Y. Dabach

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.

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.

Cholesteryl ester transfer activity in hamster plasma: increase by fat and cholesterol rich diets

We investigated the presence of cholesteryl ester transfer activity (CETA) in plasma of hamsters kept on various dietary regimens. In hamsters kept on a regular diet, CETA activity was about 5 units/4 mg protein of d greater than 1.21 g/ml fraction of plasma, as compared to about 35 units present in human d greater than 1.21 g/ml fraction. Addition of 15% margarine or butter alone or together with 2% cholesterol resulted in a 2-3-fold increase in plasma CETA. The increase in plasma CETA was correlated with plasma cholesterol levels (r = 0.78; P less than 0.001) and plasma triacylglycerol levels (r = 0.56, P less than 0.001). Hamsters consuming the cholesterol + butter-supplemented diets had the highest plasma CETA, cholesterol and triacylglycerol levels, while CETA in plasma of rats and mice remained nondetectable even after 4 weeks on the diet. The causal relation between hypercholesterolemia, hypertriglyceridemia and evaluation in CETA in hamsters remains to be elucidated.

Modulation of sphingomyelinase-induced cholesterol esterification in fibroblasts, CaCo2 cells, macrophages and smooth muscle cells.

The present study has focused on three questions concerning the effect of sphingomyelinase on release of free cholesterol from the plasma membrane and its intracellular translocation: (i) Can one change the direction of the flow of cholesterol? (ii) Can one modulate the flow? (iii) May such a mechanism be relevant in atherogenesis? (i) The results obtained show that even in the presence of potent nonlipoprotein cholesterol acceptors in the medium, the intracellular flow of cholesterol is not reduced as measured by cholesterol esterification. Moreover, in sphingomyelinase-treated cells, cholesterol efflux in presence of nonlipoprotein acceptors was not enhanced even when intracellular esterification was inhibited. (ii) Modulation of the sphingomyelinase induced cholesterol flow can be obtained by 100 microM verapamil which reduces it. In human skin fibroblast, interference with the delivery of free cholesterol to its site of esterification was found in the presence of brefeldin A. (iii) Aortic smooth muscle cells in culture are sensitive to low concentrations of sphingomyelinase and the increase in esterified cholesterol is evident also after exposure to the enzyme for 24 h. The present results suggest that in the plasma membrane, free cholesterol bound to sphingomyelin may be in a compartment which renders it more available for transport to the cell interior than for efflux. In view of the sensitivity of aortic smooth muscle cells to sphingomyelinase, this mechanism for enhanced esterification of cholesterol could be relevant to the transformation of arterial smooth muscle cells into foam cells in the process of atherogenesis.

Scavenger receptor activity and expression of apolipoprotein E mRNA in monocyte-derived macrophages of young and old healthy men

The aim of this study was to compare some aspects of lipid metabolism in monocyte-derived macrophages isolated from young males, aged 18-24 years, and old males, aged 74-90 years, who were found healthy in accordance with the Senieur protocol. The parameters tested were metabolism of 125I-acetylated low-density lipoproteins (LDL) and oxidized LDL, incorporation of [3H]cholesterol into cholesteryl ester and expression of apolipoprotein E (apo E) mRNA. Cell association and degradation of 125I-acetylated LDL by macrophages of old and young subjects, respectively, was 15,978 +/- 2492 and 9300 +/- 1416 ng/mg cell protein per 24 h. Incorporation of [3H]cholesterol into cellular [3H]cholesteryl ester in the presence of acetylated LDL in cells isolated from old subjects was twice that in cells from young subjects. The macrophages from both age groups metabolized less 125I-oxidized LDL than 125I-acetylated LDL. Cell association and degradation of 125I-oxidized LDL in cells from old and young subjects, respectively, was 6779 +/- 1398 and 3219 +/- 643 ng/mg cell protein per 24 h. Expression of apo E mRNA was determined by reverse transcriptase polymerase chain reaction. In the basal state, it was 5.8 +/- 0.4 and 2.4 +/- 0.2 photo-stimulated luminescence (PSL) units in cells from the old and young subjects, respectively, and increased after exposure to acetylated LDL. In conclusion, these findings suggest that a combination of higher scavenger receptor activity and increased expression of apo E mRNA in macrophages could contribute to (a) enhanced metabolism of modified LDL and (b) more efficient removal of cholesterol from arteries, thus leading to healthy old age.

Reverse cholesterol transport in mice expressing simian cholesteryl ester transfer protein

The role of cholesteryl ester transfer protein (CETP) in atherogenesis remains ambiguous, as both pro and antiatherogenic effects have been described. Expression of CETP increases HDL-cholesteryl ester turnover, but there is no direct evidence whether CETP mobilizes cholesterol in vivo. The rate of cholesterol removal injected into a leg muscle as cationized low density lipoprotein (cat-LDL) was compared in CETP transgenic and control mice. Four days after injection the exogenous cholesterol mass retained in muscle was 65% in CETP transgenic and 70% of injected dose in controls; it decreased to 52-54% by day 8 and negligible amounts remained on day 28. The cat-LDL was labeled with either 3H-cholesterol oleate (3H-CE) or 3H-cholesteryl oleoyl ether (3H-COE), a nonhydrolyzable analog of 3H-CE. After injection of 3H-CE cat-LDL, clearance of 3H-cholesterol had a t(1/2) of 4 days between day 4 and 8 but there was little loss of 3H-COE between day 4 and 51. Liver radioactivity on day 4 was 1.7% in controls and 3.4% in CETP transgenics; it was 2.8 and 4.6%, respectively, on day 8. 3H-COE in liver accounted for 60% of label in CETP transgenics. In conclusion, high levels of plasma CETP in mice do not enhance reverse cholesterol transport in vivo but may act on extracellularly located cholesteryl ester.

High levels of human apolipoprotein A-I and high density lipoproteins in transgenic mice do not enhance efflux of cholesterol from a depot of injected lipoproteins Relevance to regression of atherosclerosis?

The role of high density lipoprotein (HDL) and apolipoprotein A-I (apo A-I)in promoting cholesterol efflux from cultured cells and attenuation of development of atherosclerosis in transgenic (tg) animals has been well documented. The aim of the present study was to determine whether high levels of human (h) apo A-I will enhance cholesterol removal in vivo. h apo A-I in sera of tg mice was 429 +/- 18 and 308 +/- 10 mg/dl in male and female mice, the ratio of phospholipid (PL) to apo A-I was 0.94 in tg and 2.4 and 1.9 in male and female controls, taking mouse apo A-I as 100 mg/dl. The removal of lipoprotein cholesterol injected in the form of cationized low density lipoprotein (cat-LDL) into the rectus femoris muscle of h apo A-I tg is compared with control mice. After injection of cat-LDL labeled with [3H]cholesterol, the labeled cholesterol was cleared from the depot with a t 1/2 of about 4 days in both control and tg mice. The clearance of the exogenous cholesterol mass was initially much slower, it approached the t 1/2 of about 4 days between day 8 and 14 but there was no difference between tg and control mice. Cholesterol efflux from cultured macrophages exposed to media containing up to 10% serum was 56% higher with serum from tg mice than controls. In conclusion, the efflux of cholesterol from a localized depot of cat-LDL was not enhanced in h apo A-I tg mice. It appears, therefore, that while an increase above physiological levels of apo A-I or plasma HDL does play a pivotal role in the prevention of initiation and progression of early stages of atherosclerosis, the effectiveness of such an increase for the regression stage remains still to be demonstrated.

Relative resistance of the hamster to aortic atherosclerosis in spite of prolonged vitamin E deficiency and dietary hypercholesterolemia. Putative effect of increased HDL

UNLABELLED Male golden hamsters were rendered hypercholesterolemic by feeding diets enriched with cholesterol and fat. In the first series of experiments, 5% butter and 1% cholesterol were added to a chow diet and plasma cholesterol levels were maintained at 350-390 mg/dl over the entire experimental period. Groups of hamsters and their age controls consuming the chow diet, were killed after 7, 15 and 20 months when the aorta was examined for atherosclerosis by determination of cholesterol mass. In the controls, aortic total cholesterol (TC) increased with age by 28% and esterified cholesterol increased to 11% of TC. In the hypercholesterolemic animals aortic TC was only 28% higher than in the controls and cholesteryl ester was also 11.5% of TC. In the second series, one group of hamsters were fed a semi-purified diet deficient in vitamin E, containing 1% cholesterol and 10% lard; a second group received the same diet, but supplemented with vitamin E. Controls consumed local chow. After 7 months on the vitamin E deficient diet plasma alpha-tocopherol was 0.05 mg/l, in those supplemented with vitamin E it was 20 mg/l, while in the controls it was 3.3 mg/l. Plasma thiobarbituric acid reactive substances (TBARS) were higher in the vitamin E deficient group and there was a greater propensity of lipoproteins (d < 1.063 g/ml) to peroxidation in vitro than in the vitamin E supplemented group. Plasma cholesterol was 366 mg/dl in the vitamin E deficient, 336 mg/dl in the vitamin E supplemented group, and 64 mg/dl in controls. Aortic cholesterol was 79.1 in vitamin E supplemented and 84.4 micrograms/10 mg dry weight in vitamin E deficient hamsters. In both series of experiments, HDL amounted to 36-41% of plasma TC in the hypercholesterolemic animals and 59-62% in the controls. IN CONCLUSION the hamster appears to be quite resistant to atherosclerosis in face of sustained hypercholesterolemia, even in the presence of increased peroxidative stress caused by vitamin E deficiency. This relative resistance could be related to commensurate increase in plasma HDL which was observed in both series of experiments. Since vitamin E deficiency did not enhance aortic cholesteryl ester deposition, the protective effect of HDL seems to be related to its role in reverse cholesterol transport, rather than in prevention of peroxidation.