Identification of a region of the ileal-type sodium/bile acid cotransporter interacting with a competitive bile acid transport inhibitor.

Drug intervention that prevents reabsorption of circulating bile acids by the apical (ileal) sodium/bile acid cotransporter (ASBT) may be a promising new therapy for lowering of plasma cholesterol. 2164U90 is a benzothiazepine-based competitive inhibitor of bile acid transport with K(i) values of approximately 10 and 0.068 microM for the homologous human and mouse apical transporters, respectively. Hybrid human-mouse and mouse-human transporters were engineered to identify regions involved in this 150-fold difference in the inhibition constant for 2164U90. A mouse-human chimera with only the most C-terminal hydrophobic domain and the C-terminus of the transporter originating from the human variant was found to have a sensitivity to 2164U90 inhibition similar to that of the human transporter. Conversely, a human-mouse hybrid transporter encompassing the same C-terminal region from the mouse sequence but now inserted into the human sequence demonstrated the greater inhibition seen with the mouse wild type ASBT. Amino acid substitutions, individually or in combinations, of six candidate nonconserved residues between mouse and human transporters in this C-terminal domain showed replacements of Thr294 by Ser and Val295 by Ile to be responsible for the difference in the sensitivity toward 2164U90 seen between the species. The hamster apical SBAT encompassing Ser/Ile in these positions shared the lower sensitivity to 2164U90, as seen with the human ASBT, even though it is identical to the mouse SBAT in the remaining four positions of this region. In addition, the rat ASBT which is identical to the mouse ASBT in this domain also had the high sensitivity to 2164U90 inhibition found for the mouse ASBT. Methanethiosulfonates (MTS) are known to inactivate the sodium/bile acid transporters through alkylation of a cysteine in the most C-terminal hydrophobic domain (1). Inactivation of the human ASBT due to MTS modification of cysteine 270 was shown to be largely abolished when the transporter was preincubated with 2164U90, suggesting that the binding of this benzothiazepine is in the vicinity of position 270. Thus, the domain containing the two most C-terminal putative transmembrane regions of the SBATs, H8-H9, previously shown to constitute part of the binding pocket for bile acids, interacts also with the bile acid transport competitive inhibitor, 2164U90.

N,N'-diacetyl-L-cystine (DiNAC), the disulphide dimer of N-acetylcysteine, inhibits atherosclerosis in WHHL rabbits: evidence for immunomodulatory agents as a new approach to prevent atherosclerosis.

Oxidation of lipoprotein-derived lipids is generally accepted to be important in atherogenesis, and lipophilic antioxidants have been suggested as potential antiatherosclerotic agents. The antiatherogenic effects observed by certain antioxidants, especially probucol, in different animal models support this suggestion. There are however also cases where other lipophilic antioxidants have not been able to support this hypothesis. This has raised the question whether the effects of probucol and similar compounds are mainly due to some other property, unrelated to their antioxidant efficacy. For example, probucol is shown to possess immunomodulatory properties. Immune reactions are known to occur during atherogenesis. We therefore tested the dimer of N-acetylcysteine, DiNAC, which is a disulfide with immunomodulating properties and enhances oxazolone-induced contact sensitivity (CS) reactions in mice, for effects on atherosclerosis. When given to male heritable hyperlipidemic rabbit (WHHL) rabbits from 10 to 22 weeks of age, this compound reduced by 50% thoracic aorta atherosclerosis (p < 0.05), without affecting plasma lipid levels. Here we also show that probucol and a close chemical analog, both known to prevent atherosclerosis in WHHL rabbits, enhance the CS reaction in mice, while two other related antioxidants did not affect the CS reaction. At least one of these is also without effect on atherosclerosis in WHHL rabbits. The results show that DiNAC might represent a new treatment modality for atherosclerosis-related disease, and suggest that some antioxidants may have antiatherosclerotic properties more related to "immunomodulatory" properties than to antioxidant properties in general.

Dissociation of atherogenesis from aortic accumulation of lipid hydro(pero)xides in Watanabe heritable hyperlipidemic rabbits.

Antioxidants can inhibit atherosclerosis, but it is unclear how inhibition of intimal lipid oxidation relates to atherogenesis. Here we tested the effect of probucol and its metabolite bisphenol on aortic lipid (per)oxidation and atherogenesis in Watanabe heritable hyperlipidemic (WHHL) rabbits. LDL and aortas from rabbits fed probucol contained bisphenol at concentrations comparable to those in bisphenol-treated animals. Bisphenol treatment increased plasma cholesterol slightly, and plasma and aortic alpha-tocopherol more substantially; these parameters were unaffected by probucol. Bisphenol and probucol treatment both enhanced the resistance of circulating LDL to peroxyl radical-induced lipid peroxidation; this was due to bisphenol, not probucol. Only probucol enhanced LDL's resistance to Cu(2+)-induced oxidation. Both bisphenol and probucol treatment strongly inhibited aortic accumulation of hydroperoxides and hydroxides of cholesteryl esters and triglycerides [LO(O)H]. Despite this, however, probucol had a modestly significant effect on the extent of lesion formation; bisphenol had no inhibitory effect. In addition, the extent of atherosclerosis did not correlate with amounts of aortic LO(O)H present, but, as expected, it did correlate with aortic alpha-tocopherol and cholesterol. Together, these results suggest that aortic accumulation of LO(O)H is not required for, nor is alpha-tocopherol depleted during, the initiation and progression of atherogenesis in WHHL rabbits.

Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice.

Antioxidants can inhibit atherosclerosis in animals, though it is not clear whether this is due to the inhibition of aortic lipoprotein lipid (per)oxidation. Coantioxidants inhibit radical-induced, tocopherol-mediated peroxidation of lipids in lipoproteins through elimination of tocopheroxyl radical. Here we tested the effect of the bisphenolic probucol metabolite and coantioxidant H 212/43 on atherogenesis in apolipoprotein E and low density lipoprotein (LDL) receptor gene double knockout (apoE-/-;LDLr-/-) mice, and how this related to aortic lipid (per)oxidation measured by specific HPLC analyses. Dietary supplementation with H 212/43 resulted in circulating drug levels of approximately 200 microM, increased plasma total cholesterol slightly and decreased plasma and aortic alpha-tocopherol significantly relative to age-matched control mice. Treatment with H 212/43 increased the antioxidant capacity of plasma, as indicated by prolonged inhibition of peroxyl radical-induced, ex vivo lipid peroxidation. Aortic tissue from control apoE-/-;LDLr-/- mice contained lipid hydro(pero)xides and substantial atherosclerotic lesions, both of which were decreased strongly by supplementation of the animals with H 212/43. The results show that a coantioxidant effectively inhibits in vivo lipid peroxidation and atherosclerosis in apoE-/-;LDLr-/- mice, consistent with though not proving a causal relationship between aortic lipoprotein lipid oxidation and atherosclerosis in this model of the disease.

Characterization of novel indenoindoles. Part I. Structure-activity relationships in different model systems of lipid peroxidation.

Structure-activity relationships are presented for some representative compounds from a novel series of potent inhibitors of lipid peroxidation. The compounds are indenoindole derivatives with oxidation potentials in organic solvents of between 0.2 and 1.5 V. Two of these compounds, cis-5,5a,6,10b-tetrahydro-9-methoxy-7-methylindeno[2,1-b]indole (H 290/51) with an oxidation potential of 0.32 V and cis-4b,5,9b,10- tetrahydro-8-methoxy-6-methylindeno[1,2-b]indole (H 290/30) with an oxidation potential of 0.30 V, have been tested more extensively and compared with reference compounds in several pharmacological models of lipid peroxidation. The inhibitory potencies (pIC50) of the compounds in respect to Fe/Ascorbate-induced production of thiobarbituric acid-reactive substances (TBARS) in a suspension of purified soybean lecithin were calculated. These data are 8.2 for H 290/51; 8.0 for H 290/30; 5.6 for vitamin E; and 6.6 for butylated hydroxytoluene (BHT). In isolated rat renal tissue subjected to hypoxia and reoxygenation, the potency for inhibition of TBARS formation is 6.9 for H 290/51, 6.9 for H 290/30, and <5 for vitamin E. In oxidative modification of low-density lipoproteins (LDL) induced by mouse peritoneal macrophages, the corresponding pIC50 values for TBARS inhibition for each compound are: 8.7, 8.3, <5, and 6.9, respectively. It is concluded that the synthetic indenoindoles are potent antioxidants. The results suggest that indenoindoles such as H 290/51 and H 290/30 could be useful as therapeutic agents in pathophysiological situations where lipid peroxidation plays an important role.

Characterisation of novel indenoindoles. Part II. Redox-recycling with ascorbate.

In the accompanying paper it was shown that the new antioxidant, H 290/51 (cis-5,5a,6,10b-tetrahydro-9-methoxy-7-methylindenol[2,1-b]indole) , is a powerful antioxidant in several pharmacological models of lipid peroxidation and could be useful as a therapeutic agent in pathophysiological situations where lipid peroxidation plays an important role. In the present study, we characterised H 290/51 as an inhibitor of peroxidation of pure methyl linoleate. H 290/51 almost completely inhibited peroxidation induced by a lipid-soluble initiator at 37 degrees C during the induction period, both in an aqueous solution of micelles in the presence of detergents and in a homogeneous ethanol solution. In both systems, the time of the induction period was linearly related to the concentration of H 290/51. In the ethanol solution, ascorbic acid had a sparing effect on H 290/51, indicating effective interference with radical chain propagation. In aqueous solution with micelles of methyl linoleate made with the nonionic detergents Triton X-100 or Lubrol PX, ascorbic acid did not inhibit peroxidation. However, in these micelles, H 290/51 showed a concentration-dependent extension of the induction period by ascorbic acid, suggesting recycling. In the presence of the zwitterionic detergent CHAPS, although a clear induction period is seen with H 290/51, no recycling by ascorbic acid was found. The ability of H 290/51 to recycle in aqueous solutions, thus depends on the micellar composition.

Lipoprotein fractionation in deuterium oxide gradients: a procedure for evaluation of antioxidant binding and susceptibility to oxidation.

Oxidative modifications of lipoproteins appear to contribute to their atherogenicity. Very low and low density lipoproteins (VLDL and LDL) are protected against these modifications by antioxidants that can be incorporated in vivo or in vitro into the particles. We describe here ultracentrifugal procedures for isolation of VLDL and LDL that do not require subsequent dialysis or buffer equilibration. Lipoproteins were isolated in buffers with physiological ionic composition prepared in D2O (deuterium oxide). This allowed measurements of the content of antioxidants and of the susceptibility to oxidation of the isolated LDL without further manipulations. Conventional ultracentrifugal methods use high salt concentrations and require additional steps to eliminate them. This introduces uncertainties in the evaluation of antioxidant binding and on measurements of their effect on VLDL and LDL oxidation. With the method described, the composition of the isolated VLDL and LDL was indistinguishable from that of fractions isolated with KBr gradients. Also, the content of alpha-tocopherol was similar. LDL isolated with KBr solutions appeared to bind 20-45% more of the probucol present in serum than LDL isolated in isotonic solutions prepared with D2O. This was the case with probucol incorporated into plasma or serum in vivo or in vitro. Five out of seven LDL isolated with the D2O procedure from different human sera appeared more resistant to Cu(2+)-catalyzed oxidation than those obtained with KBr gradients from the same serum. In addition to the gradient procedure, we also describe a preparative version of the method that can be used with multiple samples.

The effect of felodipine on the uptake and degradation of acetylated LDL in mouse peritoneal cells and on the distribution of acetylated LDL in macrophage-rich organs of the rat.

The effect of felodipine on lipoprotein metabolism ex vivo and in vivo was investigated. In the ex vivo studies mice were given felodipine (40-125 mumol/kg body weight) or vehicle for one week. Peritoneal macrophages from these animals and controls were isolated and used in binding and degradation studies with human iodinated acetylated LDL (Ac-LDL). Macrophages from felodipine-treated mice showed a significant decrease of binding and degradation of Ac-LDL compared to macrophages from control animals (P < 0.05). The in vivo studies were performed in rats pretreated with felodipine or vehicle. To determine the distribution and plasma turnover of LDL and Ac-LDL, 125I-tyramine cellobiose labelled LDL or Ac-LDL were given i.v. No differences in the removal rate of Ac-LDL or LDL were observed between felodipine-treated or untreated rats. However, an increased uptake of Ac-LDL could be seen in the liver of the felodipine-treated rats. This increased uptake could be ascribed to the parenchymal cells because no differences in uptake could be seen in the liver endothelial cells. However, a significant decreased uptake was seen in the Kupffer cells and in the spleen, a macrophage-rich organ, of the felodipine-treated rats. The present study suggests a possible mechanism behind the antiatherogenic effects of calcium antagonists, a decreased uptake of atherogenic modified lipoproteins by peripheral macrophages and an increased uptake by the liver.

Comparison of dietary casein and soybean protein effects on plasma lipid and gastrin levels, hepatic delta 6-desaturase activity and coronary arteriosclerosis in male Sprague-Dawley rats. A 9-month study.

An increased concentration of gastrin was observed in plasma of male Sprague-Dawley strain rats fed on soybean protein diet for a 9-month period, compared with rats fed on casein diet. Both diets contained 0.5% (w/w) cholesterol. Protein-dependent differences were also observed in the fatty acid pattern of hepatic phospholipids, hepatic delta 6-desaturase activity, and plasma cholesterol. No signs of arteriosclerosis were observed in the aortas. Sixty percent of the hearts showed various degrees of lipid staining in coronary arterial branches of different sizes. Despite a large difference in plasma cholesterol level, there was no quantitative or qualitative difference between groups in the occurrence of coronary lipid staining.

Plasma very low density lipoproteins from male rats fed casein or soybean protein diets: a comparison of fatty acid composition and influence on prostanoid production.

In studies with male rats fed for 4 wk semipurified diets containing olive oil and casein or soybean protein, protein-dependent effects were observed in the fatty acid composition of the VLDL lipids, especially with regard to the pattern of polyunsaturated fatty acids. Compared with VLDL from rats fed soybean protein diet (S-VLDL), VLDL from casein-fed rats (C-VLDL) contained a greater level of oleic acid, and a reduced level of linoleic acid and arachidonic acid, in the phosphatidylcholine fraction. The proportion of 5,8,11-eicosatrienoic acid, 20:3 (n-9), varied among the different lipid classes. The highest concentration of this fatty acid (13% by weight of total fatty acids) was observed in the phosphatidylinositol fraction of C-VLDL. The level of linoleic acid was approximately halved in the triacylglycerol and cholesteryl ester fractions of C-VLDL compared with S-VLDL. When mouse peritoneal macrophages were incubated with different concentrations of S-VLDL, a saturable accumulation of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and thromboxane B2 (TXB2) was observed in the cell medium. In contrast, very low levels of 6-keto-PGF1 alpha and TXB2 were observed in the cell medium of macrophages incubated with C-VLDL at different lipoprotein concentrations, suggesting that the composition of VLDL may play an important role in relation to cellular prostanoid metabolism.

Tissue accumulation of lipoprotein associated toxaphene in normo- and hypolipidemic mice.

Normo- and hypolipidemic mice were given a single i.v. injection of 14C-toxaphene associated with low density lipoprotein (LDL), high density lipoprotein (HDL) or dimethyl sulfoxide (DMSO). The tissue distribution of radioactivity was studied 20 min and 4 h after the application. In the normolipidemic mice at 20 min postinjection there was high uptake of the 14C-toxaphene preparations in the liver and adrenals followed after 4 h by a redistribution to the adipose tissues. In the hypolipidemic mice, proportionally less label accumulated initially in the liver and adrenals while more radioactivity was seen in the kidneys and heart. The radioactivity then redistributed to the liver with a very small uptake in the adipose tissue compared to the normolipidemic mice after 4 h. The results indicate that changes in the lipid pattern, e.g. hypolipidemic conditions, may influence the tissue distribution of lipophilic xenobiotics.

Distribution of toxaphene, DDT, and PCB among lipoprotein fractions in rat and human plasma.

The distribution of 14C-toxaphene, 14C-DDT, and 14C-PCB among lipoprotein fractions was studied in vitro and in vivo using rat and human plasma. The association of these substances with rat plasma fractions was similar in both in vitro and in vivo experiments. Thirty-seven to fifty-two per cent of the total radioactivity was associated with the cholesterol-rich high density lipoproteins (HDL2, d = 1.075-1.21 g/ml) and 18-52% was recovered in the albumin-rich bottom fraction (BF, d greater than 1.21 g/ml). A time-dependent redistribution of the radioactivity from the lipoprotein fractions to the BF was also observed in the in vivo studies. In human plasma, the distribution of the three compounds was different and uncorrelated to the cholesterol level of the individual lipoprotein fractions. Toxaphene was almost equally distributed between BF (d greater than 1.21 ml), HDL (d = 1.063-1.21 g/ml) and low density lipoproteins (LDL, d = 1.006-1.063 g/ml) (26%, 27% and 29%, respectively), while only 18% appeared in the very low density lipoprotein (VLDL, d less than 1.006) fraction. In contrast, a large proportion of DDT and PCB radioactivity was recovered in the BF (52% and 62%, respectively) while only 38-48% was present in lipoprotein fractions. The complex nature of the interaction between xenobiotics and plasma lipoproteins is discussed.

Effect of metoprolol on plasma lipids and arterial intimal lipid deposition in spontaneously hypertensive rats.

The purpose of the present study was to characterize possible effects of dietary-induced plasma lipid elevations on the development of arterial lesions in spontaneously hypertensive rats (SHR) and to reveal any influence of treatment with metoprolol on these parameters. Metoprolol treatment caused an 8% decrease in heart rate and a 13% decrease in blood pressure and led to a rise in plasma triglycerides, 24%, 17% and 34% after 1, 3 and 6 months of metoprolol treatment, respectively. However, no effect on plasma triglycerides was observed after 9 months of metoprolol treatment while a reduced cholesterolemic response was observed. Intimal proliferations containing accumulations of lipids were observed in small intramural branches of coronary arteries (greater than 100 microns) in 11 of 31 control rats fed the atherogenic diet for 9 months. In contrast, similar changes were observed in only 1 of 34 metoprolol-treated rats fed an otherwise identical diet. The corresponding figures for the frequency of lipid containing intimal plaques in aorta were 6/19 in controls and 2/24 in the metoprolol-treated group.

Effects of diet and metoprolol on lipid levels in the blood plasma and morphology of the heart and intramural branches of coronary arteries of spontaneously hypertensive male rats. A 9-month study.

Semipurified diets containing 0.5% cholesterol were used in a 9-month study with spontaneously hypertensive male rats to characterize the effects of the protein source (casein vs. soybean protein), and the selective beta 1-adrenoceptor antagonist metoprolol on both lipid levels in blood plasma and the aorta, and on the morphology of intramural branches of coronary arteries. Raised blood lipid levels were observed in these rats. A significant decline in HDL2 cholesterol took place, while plasma cholesterol belonging to lipoprotein fractions of lower density increased. Metoprolol treatment led to a substantial elevation of the plasma triacylglycerol level and, with time, a reduced cholesterolemic response. The use of soybean protein instead of casein had a persistent plasma lipid-lowering effect. Arteriosclerotic changes in the form of musculo-elastic thickenings, intimal cushions and homogeneous hyalin deposits appeared in the intramural coronary arteries of rats in all groups after 9 months on the diet. However, intimal deposition of lipid was only present in rats belonging to the casein group not treated with metoprolol. Rats of this group also showed more severe myocardial lesions in the form of scar tissue with or without inflammatory cell reaction.

Apolipoprotein-E-binding proteins of rat liver endothelial cells.

In an attempt to characterise the apolipoprotein-E-binding proteins of rat liver endothelial cells, we prepared membranes from monolayer cultures of liver endothelial cells as an enriched source of membrane receptors. The membranes could specifically bind iodinated very-low-density lipoproteins (VLDL) and the binding could be inhibited effectively by unlabelled VLDL and high-density lipoproteins, but only moderately by low-density lipoproteins. To identify the binding proteins, we performed immunoprecipitation studies of solubilised iodinated liver endothelial cells and cell membranes, respectively, using purified apolipoprotein E and monospecific polyclonal IgG directed towards this apolipoprotein. The antibodies together with the bound apolipoprotein E and iodinated liver endothelial cell proteins were harvested with staphylococcal protein A-Sepharose. The immunoprecipitates were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and after autoradiography of the dried gel, the Mr of the liver endothelial cell proteins bound to apolipoprotein E could be determined. Two protein bands with molecular masses of 55-60 and 110, and a weak band of 170 kDa could be detected from intact cells. These proteins were specifically precipitated only in the presence of divalent cations, and might represent cell-surface receptors for apolipoprotein-E-containing lipoproteins. Additional bands were seen when cell membranes were used, the most prominent ones having molecular masses of 32 and 35 kDa. These proteins could be of intracellular origin, or they may be degradation products of the other apolipoprotein-E-binding proteins.

Atherosclerosis in rabbits identified as high and low responders to an atherogenic diet and the effect of treatment with a beta 1-blocker.

In order to investigate whether initial plasma lipid concentrations could be used to distinguish between high and low responders to an atherogenic diet, rabbits were divided into 3 groups according to their plasma concentrations of cholesterol and phospholipids after 4 weeks on a standard rabbit diet. Plasma cholesterol and phospholipid levels were less than 0.5 mM, less than 1.1 mM, respectively, in group 1 (n = 17), greater than 0.5 mM, less than 1.1 mM, in group 2 (n = 13), and greater than 0.5 mM, greater than or equal to 1.1 mM, in group 3 (n = 14). After 7 weeks on a diet containing 0.25% cholesterol and 3% coconut oil, animals in groups 1 and 2 had a lower increase in their plasma lipid levels compared with group 3. Half of each group was then treated with the beta 1-adrenoceptor antagonist metoprolol during the next 14 weeks on the atherogenic diet. At the end of the study, the extent of atherosclerosis both in the aortas and in the coronary arteries of the control animals showed a positive correlation to plasma cholesterol and to plasma phospholipid concentrations integrated over time. The metoprolol-treated animals in groups 1 and 2 had a reduction of atherosclerosis compared with their respective controls. We conclude that subpopulations of rabbits that react differently on an atherogenic diet can be identified by their initial plasma lipid levels, and that metoprolol treatment of low responders to an atherogenic diet significantly reduces atherosclerotic lesions of the aorta.

The role of sympathetic activity in atherogenesis: effects of beta-blockade.

Clinical and experimental evidence points to potential antiatherosclerotic effects of certain beta-adrenoreceptor antagonists. Long-term treatment with metoprolol resulted in significant reductions of total and cardiovascular mortality or morbidity due to decreased incidence of coronary and cerebrovascular complications both in a primary prevention trial in hypertensive patients and in a secondary prevention trial in patients surviving myocardial infarction. The observations suggest that a retardation of atherosclerosis development might have contributed to the reduced incidence of cardiovascular complications. An antiatherosclerotic effect of beta-blockers has been directly demonstrated in animal studies. In cholesterol-fed rabbits, metoprolol significantly reduced the development of atherosclerotic plaques in the aortic intima in the absence of any changes in blood lipids. Similar findings were reported for propranolol, which prevented psychosocial stress-induced atherosclerosis of the coronary artery in monkeys. Furthermore, beta-blockers have been shown to prevent stress-induced endothelial injury and platelet accumulation to intima at atherosclerotic predilection sites in animal models. These antiatherogenic effects may be due to biochemical and hemodynamic factors. Two biochemical effects of beta-blockade may lead to reduced cholesterol accumulation in arterial intima at unchanged serum cholesterol levels. One is a beta-blocker-induced increase of prostacyclin biosynthesis, and the other a metabolic change of low-density lipoprotein, reducing its potential for deposition in the arterial wall. The antiatherogenic effect of these factors may be reinforced by beta-blocker-induced hemodynamic changes leading to reductions of arterial flow aberrations and pressure-related wall stress.

Effect of metoprolol on diet-induced atherosclerosis in rabbits.

The effect of metoprolol, a beta 1-blocker, on atherogenesis was evaluated in rabbits fed a diet supplemented with 0.25% cholesterol and 3% coconut oil for 21 weeks. After 7 weeks on the diet, the rabbits were randomly divided into treated (n = 22) and untreated (n = 22) groups. Treated animals received metoprolol subcutaneously by an osmotic pump for 14 weeks, resulting in a plasma level of 774 +/- 69 nM during the investigation. Plasma concentrations of cholesterol, triglycerides, and phospholipids did not differ between the two groups. Nor were there any significant differences between the two groups in plasma concentrations of apolipoprotein A-I, apolipoprotein B, apolipoprotein C-III, and apolipoprotein E measured by electroimmunoassay. At the end of the study, the aortas were cut into three portions and the extent of atherosclerosis was determined by morphometry. The group that had received metoprolol had significantly (p less than 0.015) less atherosclerosis in the aorta (ascending plus arch 37.8 +/- 6.8%, thoracic 32.9 +/- 6.1%, abdominal 19.8 +/- 6.1% of total intimal area; mean +/- SEM) than the controls (ascending plus arch 54.9 +/- 7.1%, thoracic 48.0 +/- 6.2%, abdominal 25.9 +/- 5.5%).

Accumulation of cholesteryl esters and triglycerides in cultured macrophages incubated with plasma very low density lipoproteins from rats fed on casein or soybean protein diets containing moderate levels of cholesterol.

Even when administered at a comparatively low level, dietary cholesterol produces significant changes in the properties of plasma lipoproteins in rats, particularly the d less than or equal to 1.006 g/ml fraction (VLDL). The occurrence of these changes is promoted by dietary casein. To test the hypothesis that these dietary-induced perturbations might include properties influencing lipoprotein-cell interactions of relevance to atherogenesis, cultured mouse peritoneal macrophages were incubated with VLDL isolated from male rats fed on diets containing either 0, 0.25 or 0.5% cholesterol with casein or soybean protein, respectively, as the sole source of protein. No increase in cholesteryl ester content, and a comparatively small rise in triglyceride content, was observed in macrophages incubated with VLDL from rats fed on cholesterol-free diets. In contrast, a significant and apparently saturable cellular accumulation of cholesteryl esters as well as triglycerides was produced by VLDL from cholesterol-fed rats. The curves showing cellular lipid accumulation versus VLDL-protein (or VLDL-cholesterol) content in the cell medium indicated different cellular affinity for VLDL from casein-fed rats in comparison with VLDL from soybean protein-fed rats. The apoprotein composition of VLDL differed between groups of rats fed on different types of dietary protein with higher proportions of apo C's in the casein-fed rats. In addition, cholesterol feeding resulted in increased proportions of apo A-I and apo A-IV in the plasma VLDL fraction.