214 Wednesday June 28, 2000: Poster Abstracts P:W26 Regulation of Endothelial Function WeP32:W26 I Vasorelaxation and tPA release induced insulin by are i impaired in women with polycystic ovary syndrome and hyperinsulinism E De Negri, R. Fioriti, L. Ferrini, A. De Giorgi, C. Giannarelli, G. Dell'trot, R. Pedrinelli, E Fruzzetti, E Carmassi. Dpt. of Internal Medicine, Dpt. of Gynecology, Univ. of Pisa, Italy Objective: Women with polycysfic ovary syndrome (PCOS) have often in- sulin-resistance (IR) and increased risk of atherosclerosis. Hyperinsulinemia associated with PCOS can stimulate Plasminogen activator inhibitor-1 (PAI-1) release and trigger hormonal changes. As vasodilatory response to insulin is blunted in IR states, impaired vasodilation and fibrinolysis may be correlated to the increased risk of atherosclerosis. Methods: PAI-1 and tissue plasminogen activator (tPA) levels were eval- uated basally and during insulin infusion in the forearm vascular bed of 8 healthy young subjects and 4 young women with PCOS and hyperinsulinemia. Insulin was infused for 120 rain in the brachial artery at a rate calculated to raise local venous concentrations of 100 #IU/mL. Blood samples were obtained from brachial artery and vein. Results: Elevated basal PAI-1 levels were found in PCOS women (p < 0.01 vs. controls), correlating with BMI and androgen levels. In PCOS women, vasodilatory response to insulin was blunted. PAI-1 balance increased both in normal subjects (p < 0.01) and PCOS patients (p < 0.05). tPA balance increased (p < 0.01) in normals, but not in PCOS women. Conclusions: In PCOS women with IR, blunted vasorelaxation and tPA release was observed during infusion of physiological doses of insulin, as well as elevated basal PAl-1 level. An impairment of vasorelaxation and fibrinolysis could be involved in the development of atherosclerosis in IR states. I WeP33:W26 ] Involvement of GATA in the VCAM-1 proteins I induction in human endothelial cdls M. Umetani, C. Mataki, T. Hamakubo, T. Kodama. Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan We established a bioassay system to detect the intensity of monocyte-endothe- lial cell adhesion and then screened newly synthesized compounds to discover cell adhesion inhibitors. One of these inhibitors, K-7174, suppressed the expression of VCAM-1 both in cell surface expression and in mRNA level, without affecting the induction of ICAM-1 or E-selectin in cytokine-stimulated endothelial cells. K-7174 had no effect on the stability of VCAM-I mRNA, and gel shift assay revealed that its inhibitory effect on VCAM-1 induction was mediated by an effect on the binding activity of GATA protein family to the VCAM-1 gene promoter. K-7174 did not influence the binding to any other binding motif including NFKB. Studies using specific antibodies and antisense oligonucleotides to GATA proteins revealed that multiple GATA proteins and their complex formation were involved in the induction of VCAM-1 by TNFo. These results provide evidence for the importance of GATA proteins in the cy- tokine induction of VCAM-1, and also indicate that GATA proteins arc hopeful as targets for development of anti-inflammatory reagent for clinical use. WeP34:W26 I succinate inhibits adhesion of monocytes to c~-tocopheryl endothelial cells under flow by inducing caspase-dependent p65 J. Neuzil, P. yon Hundelshausen, N. Gellert, C. Weber. Institute for Prevention of Cardiovascular Diseases, Ludwig Maximilians University, Munich, Germany Activation of endothelial cells by an inflammatory stimulus renders their adhesiveness for monocytes. Pre-treatment of human umbilical vein en- dothelial cells (HUVEC) with ¢z-tocopheryl suceinate (a-TOS), hut not with ~-tocopherol (~-TOH) or ~-tocopheryl acetate (c~-TOA), inhibited adhesion of monocytic cells to TNF~- or IL- l~-stimulated HUVECs, and the inhibitory effect was suppressed by co-treatment of the cells with a caspase inhibitor. Moreover, transfection of HUVEC with a gain-of-function bcl-2 gene pre- vented the anti-adhesive effect of a-TOS, t~-TOS, but not that ot-TOH or a-TOA, treatment of HUVECs exhibited features of early apoptosis, caspase activation and specific cleavage of p65, a subunit of NF-tcB. a-TOS-pre-treated cells showed neither ItcB degradation nor nuclear translocation of p65 follow- ing TNFot stimulation. Expression of VCAM- 1 was lower in TNFa-stimulated, c¢-TOS-pre-treated cells. Transfection of endothelial cells with a VCAM-1 gene suppressed the inhibitory action of ct-TOS. The role of a caspase in p65 scission was confirmed by experiments in which HUVECs were transfected with a caspase non-clearable p65 gene. These data explain why the succinyl analogue of vitamin E inhibits adhesion of monocytes to activated endothelial cells, and suggest a relation between induction of apoptosis and NF-KB activation in endothelial cells. I WeP35:W26 [ Myocardial vascular responsiveness improves after cholesterol reduction by selective LDL-apheresis T. Sampietro, E Bigazzi, B. Dal Pint, S. Fusaro, G. Sassi, C. Marchetti, G. Sambuceti, M. Tuoni, O. Parodi, A. Bionda. CNR Institute of Clinical Physiology, Pisa; Department of lnternal Medicine, University of Pisa, /taly Our previous work showed a regulatory role of plasma cholesterol (CH) on vascular function regards endothelial adhesiveness and cutaneous microcir- culatory blood flow. To answer the question whether massive CH removal by selective LDL apheresis (dextran sulfate columns, treated plasma = 2.5-3 times the patient plasma volume) could acutely affect coronary circulation reactivity, we studied myocardial perfusion in 7 consenting, heterozygous patients (6 M and 1 E mean age 47.4 =l=6.3 years) with familial hypercholesterolemia (FH); all but one had coronary artery disease undergoing apheresis therapy. Just before and soon after apberesis, myocardial blood flow at rest (MBFrest) and after adenosine stimulation (MBFade) was assessed with 13N-ammonia and positron emission tomography. After apheresis, the mean percent reductions ofCH, LDL-CH, ApoB, HDL-CH, TG and Lp(a) were 77%, 91.5%, 87%, 6%, 64% and 89%, respectively. Adhesion molecule values (sELAM-1, sICAM-1 ) were significantly reduced, p < 0.003 and p < 0.0001, respectively. MBFrest values showed no significant change after apberesis (baseline values were 0.71 4- 0.06 ml/min/g and after apheresis were 0.86 -4- 0.25 ml/min/g). Following apheresis, MBFade increased significantly (from 1.31 4- 0.2 to 2.05 =E 0.8 ml/min/g, p < 0.05), while blood pressure rate was unmodified. These results demonstrate a direct role for CH in regulating coronary vascular reactivity, at least in FH patients, and they suggest that 'aggressive' LDL apheresis may be a useful tool to acutely reverse coronary vascular dysfunction. WeP36:W26 [ Significance of up and down regulation vascular I of endothelial growth factor receptor in angiogenesis S. Murota, M. Onodera, J. Wang, I. Morita. Tokyo Medical and Dental University, Tokyo, Japan Objective: Vascular endothelial growth factor (VEGF) is an endothelial cell specific growth factor. The growth signal of VEGF is transfered through its specifc tyrosine kioase receptor, VEGF-R2. VEGF production is known to he regulated by various substances and conditions in several tissues and cells. By contrast, the regulation of VEGF receptor remains unknown. Methods: Endothelial cells were examined for the tube forming activity and the VEGF-R2 expression. The former was assayed in vitro by the Type 1 collagen gel method and the latter was quantified by the fluorescence image analysis. Results: 1) Endothelial cells exposed to high glucose concentration (33 mM) for 30 days increased the tube formation induced by VEGE but not by serum and bFGE Immunohistochemical study showed that VEGF-R2 expression was up regulated by the high dose glucose treatment. 7) Delec- tion assay of the VEGF-R2 promoter in endothelial cells treated with long term high glucose and VEGF showed that the 4 th SP1 site located between positions -116 and -95 in the VEGF-R2 promoter plays a very important role in VEGF-R2 gene expression. 3) Collagen and osteoblast conditioned medium caused up regulation of VEGF-R2 expression in endothelial cells and to enhance the tube formation. 4) Eicosapentaenoic acid (EPA) pretreatment caused down regulation of VEGF-R2 expression in endothelial cells and to inhibit the tube formation. Conclusions: Up and down reguration of VEGF recepter in endothelial cells is as important as the change in VEGF production in tissues near by, and both changes can affect the angiogenesis very much. WeP37:W26 I A Mediterranean diet, in monounsaturated-fat and high a low fat diet improve endothelial function in hypercholesterolemic patients Jos6 L6pez-Miranda, Francisco Fuentes, Francisco Sfmchez, Purificaci6n G6mez, Elier Paz, Pablo Ptrez Martfnez, Carmen Marin, Pedro Castro, Jos6 M. Ordov~is, Jos6 Jimtnez Ptreperez, Francisco Pdrez-Jimfnez. Hosp. Reina Sofia, Cdrdoba, Hosp. Alto Guadal- quivir, And~jar, Spain; USDA HNRCA Tufts University, Boston MA, USA Endothelial dysfunction is an early event in atherogenesis and it is present Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000