Homozygous Watanabe Heritable Hyperlipidemic Rabbits Research Articles

Effects of NO-1886, a Lipoprotein Lipase Promoting Agent, on Homozygous and Heterozygous Watanabe Heritable Hyperlipidaemic Rabbits

The novel compound NO-1886 ([4-(4-bromo-2-cyano-phenylcarbamoyl)-benzyl]-phosphonic acid diethyl ester, CAS 133208-93-2) is a lipoprotein lipase (LPL) activator, and long term administration of NO-1886 protects against the development of experimental atherosclerosis in rats and rabbits. In the present experiments, the effects of this compound were examined in Watanabe heritable hyperlipidaemic (WHHL) rabbits, an animal model for familial hypercholesterolemia lacking low density lipoprotein (LDL) receptors. NO-1886 increased postheparin plasma LPL activity, resulting in a reduction of plasma triglycerides with concomitant elevation of HDL cholesterol in heterozygous WHHL rabbits. However, the compound did not cause any changes in plasma lipids and postheparin plasma LPL activity in homozygous WHHL rabbits. The different responses suggest that the effects of NO-1886 may be either mediated by LDL receptors, or that persistent exposure to extreme hypercholesterolemia might affect the cellular response to this particular compound in homozygous WHHL rabbits.

HMG-CoA reductase inhibitor augments the serum total cholesterol-lowering effect of human adipose tissue-derived multilineage progenitor cells in hyperlipidemic homozygous Watanabe rabbits

Familial hypercholesterolemia (FH) is an autosomal codominant disease characterized by high concentrations of proatherogenic lipoproteins secondary to deficiency in low-density lipoprotein (LDL) receptor. We reported recently the use of in situ stem cell therapy of human adipose tissue-derived multilineage progenitor cells (hADMPCs) in lowering serum total cholesterol in the homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model of homozygous FH. Here we demonstrate that pravastatin, an HMG-CoA reductase inhibitor, augmented the cholesterol-lowering effect of transplanted hADMPCs and enhanced LDL clearance in homozygous WHHL rabbit. The results suggest the potential beneficial effects of in situ stem cell therapy in concert with appropriately selected pharmaceutical agents, in regenerative medicine.

L-Arginine plus atorvastatin for prevention of atheroma formation in genetically hypercholesterolaemic rabbits

We investigated the combined effect of dietary supplementation with L-arginine, which is the precursor of NO, and pharmacological treatment with atorvastatin, which is a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor, on the development of atherosclerosis in homozygous Watanabe heritable hyperlipidaemic rabbits. Rabbits were fed either standard rabbit chow (group C; n 9) as control, a 1.5 % L-arginine diet (group A; n 9), standard rabbit chow plus atorvastatin (2.5 mg/kg per d) in drinking water (group S; n 8), or standard rabbit chow plus a 1.5 % L-arginine diet with atorvastatin (group SA; n 8). Blood was sampled at 2-week intervals. After 8 weeks (T8), the aorta was harvested for topographic and histological analysis. Only the SA group showed decreases in total area of lesions (21 %) and the area of abdominal lesions (44 %) compared with the control group (P = 0.019). Furthermore, plaques in the SA group were smaller and less thick than those observed in the S group. Unexpectedly, plasma nitrite + nitrate levels were not modified under either the L-arginine diet alone or under L-arginine plus atorvastatin. The present study is the first to demonstrate that diet supplementation with L-arginine associated with a statin (atorvastatin) is more efficient in reducing lesion size than treatment with L-arginine or a statin alone. This is a relatively novel therapeutic approach associating a macronutrient and a drug.

Pravastatin Inhibits Expression of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 (LOX-1) in Watanabe Heritable Hyperlipidemic Rabbits

LOX-1, a receptor for oxidized low-density lipoprotein (OxLDL), seems to play a critical role in foam cell formation of macrophages (Mphis) and smooth muscle cells (SMC). Inhibition of LOX-1 expression reduces foam cell formation and might influence lipid core formation in atherosclerotic lesions. Because statins are able to downregulate LOX-1 expression in vitro, we examined if pravastatin can be used to reduce LOX-1 expression and lipid core formation in lesions of Watanabe heritable hyperlipidemic (WHHL) rabbits. Pravastatin downregulated LOX-1 expression in cultured human Mphis and in cultured human aortic SMCs. Homozygous WHHL rabbits were treated with 50 mg kg(-1) d(-1) pravastatin for 32 weeks. Immunohistochemical studies revealed that LOX-1 was expressed in intimal Mphis and SMCs of atherosclerotic lesions. The pravastatin-treated rabbits showed, compared with untreated rabbits, a significantly reduced LOX-1 protein and mRNA expression in the aortic arch. Lipid labeling of this aorta region also demonstrated a strong reduction of the ratio of lipid core area/total lesion area in pravastatin-treated rabbits. The in vivo inhibition of LOX-1 expression by pravastatin demonstrated here represents a new pleiotropic effect of pravastatin. This in vivo inhibition of LOX-1 might be one mechanism for the lipid core reducing effect of pravastatin in atherogenesis.

Effects of a novel antihyperlipidemic agent, S-2E, on the blood lipid abnormalities in homozygous WHHL rabbits

To improve mixed hyperlipidemia in the low-density lipoprotein (LDL) receptor—deficient state, suppression of very-low-density lipoprotein (VLDL) particle production may be an important approach. We previously reported that S-2E, (+)-(S)- p-[1-( p-tert-butylphenyl)-2-oxo-4-pyrrolidinyl] methoxybenzoic acid, suppressed VLDL particle production by inhibiting the biosynthesis of both sterol and fatty acids in the liver. We therefore examined whether S-2E lowered the blood cholesterol and triglyceride (TG) levels simultaneously in homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, which correspond to human familial hypercholesterolemia. S-2E given orally at doses of 30 to 300 mg/kg significantly lowered serum total cholesterol (TC) levels at 1 week as well as TG at 2 weeks, and the lowering of TC and TG levels by S-2E reached a maximum at 3 to 4 weeks. In contrast, oral administration of pravastatin at doses of 10 to 100 mg/kg resulted in a significant suppression of TC levels (100 mg/kg) but not TG levels. Further analysis of the TC content in fractionated serum of control and S-2E—treated animals showed that suppression of TC level by S-2E is attributable to a decrease in the proportions of VLDL, intermediate-density lipoprotein (IDL), and LDL. It is, therefore, reasonable to assume that S-2E may be useful to improve the blood lipid abnormalities in the LDL receptor—deficient state.

Long-term estradiol treatment improves VIP-mediated vasodilation in atherosclerotic proximal coronary arteries

The aim of the present study was to evaluate the impact of long-term estrogen replacement therapy (ERT) on the vasodilatory effect of the two peptides vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) in atherosclerotic coronary and cerebral arteries. Female ovariectomized homozygous Watanabe heritable hyperlipidemic rabbits were randomized to 16 weeks treatment with 17β-estradiol or placebo. The diet was semisynthetic, thereby avoiding the influence of phytoestrogens. Artery ring segments were mounted for isometric tension recordings in myographs. Following precontraction, the dose–response relationships for VIP and PACAP were evaluated. Treatment with 17β-estradiol significantly improved the maximum VIP-mediated vasodilation ( E max, percentage of precontraction) in proximal coronary arteries (45.8±9.6% vs. 24.1±3.7%, p<0.05). In the same artery segment, 17β-estradiol induced a significant decrease in the relative ratio between the repeated contractile response to potassium 30 and 120 mM (100±7% vs. 132±11%, p<0.05). For distal coronary arteries, there was a tendency to similar changes, but no statistical differences for the potassium or VIP responses in cerebral or distal coronary arteries were found between the two groups. 17β-estradiol induced no changes in the PACAP-mediated vasodilation. These results suggest that long-term treatment with 17β-estradiol improves the VIP-mediated but not the PACAP-mediated vasodilation in atherosclerotic proximal coronary arteries.

Lipid-lowering effects of TAK-475, a squalene synthase inhibitor, in animal models of familial hypercholesterolemia

The lipid-lowering effects of 1-{2-[(3 R,5 S)-1-(3-acetoxy-2,2-dimethylpropyl)-7-chloro-1,2,3,5-tetrahydro-2-oxo-5-(2,3-dimethoxyphenyl)-4,1-benzoxazepine-3-yl] acetyl} piperidin-4-acetic acid (TAK-475), a novel squalene synthase inhibitor, were examined in two models of familial hypercholesterolemia, low-density lipoprotein (LDL) receptor knockout mice and Watanabe heritable hyperlipidemic (WHHL) rabbits. Two weeks of treatment with TAK-475 in a diet admixture (0.02% and 0.07%; approximately 30 and 110 mg/kg/day, respectively) significantly lowered plasma non-high-density lipoprotein (HDL) cholesterol levels by 19% and 41%, respectively, in homozygous LDL receptor knockout mice. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, simvastatin and atorvastatin (in 0.02% and 0.07% admixtures), also reduced plasma levels of non-HDL cholesterol. In homozygous WHHL rabbits, 4 weeks of treatment with TAK-475 (0.27%; approximately 100 mg/kg/day) lowered plasma total cholesterol, triglyceride and phospholipid levels by 17%, 52% and 26%, respectively. In Triton WR-1339-treated rabbits, TAK-475 inhibited to the same extent the rate of secretion from the liver of the cholesterol, triglyceride and phospholipid components of very-low-density lipoprotein (VLDL). These results suggest that the lipid-lowering effects of TAK-475 in WHHL rabbits are based partially on the inhibition of secretion of VLDL from the liver. TAK-475 had no effect on plasma aspartate aminotransferase and alanine aminotransferase activities. Thus, the squalene synthase inhibitor TAK-475 revealed lipid-lowering effects in both LDL receptor knockout mice and WHHL rabbits.

Squalene synthase inhibitors reduce plasma triglyceride through a low-density lipoprotein receptor-independent mechanism

Inhibitors of squalene synthase are considered to be candidate drugs to reduce both plasma cholesterol and triglyceride. However, little is known about the mechanism of squalene synthase inhibitor-specific effect on plasma triglyceride. In this study, we confirmed the triglyceride-lowering effect of ER-27856, a potent squalene synthase inhibitor prodrug, in rhesus monkeys. To determine the role of low-density lipoprotein (LDL) receptor in the triglyceride-lowering effect of squalene synthase inhibitors, we intravenously administered ER-28448, the active form of ER-27856, to Watanabe heritable hyperlipidemic (WHHL) rabbits for 4 days. In heterozygotes, ER-28448 reduced plasma cholesterol and triglyceride by 52% and 37%, respectively. In homozygous rabbits, in contrast, ER-28448 lowered plasma triglyceride by 40% but did not lower plasma cholesterol. Orally administered ER-27856 reduced plasma triglyceride in homozygous animals but atorvastatin and bezafibrate did not. In hepatocytes isolated from homozygous WHHL rabbits, squalene synthase inhibitors but not atorvastatin reduced triglyceride biosynthesis. These data demonstrate that squalene synthase inhibitors reduced plasma triglyceride through an LDL receptor-independent mechanism, which was distinct from that of the triglyceride-lowering action of atorvastatin or bezafibrate. The reduction of hepatic triglyceride biosynthesis may play an important role in the hypotrigyceridemic action of squalene synthase inhibitors.

Age-associated decrease in plasma cholesterol and changes in cholesterol metabolism in homozygous watanabe heritable hyperlipidemic rabbits

We examined the cholesterol metabolism of homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model deficient in low-density lipoprotein (LDL) receptors, to clarify the mechanism of the age-associated decrease of plasma total cholesterol, one of the properties of WHHL rabbits. The rabbits were examined at several ages: after weaning at 3 months, at sexual maturation at 6 months, at 12 months, and at 24 months, equivalent to about 35 years of age in humans. Plasma total cholesterol, triglyceride, and phospholipid levels decreased with aging by about 45%. These reductions were mainly dependent on a decrease in the LDL fraction. In the liver microsomal fraction, although there were no age-related changes in the cholesterol concentration and cholesterol 7alpha-hydroxylase (C7H) activity, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity increased and acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity decreased with aging. The lipolytic activity varied with aging. The secretion rate of very-low-density lipoprotein (VLDL) cholesterol as determined by injection of Triton WR-1339 decreased significantly with aging, while the catabolic rate of VLDL cholesterol was about 2-fold higher in the oldest group versus the young groups. From these results, we conclude that the age-associated decrease in plasma cholesterol in WHHL rabbits is related not only to a decrease in the secretion rate of VLDL cholesterol but also to an increase in the catabolic rate.

Measurement of expired carbon dioxide to assess the metabolism of remnant lipoproteins.

The cholesteryl esters transported from the intestine in chylomicrons are delivered to the liver. Hepatocytes take up chylomicron remnants by receptor-mediated endocytosis and the cholesteryl esters are subsequently degraded. In this study we measured the appearance in breath of labeled carbon dioxide after injection of chylomicron-like emulsions labeled with radioactive cholesteryl [1-14C]oleate. Measurements by the breath test provide an integrated assessment of capacity for clearance and subsequent metabolism of the remnants of the triglyceride-rich lipoproteins. In normal rats, mice, and rabbits injected with the radioactive emulsions, label appeared in the breath after a delay of approximately 30 min, appreciably slower than the appearance of label after injection of emulsions labeled with [14C]triolein or of [14C]oleic acid complexed with albumin. To test for the ability of the procedure to detect defects in remnant clearance, labeled emulsions were injected into diabetic rats, apoE-deficient mice, low density lipoprotein receptor (LDLr)-deficient mice, and Watanabe heritable hyperlipidemic (WHHL) rabbits with defective low density lipoprotein receptors. In rats made diabetic by treatment with streptozotocin the appearance of 14CO2 in breath was slower than in normal control rats. This finding was consistent with previous evidence from our laboratory that remnant clearance is defective in diabetic rats. In LDLr-deficient mice the appearance of 14CO2 was slower when compared with control mice and in apoE-deficient mice the appearance of 14CO2 was extremely small. In homozygous WHHL rabbits, the appearance of 14CO2 in breath was much slower than in normal control rabbits, while in heterozygous WHHL rabbits an intermediate level of appearance was found, consistent with our previous findings of defective remnant clearance in WHHL rabbits. Emulsions with cholesterol omitted, previously found to be cleared from plasma much slower than chylomicron-like emulsions, had much slower appearance of label in breath. The breath test as described is consistent with predicted metabolism of chylomicron remnants and therefore provides a useful means of assessment of remnant catabolism in the intact animal.

High dose of fluvastatin sodium (XU62-320), a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, lowers plasma cholesterol levels in homozygous Watanabe-heritable hyperlipidemic rabbits

The effects of fluvastatin sodium (XU62-320), a new type of inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on plasma cholesterol and triacylglycerol levels were investigated using homozygous Watanabe-heritable hyperlipidemic (WHHL) rabbit, an LDL-receptor-deficient animal which expresses a hepatic LDL receptor activity less than 5% that of control rabbits. Plasma levels of total, VLDL- and LDL-cholesterol were decreased profoundly after oral administration of fluvastatin at a dose of 50 mg/kg per day for 4 weeks. Plasma triacylglycerol levels were not affected by fluvastatin. Hepatic HMG-CoA reductase activity increased by 3-fold and hepatic LDL receptor activity increased by only 3.7-fold, as calculated by Scatchard plot analysis, with fluvastatin administration for 4 weeks, and the hepatic mRNA level for the rabbit LDL receptor was increased by 3-fold. Combined administration of fluvastatin (50 mg/kg per day) and cholestyramine, a bile acid sequestrant resin, at a level of 2% of the diet for 4 weeks more profoundly decreased plasma total, VLDL- and LDL-cholesterol levels with induction of hepatic cholesterol 7α-hydroxylase and no further induction of the hepatic LDL receptor. Plasma triacylglycerol levels were increased by the combination treatment. These results suggest that high dose of fluvastatin sodium is effective in lowering plasma cholesterol levels in homozygous WHHL rabbits through the shared mechanisms involving decrease in production and secretion of cholesterol from the liver and the induction of hepatic LDL receptor. Additional effect of cholestyramine on decrease in plasma cholesterol levels seems to be due to the further decrease in hepatic cholesterol secretion by up-regulation of hepatic cholesterol 7α-hydroxylase.

Development and progression of atherosclerosis in aorta from heterozygous and homozygous WHHL rabbits. Effects of simvastatin treatment.

This study was conducted to define progression of atherosclerosis in both homozygous and heterozygous Watanabe heritable hyperlipidemic (WHHL) rabbits and to investigate the ability of the HMG CoA reductase inhibitor simvastatin to attenuate progression of the disease. We examined contractile responses to phenylephrine and endothelium-dependent relaxation in response to carbachol in thoracic aorta at 3, 6, 9, and 12 months in control New Zealand White (NZW) rabbits, homozygous WHHL rabbits, and heterozygous WHHL rabbits. Homozygous and heterozygous rabbits were treated with simvastatin (10 mg/kg per day) from 3 to 6 months and from 9 to 12 months of age. Simvastatin significantly reduced serum cholesterol levels in young heterozygotes, with a nonsignificant trend toward a reduction in older heterozygotes. In homozygotes, no significant fall was observed. Contractile function declined progressively with age in all groups--most in homozygotes and least in NZW rabbits. Relaxation was unaffected by age in NZW rabbits; relaxation declined in the heterozygotes and declined to a greater extent in homozygotes. Simvastatin retarded the loss of function in the young heterozygotes. Similar trends were observed in young homozygotes and older heterozygotes, with no effect in older homozygotes. Histological studies revealed the progressive development of early atherosclerosis in heterozygotes, and more advanced atherosclerosis was observed in homozygotes. Simvastatin did not inhibit development of atheroma. A correlation was observed between vascular function and structure. However, functional changes preceded the development of atheroma. In addition, we have demonstrated that simvastatin can help to reduce the loss of vascular function associated with the progression of atherosclerosis in the heterozygous WHHL rabbit.

HMG-CoA reductase inhibitor and cholestyramine induce hepatic LDL receptor and are effective for lowering plasma cholesterol in homozygous Watanabe heritable hyperlipidemic rabbits

Male Fisher 344 rats were fed diets containing either 20% corn oil, 20% coconut oil or 18% cocomnut oil plus 2% corn oil for 3 wk. A single dose of [3H]aflatoxin B1 ([3H]AFB1) was administered ip and the biliary excretion of afflatoxin metabolites and the binding of 3H to nucleic acids were studied. In other experiments the in vitro metabolism of AFB1 by liver postmitochondrol supernatants prepared from rats fed the different sources of dietry fat was determined. The major nonextractable aqueous metabolite of AFB1 was the aflatoxin B1-glutathione conjugate (AFB1-GSH). Variation in the source of dietry fat did not affect production of the conjugate, nor was in vivo binding of AFB1 to nucleic acids affected. Aflatoxin P1 (AFP1)—mostly conjugated—and aflatoxin M1 (AFM1) were also identified in the bile, and the quantities of these metabolites produced were unaffected by the dietary treatments. The metabolites recovered in the in vitro study included aflatoxins Q1, P1 and M1. The corn oil diet produced a higher microsomal cytochrome P-450 level than the coconut oil diet. Associated with the higher level of cytochrome P-450 was increased in vitro coversion of AFB1 and AFM1, but not ot AFP1. The in vitro production of aqueous metabolites and the covalent binding of metabolites to protein was unaffected by the dietary treatments. The results of the in vivo studies suggest that the formation of the putative carcinogenic metabolite, AFB1-epoxide, which undergoes detoxification through glutathione conjugation, is not affected by the type of dietary fat. These observations further suggest that dietary fat, in particular unsaturated fat, affects AFB1 carcinogenisis through a mechanism other than by alteration of the metabolic activaqtion of AFB1.

Pravastatin sodium, a competitive inhibitor of hepatic 3-hydroxy-3-methylglutaryl coenzyme a reductase, decreases the cholesterol content of newly secreted very-low-density lipoprotein in Watanabe heritable hyperlipidemic rabbits

We examined the secretion of very-low-density lipoprotein (VLDL) when hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme of cholesterol biosynthesis, was inhibited. To inhibit HMG-CoA reductase in liver, pravastatin sodium, a competitive inhibitor of HMG-CoA reductase, was administered to homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, a low-density lipoprotein receptor-deficient animal model, at a dosage of 50 mg/kg per day for 5 weeks. Although triglyceride levels were not changed, total cholesterol levels of sera and each atherogenic lipoprotein were decreased by approximately 30%. As a result, the percentage of cholesterol concentration in newly secreted VLDL was significantly decreased by 24%. The VLDL secretion rate was determined by intravenous injection of Trion WR-1339. The VLDL secretion rate was significantly decreased by 23% using cholesterol as an index, but it did not change using triglyceride, phospholipid, or protein as an index. It is concluded that one of the mechanisms of serum total cholesterol decrease due to reduction of the putative cholesterol pool of the liver in homozygous WHHL rabbits is caused by a decrease of cholesterol content in newly secreted VLDL particles.

In vivo kinetics of lipoprotein(a) in homozygous Watanabe heritable hyperlipidaemic rabbits

In vivo kinetics of lipoprotein(a) [Lp(a)] were investigated in homozygous Watanabe heritable hyperlipidaemic (WHHL) rabbits (an animal model of familial hypercholesterolemia (FH)), and in normolipidemic Japanese White rabbits (controls). 125I-labelled Lp(a) and 131I-labelled LDL were simultaneously injected intravenously. Blood samples were then taken periodically. Kinetic parameters were calculated from the plasma radioactivity decay curves. The fractional catabolic rates (FCRs) of both Lp(a) and LDL (1.355 +/- 0.189 pools per day and 1.278 +/- 0.397 pools per day, respectively) in the WHHL rabbits were significantly (P < 0.005) smaller than those in the control rabbits (2.008 +/- 0.083 pools per day and 2.855 +/- 0.759 pools per day, respectively). In WHHL rabbits, the FCRs of Lp(a) and LDL were similar. However, in control rabbits, the FCR of Lp(a) was significantly (P < 0.01) smaller than that of LDL. In WHHL rabbit organs, the mean ratio of 125I-Lp(a): 131I-LDL, 48 h after injection, normalized to the corresponding isotope ratio in plasma, were 1.525, 1.020, 1.819 and 1.967, in liver, kidney, spleen and bile, respectively. These values were significantly higher than the corresponding values in control rabbits (0.590, 0.677, 0.862 and 0.766, respectively). Our data strongly suggest that Lp(a) clearance is not entirely dependent upon LDL receptors and may be mediated by some other mechanisms.

Effects of Pravastatin Sodium Alone and in Combination with Cholestyramine on Hepatic, Intestinal and Adrenal Low Density Lipoprotein Receptors in Homozygous Watanabe Heritable Hyperlipidemic Rabbits

Pravastatin sodium (pravastatin), a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, was administered alone (50 mg/kg) or in combination with cholestyramine, a bile acid sequestrant resin, at the level of 2% in the diet to homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits for 4 weeks. The low density lipoprotein (LDL)-cholesterol levels were reduced by 29% and 56% with pravastatin alone and the combination treatment, respectively. Hepatic LDL receptor activity was increased by 11.2- and 13.9-fold with pravastatin alone and the combination treatment, respectively. The LDL receptor activity in the untreated homozygous WHHL rabbits was only 2.5% of that in the normal rabbits. mRNA for the LDL receptor in the liver was also increased by 2.1- and 3.4-fold with pravastatin alone and the combination treatment, respectively. On the other hand, mRNA for the LDL receptor in the adrenal gland was not affected by pravastatin and the combination treatment, whereas the mRNA in the intestine was increased in both groups. These results suggest the following: 1) the induction of hepatic LDL receptor activity by the treatment of pravastatin alone or in combination with cholestyramine is the main cause of the reduction of serum cholesterol levels by these treatments even in LDL receptor-deficient animals. 2) The induction of the mRNA for the LDL receptor in the liver and intestine, but not that in the adrenal gland, might be a reflection of the tissue-selective inhibition of cholesterol synthesis by pravastatin.

Increased removal of beta-very low density lipoproteins after ethinyl estradiol is associated with increased mRNA levels for hepatic lipase, lipoprotein lipase, and the low density lipoprotein receptor in Watanabe heritable hyperlipidemic rabbits.

The mechanism by which ethinyl estradiol (EE) decreases the concentration of lipids in the d less than 1.019 g/ml fraction (beta-very low density lipoprotein [beta-VLDL]) of homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits was studied. Treatment with EE increased the activity of hepatic lipase (HL) twofold to threefold in postheparin plasma and in liver biopsies. Postheparin plasma and adipose tissue lipoprotein lipase (LPL) activities were also increased twofold to fourfold after EE. The effects of EE on HL and LPL activities were associated with a threefold to sixfold elevation in liver HL mRNA and a fourfold elevation in adipose tissue LPL mRNA steady-state levels, pointing to an effect of EE on HL and LPL gene transcription. EE also increased liver low density lipoprotein (LDL) receptor mRNA levels threefold to fivefold. These results suggest a concerted action of LPL, HL, and the LDL receptor in the removal of beta-VLDL in homozygous WHHL rabbits with a defective LDL receptor. In addition, the content of apolipoprotein E in the d less than 1.019 g/ml fraction changed toward normal after EE. Because the remaining particles contained apolipoprotein B-100 almost exclusively, it is likely that apolipoprotein E-containing beta-VLDLs are preferentially removed. This may be the result of the increased activity of LPL and HL influencing the conformation of apolipoprotein E on the beta-VLDL particle in such a way that it is directly removed from the circulation, possibly by the induced LDL receptor.

Putative mechanisms of the impairment of endothelium-dependent relaxation of the aorta with atheromatous plaque in heritable hyperlipidemic rabbits.

Attenuation of acetylcholine-induced endothelium-dependent relaxation of thoracic aortas excised from Watanabe heritable hyperlipidemic (WHHL) rabbits linearly correlated with the percent area coated with atheromatous plaque. To elucidate mechanisms related to this reduced endothelium-dependent relaxation in the presence of atherosclerosis, the acetylcholine-induced release of endothelium-derived relaxing factor (EDRF) was assessed functionally as a percent relaxation of the precontracted detector strips obtained from the tunica media beneath the intact intima or the atheromatous plaque in the same aortic ring preparation. Relaxations of the normal detectors to effluents containing EDRF of thoracic aortas during stimulation by acetylcholine (3 x 10(-6) M) in heterozygous and homozygous WHHL rabbits were 73 +/- 5% and 59 +/- 9% (p less than 0.01) of the phenylephrine-induced precontraction, respectively. Relaxations of the atherosclerotic detectors to effluents (EDRF) through the aortas during stimulation by acetylcholine (3 x 10(-6) M) in heterozygous and homozygous WHHL rabbits were 16 +/- 4% and 14 +/- 5%, respectively--values significantly smaller than those seen in the normal detectors. When superoxide dismutase was added to the perfusate of the donors from homozygous and heterozygous WHHL rabbits, atherosclerotic detectors relaxed by effluents stimulated by acetylcholine to 73% and 65% (p less than 0.01 versus before the addition of superoxide dismutase) of the normal detector, respectively. Relaxations induced by sodium nitroprusside as well as the contractions by acetylcholine, phenylephrine, and KCl (118 mM) were comparable in detector strips from the normal and atherosclerotic portions. Thus, not only is the amount of EDRF released by acetylcholine reduced in the presence of atherosclerosis, the tunica media beneath the atheromatous plaque is also to some extent responsible for the superoxide-induced inactivation of EDRF.

Increased hepatic lipase activity and increased direct removal of very-low-density lipoprotein remnants in Watanabe heritable hyperlipidaemic (WHHL) rabbits treated with ethinyl oestradiol

We studied the effects of ethinyl oestradiol on the serum concentrations and metabolism of very-low- and low-density lipoproteins (VLDL and LDL) in Watanabe heritable hyperlipidaemic (WHHL) homozygous rabbits, an animal model for familial hypercholesterolaemia. The results were compared with those in untreated homozygotes as well as in heterozygotes treated or not with ethinyl oestradiol. The gain in body weight was similar in all groups. Treatment with ethinyl oestradiol resulted in the homozygotes in an approx. 80% decrease in the concentrations of lipids and apoprotein B in the d less than 1.019 lipoprotein fraction; those in the LDL fraction did not change. In the heterozygotes, basal serum lipids and apoprotein B levels in the d less than 1.019 fraction were low; ethinyl oestradiol treatment especially affected the LDL fraction (cholesterol -84%, apoprotein B -64%). Turnover experiments with 125I-labelled VLDL revealed that, on treatment with ethinyl oestradiol, the fractional catabolic rate in homozygous rabbits increased 2-fold. The secretion rates of lipids and protein in the d less than 1.019 fraction as estimated after injection of Triton WR-1339 was not decreased. In homozygotes and heterozygotes increases in post-heparin hepatic lipase activity of 62 and 80% respectively were observed, with no changes in lipoprotein lipase activity. We conclude that ethinyl oestradiol induces in homozygous WHHL rabbits a direct removal of VLDL and VLDL remnants from the plasma, apparently due to an increase in hepatic lipase activity.

Cholesterol-fed heterozygous Watanabe heritable hyperlipidemic rabbits: a new model for atherosclerosis

Homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits are used widely to study atherosclerosis, but the WHHL heterozygous rabbit has received little attention. To study their potential as a model for atherosclerosis, heterozygous WHHL and New Zealand white (NZW) rabbits were fed diets containing 0%, 0.5% and 1.0% cholesterol. Plasma lipids were analyzed at 0, 4, 8, 12, 16 and 24 weeks, and animals were killed at 12 and 24 weeks. Plasma cholesterol levels were significantly higher in cholesterol-fed WHHL heterozygotes at 8 weeks compared with NZW rabbits, but no differences were apparent at other times. Atherosclerotic plaques in the aortas of cholesterol-fed WHHL heterozygous rabbits differed from those in NZW rabbits, in that the WHHL had complicated lesions with necrosis, cholesterol clefts, fibrous caps and calcification, similar to that found in humans and homozygous WHHL rabbits. In contrast, NZW rabbits had predominantly foam cell lesions. Heterozygous WHHL rabbits also had less extensive extravascular foam cell deposits. Our results suggest that the cholesterol-fed heterozygous WHHL rabbit may provide a promising model for studying the pathogenesis of atherosclerosis.