Richard W. St. Clair

Influence of Duration of Cholesterol Feeding on Esterification of Fatty Acids by Cell-Free Preparation of Pigeon Aorta

Influence of duration of cholesterol feeding on esterification of fatty acids and hydrolysis of cholesteryl esters was studied in cell-free preparations of aorta from White Carneau pigeons. Esterification of fatty acids required ATP and CoA; greater than 80% of the esterifying activity was located in the particulate fraction obtained by centrifugation at 105,000×g (after a preliminary centrifugation at 1000×g). Fatty acids were incorporated most efficiently into phospholipid, primarily (82%) lecithin. Greater than 87% of the fatty acid was esterified at the 2-position. During 8 months of cholesterol feeding, incorporation of oleic acid into phospholipids and triglycerides increased relatively little (less than double that of controls); no changes were seen before 1 month. Esterification of oleic acid to cholesterol was increased after 2 weeks of cholesterol feeding (before gross lesions were seen), eventually reaching a maximum increase of 30- to 50-fold. Cholesterol was esterified by transfer of fatty acyl-CoA to cholesterol, a mechanism similar to that described for liver and adrenal cortex. Little if any cholesterol esterification occurred when lecithin labeled at the 2-position with oleic acid-l-14C was used as substrate. The relationship between duration of cholesterol feeding and hydrolysis of cholesteryl oleate could not be evaluated since results depend directly on an unknown extent of equilibration of substrate with pre-existing cholesteryl ester pools.

Macrophage-specific transgenic expression of cholesteryl ester hydrolase significantly reduces atherosclerosis and lesion necrosis in Ldlr–/– mice

Accumulation of cholesteryl esters (CEs) in macrophage foam cells, central to atherosclerotic plaque formation, occurs as a result of imbalance between the cholesterol influx and efflux pathways. While the uptake, or influx, of modified lipoproteins is largely unregulated, extracellular acceptor-mediated free cholesterol (FC) efflux is rate limited by the intracellular hydrolysis of CE. We previously identified and cloned a neutral CE hydrolase (CEH) from human macrophages and demonstrated its role in cellular CE mobilization. In the present study, we examined the hypothesis that macrophage-specific overexpression of CEH in atherosclerosis-susceptible Ldlr(-/-) mice will result in reduction of diet-induced atherosclerosis. Transgenic mice overexpressing this CEH specifically in the macrophages (driven by scavenger receptor promoter/enhancer) were developed and crossed into the Ldlr(-/-) background (Ldlr(-/-)CEHTg mice). Macrophage-specific overexpression of CEH led to a significant reduction in the lesion area and cholesterol content of high-fat, high-cholesterol diet-induced atherosclerotic lesions. The lesions from Ldlr(-/-)CEHTg mice did not have increased FC, were less necrotic, and contained significantly higher numbers of viable macrophage foam cells. Higher CEH-mediated FC efflux resulted in enhanced flux of FC from macrophages to gall bladder bile and feces in vivo. These studies demonstrate that by enhancing cholesterol efflux and reverse cholesterol transport, macrophage-specific overexpression of CEH is antiatherogenic.

MEtabolism by cells in culture of low-density lipoproteins of abnormal composition from non-human primates with diet-induced hypercholesterolemia

Diet-induced hypercholesterolemia in non-human primates results in the production of a low-density lipoprotein (LDL) of abnormal size and composition. This LDL from hypercholesterolemic monkeys has been shown to be more atherogenic than the same amount of LDL from normocholesterolemic animals. Previous studies have demonstrated that hypercholesterolemic LDL is approximately twice as effective as normal LDL in stimulating cholesterol accumulation and esterification in arterial smooth muscle cells in culture. The purpose of the present study was determine whether this effect was secondary to differences in metabolism of the normal and hypercholesterolemic LDL. for this, the metabolism of 125I-labeled normal and hypercholesterolemic LDL from rhesus and cynomolgus monkeys was compared in several lines of skin fibroblasts and smooth muscle cells. Both normal and hypercholesterolemic LDL bound with high affinity to the same cell surface receptor. However, the affinity for binding of hypercholesterolemic LDL was about twice that of normal LDL (apparent dissociation constant for binding, Kd, was 2.63 micrograms protein/ml and 4.35 micrograms protein/ml, respectively). Conversely, only about 50% as many particles of hypercholesterolemic were able to bind to the receptor, compared with normal LDL. Those cells with the greatest capacity to metabolize LD generally accumulated the most cholesterol with either hypercholesterolemic or normal LDL. In all cell lines, nearly twice as much cholesterol accumulated in cells incubated with hypercholesterolemic LDL compared with normal LDL, and this differential could not be explained by differences in metabolism of the two lipoproteins, suggesting that some cholesterol entered the cells independent of the uptake of the intact LDL molecule. LDL receptors appear necessary for this to occur, since no difference in cholesterol accumulation was observed in cells genetically deficient in LDL receptors.

Histone Deacetylase 9 Represses Cholesterol Efflux and Alternatively Activated Macrophages in Atherosclerosis Development

Objective— Recent genome-wide association studies revealed that a genetic variant in the loci corresponding to histone deacetylase 9 (HDAC9) is associated with large vessel stroke. HDAC9 expression was upregulated in human atherosclerotic plaques in different arteries. The molecular mechanisms how HDAC9 might increase atherosclerosis is not clear. Approach and Results— In this study, we show that systemic and bone marrow cell deletion of HDAC9 decreased atherosclerosis in LDLr−/− (low density lipoprotein receptor) mice with minimal effect on plasma lipid concentrations. HDAC9 deletion resulted upregulation of lipid homeostatic genes, downregulation of inflammatory genes, and polarization toward an M2 phenotype via increased accumulation of total acetylated H3 and H3K9 at the promoters of ABCA1 (ATP-binding cassette transporter), ABCG1, and PPAR-&ggr; (peroxisome proliferator-activated receptor) in macrophages. Conclusions— We conclude that macrophage HDAC9 upregulation is atherogenic via suppression of cholesterol efflux and generation of alternatively activated macrophages in atherosclerosis.

Serum cholesterol efflux potential is an independent predictor of coronary artery atherosclerosis

The efflux of cholesterol from cells and its incorporation into HDL is believed to be the initial step in reverse cholesterol transport. This report addresses the question of whether there is a relationship between the ability of serum to promote efflux of cholesterol from cells in culture and the severity of coronary artery atherosclerosis (CAA). Surgically postmenopausal cynomolgus monkeys (n=142) were treated for 2-years with conjugated equine estrogens (CEE), CEE plus medroxyprogesterone acetate, or two different doses of tibolone, a synthetic steroid. CAA was determined at necropsy, and the cholesterol efflux potential of serum from each animal was determined using 3H-cholesterol-labeled Fu5AH cells and human skin fibroblasts in culture. A significant negative correlation was seen between CAA and cholesterol efflux from Fu5AH cells (r=-0.44, P< or =0.0001), but not skin fibroblasts. Although there was a wide range of plasma HDL cholesterol concentrations in these animals (10-81 mg/dl), using multiple regression analysis, LDL+VLDL cholesterol and the serum cholesterol efflux potential were the only significant independent predictors of CAA, explaining 41.6 and 10.7% of the variability (P<0.0001), respectively. Thus, the potential of serum to promote cholesterol efflux from Fu5AH cells may represent a useful independent measure for improving the assessment of CAA risk.

Effects of Regression of Atherosclerotic Lesions on the Content and Esterification of Cholesterol by Cell-Free Preparations of Pigeon Aorta

This study was designed to determine the effect of regression of atherosclerotic lesions on the incorporation of 1-14C-oleic acid into phospholipids, triglycerides, and cholesteryl esters and to compare these metabolic alterations with changes in the extent of atherosclerosis and the content of cholesterol and cholesteryl esters in the lesions. Aortic atherosclerosis was produced in White Carneau pigeons by feeding them an atherogenic diet for 1−8 months. The birds were then switched to a cholesterol-free diet for 6 months for the regression phase of the experiment. Following the regression phase, no changes were noted in the atherosclerotic index or the free-cholesterol content of aortas from pigeons that had received the atherogenic diet for 1−5 months; however, a reduction in both of these parameters was seen in aortas from pigeons fed the atherogenic diet for 8 months. There was a marked reduction in the content of cholesteryl esters in the aortas following the regression phase paralleled by a decrease in the rate of cholesterol esterification. No change was seen in the rate of incorporation of fatty acid into phospholipids or triglycerides. These studies suggest that local cholesterol esterification might be of considerable importance in maintaining the large amount of cholesteryl esters found in the atherosclerotic lesion and that changes in the rate of cholesterol esterification are associated with the early events in both progression and regression of atherosclerosis.

Synthesis of squalene and sterols by isolated segments of human and pigeon arteries

Abstract The ability of isolated arterial segments from man and pigeon to synthesize squalene and sterols from dl -mevalonate-2-14C has been studied by an in vitro perfusion technique. Following a 4-hour perfusion, more than 99% of the lipid radioactivity was contained in two major fractions after chromatography on alumina. The less polar fraction, accounting for 30–60% of the total lipid radioactivity in arterial segments from pigeon and man respectively, was shown to be more than 95% squalene. Squalene synthesis in the pigeon was positively and significantly correlated with the severity of atherosclerosis. Because of the highly variable postmortem interval, no such correlation was attempted for human arterial segments. The polar fraction from the alumina column, although possessing properties similar to cholesterol, contained on the order of 1% cholesterol when purified through the dibromide. The identity of the major portion of radioactivity in this fraction remains unknown.

Stimulation of cholesterol esterification in vitro in organ cultures of normal pigeon aorta

Abstract The ability of cholesterol esterification to be stimulated in vitro was studied in organ cultures of White Carneau pigeon aorta. Aortic segments from pigeons, less than four months of age and without grossly visible atherosclerosis, were maintained in organ culture with tissue culture medium containing from 5–60% normo- (NCS) or hypercholesterolemic (HCS) pigeon serum. Esterification of oleic acid-1-14C and cholesterol-1,2-3H was measured after 1–9 days in culture. Incorporation of oleic acid-1-14C into phospholipids and triglycerides was stimulated a maximum of 2-fold in cultures containing HCS. This stimulation was influenced by the oleic acid content of the culture medium with increased incorporation into phospholipids and triglycerides occurring when cultured in media containing higher concentrations of oleic acid. Under the same conditions, esterification of oleic acid-1-14C to cholesterol was stimulated as much as 7-fold and was independent of both the concentration of oleic acid in the culture medium and the uptake of oleic acid-1-14C into the arterial segments. Stimulation of esterification to cholesterol was seen as early as 1 day after culture with HCS. When different concentrations of NCS or HCS were added to the culture medium the stimulation of cholesterol esterification was proportional to the concentration of cholesterol in the culture medium up to a concentration of 2–4 mg/ml. Associated with the increased cholesterol esterification was an increase in the cholesteryl ester content of the arterial segments and the appearance of both intra- and extracellular lipid droplets.

Effect of sucrose polyester on plasma lipids and cholesterol absorption in African green monkeys with variable hypercholesterolemic response to dietary cholesterol

Abstract The effect of sucrose polyester (SPE), a nonabsorbable fat, on plasma lipids and cholesterol absorption was studied in 29 adult male African green monkeys whose plasma cholesterol concentrations ranged from 133 to 358 mg/dl while consuming a diet containing 0.36 mg cholesterol/kcal. SPE was added to this diet at concentrations equivalent to doses for human beings of 15, 30, and 45 g/day. All 29 animals were fed either no SPE or one of the three concentrations of SPE for 2-mo intervals in a Latin square experimental design which ultimately allowed each animal to receive all dietary treatments. The lowest concentration of SPE reduced absorption of dietary cholesterol in all animals by an average of 16%. Increasing the concentration of SPE did not further decrease cholesterol absorption. Plasma cholesterol concentrations, however, were reduced by SPE only in the most hypercholesterolemic animals. The plasma cholesterol lowering effect was progressively less in animals that were less responsive to dietary cholesterol, such that in animals with plasma cholesterol concentrations less than 200 mg/dl there was no effect of SPE. The cholesterol lowering effect of SPE was due entirely to a lowering in LDL, with no effect on HDL cholesterol or triglycerides. Animals with the greatest plasma cholesterol response (hyperresponders) absorbed a significantly higher percentage of dietary cholesterol than did hyporesponders (56% vs. 37%). When cholesterol absorption was decreased in hyperresponders by SPE to levels equivalent to hyporesponders, the hyperresponders still maintained higher plasma cholesterol concentrations. Thus, the difference in cholesterol absorption appears to only partially explain the individuality in plasma cholesterol response to dietary cholesterol. These results also suggest that SPE may be a particularly useful drug in lowering plasma cholesterol concentrations in individuals that are hyperresponsive to dietary cholesterol.

Cholesterol esterification and cholesteryl ester accumulation in cultured pigeon and monkey arterial smooth muscle cells

Abstract Aortic smooth muscle cells from atherosclerosis-susceptible White Carneau and resistant Show Racer pigeons were grown in culture utilizing conditions identical to those developed for the culture of rhesus monkey arterial smooth muscle cells and skin fibroblasts. Pigeon smooth muscle cells had ultrastructural and growth characteriscis similar to mammalian smooth muscle cells in culture including growth in multiple overlapping layers, numerous pinocytic vesicles, and abundant myofilaments. Cells were incubated for 24 to 72 hr with culture medium containing either lipoprotein deficient serum, fetal calf serum, normocholesterolemic pigeon serum or hypercholesterolemic pigeon serum, and differences in cholesterol content and in the rate of cholesterol esterification were studied. Although there was substantial variability in the absolute cholesterol content among different cell lines, cells of the same type behaved similarly in their response to the various test sera. Generally, the higher the cholesterol content of the culture medium the greater the cholesterol content of the cells. There were, however, considerable differences in the magnitude and pattern of response among the different cell types. Incubation with 10% hypercholesterolemic serum resulted in an increase of similar magnitude in the free cholesterol content of all cell lines. This was not true, however, for the accumulation of cholesteryl esters. Incubation with 10% hypercholesterolemic serum produced a marked increase in the cholesteryl ester content of monkey skin fibroblasts and smooth muscle cells while producing only a slight increase in the cholesteryl ester content of pigeon smooth muscle cells unless very high concentrations of hypercholesterolemic serum were used. Monkey skin fibroblasts were the most responsive to cholesteryl ester accumulation with a greater than 28-fold increase in cholesteryl ester content occurring when incubated with hypercholesterolemic serum. Incubation for 24 hr with hypercholesterolemic serum stimulated cholesterol esterification 4 to 8-fold in monkey cells in a manner that paralleled the time course of accumulation of cholesteryl esters, while no stimulation in cholesterol esterification occurred in pigeon cells, consistent with their lack of ability to accumulate large amounts of cholesteryl esters. No differences in the response to either normal or hypercholesterolemic serum were seen between smooth muscle cells from atherosclerosis-susceptible White Carneau and resistant Show Racer pigeons. There were, however, major differences between pigeon and monkey cells. Although results indicate that the mechanism of accumulation of cholesteryl esters by pigeon smooth muscle cells may be different than for mammalian cells, no differences were seen in any of the parameters measured that might help to explain the difference in susceptibility to atherosclerosis of the two breeds of pigeons.