thoracic aorta in patients with stroke A five year survey in Norway

I. Asmussen. Countyhospital Førde, Norway Atherosclerosis are found in patients with stroke or other ischemic episodes. We present a study utilizing the transesophageal (TEE) approach. Data represent a 64 month period. 1017 TEE had been performed by one investigation, 389 in patients with stroke or other ischemic episodes (38%) Apart from cardiac sources of emboli, atherosclerosis of the thoracic aorta was the major finding, with severe lesions in approximately 25%. Atherosclerotic lesions were present at 3 distinct locations: around the diaphragma, at a location 35 cm from the teeth, and in the aortic arch. Lesions were, apart from one, never seen in the ascending aorta. The lesions were the classical raised or elevated plaques in with rupture, ulcerations, fissuring and even craching of calcified intima, that would protude like a stick into the lumen. Waving thrombi would decorate the volcanic crater edges, and in a few, cm long thrombi would “wag the tail” in the bloodstream. A distinct lesion was found: single plaques with a very tiny base. At control TEE they all had disappeared. These lesions could be demonstrated in the young and in otherwise were active people. Classical atherosclerosis represent a source of emboli to the brain. However, a distinct lesion can be demonstrated predominantly in the young and in otherwise very physically active people: a single localized thrombus or plaques. They were found in young that had had their ischemic episode after a long day of skiing (and falling) or in two men, the history was a long day with jumping. Minor rupture could be the explanation. These lesions occurred at the site of the type B aortic dissection. The outcome was good. Ischemic episodes are caused by lesions of the aortic arch. However, single located plaques or thrombi in the thoracic aorta can be found in otherwise healthy persons, mainly in very active people, probably representing aortic wall rupture with minor aortic dissection. 3P-0885

The antiproliferative effect of rapamycin is associated with inhbition of cholesterol ester synthesis

M. Mulas 1 , P. Petruzzo 2 , A. Cappai 2 , C. Mulas 1 , G. Demuro 1 , B. Batetta 1 , S. Dessi 1 . 1 Dept. of Biomedical Science and Biotechnology, 2 Dept. of Surgery Science, Cagliari, Italy Objective: Recently, we provided a link between cholesterol esterification and the control of cell cycle G1/S transition of VSMC, indicating that cholesterol esterification may accelerate the progression of vascular proliferative diseases by modulating the rate of the VSMC proliferation. Rapamycin, a macrolide antifungal agent used as an antirejection drug in organ-transplant recipients, is a powerful inhibitor of several regulators of cell-cycle progression and the migration of VSMC. The drug also has antiproliferative effects on a variety of other cell types. Recently, implantation of rapamycin (sirolimus)-coated stents in patients with angina pectoris was shown to be safe and effective in inhibiting neointimal formation. Methods: In the present study we measured 14 C-acetate incorporated into

free and esterified cholesterol as well as 14 C-oleate into cholesterol esters during the growth of VSMC treated or not with rapamycin (100ng/ml). Results: Our results showed that the inhibition of VSMC proliferation produced by rapamycin was associated with a strong decrease of both 14 Cacetate and 14 C-oleate incorporated into cholesterol esters, (about 90-95%). Rapamycin treatment also results in a decrease of de novo cholesterol synthesis as indicated by 14 C-acetate incorporated into free cholesterol, however in this case the inhibition was about 25-30%. Results obtained with VSMC were reproduced in a model of murine tumoral macrophages and in that of human lymphobastic T cells (CEM). Conclusions: Our results suggested that cholesterol esterification closely paralleled (and actually preceded) DNA synthesis whether occurring in neoplastic or non-neoplastic populations, supporting the idea that cholesterol esterification might be a target of proliferative diseases by functioning as a critical regulator of cell proliferation.

GENE THERAPY 3P-0886

Ultrasound increases retrovirus-mediated gene transfer to endothelial cells and vascular smooth muscle cells

T. Naka, T. Sakoda, T. Abe, Y. Fujioka, T. Tsujino, M. Ohyanagi. Dept. of Internal Medicine, Cardiovascular Div., Hyogo College of Medicine, Japan Background: Retrovirus-based vector integrates the transgene into the genome of the target cells, which can sustain long-term expression. However, the transduction efficiency is known to be low. Ultrasound was reported to increase gene expression using plasmid DNA. However, there are no reports which show ultrasound effect to virus-mediated gene transfer efficiency. Methods: Retrovirus-mediated gene transfer system was used to transfect cultured 293T cells, bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and rat skeletal muscle myoblasts (L6 cells) with the LacZ gene combined with ultrasound exposure. A cell viability assay was performed on cultured 293T cells that were exposed to varying durations (5 sec to 30 sec) and power levels (1.0 watts/cm2 to 4.0 watts/cm2 ) of ultrasound after transduced by retrovirus. Effect of ultrasound to virus itself was evaluated by measuring titers (Transducing Units/ml (TU/ml)) of virus to 293T cells after exposure of ultrasound to virus only. Results: Below 1.0 watts/cm2 and 5 seconds exposure, ultrasound showed no cytotoxicity to 293T cells transduced by retrovirus, and no toxicity to virus itself. At the same condition, ultrasound resulted in significant increases in retrovirus-mediated gene expression at 6.6-fold, 4.8-fold, 2.3- fold, and 3.2-fold in 293T cells, BAECs, RASMCs, and L6 cells respectively. Conclusions: Ultrasound associated local gene therapy has potential for not only plasmid-DNA-, but also virus-mediated gene transfer. 3P-0887

LDL receptor gene therapy protects against atherosclerosis in a mouse model of familial hypercholesterolemia

S. Nomura, A. Merched, K. Oka, L. Pastore, A. Beaudet, L. Chan. Baylor College of Medicine, USA Objectives: Familial hypercholesterolemia (FH) is caused by LDL receptor (LDLR) deficiency. Most FH patients with complete LDLR deficiency are resistant to conventional therapies. LDLR gene therapy using adeno-associated virus has proven ineffective due to inadequate transgene expression and stimulation of antibodies against newly synthesized LDLR. However, production of neutralizing antibodies against neoantigens does not always occur with gene therapy as we have shown in apoAI and apoE null mice. In this study, we tested the efficacy of in vivo LDLR gene therapy using helper-dependent adenovirus (HDAd). Methods: LDLR deficient mice were fed a high cholesterol diet and treated with HDAd expressing monkey LDLR driven by PEPCK promoter, or PBS (control), and plasma lipoproteins, anti-LDLR antibody titer, and atherosclerotic lesions were analyzed. Results: At 24 weeks after HDAd-LDLR injection at a dose of 1.5 × 1013 vector particles (VP)/kg, mice had plasma cholesterol level of 131 mg/dl (vs. 637 mg/dl in controls, p<0.001) and mean atherosclerotic lesion area of 6.6 mm2 (vs. 16.6 mm2 in controls, p<0.001). Comparing these results to those obtained from base line group (3 mm2 ), the lesions in control group progresses 5-fold, whereas those in the treated group only 2-fold. Therefore, LDLR gene therapy markedly inhibited lesion progression. In 2 of 9 treated mice, we detected anti-LDLR antibody titer in plasma. We next studied the long-term efficacy of treatment using a lower dose (5 × 1012 VP/kg). At the time of

XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan