O. Stein

Atheroprotective mechanisms of HDL

The aim of this review was to bring together results obtained from studies on different aspects of HDL as related to CHD and atherosclerosis. As atherosclerosis is a multistep process, the various components of HDL can intervene at different stages, such as induction of monocyte adhesion molecules, prevention of LDL modification and removal of excess cholesterol by reverse cholesterol transport. Transgenic technology has provided a model for atherosclerosis, and permitted evaluation of the contributions of different HDL components towards the global effect. The availability of apo AIV transgenic mice amplified the results obtained from apo AI overexpressors with respect to prevention of atherosclerosis. Prevention of atherosclerosis in apo E deficient mice by relatively small amounts of macrophage derived apo E may open new possibilities for therapeutic intervention. Contrary to early notions, increased plasma levels of CETP, even in the presence of low but functionally normal HDL, were atheroprotective. The extent to which paraoxonase and apo J participate in prevention of human atherosclerosis needs further evaluation. The findings that LCAT overexpression in rabbits was atheroprotective in contrast to increase in atherosclerosis in h LCAT tg mice, which was only partially corrected by CETP expression, call for some caution in the extrapolation of results from transgenic animals to humans. The important discovery of SR-BI as the receptor for selective uptake of CE from HDL revived interest in the clearance of CE from plasma. This pathway supplies also the vital precursor for steroidogenesis in adrenals and gonads and was shown to be dependent on apo AI.

The role of apolipoprotein A-IV in reverse cholesterol transport studied with cultured cells and liposomes derived from an ether analog of phosphatidylcholine.

Cholesterol efflux was studied in a model system in culture using apolipoproteins and phospholipids added in the form of liposomes at concentrations expected to be present in the extracellular fluid. Fibroblasts were seeded in medium containing [3H]cholesterol-labeled serum, grown till confluent, and the [3H]cholesterol efflux was studied in serum-free medium. Addition of delipidated HDL apolipoprotein resulted in a very low release of [3H]cholesterol, which did not increase with time of exposure or concentration of apolipoproteins. Addition of increasing amounts of HDL apolipoprotein to liposomes prepared from either dioleoylphosphatidylcholine (PC) or its nonhydrolysable ether analog, dioleylphosphatidylcholine (DOEPC) resulted in a 3-5-fold increase of [3H]cholesterol efflux, over that achieved with liposomes alone. This model system permitted the test of the putative role of apolipoprotein A-IV in cholesterol removal from cells. The ability of apolipoprotein A-IV to enhance [3H]cholesterol efflux from cells by DOEPC liposomes was compared to that of apolipoproteins A-I, E and C, which were added at equimolar concentrations. At nM concentrations, apolipoproteins A-IV, A-I and E were equally able to enhance cholesterol efflux, while C apolipoproteins were less effective at these low concentrations. Mixtures prepared from apolipoprotein A-IV, A-I and E and PC or DOEPC liposomes were equally effective in cholesterol removal, while phosphatidylethanolamine liposome apolipoprotein mixtures had a much lower capacity. The present study provides the first evidence that apolipoprotein A-IV can play a role in reverse cholesterol transport as was suggested on the basis of high concentrations of this apolipoprotein in nonlipoprotein form in plasma and extracellular fluid. The efficacy of DOEPC liposomes to serve as cholesterol acceptors might be of potential value for enhancement of reverse cholesterol transport in vivo.

Cigarette smoking renders LDL susceptible to peroxidative modification and enhanced metabolism by macrophages

The effect of cigarette smoking on peroxidation of plasma low density lipoprotein (LDL) was studied in 16 smokers aged 23-56 years; 12 nonsmokers of similar age served as controls. The smokers were asked to refrain from smoking 24-40 h prior to testing. Peroxidation was assessed by determination of thiobarbituric acid reactive substances (TBARS) in fresh plasma and LDL and by an increase in 125I-LDL metabolism by peritoneal macrophages. There was no difference in TBARS in freshly prepared plasma or LDL of smokers and nonsmokers. However, LDL of smokers, conditioned by incubation with bovine aortic smooth muscle cells (SMC), had 2-fold or higher TBARS values when compared to SMC conditioned LDL of nonsmokers. 2-4-fold higher TBARS values were seen also in SMC conditioned LDL when the comparison was made between LDL isolated from plasma before smoking of 6-7 cigarettes (time 0), and LDL of the same individual isolated from plasma 90 min thereafter. The metabolism of SMC conditioned 125I-LDL by peritoneal macrophages was examined; LDL isolated from plasma of smokers at time 0 was metabolized twice as avidly as LDL of nonsmokers and a further increase was seen with LDL isolated 90 min after acute smoking. The present results indicate that cigarette smoking renders plasma LDL more susceptible to subsequent peroxidative modification by cellular elements.

Bovine aortic endothelial cells display macrophage-like properties towards acetylated 125I-labelled low density lipoprotein

Bovine aortic endothelial cells in culture were shown to take up and degrade acetylated 125I-labelled low density lipoproteins (125I-acetylated LDL) in preference to 125I-labelled low density lipoproteins (LDL). The confluent cultures of endothelial cells had a higher rate of degradation of 125I-acetylated LDL than did subconfluent cells. The ratio of degradation of 125I-acetylated LDL to 125I-LDL was 3--9 in the case of the endothelial cells, 0.06--0.11 for aortic smooth muscle cells and 18 for mouse peritoneal macrophages. The uptake and degradation of acetylated LDL by the endothelial cells was accompanied also by an increase in cellular cholesterol. The present findings indicate that cultured endothelial cells display certain macrophage-like properties towards serum lipoproteins.

Effect of vitamin C and E supplementation on susceptibility of plasma lipoproteins to peroxidation induced by acute smoking

The effect of acute smoking on plasma lipoproteins was studied in seventeen smokers. In study 1, 7 subjects were examined prior to and 2 weeks after supplementation with vitamin C. In study 2, the effect of acute smoking was first determined in 10 additional subjects and subsequently they were divided into 3 groups, 3 and 4 subjects were supplemented with vitamin C or E, respectively, for 4 weeks, and 3 remained untreated. Plasma and LDL TBARS were examined at time zero (i.e., 40-48 h after total abstention from smoking) and at 90 min after acute smoking (5-7 cigarettes). In all 17 subjects examined prior to vitamin supplementation, significantly higher TBARS values were found in plasma, native LDL and LDL conditioned with smooth muscle cells (SMC) when the 90 min values were compared to 0 time. The LDL isolated after 90 min and conditioned with SMC was metabolized more extensively by mouse peritoneal macrophages than its zero time counterpart. The differences between the 0 time and 90 min values were not seen after the subjects had been supplemented with vitamin C for 2 or 4 weeks or with vitamin E for 4 weeks. The present results indicate that acute smoking exerts an oxidative stress on plasma lipoproteins and that higher plasma levels of natural antioxidants, such as vitamins C and E have a protective role.

Metabolism of HDL-cholesteryl ester in the rat, studied with a nonhydrolyzable analog, cholesteryl linoleyl ether

Intralipid was sonicated with [3H]cholesteryl linoleyl ether (a nonhydrolyzable analog of cholesteryl linoleate) and incubated with rat HDL and d greater than 1.21 fraction of rabbit serum at a ratio of 0.012 mg triacylglycerol to 1 mg HDL protein. 25% of [3H]cholesteryl linoleyl ether was transferred to HDL. The labeled HDL was injected into donor rats and was screened for 4 h. [125I]HDL was subjected to the same protocol as the 3H-labeled HDL, including screening. The screened, labeled sera were injected into acceptor rats and the disappearance of radioactivity from the circulation was compared. The t1/2 in the circulation of [125I]HDL was about 10.5 h, while that of [3H]cholesteryl linoleyl ether-HDL was about 8 h. The liver and carcass were the major sites of uptake of [3H]cholesteryl linoleyl ether-HDL and accounted for 29-41% (liver) and 30% (carcass) of the injected label. Maximal recovery of [3H]cholesteryl linoleyl ether in the liver was seen 48 h after injection, and thereafter there was a progressive decline of radioactivity, which reached 7.8% after 28 days. The maximal recovery of [125I]HDL in the liver was about 9%. Pretreatment of the acceptor rats with estradiol for 5 days resulted in a 20% increase in the hepatic uptake of [3H]cholesteryl linoleyl ether-HDL and a 5-fold increase in adrenal uptake. The present findings indicate that in the rat the liver is the major site of uptake of HDL cholesteryl ester and that part of the HDL cholesteryl ester may be cleared from the circulation separately from the protein moiety. On the basis of our previous findings (Stein, Y., Kleinman Y, Halperin, G., and Stein, O. (1983) Biochim. Biophys. Acta 750, 300-305) the loss of the [3H]cholesteryl linoleyl ether from the liver after 14-28 days was interpreted to indicate that the labeled [3H]cholesteryl linoleyl ether had been taken up by hepatocytes.

Fish oil ingestion in smokers and nonsmokers enhances peroxidation of plasma lipoproteins

The effect of fish oil ingestion (10 g MaxEPA/day) on the susceptibility of plasma lipoproteins to peroxidation was examined in 20 smokers (study A and B) and 22 nonsmokers (study C). The subjects were examined at the onset of each study (baseline values), divided into control and experimental groups and reexamined 4 weeks later. Smokers were examined 40 h after abstention from smoking (0 time) and 90 min after acute smoking (4-6 cigarettes). The parameters studied were TBARS, which provide an indication of peroxidative injury, and metabolism of conditioned LDL by macrophages as a biological indicator. These parameters were significantly higher (P less than 0.05-0.001) when the 90 min values of smokers were compared to time 0. After 4 weeks of fish oil ingestion, a significant rise above baseline values (33-50%) in plasma and LDL TBARS was found in smokers examined at time 0 and after acute smoking. Peroxidative modification of LDL isolated from smokers fed fish oil resulted in significantly higher TBARS (34-41%) and its metabolism by macrophages was higher (65-139%) compared to baseline values. In nonsmokers, the baseline values of the above parameters were lower than in smokers. Ingestion of fish oil resulted in a significant rise in TBARS in plasma (33%), LDL (137%), conditioned LDL (36-40%) and metabolism of conditioned LDL (70%) by macrophages. In 6 nonsmokers and 4 smokers, 400 mg of vitamin E/day were given with the fish oil. In the nonsmokers, vitamin E counteracted the effect of fish oil more effectively than in the smokers. In the light of the present results, indiscriminate recommendation of fish oil supplementation to the population at large should be cautioned.

High density lipoproteins reduce the uptake of low density lipoproteins by human endothelial cells in culture

Endothelial cells, explanted from human umbilical veins and cultured, maintained morphological characteristics of vascular endothelium. When exposed to human serum lipoproteins, the cells bound and took up low density lipoproteins in preference to high density lipoproteins. High density lipoproteins reduced markedly the uptake of low density lipoproteins and affected surface binding to a lesser extent. These data suggest that the different levels of high density lipoprotein encountered in normal plasma of males and females could modulate differently the transendothelial transport of low density lipoproteins and provide a possible explanation for the lesser severity of atheromatosis in the aortic intima of premenopausal females.

Colchicine-induced inhibition of very low density lipoprotein release by rat liver In vivo

Abstract The role of microtubules in the secretion of very low density lipoproteins by rat liver was studied with the help of colchicine. Pretreatment of female rats for 180 min with 0.1 and 1.0 mg/100 g of body weight of colchicine resulted in a 50% and 80% inhibition, respectively, of the release of labeled triglyceride into the serum, 90 min after administration of [ 14 C] palmitic acid and Triton WR 1339. In the colchicine-treated rats serum triglycerides levels were 50–80% lower than in the controls. In the liver, accumulation of Golgi-derived secretory vesicles containing lipoprotein granules indicated that colchicine had blocked the final step of lipoprotein release from the liver, which might be dependent on integrity of the microtubular system.

Reassembly of Mycoplasma membranes disaggregated by detergents.

Abstract Plasma membranes of Mycoplasma laidlawii and Mycoplasma gallisepticum were solubilized by several ionic and non-ionic detergents. Solubilization of the membranes by sodium dodecyl sulfate (SDS) separated membrane lipid from protein as demonstrated by polyacrylamide gel electrophoresis of the solubilized material. The solubilized membrane material reaggregated spontaneously on removal of the detergent by dialysis or by Sephadex G-25, and formed vesicles limited by a triplelayered membrane of about the same thickness as the original Mycoplasma membrane. A divalent cation (e.g., Mg2+) was essential for membrane reassembly. The ratio of lipid to protein in membrane reaggregates varied considerably according to the Mg2+ concentration. At a low Mg2+ concentration reaggregates contained a higher percentage of lipid. The present results seem to bear out the suggestion of Engelman et al. [Biochim. Biophys. Acta135, 381 (1967)] that the SDS-solubilized membrane material does not consist of homogeneous lipoprotein subunits but of separate SDS-lipid and SDS-protein complexes. The reassembly of solubilized membrane lipid and protein on removal of the detergent indicates that these components contain sufficient structure-determining information to interact spontaneously in the presence of Mg2+ and produce membraneous structures.