Inhibition of cholesteryl ester transfer protein activity in hamsters alters HDL lipid composition

Monday 10 October 1994: Poster Abstracts Animal models 30 min of ischemia, the non-ischemic and ischemic myocardial samples were taken from the circumflex region and LAD region, respectively. The myocardiai mitochondtia isolated from each sample were used for determination of respiratory function, which was assessed in terms of respiratory control index (RCI) and ADP/O ratio. When glutamate was used as substrate, ischemia significantly decreased RCI in control and simvastatin groups, but not in the pravastatin group. ADP/O ratio in all three groups was not altered by ischemia. When succinate was used as substrate, RCI and ADP/O ratio did not change during ischemia in control and pravastatin groups. However, RCI and ADP/O ratio in simvastatin group were significantly decreased by ischemia. These results suggest that simvastatin may enter the myocardial cell, prevent ubiquinone biosynthesis, and cause dysfunction of mitochondrial respiration. This may be one of the reasons for worsening of the myocardial stunning during reperfusion.

Influence of HMG-CoA reductase inhibitors on mito151 chondrial respiration in rat liver and heart &&&& Ichihara K, Dept. of Pharmacology, Hokkaido College of Pharmacy, 7-l Katsuraoka, Otaru 047-02, Japan

A 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor prevents mevalonic acid formation, and reduces cholesterol biosynthesis. Ubiquinone, an important member of the mitochondrial respiratory chain, is also synthesized from mevalonic acid. Inhibition of mevalonic acid formation may affect mitochondrial respiration because of reduction of ubiquinone synthesis. Therefore, we examined the effect of HMG-CoA reductase inhibitors, pravastatin and simvastatin, on mitochondrial respiration in rat liver and heart, particularly during ischemia. Vehicle, pravastatin (2 or 4 mg/kg per day) or simvastatin (1 or 2 mg/kg per day) was given orally for 3 weeks. Rats were anesthetized and ventilated with a respirator. Ischemia was induced by ligation of the aorta to cease the systemic circulation for 60 min. After isolation of hepatic and myocardial mitochondria, respiratory function was assessed in terms of QG3, QG4, respiratory control index (RCI) and ADP/O ratio. In liver mitochondria, ischemia reduced QC+, QG4, RCI, and ADP/O ratio in vehicletreated group. Both HMG-CoA reductase inhibitors appeared to enhance the worsening of respiratory function due to ischemia. In heart mitochondria, ischemia reduced QGg, QG4, RCI, and ADP/O ratio in vehicle-treated group. Simvastatin but not pravastatin further worsened respiratory function in ischemic heart. The effects of HMG-CoA reductase inhibitors on mitochondrial respiration (determined with succinate as a substrate) were more potent than those with glutamate. In conclusion, HMGCoA reductase inhibitors may enhance the impairment of mspiratory function due to ischemia in hepatic and myocardial mitochondria. Particularly, simvastatin may potentiate the worsening of mitochondrial respiration in ischemic hearts. LS2904, a synthetic HMG-CoA reductase inhibitor: 161 preliminary results on Watanabe atherosclerosis f&&&2, Guillemain .I*, Gerard P*, Pomies JP*, Centre de Recherche et Developpement Lipha, II5 avenue Lacassage, 69003 Lyon, France; VMT Pharmatox Histotox, rue J. Cugnot, BP 253,37702 St Pierre des Corps Cedex, France LS2904, a new synthetic HMG-CoA reductase inhibitor, has been shown to be hypocholesterolemic (62nd EAS Congress, Jerusalem, September 1993). The clinical objective of drug treatment remains the regression of atherosclerosis. The Watanabe Heritable Hyperlipidemic Rabbit (WHHL) is an appropriate model: atberosclerotic lesions in WHHL closely resemble those in humans. We have examined the activity of LS2904 in WHHL. A preliminary study was conducted in 3-month-old WHHL rabbits (breeding center: Centre de Production R. Jeanrot, Olive&

5

France). Animals received placebo (n = 4) or LS2904 (n = 5) at a dose of 25 mg/kg by gavage once a day for 4 months. Serum lipid levels were measured before and after 2 and 4 months of treatment and in aorta at the end of the study. At 7 months, rabbits were killed and aortas were analyzed by morphometry. LS2904 decreased serum total cholesterol by 26% and Tg by 36% after 4 months of treatment. A potent effect was also demonstrated on aorta cholesterol: lOrmol/g in the control group, 3pmoVg in the LS2904 group. Morphometric analysis of the extent of the plaque formation (% of total surface covered with fatty streaks) showed a protective effect of LS2904 on atherosclerosis (38.5% in controls, 16% after LS2904). These preliminary results in WHHL rabbits suggest that the hypocholesterolemic drug may have potential against the development of atherosclerosis. Susceptibility to early atherosclerosis in transgenic 171 mice expressin human apolipoprotein A-II Marzal-Casacuberta Aa,%, Kim H-Sa, Shen Ja, Gonzalez-Sastm Fbc Blanco-Vbaca F sc,d , Chan La, aBaylor Coil. of Medicine, Houston, TX; Univ. Autonoma de Barcelona, Barcelona, Spain; CServei de Bioquimica, Hospital de la Santa Creu i Sant Pau. C/ Antoni M. Claret 167, 08025 Barcelona, Spain; dlnst. de Recerca de I’Hospital de la Santa Creu i Sant Pau, Barcelona, Spain

Apolipoprotein (apo) A-II is the second most abundant protein in HDL. Its function and relation to atherosclerosis remain unclear. We have developed three lines of human apo A-II transgenic mice by microinjection of a 3 kb fragment of genomic DNA that contained the natural human apo A-II gene into C57BIf6 embryos. These lines were called 11.1, 25.3 and 21.5. The 11.1 line had the highest (47 + 2 mg/dl) and the 25.3 line the lowest (19.3 + 1 mg/dl) plasma level of human apo A-II. These two lines were selected to study atherosclerosis susceptibility. The development of atherosclerotic lesions in transgenics (T) and control (C) mice after 3 months of regular chow diet or high-fat highcholesterol diet (HFHCD) was studied by Oil Red 0 staining of the aortic sinus wall. There were essentially no atherosclerotic lesions in T or C mice after 3 months of regular chow diet. In contrast, 3 months of HFHCD feeding caused the appearance of atherosclerotic lesions in both C and T animals. There was no significant difference (U of Mann Whitney, P < 0.05) in the extent of the atherosclerotic lesions (measured in pm2) following HFHCD between C and T animals, or between either transgenic line and the C group. The potential role of apo A-II in atherogenesis requires further studies. Inhibition of cholesteryl ester transfer protein activity 181 in hamsters alters HDL lipid composition Gavnor BJ, Sand TM, Clark RW, Aiello RJ, Bamberger MJ, Moberly JB, Pfizer Central Research, Groton, CT 06340, USA We investigated the role of CETP in hamsters by using a monoclonal antibody (Mab) that inhibited CETP activity. Mabs prepared against artially purified human CETP were screened for inhibition of [‘:Hlcholesteryl oleate (CE) transfer from LDL to HDL in the presence of human plasma bottom fraction (d > 1.21 g/ml). Mab lC4 inhibited CE transfer in both human bottom fraction (IC5o = -4pg/ml) and in whole plasma from male Golden Syrian hamsters (IC5o = -3.O,&ml). Purified Mab lC4 was injected into chow- and cholesterol-fed hamsters, and blood was collected for analysis of plasma CETP activity and HDL lipid composition. Plasma CETP activity was inhibited 70-808 up to 24 h following injection of 5OOpg lC4 (-3.7 mg/kg). The amount of antibody required for 50% inhibition at 24 h post-injection was 2OOyg (- 1.5 mg/kg). Inhibition of CETP in vivo increased hamster HDL-cholesterol by 33% (P c O.OOOl),increased HDL-CE by 3 1% (P < O.OOOl),and decreased HDL-triglyceride by 42% (P <

Atherosclerosis X, Montreal, October 1994

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Monday 10 October 1994: Poster Abstracts Animal models

O.OOOl),as determined following HDL isolation by ultracentrifugation (n = 36). An increase in HDL cholesterol and a mdistribution of cholesterol to a larger HDL particle was also observed following FPLC gel filtration of plasma lipoproteins. These rcsubs demonstrate that inhibition of CETP activity in hamsters alters HDL lipid composition and particle size and further demonstrate the utility of hamsters as a model for CETP inhibition. Effect of E5324, a potent ACAT inhibitor, on athero191 genesis in WHHL rabbits a, Ohtsuka I, Kogushi M, Saeki T, Kobayashi H, Yamada T, Hiyoshi H, Takada M, Saitou I, Tsukuba Res. Labs, Eisai Co. Ltd, Tokodai - 5, Tsukuba, Ibaraki 300 - 26, Japan E5324, N-butyl-N’-[2-[3-(5-ethyl-4-phenyl-lH-imidazol-l-yl)propoxy]-6-methylphenyllurea, is a potent and orally absorbable ACAT (acyl-CoA:cholesterol acyltransferase) inhibitor. E.5324 inhibited ACAT activity in microsomes or homogenate prepared from various tissues (intestine, liver and aorta) of several species including human, with IC5o values of 30-190 nmol/I. In this study, we examined the anti-atherosclerotic and hypolipidemic effect of E5324 in WHHL rabbits. Male WHHL rabbits were fed a standard rabbit chow diet concurrently with E5324 (0.02% and 0.1% in the diet) or probucol (1% in the diet) for 16 weeks. Probucol reduced plasma total cholesterol and HDLcholesterol compared to the control group. However, E5324 did not show a hypocholesterolemic effect in either dose. On the other hand, E5324 at a dose of 0.1% and probucol significantly reduced atherosclerotic plaque formation, cholesterol content and ACAT activity in the aortic arch and the thoracic aorta compared to the control group. These results indicate that E5324 may have a direct effect on the arterial wall in WHHL rabbits.

Effect of simvastatin on plasma cholesterol, on fecal sterol balance and on whole body cholesterol synthesis as measured by incorporation of D20 in hamsters Lenesfeld, Miiller St, Gesterhelt G, Bubendorf A, Grosjean M, 1

Hoffmann-La Roche, Preclinical Phmma Research, Grenzacherstrasse 124, CH-4002 Basel, Switzerland

We studied the relationship between the effect of an HMGCoAreductase inhibitor on plasma cholesterol and its effect on whole body cholesterol synthesis. Male hamsters on a 40 Cal% fat diet (9 g/animal per day containing 0.6 mg cholesterol/day and 3.2 mg sitosterobday) received 0, 10 and 30ymoblcg per day simvastatin in the diet for 7 days and were intubated with 3 ml D20 on days 3 and 5. Plasma cholesterol was measured calorimetrically. To determine steroid balance, we measured dietary and fecal neutral sterols (GLC) and fecal bile salts (3a-steroid-dehydrogenase). In order to calculate newly formed cholesterol, we measured (i) the 4591458 mass ratio of the TMS cholesterol ether (corrected by controls receiving H20) by GC/MS, (ii) cholesterol concentration by GLC, and (iii) D enrichment of body water by IR-ATR spectroscopy. Simvastatin at doses of 10 and 30pmol/kg per day, respectively, reduced plasma cholesterol from 100 + 9SD to 87 + 1 and 71 + 8% and increased total sterol excretion from 3.6 It 0.2 to 4.1 kO.2 and 5.OkO.8 mg/animal per day. At 30pmobkg per day, simvastatin increased cholesterol synthesis (D20 incorporation) from 3.5 + 1.O (control level) to 3.9 * 1.Omg/animal per day. In hamsters, simvastatin lowered plasma cholesterol but, surprisingly, did not reduce overall cholesterol synthesis, as demonstrated by two independent methods. The reduction of plasma cholesterol may therefore be related to a mechanism other than reduction of overall cholesterol synthesis.

ELI &ZLL

A major familial hypercholesterolemia subphenotype (FHC-r) in swine is inherited as an autosomal recessive trait and its gene is not linked to the apo B locus Dentine MR, Hasler-Rapacz JO, Rapacz JM Jr, Depts.

of Genetics, Meat & Animal Science and Dairy Science, 666 Animal Sciences Building, Univ. of Wisconsin, WI 53706, USA

The objective of this study was to determine in swine the mode of inheritance of a quantitative hypercholesterolemia variant dissected from a complex familial hypercholesterolemia (FIX) closely resembling human familial combined hyperlipidemia and to test whether the gene locus for this disorder is linked with the apo B locus. Genetic analysis of segregation data on cholesterol variations in plasma of 418 progenies from 58 designed matings has indicated that the isolated dyslipidemia represents a qualitative FHC subphenotype which showed a monogenic autosomal recessive mode of inheritance, designated FHC-r. Data were tested for goodness-of-fit to three genetic models (additive, dominant, recessive) and the recessive model was a significantly better fit (R* = 0.91). Data from three mating types (FIX-r x FHC-r; N x N; FHCr x N) resulted in the following means: 222.5 f 25, 98.8 * 12.5 and 103.4 f 13.3 mg/dl for the three genotypes r/r, N/N and N/r, respectively, determined by two alleles designated r for the recessive hypercholesterolemia and N for the dominant normolipidemic phenotype. Segregation data on the quantitative cholesterol phenotypes, specified by r and N alleles at the FIX-r locus, and the Lpb5 allotypic marker determined by the apo B allelic gene were used for linkage analysis. A very low lod score (Z= -21.8) obtained at the recombination fraction (0) of 0.05 indicated that the apo B and FHC-r loci are not closely linked. Pathological findings after intra-arterial stenting in Watanabe heritable hyperlipidemic (WHHL) and normal rabbits Takagi M, Ueda M, Kasayuki N, Naruko T, Takeuchi K, Takeda T, Matsuo R, Nakamura K, Shiomi M, Becker AE, Is? Dept. of

1121

Medicine, Osaka City Univ. Med. Abeno-ku, Osaka, Japan, 545

Sch.,

I-5-7

Asahi-machi,

The WHHL rabbit produces atherosclerotic lesions very similar to those in humans and may serve as an animal model for interventional procedures. We used the WHHL rabbit to study the atherosclerotic wall response after stenting. Normal rabbits served as controls, The self-expandable metallic stems (outer diameter 12 mm, length 13 mm) were implanted transluminally into the thoracic aorta. Five rabbits (3 WHHL, 2 controls) were sacrificed 1, 2, 3,4, and 8 weeks after implantation. The site of stenting was examined histologically. The maximal thickness of the neo-intima at the site of stenting was measured. The cellular characteristics were studied electron microscopically and immunocytochemitally, using monoclonal antibodies against smooth muscle actin (HHF35, CGA7), macrophages (RAM1 1), and vimentin. Neo-intimal thickness was significantly higher in WHHL rabbits at 4 and at 8 weeks (P
Atherosclerosis X, Montreal, October 1994