Sandra R. Bates

Very low density lipoproteins promote triglyceride accumulation in macrophages.

Incubation of mouse peritoneal macrophages with very low density lipoproteins (VLDL) from normal rats or rhesus monkeys markedly increased the levels of intracellular trlglycerldes by 10-to 56-fold and was accompanied by the production of oil red 0 positive vacuoles. The stimulation of triglyceride accumulation in macrophages was timeand concentration-dependent and was specific for VLDL. Three possible mechanisms for the VLDL-stlmulated triglyceride accumulation In macrophages were explored: receptor-mediated uptake, action of llpoprotein llpase, and phagocytosis. Macrophage uptake and degradation of 125I-monkey and rat VLDL demonstrated saturable and nonsaturable components. Uptake of 125I-VLDL could be inhibited by unlabeled normal VLDL, although hyperllpemlc VLDL was more effective. HDL did not compete to a significant extent. Heparln released llpoprotein lipase-llke activity from peritoneal macrophages. Addition of heparln with VLDL resulted in a greater, more rapid elevation in Intracellular triglycerides, which was partially Inhibited by albumin. Free fatty acid and Intrallpid also produced triglyceride accumulation In macrophages. The data showed that all three of the mechanisms examined could contribute to the metabolism of VLDL by macrophages and cause the production of trlglycerlderlch cells with a “foamy” appearance, although the evidence suggested that the action of llpoprotein llpase was probably the most important In this process.

Effect of hyperlipemic serum on cholesterol accumulation in monkey aortic medial cells.

The effect of hyperlipemic monkey serum on cholesterol ester formation and accumulation in monkey aortic medial cells grown in tissue culture was studied. The cellular incorporation and esterification of free cholesterol was followed using the specific activity of serum labeled with free [14C]cholesterol while the cellular sterol content was analyzed by gas-liquid chromatographic techniqyes. The effects produced by hyperlipemic monkey serum (HMS) and normal monkey serum (NMS) were evaluated at both comparable percentage levels in the media and at equivalent exogenous cholesterol concentrations. When the two sera were adjusted to equal exogenous free cholesterol levels, the incorporation of free cholesterol by the aortic medial cells was related to the free cholesterol concentration of the culture media whether supplied by normal or hyperlipemic serum cholesterol. Under these conditions the total cholesterol content of the HMS-grown cells was 35% greater than that of NMS-grown cells, due to an elevation in free cholesterol of approximately 3 mug/mg cell protein and a 2- to 4-fold increase in esterified cholesterol. At similar percentage levels, the hyperlipemic serum stimulated a greater incorporation of free cholesterol into the monkey medial cells, accompanied by a 2-fold increase in the cellular esterification of this free cholesterol.

Structural and functional changes of rhesus serum low density lipoproteins during cycles of diet-induced hypercholesterolemia.

Over the course of a 2-year study, two male rhesus monkeys underwent episodes of diet-induced hypercholesterolemia (from a diet supplemented with 25% coconut oil and 2% cholesterol) followed by regression phases in which the animals received a low fat Purina chow diet. During the induction of hypercholesterolemia, serum cholesterol, apo B, saturation of low density lipoprotein (LDL) cholesteryl ester fatty acyl chains, and the ability of the serum to stimulate cholesterol esterification by smooth muscle cells rose immediately and in parallel, whereas there was a lag period before the serum became mitogenic to smooth muscle cells. Concurrently, there were important changes in the density, size, chemistry, and concentration of the LDL species in the rhesus serum; induced LDL shifted from the LDL-II to the LDL-I density region with increasing cholesterol concentration. Both structural and functional changes were reversed upon return to a normal Purina chow diet, although at different rates. Serum cholesterol, apo B, and the rate of cholesterol esterification in smooth muscle cells promoted by the serum declined in parallel while the mitogenicity of the serum to smooth muscle cells and the degree of saturation of LDL cholesteryl ester fatty acids took longer to return to normal values. In fact, there was an immediate and dramatic rise in saturation upon reversal before the LDL cholesteryl ester fatty acyl chains returned to their normal composition. The Lp(a) particles did not increase in either concentration or size in response to the test diet, although the change in their lipid composition was similar to those of the other LDL species. The studies indicate that dietary manipulations affect the physicochemicai properties of the LDL particles, and that the resultant structural alterations are accompanied by changed in vitro cellular response, suggestive of a greater atherogenicity.

Accumulation and loss of cholesterol esters in monkey arterial smooth muscle cells exposed to normal and hyperlipemic serum lipoproteins

The effects of high low and very low density lipoprotein fractions from normal or hyperlipemic rhesus monkey serum on the accumulation or removal of cholesterol esters from rhesus monkey smooth muscle cells in tissue culture were determined. Serum or serum lipoproteins were labeled with [14C] free cholesterol and adjusted to the same free cholesterol level in the incubation medium. Of the two normal lipoproteins examined, the LDL fraction caused more esterification than the HDL. Cells incubated in hyperlipemic serum showed a 2-fold stimulation in esterification as compared to cells in normal serum. This was contributed by hyperlipemic VLDL and LDL and led to a concomitant increase in cellular cholesterol ester content. Both hyperlipemic LDL and HDL stimulated esterification when compared to their normal counterparts. Cholesterol ester removal was examined by incubating the serum or lipoprotein fractions with cells enriched in cholesterol ester through a prior exposure to hyperlipemic serum. The cells incubated in normal or hyperlipemic HDL or lipoprotein-deficient serum had the lowest cholesterol ester content. Thus, the lipoprotein fractions which caused the lowest levels of cholesterol esterification were also the most efficient in the removal of cellular cholesterol esters.

Effect of an elastin growth substrate on cholesteryl ester synthesis and foam cell formation by cultured aortic smooth muscle cells

Exposure of smooth muscle cells cultured on plastic or glass to hyperlipidemic serum did not result in the formation of foam cells. Since elastin binds serum lipids, and vascular smooth muscle cells are normally closely associated with elastin, we investigated the effects of an elastin substrate on lipid metabolism and on the accumulation of lipid vacuoles by rabbit aortic smooth muscle cells in culture. When cells were grown in plastic petri dishes, cholesteryl ester synthesis, as measured by [14C]oleate incorporation into cholesteryl esters, was 3 times greater in rabbit hyperlipidemic serum (HLS) than in normolipemic serum (NLS) (P less than 0.001). For cells of the same subculture grown on the elastin substrate, the synthetic rate was 6-fold greater in HLS compared to NLS (P less than 0.005). The cells grown on the elastin membranes in the presence of HLS contained large numbers of Oil red O stainable lipid vacuoles and resembled foam cells, while those grown in petri dishes and exposed to HLS showed only an occasional cell containing a few vacuoles. Pre-incubation in lipoprotein-deficient serum markedly enhanced the stimulatory effect of HLS on cholesteryl ester synthesis for cells growing in plastic petric dishes but had much less stimulatory effect on the cells growing on elastin membranes. These studies indicate that close association with elastin modulates the response of smooth muscle cells to hyperlipidemia and suggest a role for elastin in the formation of foam cells of smooth muscle origin during atherogenesis.

Interaction of low density lipoproteins from normal and hyperlipemic rhesus monkeys with arterial smooth muscle cells in culture

Abstract Monkey arterial smooth muscle cell binding, incorporation and degradation of low density lipoproteins (LDL) from normal or hyperlipemic rhesus monkey plasma were examined by labeling lipoproteins with 125 I. Specific and nonspecific binding of both lipoproteins occurred. Although the dissociation constant for the normal LDL was twice as high as for hyperlipemic LDL, indicating a higher binding affinity for hyperlipemic LDL, the maximal binding capacity of the smooth muscle cells for normal [ 125 I]LDL was 2-fold higher than for hyperlipemic [ 125 I]LDL. In competition experiments where the two types of LDL competed with normal [ 125 I]LDL for binding to smooth muscle cells, LDL isolated from hyperlipemic plasma was a more effective competitor than normal LDL. Receptors for normal LDL were reduced to a greater extent by preincubation with hyperlipemic LDL than by the same protein concentration of normal LDL. Under conditions where nonspecific uptake of lipoproteins would occur predominantly, the cells showed higher incorporation and degradation of normal LDL than hyperlipemic LDL. The corresponding cellular cholesterol ester levels showed an inverse pattern, with higher accumulation of cholesterol ester in the cells grown in hyperlipemic LDL. The results suggest that binding and incorporation of LDL by specific receptors or through the nonspecific pathway play a role in the changes of cellular cholesterol content but cannot totally account for the enhanced accumulation of cellular cholesterol esters produced by hyperlipemic monkey LDL.

Effect of normolipemic and hyperlipemic serum on biosynthetic response to cyclic stretching of aortic smooth muscle cells.

Arterial smooth muscle cells synthesize matrix macromolecules in response to mechanical stimulation. Exposure to serum lipids also stimulates connective tissue fiber accumulation. To assess the effect of serum lipids on the biosynthetic response to tensile stress, we subjected rabbit aortic smooth muscle cells that were cultured on purified elastin membranes to cyclic stretching and relaxation 50 times per minute in the presence of serum-free medium (SFM), normolipemic serum (NLS), or hyperlipemic serum (HLS). Incorporation of 14C-proline into proline and into hydroxyproline was taken as a measure of protein and collagen synthesis. When cells were grown in plastic Petri dishes, exposure to NLS or HLS increased both protein and collagen production to the same extent compared to synthesis in SFM (1.7 times for NLS and 1.6 times for HLS; p less than 0.001 compared to SFM). For cells grown on stationary elastin membranes, NLS and HLS also increased protein and collagen synthesis compared to SFM. The effect of NLS was 1.35 times that of HLS for protein and 1.43 times greater for collagen (p less than 0.03). Cyclic stretching in SFM doubled synthesis for both protein (p less than 0.002) and collagen (p less than 0.002) compared to stationary controls, but had no effect on synthesis in NLS. In HLS, however, cyclic stretching elevated synthesis to the same level as was found in NLS (p less than 0.003). We conclude that the relative inhibition of synthesis on stationary membranes by HLS was not due to a toxic effect, since HLS increased synthesis both in Petri dishes and on elastin membranes, and the amplifying effect of cyclic stretching in HLS was similar to that seen in SFM.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of the hyperlipidemic serum or LDL-induced cellular cholesterol ester accumulation by 22-hydroxycholesterol and its analogue

Incubation of monkey arterial smooth muscle cells with hyperlipidemic serum or low-density lipoproteins (LDL) produces intracellular cholesterol ester accumulation. Increased esterification of free cholesterol within the cell may account for this effect. To examine such a possibility, oxygenated sterols were used to block cholesterol esterification. The increased esterification of free cholesterol by smooth muscle cells and human skin fibroblasts, through their exposure to hyperlipidemic lipoproteins, was inhibited by 22-hydroxycholesterol and its analogue, SC-32561 (22-hydroxy-25-fluorocholesterol). The hyperlipidemic LDL-stimulated elevation in the cholesterol ester content of the smooth muscle cells was also prevented by these sterols. This reduction in cellular cholesterol ester did not coincide with an increase in free cholesterol. 22-Hydroxycholesterol also blocked the stimulation of the esterification of cholesterol due to 25-hydroxycholesterol. In the absence of lipoproteins, 22-hydroxycholesterol and SC-32561 had a minor effect on the incorporation of [14C]oleate into cholesterol esters, and efficiently reduced sterol synthesis in fibroblasts and smooth muscle cells. 22-Hydroxycholesterol and SC-32561 had the additional effect of lowering the number of cell-surface LDL receptors to a greater extent than did hyperlipidemic LDL. The presence of 22-hydroxycholesterol did not alter the interaction of normal LDL with the receptor. Oxygenated sterols recovered in the cell represented 4-21% of the total sterol content. The level of intracellular oxygenated sterols was significantly reduced by the presence of lipoproteins in the culture media. Due to the multiple effects of the oxysterols, they were not effective as tools in determining the contribution of acyl-coenzyme A:cholesterol acyltransferase enzyme activity to the intracellular pool of cholesterol esters. These results indicated that 22-hydroxycholesterol and SC-32561 effectively blocked the hyperlipidemic LDL-stimulated increase in smooth muscle cell cholesterol ester content by lowering cholesterol esterification, reducing cholesterol synthesis and down-regulating LDL cell-surface receptors.

Arterial Smooth Muscle Cells in Tissue Culture

Smooth muscle cells are among the most remarkable of the mesenchymal cells. They have long been of interest to biologists and physiologists. It is not surprising that they were grown in vitro by Carrell, Maximow, and other pioneers who first cultured diploid cells, since it is relatively easy to grow the cells in pure culture from explants (Carrell and Burrows, 1910; Maximow, 1925). The outgrowths from these explants in turn provide cells which are readily subcultured.

15( S )-Lipoxygenase-1 associates with neutral lipid droplets in macrophage foam cells: evidence of lipid droplet metabolism

15(S)-lipoxygenase-1 (15-LO-1) was present in the whole-cell homogenate of an acute human monocytic leukemia cell line (THP-1). Additionally, 15-LO-1 was detected on neutral lipid droplets isolated from THP-1 foam cells. To investigate if 15-LO-1 is active on lipid droplets, we used the mouse leukemic monocytic macrophage cell line (RAW 264.7), which are stably transfected with human 15-LO-1. The RAW 15-LO-1 cells were incubated with acetylated low density lipoprotein to generate foam cells. 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], the major 15-LO-1 metabolite of arachidonic acid, was produced in the 15-LO-1 RAW but not in the mock transfected cells when incubated with arachidonic acid. Lipid droplets were isolated from the cells and incubated with arachidonic acid, and production of 15(S)-HETE was measured over 2 h. 15(S)-HETE was produced in the incubations with the lipid droplets, and this production was attenuated when the lipid droplet fraction was subjected to enzyme inactivation through heating. Efflux of 15(S)-HETE from cholesteryl ester-enriched 15-LO RAW cells, when lipid droplets are present, was significantly reduced compared with that from cells enriched with free cholesterol (lipid droplets are absent). We propose that 15-LO-1 is present and functional on cytoplasmic neutral lipid droplets in macrophage foam cells, and these droplets may act to accumulate the anti-inflammatory lipid mediator 15(S)-HETE.