Development and partial metabolic characterization of a dietary cholesterol-resistant colony of rabbits

A colony of New Zealand white rabbits has been developed which, when fed a cholesterol-supplemented diet, exhibit unusual resistance to hypercholesterolemia and atherosclerosis, disorders usually observed in normal cholesterol-fed rabbits. When resistant rabbits (RT) were fed a normal low cholesterol diet (ND), their plasma lipoprotein patterns were significantly different from those of normal rabbits (NR) fed the same diet. The low density lipoprotein cholesterol (LDL-c)/high density lipoprotein cholesterol (HDL-c) ratio and LDL-c/very low density lipoprotein cholesterol (VLDL-c) ratio were lower in the resistant rabbits. The hydrated density of HDL of the normal-responsive rabbits was greater than that of the resistant rabbits. LDL from resistant rabbits contained a lower proportion of esterified cholesterol and protein than LDL from normal rabbits. Peripheral mononuclear cells from resistant rabbits bound about 30% more 125I-labeled rabbit LDL than mononuclear cells from normal rabbits. These results demonstrate that the plasma cholesterol levels of these animals is at least partly under genetic control and that compositional differences exist between the major plasma lipoprotein classes of normal and resistant rabbits even during the ingestion of low-cholesterol diet. The results indicate that at least a part of the difference in the cholesterolemic responses between the two rabbit groups is due to an enhanced LDL uptake by the mononuclear cells, and presumably by other somatic cells of the resistant group.     

Evidence for the preferential utilization of esterified cholesterol in progesterone production by rabbit corpora lutea

Our previous studies show that lipoproteins stimulate progesterone secretion by rabbit luteal cells in vitro and that estradiol modifies this effect. This study examines the relationship between estradiol and serum lipoproteins for progesterone production by rabbit corpora lutea in vivo. Using morphometric analysis, we determined that estrogen treatment of hysterectomized pseudopregnant (E-hyst) rabbits increased luteal lipid volume by mid-pseudopregnancy without altering serum progesterone levels. Treatment of E-hyst rabbits with 4-amino-3,4,pyrazolo pyrimidine (APP) during early to mid-pseudopregnancy reduced serum cholesterol levels without decreasing serum progesterone concentrations. However, 3-hydroxy-3 methyl glutaryl-CoA reductase activity was increased. Thus, in the presence of exogenous estrogen, serum cholesterol is esterified and stored rather than converted directly into progesterone. APP-treatment of E-hyst rabbits during late-pseudopregnancy, when estrogen receptor levels are low, increased serum progesterone levels and reduced intracellular lipid content. Thus, stored lipid is the primary source of cholesterol for progesterone synthesis. In addition, estrogen, via estrogen receptor, is important in maintaining steady progesterone output despite fluctuations in serum lipoprotein levels. A working model for cholesterol utilization by rabbit luteal cells is presented, which suggests that stored cholesterol esters, derived from both endogenous and exogenous sources, is the key source or cholesterol for progesterone production. Furthermore, we propose that estradiol regulates the uptake and storage of cholesterol and its rate of metabolism into progesterone.     

Hyaluronan accumulation is elevated in cultures of low density lipoprotein receptor-deficient cells and is altered by manipulation of cell cholesterol content

The extracellular matrix molecule hyaluronan (HA) accumulates in human atherosclerotic lesions. Yet the reasons for this accumulation have not been adequately addressed. Because abnormalities in lipid metabolism promote atherosclerosis, we have asked whether disrupted cholesterol homeostasis alters HA accumulation in low density lipoprotein receptor-deficient cell cultures. Cultured aortic smooth muscle cells (ASMC) from Watanabe heritable hyperlipidemic (WHHL) rabbits and skin fibroblasts from homozygous patients with familial hypercholesterolemia accumulated 2-4-fold more HA than corresponding cells from age- and sex-matched normolipidemic rabbits and individuals. This occurred in both cell-associated and secreted HA fractions and was independent of cell density or medium serum concentration. WHHL ASMC cultures synthesized twice the proportion of high molecular mass HA (>2x10(6) Da) as normal rabbit ASMC but showed a lower capacity to degrade exogenous [3H]HA. Most importantly, cholesterol depletion or blocking cholesterol synthesis markedly reduced HA accumulation in WHHL ASMC cultures, whereas cholesterol replenishment or stimulation of cholesterol synthesis restored elevated HA levels. We conclude the following: 1) maintaining normal HA levels in cell cultures requires normal cell cholesterol homeostasis; 2) HA degradation may contribute to but is not the predominant mechanism to increase high molecular mass HA accumulation in low density lipoprotein receptor-deficient WHHL ASMC cultures; and 3) elevated accumulation of HA depends on cellular or membrane cholesterol content and, potentially, intact cholesterol-rich microdomains.     

Dissociation between cholesterol secretion and plasma lipid transfer activity in rabbits

Human and rabbit plasma contains a lipid transfer protein that transfers cholesteryl esters and triglycerides among the plasma lipoproteins and may also have a role in the movement of lipids into and out of cells. Little is known about the regulation of the activity of the lipid transfer protein, but in the rabbit, hypercholesterolemia is associated with increased plasma lipid transfer activity (LTA). Perfused rabbit livers secrete LTA, and hepatic cholesterol secretion is increased in rabbits with diet-induced hypercholesterolemia. Thus, experiments were performed with rabbits to determine if LTA is regulated by a concerted hepatic secretion of lipoprotein protein cholesterol and LTA. Rabbits were fed chow or chow plus coconut oil (14% wt/wt), and plasma lipids, LTA, and the rate of secretion of cholesterol into plasma were determined. Coconut oil feeding increased plasma cholesterol by 68%, LTA by 42%, and hepatic cholesterol secretion by 69%. Mevinolin (75 mg/day), an inhibitor of cholesterol biosynthesis, lowered LTA and plasma cholesterol without affecting the rate of secretion of cholesterol into plasma. These studies provide further evidence that, in the rabbit, plasma cholesterol and LTA are closely related, and the association is not likely to be caused by a concerted hepatic secretion of cholesterol and LTA.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and the esterification of cholesterol in human long term lymphoid cell lines.

The regulation of the rate-controlling enzyme in cholesterol biosynthesis and of the incorporation of [14C]oleate into cholesterol esters were studied in established lymphoid cell lines from normal subjects and compared with that of eight patients with genetic abnormalities of lipid metabolism. The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme in cholesterol biosynthesis, increases in lymphoid cell lines derived from normal subjects after the culture medium is changed to a lipid deficient medium and reaches peak activity after 48 hr. The addition of whole serum and of low density lipoproteins to cell lines derived from normal subjects suppressed 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by 50%, but failed (almost completely) to suppress the activity in the lymphoid cell lines derived from two patients with homozygous familial hypercholesterolemia. When 7-ketocholesterol was added, the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase was markedly suppressed in both normal and abnormal lymphoid cell lines. Lymphoid cell lines derived from patients presumably heterozygous for familial hypercholesterolemia were difficult to distinguish from normal cells in these studies. The incorporation of [14C]oleate into the fatty acid fraction of cholesteryl esters was stimulated by the addition of the low density lipoproteins to the culture media of the lymphoid cell lines derived from the normal human subjects. The lymphoid cell lines derived from the patients with homozygous familial hypercholesterolemia showed no increase in [14C]oleate incorporation into cholesteryl esters even when a fourfold amount of low density lipoprotein was added to the media; a modest increase in [14C]oleate incorporation was observed in lymphoid cell lines from patients with heterozygous familial hypercholesterolemia. The results of these studies in lymphocyte cell lines are compared with the findings in cultured human fibroblasts obtained from normal subjects and from patients with homozygous familial hypercholesterolemia. Studies of the regulation of cholesterol biosynthesis in the apparently permanent lymphoid cell line maintained in suspension culture offer certain advantages over cultured skin fibroblasts, and, in addition, provide a second tissue for the study of genetic abnormalities from the same patient.     

Similar content of phospholipids and gangliosides in normal and homozygous familial hypercholesterolemia fibroblasts.

The cellular content of total and individual phospholipids and gangliosides was measured in fibroblasts cultured from four normal subjects, three patients with lysosomal lipid storage diseases, and two subjects with homozygous familial hypercholesterolemia. Measurements were made on cells grown in medium containing fetal calf serum under conditions in which normal cells derive cholesterol for cell growth from low density lipoprotein present in the fetal calf serum, whereas familial hypercholesterolemia homozygote cells, which lack cell surface low density lipoprotein receptors, derive cholesterol from endogenous synthesis. No difference was observed in the cellular content of total or individual phospholipids and gangliosides in the normal and familial hypercholesterolemia homozygote cells. In contrast, cells from a patient with Niemann-Pick disease and a patient with Sandhoff disease showed elevations in the content of sphingomyelin and complex gangliosides, respectively.     

Acute ingestion of different dietary fatty acid species modulates postprandial lipid responses in New Zealand white rabbits

Although several investigations have linked the degree of fatty acid saturation to plasma lipid responses in the postprandial state, further evaluation is necessary. In this study, we compared the effect of saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids on postprandial lipid metabolism using complementary in vivo and in vitro approaches. Fat (10 g) cholesterol (0.5 g) test meals that provided either lard (SFA), olive oil (MUFA), or sunflower oil (PUFA) were ingested by chow-fed New Zealand white rabbits (n = 8). In addition, hepatic uptake of triglyceride-cholesterol-rich lipoproteins (TCRL) isolated from rabbits chronically ingesting SFA, MUFA, or PUFA diets was measured using freshly isolated chow-fed rabbit hepatocytes. Whatever dietary fatty acids ingested, postprandial triglyceridemia and occurrence of radiolabelled dietary lipids in plasma were not markedly different. Conversely, SFA induced higher postprandial cholesterolemia and phospholipemia than MUFA (P < 0.05) whereas PUFA prevented postprandial cholesterol increase. TCRL disappearance from cultured liver cell media was delayed with SFA-rich TCRL and faster with PUFA whereas MUFA-rich TCRL showed an intermediate figure. From these data, we conclude that SFA, MUFA, and PUFA elicited different postprandial plasma and lipoprotein lipid responses. The fatty acid composition of TCRL had a major impact on their subsequent metabolism, especially uptake by cultured hepatocytes. The SFA-induced hypercholesterolemia could be related to an altered hepatic uptake whereas a faster clearance and hepatic uptake could explain the cholesterol-lowering effect of PUFA in rabbits. MUFA, like PUFA, accelerate uptake by hepatocytes but favor cholesterol ester enrichment of TCRL.     

Removal of apoprotein-B-containing lipoproteins by plasmapheresis using immobilized phosphorylcholine-binding protein affinity adsorbent

Rat serum phosphorylcholine binding protein was earlier shown to bind lipoproteins containing apoproteins B and E from human very low and low density lipoproteins. The present studies were undertaken to show the effectiveness of rat serum phosphorylcholine-binding protein immobilized on Sepharose affinity column to remove apoprotein-B-containing lipoproteins from normal and hypercholesterolemic rabbit plasma, when used in a plasmapheresis system. The maximum in vitro binding of very low and low density lipoproteins from hypercholesterolemic rabbit plasma to the affinity adsorbent was Ca2+ dependent, and the cholesterol bound to the column at the optimum calcium concentration (2.5 mM) was 21% of the total plasma cholesterol applied. The in vivo binding of total cholesterol from normal and hypercholesterolemic rabbit plasma during plasmapheresis ranged from 0.22 to 7.7%. Total mass of cholesterol bound ranged from 3.86 and 27.52 mg at plasma cholesterol concentrations 13.8 and 282 mg/dL, respectively. Most (greater than 95%) of the bound cholesterol was associated with very low and low density lipoproteins. These studies show the ability of immobilized rat serum phosphorylcholine-binding protein to lower the atherogenic apoprotein-B-containing lipoproteins from plasma of hypercholesterolemic rabbits.     

Hypercholesterolemia boosts joint destruction in chronic arthritis. An experimental model aggravated by foam macrophage infiltration

Objective

The aim of this study was to determine whether hypercholesterolemia increases articular damage in a rabbit model of chronic arthritis.

Methods

Hypercholesterolemia was induced in 18 rabbits by administrating a high-fat diet (HFD). Fifteen rabbits were fed normal chow as controls. Chronic antigen-induced arthritis (AIA) was induced in half of the HFD and control rabbits, previously immunized, by intra-articular injections of ovalbumin. After sacrifice, lipid and systemic inflammation markers were analyzed in blood serum. Synovium was analyzed by Krenn score, multinucleated cell counting, immunohistochemistry of RAM11 and CD31, and TNF-α and macrophage chemoattractant protein-1 (MCP-1) gene expression. Active bone resorption was assessed by protein expression of receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) and quantification of cathepsin K, contact surface and the invasive area of pannus into bone.

Results

Rabbits receiving the HFD showed higher total serum cholesterol, HDL, triglycerides and CRP levels than rabbits fed a normal diet. Synovitis score was increased in HFD, and particularly in AIA and AIA + HFD groups. AIA + HFD synovium was characterized by a massive infiltration of RAM11+ cells, higher presence of multinucleated foam cells and bigger vascularization than AIA. Cathepsin K+ osteoclasts and the contact surface of bone resorbing pannus were also increased in rabbits with AIA + HFD compared with AIA alone. Synovial TNF-α and MCP-1 gene expression was increased in AIA and HFD rabbits compared with healthy animals. RANKL protein expression in AIA and AIA + HFD groups was higher compared with either HFD or normal groups.

Conclusions

This experimental model demonstrates that hypercholesterolemia increments joint tissue damage in chronic arthritis, with foam macrophages being key players in this process.     

Metabolism of low-density lipoproteins by cultured hepatocytes from normal and homozygous familial hypercholesterolemic subjects

The profoundly elevated concentrations of low-density lipoproteins (LDL) present in homozygous familial hypercholesterolemia lead to symptomatic cardiovascular disease and death by early adulthood. Studies conducted in nonhepatic tissues demonstrated defective cellular recognition and metabolism of LDL in these patients. Since mammalian liver removes at least half of the LDL in the circulation, the metabolism of LDL by cultured hepatocytes isolated from familial hypercholesterolemic homozygotes was compared to hepatocytes from normal individuals. Fibroblast studies demonstrated that the familial hypercholesterolemic subjects studied were LDL receptor-negative (less than 1% normal receptor activity) and LDL receptor-defective (18% normal receptor activity). Cholesterol-depleted hepatocytes from normal subjects bound and internalized 125I-labeled LDL (Bmax = 2.2 micrograms LDL/mg cell protein). Preincubation of normal hepatocytes with 200 micrograms/ml LDL reduced binding and internalization by approx. 40%. In contrast, 125I-labeled LDL binding and internalization by receptor-negative familial hypercholesterolemic hepatocytes was unaffected by cholesterol loading and considerably lower than normal. This residual LDL uptake could not be ascribed to fluid phase endocytosis as determined by [14C]sucrose uptake. The residual LDL binding by familial hypercholesterolemia hepatocytes led to a small increase in hepatocyte cholesterol content which was relatively ineffective in reducing hepatocyte 3-hydroxy-3-methylglutaryl-CoA reductase activity. Receptor-defective familial hypercholesterolemia hepatocytes retained some degree of regulatable 125I-labeled LDL uptake, but LDL uptake did not lead to normal hepatocyte cholesterol content or 3-hydroxy-3-methylglutaryl-CoA reductase activity. These combined results indicate that the LDL receptor abnormality present in familial hypercholesterolemia fibroblasts reflects deranged hepatocyte LDL recognition and metabolism. In addition, a low-affinity, nonsaturable uptake process for LDL is present in human liver which does not efficiently modulate hepatocyte cholesterol content or synthesis.     

Fluorescence-polarization measurements on normal and mutant human skin fibroblasts

Measurements of fluorescence polarization in intact diploid skin fibroblasts after exposure to 1,6-diphenyl-1,3,5-hexatriene were used to estimate the fluidity of the lipid phase(s) of cellular membranes. The membrane lipids of cells derived from four patients with homozygous familial hypercholesterolemia were in a more fluid state than those of cells obtained from 13 other individuals of normal and nonrelated mutant genotypes when all cultures were grown on medium with native serum. The only other cell type having membrane lipids of increased fluidity under these conditions was one fibroblast line derived from a patient with the Lesch-Nyhan syndrome. Examination of two additional nonconsanguinous lines of Lesch-Nyhan fibroblasts, however, revealed that an abnormally high level of lipid fluidity was not a common property of the membranes of cells of this genotype. Incubation of cultures in medium containing lipid-depleted serum (virtually devoid of lipoprotein-bound sterol) caused a reversible increase in the fluidity of the membranes of normal cells to values similar to those of the hypercholesterolemic cells, but had no effect on the membranelipid fluidity of the latter. By contrast, exposure of cultures to cholesterol not bound to lipoprotein in serum-free medium resulted in a decrease in the lipid fluidity of the membranes of both normo- and hypercholesterolemic fibroblasts.     

Effects of hyperlipidemia on the nursing ability of WHHL rabbits

The homozygous Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for human familial hypercholesterolemia, which has been maintained in a closed colony, has a reproductive ability which is remarkably lower than that of normal rabbits. The present study was undertaken to determine whether this low reproductive ability was associated with hyperlipidemia, since it is not associated with inbreeding depression. WHHL dams with over 600 mg/dl of serum cholesterol level showed a weaning rate of only about 20%, while dams with about 300 mg/dl of cholesterol showed a 64% weaning rate. Both conception and weaning rate seemed to decrease with a rise in serum triglyceride. The weaning age of homozygous offspring from the homozygous WHHL dams was significantly higher than homozygous offspring from heterozygous WHHL dame. The rate of increase in body weight of the offspring from WHHL dams was significantly lower than that of the offspring from heterozygous dams under 24 days of age. We concluded that the low reproductive ability, especially low nursing ability, was associated with hyperlipidemia due to the deficiency of low density lipoprotein receptors.     

Mutations affecting the binding,internalization, and lysosomal hydrolysis of low density lipoprotein in cultured human fibroblasts,lymphocytes, and aortic smooth muscle cells

Studies comparing the metabolism of low density lipoprotein (LDL) in normal cells and in cells cultured from patients with homozygous familial hypercholesterolemia have disclosed the existence of a receptor for plasma LDL. This receptor has been identified on the surface of human fibroblasts, lymphocytes, and aortic smooth muscle cells. An extension of these studies to cell strains derived from patients with other single gene defects in cholesterol metabolism has provided additional insight into the normal mechanisms by which cells regulate their cholesterol content and how alterations in these genetic control mechanisms may predispose to atherosclerosis in man.     

The effect of dietary pectin on serum lipoprotein cholesterol in rabbits.

The effect of pectin on serum lipoprotein cholesterol during and after induction of hypercholesterolemia in rabbits was studied. The addition of 5% pectin to rabbit diets containing 5% fat and 1% cholesterol partly repressed the rise of serum β- and α-lipoprotein cholesterol. Interestingly, the addition of pectin to the basal diet after the induction of hypercholesterolemia actually delayed the return of serum cholesterol levels to baseline values. Several mechanisms to explain the hypocholesterolemic action of pectin are discussed.     

Concentration and distribution of apolipoproteins A-I and E in normolipidemic, WHHL and diet-induced hyperlipidemic rabbit sera.

Two sandwich-type enzyme immunoassays have been developed to measure apolipoproteins A-I and E in rabbit serum. Specific goat antibodies were purified by affinity chromatography and used both for coating and for preparing antibody-peroxydase conjugates. The sensitivity of these assays is sufficient to allow studies of apo A-I and E distribution in lipoproteins fractionated by gel filtration from 50 microliters of serum. In WHHL rabbits, apo A-I is 5-fold lower (5.2 +/- 2.5 mg/dl) and apo E is 8-fold higher (9.9 +/- 3.5 mg/dl) than in normolipidemic rabbits (29 +/- 4.3 mg/dl and 1.3 +/- 0.5 mg/dl, respectively). In hyperlipidemic rabbits, fed 2 months on a 0.5% cholesterol diet, the apo A-I level was similar (32 +/- 12 mg/dl) to that of normolipidemic rabbits, but the apo E level is 12-fold higher (15.1 +/- 5.5 mg/dl). In addition, HDL particles were enriched with cholesterol and apo E. The bulk of apo E and cholesterol is located in large beta-VLDL in diet-induced hyperlipidemia, whereas they are mainly located in smaller size beta-VLDL in WHHL rabbits. In normolipidemic rabbits apo E occurs mainly in HDL, and cholesterol is distributed in the main three lipoprotein fractions VLDL, LDL and HDL. Interestingly, HDL of WHHL rabbit are deficient in apo A-I. These results are compatible with profound perturbations of lipoprotein composition and metabolism in atherogenic hyperlipidemia.     

A novel mechanism by which probucol lowers low density lipoprotein levels demonstrated in the LDL receptor-deficient rabbit

Treatment of low density lipoprotein (LDL) receptor-deficient rabbits (WHHL rabbits) with probucol (1% w/w in a chow diet) lowered their LDL-cholesterol levels by 36%, consonant with the reported effectiveness of the drug in patients deficient in the LDL receptor. Initial studies of LDL fractional catabolic rate (FCR) using 125I-labeled LDL prepared from the serum of untreated WHHL rabbits showed no difference between probucol-treated WHHL rabbits and untreated WHHL rabbits. When, however, 125I-labeled LDL was prepared from donor WHHL rabbits under treatment with probucol and injected back into them, the FCR was found to be increased by about 50% above that measured simultaneously using 131I-labeled LDL prepared from untreated WHHL donors. The labeled LDL from probucol-treated donors was also metabolized more rapidly than that from untreated donors when injected into untreated WHHL rabbits or into untreated wild-type New Zealand White rabbits. Finally, it was shown that rabbit skin fibroblasts in culture degraded labeled LDL prepared from probucol-treated WHHL rabbits more rapidly than that prepared from untreated WHHL donors. This was true both for normal rabbit fibroblasts and also for WHHL skin fibroblasts, although the absolute degradation rates in the latter were, of course, much lower for both forms of LDL. The data indicate that a major mechanism by which probucol lowers LDL levels relates not to changes in the cellular mechanisms for LDL uptake or to changes in LDL production but rather to intrinsic changes in the structure and metabolism of the plasma LDL of the probucol-treated animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adipophilin在动脉粥样硬化病变和脂质负荷细胞中的作用研究(英文)

Adipophilin是细胞内脂质聚集和与脂质聚集有关疾病的标志物 ,巨噬细胞源性泡沫细胞的形成是动脉粥样硬化性疾病发生的重要环节 .为了探讨adipophilin在动脉粥样硬化性疾病的作用 ,通过高胆固醇饲料喂养新西兰白兔 12周 ,复制动脉粥样硬化疾病模型 ,同时测定血脂的变化和动脉壁胆固醇 ,使用HE染色、苏丹Ⅳ染色观察动脉粥样硬化病变的形成 ,使用免疫组织化学的方法观察动脉粥样硬化病变处和动物肝脏中adipophilin的表达 .结果发现 ,高胆固醇饲料喂养组血清总胆固醇、低密度脂蛋白胆固醇和动脉壁胆固醇明显增高 ,动脉粥样硬化病变面积增加到 (40 0 6± 7 2 9) % ,动脉粥样硬化病变处adipophilin表达呈阳性 ;而adipophilin在肝脏中的表达无论是高胆固醇饲料喂养组或对照组均为阴性 .使用80mg/L OxLDL与小鼠腹膜巨噬细胞共孵育 ,复制脂质负荷细胞 ,然后把构建的 1mmol/Ladipophilin反义寡核苷酸与该细胞共孵育 .结果发现 ,使用油红O染色观察的细胞内脂滴明显减少 ,生化测定细胞内胆固醇酯显著降低 ,与对照组相比 ,差别有显著性 .说明adipophilin与动脉粥样硬化病变有密切的关系 ,控制adipophilin的表达能够减少巨噬细胞细胞内胆固醇酯的聚集     

Elimination of the atherogenic effect of beta blocker propranolol by papaverine]

Recently, the ability of beta-blockers to stimulate proliferative activity and induce lipid accumulation in cultured human aortic intimal cells has been demonstrated. Moreover, the addition of calcium antagonists completely blocked the increase in proliferative activity and abolished cholesterol accumulation caused by propranolol. In this study blood serum of rabbits treated with 20 mg of propranolol induced 2-fold cholesterol accumulation in mouse peritoneal macrophages. Papaverin did not influence this effect. In case of simultaneous administration of propranolol and papaverin rabbit serum did not exhibit the ability to accumulate intracellular lipids. Propranolol substantially stimulated the formation of myointimal thickening and neutral lipid accumulation in denuded rabbit aorta. Papaverin completely blocked the propranolol-produced atherogenic changes. The data suggest that in vitro and in vivo atherogenic effects of beta-blockers may be prevented by papaverin.     

Uptake of 2,3,7,8-tetrachlorodibenzo-p-dioxin from plasma lipoproteins by cultured human fibroblasts

Tritiated 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) added to human plasma in vitro associated with the plasma lipoproteins. The effects of plasma and lipoproteins on cellular uptake of dioxin were studied using normal human skin fibroblasts and mutant fibroblasts from a patient with homozygous familial hypercholesterolemia. The latter cells lack the normal cell membrane receptor for low density lipoprotein (LDL). The time- and temperature-dependent cellular uptake of [3H]dioxin was greatest from LDL, intermediate from high density lipoprotein (HDL) and least from serum. A significantly greater uptake from LDL by the normal cells compared to the mutant cells indicated the involvement of the LDL receptor-mediated pathway. Concentration-dependent studies indicated that the cellular uptake at 37 degrees C of [3H]dioxin varied linearly with dioxin concentration at constant LDL concentration. Thin-layer chromatography (TLC) showed that conversion to more polar compounds may have occurred after 24-h incubation with cells. [3H]Dioxin could be removed from cells efficiently by incubation with 20% serum greater than HDL greater than LDL. Since the vehicle of delivery may influence subsequent location and metabolism of this compound in cells, it is concluded that the physiologic vehicles (either serum- or LDL-associated dioxin), rather than organic solvents, should be used in experiments with cultured cells or perfused organs.     

A defect in mobilization of cholesteryl esters in rabbit macrophages

Macrophages provide an important way for cholesteryl esters to accumulate in tissues in pathologic amounts. We studied cholesteryl ester metabolism in thioglycollate-induced peritoneal macrophages obtained from normocholesterolemic and hypercholesterolemic rabbits. The macrophage preparations from normocholesterolemic rabbit (MN cells) had 26 nmol esterified cholesterol/mg cellular protein, incorporated 1 nmol of labeled oleate into cholesteryloleate/2 h per mg cellular protein and had an acyl-coenzyme A:cholesterol acyltransferase activity of 22 pmol cholesterylpalmitate formed/min per mg protein in isolated membranes. The macrophage preparations from hypercholesterolemic rabbits (MHC cells) contained a 12-fold greater mass of cholesteryl ester, had an 8-times higher rate of formation of cholesteryloleate, and had 3-times more acyl-coenzyme A:cholesterol acyltransferase activity in the isolated membranes. When a cholesterol acceptor (10% fetal bovine serum or 10 mg of lipid-free fetal bovine serum protein) was added to the culture medium of rabbit MHC cells, the MHC cells retained more than 70% of their cholesteryl esters after 48 h of incubation. In contrast, when a cholesterol acceptor (10% fetal bovine serum) was added to the medium of thioglycollate-induced, cholesterol-enriched macrophages from mice, the mice macrophages retained only 19% of their cholesteryl esters after 48 h of incubation. The limited capacity of rabbit macrophages to release unesterified cholesterol from stored cytoplasmic cholesteryl esters to an exogenous acceptor may be related to the propensity of rabbits to develop atherosclerotic lesions.