58 Thursdar 27 May 1999

Poster session: The metabolic ,~3,ndronw

cluster (Xmnl, Mspl/AI and Sstl), there were no differences in frequency distributions of these two variants between probands in = 34), affected- (n = 220) and non-affected (n = 300) family members and spouses in = 236). No significant associations between plasma lipid traits, plasma apo CIII, plasma insulin levels and the two DNA variants were observed. In conclusion there is no in uioo evidence that these variants in the insulin response element are involved in the insulin-dependent regulation of apo CIII gene expression.

In vitro transcription studies in both HepG2-cells and CaCo2-cells us- ing experimental reporter constructs with the apo CIII promoter region (+14/-237) and the apo CIII enhancing region (-237/-1342) were performed to further explore the role of the IRE variants in apo CIII gene expression.

Table: Effect of',ariation in the IRE on insulin dependent aIx'~ CI[I transcription activity.

genotype -455T/-.182C ~55C:-482T medium -insulin +insulin -insulin +insulin

HepG2 1939+383 t 335::t:229" 19OI :t_371 1365~31~4 ° Caco2 12944-502 1698 ±585" 12 t 04-448 15574-198

Mean::t:SD. Values are presenting percentage enhancement compared to contrcd promoter wlthoul enhancing n,."gion 1100%1. P-values ',.~zr¢ lgSlgd using Studentg T-test. ": P < 0.05

It was found that I) ApoClll gene expression is regulated by insulin, independent of variants in the insulin response element. 2) Insulin promotes apo CIII gene expression in CaCo2-cells but decreases gene expression in HepG2-cells.

IN VIVO GENE TRANSFER OF ENDOTHELIAL NITRIC OXIDE SYNTHASE TO CAROTID ARTERIES FROM HYPER- C H O L E S T E R O L E M I C RABBITS ENHANCES ENDOTHELIUM- DEPENDENT RELAXATIONS BUT DOES NOT REVERSE VASCULAR DYSFUNCTION

T. O'Brien, J. Sato, T. Mohasci, A. Noel, E Gloviczki, G. Mozes, Z.S. Katusic. Departments of EndocrinoloKv and Vascular Surgery. Maro Clinic and Foundation, Rochester, Minnesota, USA

Hypereholesterolemia is associated with abnormal endothelium-dependent vasodilatation. This is believed to be due to decreased nitric oxide bioavail- ability. Adenoviral vectors have been used to transfer genes to the vascular wall with high efficiency. The aim of the current study was to examine the effect of adenoviral-mediated gene transfer of endothelial nitric oxide synthase (eNOS) to the hypercholesterolemic rabbit carotid artery in vivo. In addition, we sought to determine whether adenoviral-mediated gene transfer per se was associated with vascular dysfunction. Rabbits were fed a 1% cholesterol diet for 10 weeks. Vascular reactivity was assessed in non- transduced carotid arteries from chow and cholesterol-fed animals (n = 16). In addition, carotid arteries were surgically isolated and adenoviral vectors encoding eNOS (AdeNOS) or ~-galactosidase (Adl~Gal) were delivered to the vessel lumen (n = 16). While atherosclerosis was not observed in the carotid artery of cholesterol-fed animals, abnormal acetylcholine-mediated endothelium dependent-vasorelaxation was detected. In contrast, responses to calcium ionophore A23187 and NO donor were normal. Vascular reac- tivity was similar in non-transduced and Ad[~-gal transduced vessels from hypercbolesterolemic animals. Thus, adenoviraI-mediated gene transfer to the hypercholesterolemic rabbit carotid artery did not result in vascular dysfunction. In vessels transduced with eNOS, transgene expression was demonstrated by RT PCR and immunostaining. Using the latter technique, expression was detected in both the endothelium and adventitia. While eNOS overexpression enhanced the sensitivity of the vessels from cholesterol-fed animals to low concentrations of acetylcholine, vascular function was not normalized. Thus, gene therapy approaches to cholesterol-induced vascu- lar dysfunction aiming at eNOS overexpression may not be sufficient to normalize vascular function.

MULTIPLEX FAMILIAL HYPERCHOLESTEROLEMIA MUTA- TION ANALYSIS USING PCR AND THE OLIGONUCLEOTIDE LIGATION ASSAY ON AN AUTOMATED DNA SEQUENCER

S.J. Scharf, H. Baron, S. Fung, EC. Luft, H. Schuster. Applied Biosystems Division, Perkin Elmer Corporation, Foster City, USA; Franz Volhard Clinic and Max Delbnick Center, Humboldt University, Berlin, German),

About 10% of individuals with lipid disorders have familial hypercholes- terolemia (FH), an autosomal dominant disease associated with premature arteriosclerosis. The identification of FH patients and estimation of LDL

receptor mutation frequencies requires a high-throughput method since Familial Hypercholesterolemia is very heterogeneous at the molecular level. We have developed a sensitive, specific PCR and oligonucleotide ligation assay (OLA) which currently allows simultaneous testing for the ApoB R3500Q mutation and 24 LDL Receptor Gene mutations which have been identified in German, Swiss, Danish, and Dutch FH patients. Each ligation product represents an individual allele and can be identified by its defined electrophoretic mobility and fluorescent color on a 4-color ABI sequencer. The combined use of three different fluorescent dyes permit multiple loci to be interrogated in one gel lane or sample injection. The analysis, including allele identification, is automated and genotypes can be reported in a format that can be downloaded to a population database. The test can also be used very effectively from blood sample spotted onto paper cards for use in genetic fieldworking to identify patients. This assay has been performed on over 1000 patient samples with close to 100 percent accuracy.

ROLE OF TRANSCRIPTIONAL FACTORS USF AND HNF4 IN THE TRANSCRIPTION OF APOLIPOPROTEIN GENES

A. Ribeiro, D. Pastier, E Cardot, J. Chambaz. U 505 INSERM, Unit~ersiti Pierre et Marie Curie, Paris, France

In the present study we establish the functional significance of the ubiqui- teously expressed upstream stimulatory factor (USF] in the hepatic-specific apoA-II and C-Ill promoter activity. We demonstrate synergism between USF and the liver-enriched factor HNF-4 in the transactivation of both promoters. This synergistic effect involves different molecular mechanisms. In apoA-II gene promoter, USF binds to the proximal element AB and the distal elements K and L, and most likely plays a crucial role in the synergistic interaction of the distal enhancer and the proximal promoter region. Mutation within the proximal E-box motif of element AB abolished the USF2a mediated transactivation. A dominant negative form of USF2a inhibits the activity of apoA-II promoter constructs containing the regulatory elements AB, K and L. The USF1/2a heterodimer, which is naturally expressed in the liver, is as efficient as the USF2a homodimer in the transactivation of apoA-II promoter/enhancer constructs. In addition, we show that HNF-4 and USF bind to their cognate sites of the apoA-ll enhancer cooperatively. In apoC-lll gene promoter, USF is able to synergize the effect of HNF-4 on apo C-Ill promoter activity, due to an action on its prosimal element B. However, the binding of USF and HNF-4 on element B is mutually exclusive and synergism between USF and HNF-4 is not suppressed by a mutation m the USF binding domain. Indeed, USF is able to stimulate the binding of HNF-4 on element B without binding to DNA. In conclusion, ubiquitously expressed USF plays a crucial role in synergy with HNF-4 in the constitutive expression of apoA-II and C-Ill genes in the liver. A potential role of USF and HNF-4 in the metabolic regulation of apoA-ll gene is under current investigation, and comparison with apoC-Ill may decipher the molecular mechanisms invovled in this metabolic regulation.

Poster sess ion: T h e m e t a b o l i c s y n d r o m e

ENDOTHELIAL DYSFUNCTION AND INSULIN RESISTANCE IN MEN WITH SMALL LDL PARTICLES

J. Vakkilainen, S. M~ikimatlila, M.-R. Taskinen, H. Yki-J~irvinen. Helsinki UniversiO, Central Hospital, Department of Medicine. Finland

Background: Both insulin resistance and small, dense LDL particles are risk factors for coronary artery disease. It is unknown whether the lipoprotein phenotype characterising insulin resistance and consisting of small LDL particles, mild hypertriglyceridemia, and low HDL cholesterol is associated with endothelial dysfunction in oivo.

Methods and Results: We determined in vivo endothelial function in 24 healthy men by measuring blood flow responses to intra-arterial infusions of acetylcholine (ACh, an endothelium-dependent vasodilator) and sodium nitroprusside (SNP, an endothelium-independent vasodilator). LDL peak particle size was measured with gradient gel electrophoresis. Men with small LDL particles (LDL diameter < 25.5 nm, n = 8), had a lower blood flow response to ACh than men with large LDL particles (LDL diameter > 25.5 nm, n = 16), mean blood flow 7.1+3.3 vs. 9.6±3.0 ml/dl.min, P = 0.003. The groups had comparable LDL cholesterol concentration (3.88 + 0.61 vs. 3.57±1.01 mmol/I) and body mass index. Men with small LDL had higher triglyceride level (1.82+0.58 us. 1.07+0.52 retool/l, P < 0.01) and lower insulin-stimulated whole-body glucose uptake (4.2+1.5 vs. 6.6+ 1.7 mg/kg body weight-minute, P < 0.01 ) than men with large LDL particles. Of these variables, only LDL particle size correlated significantly with ACh-induced

71st EAS Congress and Satellite Symposia