Physical and chemical characteristics of apolipoprotein A-I-lipid complexes produced by Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene

Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene linked to the human metallothionein gene promoter region secrete large quantities of apolipoprotein A-I (7.1 +/- 0.4% total secreted protein) in the presence of zinc. Approx. 16% of the secreted apolipoprotein A-I is complexed with lipid and can be isolated ultracentrifugally at d less than or equal to 1.21 g/ml. The latter complexes are composed of discs and vesicles as judged by electron microscopy and can be further separated by column chromatography into three fractions: fraction I, mostly vesicles (60-260 nm) and large discs (18-20 nm diameter); fraction II, discs 14.2 +/- 2.6 nm diameter; and fraction III, nonresolvable by electron microscopy. The latter fraction is extremely lipid-poor (94% protein, 6% phospholipid); in contrast, the protein, phospholipid and unesterified cholesterol content for the other fractions are 43, 33 and 24%, respectively, for fraction I and 53, 33 and 14%, respectively, for fraction II. Fraction II particles contain three and four apolipoprotein A-Is per particle as determined by protein crosslinking while large structures in fraction I contain primarily six to seven apolipoprotein A-Is per particle. Following incubation with purified lecithin: cholesterol acyltransferase, discoidal particles were transformed into apparent spherical particles 12.9 +/- 3.4 nm diameter; this transformation coincided with 19-21% conversion of unesterified cholesterol to esterified cholesterol. The apolipoprotein A-I-lipid complexes isolated from Chinese hamster ovary cell media are similar to nascent HDL found in plasma of lecithin:cholesterol acyltransferase-deficient patients and those secreted by the human hepatoma line, Hep G2. The ability of the Chinese hamster ovary cell nascent HDL-like particles to undergo transformation in the presence of purified lecithin:cholesterol acyltransferase indicates that they are functional particles.     

Cholesterol content and sterol synthesis in human skin fibroblasts and rat aortic smooth muscle cells exposed to lipoprotein-depleted serum and high density apolipoprotein/phospholipid mixtures.

Confluent cultures of human skin fibroblasts and rat aortic smooth muscle cells were shown to lose 15-27% of their cellular cholesterol upon replacement of the fetal calf serum with human high density lipoprotein (50 mug cholesterol/ml) or lipoprotein-depleted serum at a concentration equivalent to 40% whole serum. Addition to the latter medium of high density apoliproprotein/phospholipid mixtures resulted in further enhancement of cellular cholesterol loss which was evident by 12 h of incubation. Human skin fibroblasts that had been enriched in cholesterol by previous incubation with low density lipoprotein lost their cholesterol in the presence of a high density apolipoprotein/sphingomyelin mixture as readily as non-enriched cells. Concomitant with the marked cholesterol depletion there was a stimulation of sterol synthesis from acetate. The more pronounced loss of cellular cholesterol induced by the presence of phosphatidylcholine or sphingomyelin resulted in a greater incorporation of acetate into sterol in both smooth muscle cells and skin fibroblasts. The present findings indicate that peripheral cells, in spite of their capacity to synthesize cholesterol, depend on exogenous cholesterol for the maintenance of normal levels. It is suggested that the native cholesterol "acceptor" in the lipoprotein-depleted serum is an apolipoprotein which under the experimental conditions can form a complex with phospholipids and might also represent the physiological cholesterol "acceptor" in peripheral lymph.     

Apolipoprotein AI efficiently binds to and mediates cholesterol and phospholipid efflux from human but not rat aortic smooth muscle cells

Aortic smooth muscle cells (SMC) from several animal species have been reported to resist depletion of cellular cholesterol by the major apolipoprotein of HDL, apoAI. Resistance of SMC to this protective action of apoAI, if present in humans, could contribute to the overaccumulation of arterial wall cholesterol seen in atherosclerosis. We investigated the ability of human aortic medial SMC to bind and be depleted of cholesterol and phospholipids by apoAI. In contrast to rat aortic SMC, but similar to human fibroblasts, human SMC were readily depleted of cholesterol by apoAI, measured by a marked depletion of intracellular cholesterol available for esterification, and an increase in cholesterol efflux to the medium. Human SMC were also actively depleted of the phospholipids phosphatidylcholine and sphingomyelin by apoAI. In contrast, rat SMC released only a small fraction of these cellular phospholipids to apoAI-containing medium. (125)I-labeled apoAI bound with high affinity and specificity to human SMC, but failed to bind to rat SMC. Similar levels of expression of class B, type I scavenger receptor (SR-BI) and caveolin in human and rat SMC suggested these proteins do not account for the differences in apoAI binding or lipid efflux seen in these cells. An enhancer of apolipoprotein-mediated cholesterol efflux, tyrosyl radical-oxidized HDL, markedly amplified the depletion of cholesterol available for esterification in human SMC compared to HDL, but had no enhanced effect in rat SMC. These results show that human SMC bind and are readily depleted of cellular lipids by apoAI, and suggest that apoAI-mediated cholesterol efflux from arterial SMC may contribute significantly to the circulating pool of HDL cholesterol in vivo. The marked difference in apoAI binding to human and rat arterial SMC provides an excellent model to study the nature of the apoAI-cell binding interaction.     

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.     

Apolipoprotein-mediated cellular lipid release requires replenishment of sphingomyelin in a phosphatidylcholine-specific phospholipase C-dependent manner

When sphingomyelin is digested by sphingomyelinase in the plasma membrane of rat astrocytes, productions of sphingomyelin, diacylglycerol, and phosphatidylcholine are stimulated. D609, an inhibitor of phosphatidylcholine-specific phospholipase C, suppressed these effects. Similarly, when apolipoprotein A-I removed cellular cholesterol, phosphatidylcholine, and sphingomyelin to generate high density lipoprotein, cholesterol synthesis from acetate subsequently increased, and sphingomyelin synthesis from acetate and serine also increased. D609 inhibited these effects again. D609 also inhibited the cholesterol removal by apoA-I not only from the astrocytes but also from BALB/3T3 and RAW264 cells. D609 decreased cholesterol synthesis, although D609 did not directly inhibit hydroxymethylglutaryl-CoA reductase. ApoA-I-stimulated translocation of newly synthesized cholesterol to cytosol was also decreased by D609. A diacylglycerol analog increased the apoA-I-mediated cholesterol release, whereas ceramide did not influence it. We concluded that removal of cellular sphingomyelin by apolipoproteins is replenished by transfer of phosphorylcholine from phosphatidylcholine to ceramide, and this reaction may limit the removal of cholesterol by apoA-I. This reaction also produces diacylglycerol that potentially triggers subsequent cellular signal cascades and regulates intracellular cholesterol trafficking.     

Role of lipoproteins in growth of human adult arterial endothelial and smooth muscle cells in low lipoprotein-deficient serum

Recently improved culture conditions for human adult arterial endothelial and smooth muscle cells from a wide variety of donors have been used to study the effects of lipoproteins on proliferation of both cell types in low serum culture medium. Optimal growth of endothelial and smooth muscle cells in an optimal nutrient medium (MCDB 107) containing epidermal growth factor, a partially purified fraction from bovine brain, and 1% (v/v) lipoprotein-deficient serum was dependent on either high- or low-density lipoprotein. High- and low-density lipoprotein stimulated cell growth by three- and five-fold, respectively, over a 6-day period. Optimal stimulation of both endothelial and smooth muscle cell growth occurred between 20 and 60 micrograms/ml of high- and low-density lipoproteins, respectively. No correlation between the activation of 3-hydroxyl-3-methylglutaryl coenzyme. A reductase activity and lipoprotein-stimulated cell proliferation was observed. Lipid-free total apolipoproteins or apolipoprotein C peptides from high-density lipoprotein were partially effective and together with oleic acid effectively replaced native high-density lipoprotein for the support of endothelial cell growth. In contrast, apolipoproteins or apolipoprotein C peptides from high-density lipoprotein alone or with oleic acid had no effect on smooth muscle cell proliferation. The results suggest a functional role of high- and low-density lipoproteins and apolipoproteins in the proliferation of human adult endothelial and smooth muscle cells.     

Triglyceride and cholesteryl ester hydrolysis in a cell culture model of smooth muscle foam cells

Cultured rabbit aortic smooth muscle cells were converted to foam cells by exposure to sonicated lipid droplets of defined composition using an inverted culture technique. Uptake of the lipid droplets by the cells was shown to be dependent on the time of exposure to the droplets and on the mass of droplets presented to the cells. A comparison of the hydrolysis of triolein and cholesteryl oleate by cells that had been exposed to isotropic lipid droplets containing equimolar amounts of the two lipids revealed that the rate of hydrolysis of triglyceride was 3 to 4 times faster than that for cholesteryl ester. The hydrolysis of cholesteryl oleate from cells loaded with the isotropic droplets was approximately 1.5 times as fast as that from cells loaded with anisotropic droplets containing only cholesteryl oleate. A comparison of the hydrolysis of cholesteryl ester in the presence and absence of Sandoz compound 58-035, an inhibitor of acyl CoA:cholesterol acyl transferase, by cells loaded with isotropic droplets showed that about 30% of the free cholesterol liberated by hydrolysis was reesterified regardless of the mass of free cholesterol. We conclude that cultured smooth muscle cells have a greater capacity to hydrolyze triglyceride than cholesteryl ester, and that the rate of hydrolysis of cholesteryl ester appears to be related to the physical state of the droplet in which the cholesteryl ester is stored. In addition, it appears that the smooth muscle cells have a cholesteryl ester cycle that is inefficient in the reesterification of excess free cholesterol.     

Localization of androgen receptors in ram epididymal principal cells

Androgen receptor was immunolocalized in the epididymal epithelium of rams and in isolated cells using an antibody against a synthetic polypeptide representing a portion of the androgen receptor. Immunostaining was predominant in the epithelium in tissue sections. Concentrations of androgen receptor were determined in cells from the central caput, distal caput, and central corpus epididymidis enzymically dissociated and elutriated to provide two fractions. On the average (n = 18), Fraction I contained 8% principal cells while Fraction II contained 71% principal cells; the stromal cells in each fraction were primarily smooth muscle and fibroblasts. For each sample, the number of DHT receptors (fmol) per 10(6) total cells was greater in Fraction II than in Fraction I. Few cells in Fraction I were immunostained for androgen receptor, whereas most cells in Fraction II were intensely stained. The numbers of DHT receptors per cell, or per principal cell, were similar for the central caput and distal caput, but lower in the central corpus epididymidis. The results support our hypothesis that most epididymal DHT receptors are localized in principal cells and confirm that the region between the central caput and proximal corpus of the ram epididymis is most dependent on androgen stimulation.     

Modulation of vascular smooth muscle cells proteoglycan synthesis by the extracellular matrix

In this study, we investigated the effect of the extracellular matrix (ECM) secreted by vascular cells on proteoglycan (PG) synthesis by vascular smooth muscle cells in culture. PG synthesis of human aortic smooth muscle cells plated on plastic or the matrices derived from vascular endothelial cells, vascular smooth muscle cells, or THP-1 macrophages was characterized. Smooth muscle cell and macrophage matrices increased both secreted and cellular smooth muscle cells PG production by 2.5-fold to 3.9-fold, respectively, over plastic and endothelial cell matrix. Macrophage matrix was more potent than smooth muscle cell matrix in this regard. Selective enzymatic removal of chondroitin sulfates, collagen, and elastin from smooth muscle cell matrix enhanced the stimulation of PG synthesis, as did the removal of chondroitin sulfates from macrophage matrix. PG turnover rates were similar for smooth muscle cells plated on the three matrices. The newly synthesized PG from cultures plated on smooth muscle cell-, and macrophage-derived matrices had greater charge density, larger molecular size, and longer glycosaminoglycan chains than those from endothelial cell matrix cultures. These data show that the ECM plays a major role in modulating vascular smooth muscle cell PG metabolism in vitro.     

Effect of reconstituted discoidal high-density lipoproteins on lipid mobilization in RAW 264.7 and CHOK1 cells

Reconstituted discoidal high‐density lipoproteins (rHDL) resemble nascent HDL, which are formed at the early reverse cholesterol transport steps, and constitute the initial cholesterol (Chol) acceptors from cell membranes. We have used different sized rHDL containing or not Chol, to test their abilities to promote cholesterol and phospholipid efflux from two different cell lines: Raw 264.7 macrophages and CHOK1 cells. All rHDL and lipid‐free apolipoprotein A‐I (apoA‐I) were found to be bound to CHO and RAW cells. In RAW cells, a positive correlation between cellular binding and Chol removal was found for 78 and 96 Å rHDL. Chol‐free rHDL were more effective than Chol‐containing ones in binding to RAW cells and promoting Chol removal. These results were more evident in the 96 Å rHDL. On the other hand, rHDL binding to CHO cells was relatively independent of disc size and Chol content. In spite of the fact that apoA‐I and rHDL promoted Chol efflux from both cellular lines, only in CHOK1 cells this result was also associated to decrease Chol esterification. Among choline‐containing phospholipids, only phosphatidylcholine (PC) (but not sphingomyelin) was detected to be effuxed from both cellular lines. With the only exception of Chol‐free 96 Å discs, the other rHDL as well as apoA‐I promoted PC efflux from RAW cells. Chol‐containing rHDL were more active than Chol‐free ones of comparable size to promote PC efflux from RAW macrophages. Regarding CHO cells, only apoA‐I and Chol‐free 78 Å rHDL were active enough to remove PC. J. Cell. Biochem. 113: 1208–1216, 2012. © 2011 Wiley Periodicals, Inc.     

Modulation of membrane composition of swine vascular smooth muscle cells by homologous lipoproteins in culture

Swine vascular smooth muscle cells were exposed to homologous low-density or high-density lipoprotein fractions for 24 h. Total cell membranes were isolated from the post-nuclear supernatant of the cell homogenates, fractionated by sucrose density gradient centrifugation and characterized by enzyme assays. The membrane fraction with the lowest density was enriched in plasma membrane marker enzymes. Cholesterol analysis showed that cells exposed to low-density lipoprotein had higher cholesterol-to-protein ratios in total cells, total cell membranes and individual membrane fractions than had the cells exposed to high-density lipoproteins. Cholesterol-to-phospholipid ratios of the plasma membrane-enriched fraction from cells exposed to low-density lipoprotein were higher than the same membrane fraction of cells exposed to high-density lipoprotein. Studies with iodinated lipoproteins showed that these compositional changes could not be due to lipoprotein contamination. Membrane microviscosity was determined by fluorescence depolarization with diphenylhexatriene and the microviscosity of the plasma membrane-enriched fraction was different in the cells exposed to the two different lipoprotein fractions. This difference in membrane microviscosity was significant only when the medium cholesterol content was 40 μg per ml or greater; cells exposed to low-density lipoprotein gave membranes with higher microviscosity.These results demonstrate that the properties of vascular smooth muscle cell membranes are influenced by exposure of the cells to homologous lipoprotein fractions.     

Effect of trifluoperazine on cholesterol metabolism of smooth muscle cells exposed to hypercholesterolemic medium in vitro

We recently demonstrated that the preventive effect of trifluoperazine (a potent inhibitor of calmodulin, protein kinase C, and phospholipase A2) on cholesterol-induced atherogenic activity of smooth muscle cells was mediated through its ability to inhibit smooth muscle cellular DNA synthesis coupled with stimulation of LDL receptor synthesis. The present study addressed the effect of trifluoperazine on cholesterol metabolism of aortic SMCs enriched with cholesterol through the nonreceptor pathway and revealed that (a) TFP caused inhibition of cholesterol synthesis compared with control cells bathed with hypercholesterolemic medium alone. (b) The drug also caused inhibition of free cholesterol and cholesteryl ester accumulation within smooth muscle cells compared to control cells. These results demonstrate that the preventive effect of TFP on atherogenic activity of smooth muscle cells may also be due to its ability to affect the altered/modified cholesterol metabolism of smooth muscle cells exposed to hypercholesterolemic medium in vitro.     

Metabolism of cytoplasmic triacylglycerol in cultured aortic smooth muscle cells

A turnover of cytoplasmic triacylglycerol was studied in cultured rat, rabbit, and bovine aortic smooth muscle cells. Cytoplasmic triacylglycerol was labeled with [3H]glycerol in the presence of oleic acid in the medium and its loss from the cell was studied in the presence of carrier glycerol. Multiple additions of Isuprel or dibutyryl cyclic AMP during the chase period did not enhance the loss of labeled triacylglycerol. The rate of hydrolysis of cellular triacylglycerol was unchanged in the absence or in the presence of 100 microM chloroquine. Modulation of cellular cholesterol content by addition of low density lipoprotein or high density apolipoprotein--sphingomyelin liposomes did not affect the residence time of the cellular triacylglycerol. We conclude that cytoplasmic triacylglycerol in cultured aortic smooth muscle cells is metabolized by an extralysosomal enzyme which is neither catecholamine responsive nor affected by modulation of cellular cholesterol.     

Effects of cholesterol oxidase on cultured vascular smooth muscle cells

Summary Cholesterol oxidase (3-hydroxy-steroid oxidase) catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. The purpose of the present study was to investigate its effects on cultured vascular smooth muscle cells. Cultured rabbit aortic smooth muscle cells were morphologically altered after exposure to cholesterol oxidase in the presence of culture medium containing 10% fetal calf serum. If fetal calf serum was absent, cells were unaffected by the treatment. The extent of morphological change of the smooth muscle cells was dependent upon the time of exposure to the enzyme and the concentration of cholesterol oxidase employed. After moderate treatment with cholesterol oxidase, cells excluded trypan blue. Further, a specific mitochondrial marker DASPMI (dimethyl aminostyryl-methyl-pyridiniumiodine) which was used as a fluorescent index of cell viability, revealed that cell viability was unchanged after moderate cholesterol oxidase treatment. Nile red, a hydrophobic probe which selectively stains intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with cholesterol oxidase. Cellular nile red fluorescence intensity increased linearly with the time and concentration of cholesterol oxidase treatment. These results demonstrate that cholesterol oxidase alters lipid deposition in the cell and changes cell morphology. The primary site of action of cholesterol oxidase appears to be independent of the cell membrane itself and instead is dependent upon the lipid content in the surrounding culture media. These changes occur prior to the cytotoxic effects of extensive oxidation. Because oxidized cholesterol may play an important role in the pathogenesis of atherosclerosis, our results have implications for intracellular accumulation of lipids in smooth muscle cells during the atherosclerotic lesion.     

Cellular cholesterol efflux. Role of cell membrane kinetic pools and interaction with apolipoproteins AI, AII, and Cs.

Efflux of [14C]cholesterol from various cells was monitored in the presence of discoidal complexes of egg phosphatidylcholine and purified apolipoproteins, containing either apoAI, AII, or Cs. Particles containing apoAI were more efficient acceptors than those containing apoAII or Cs when the donor cells were J774 macrophages. No differences were observed when the same acceptor preparations were exposed to Fu5AH rat hepatoma or rabbit aortic smooth muscle cells. The differential efficiency of apolipoproteins in stimulating cholesterol removal from J774 cells was maintained in a plasma membrane-enriched fraction isolated from the same cells. Nonlinear regression analysis of kinetic data obtained from J774 cells exposed to apoAI complexes indicated that cholesterol efflux was best fitted to a curve describing the release from two kinetic compartments. Approximately 10% of cholesterol was transferred from a rapidly exchangeable pool with a t1/2 ranging between 1.5 and 3 h, and the remaining fraction was released from a slower pool with a t1/2 of about 20 h. Modulation of cholesterol efflux from J774 cells by either varying the concentration or the apolipoprotein composition of the acceptors influenced the size of the pools and the t1/2 of the slow pool. Kinetics of cholesterol efflux from membranes isolated from J774 cells also best fit a two-compartment model and modification of the apolipoprotein composition of the acceptor induced a pattern of changes in pool size and half-time similar to that described for whole cells. In the three cell lines studied, we consistently resolved a slow pool with a half-time ranging between 15 and 20 h. In smooth muscle cells only the slow pool was evident, whereas in Fu5AH a very large fast pool was also resolved. In contrast to J774 cells, apolipoprotein composition of the acceptor did not influence the pools in these two cell lines. These results led us to propose a new model regarding the influence of multiple kinetic pools of cholesterol on the regulation of cholesterol desorption from the cell membrane.     

Expression of apolipoprotein E by cultured vascular smooth muscle cells is controlled by growth state

Rat vascular smooth muscle cells (SMC) in culture synthesize and secrete a approximately 38,000-Mr protein doublet or triplet that, as previously described (Majack and Bornstein. 1984. J. Cell Biol. 99:1688-1695), rapidly and reversibly accumulates in the SMC culture medium upon addition of heparin. In the present study, we show that this approximately 38,000-Mr heparin-regulated protein is electrophoretically and immunologically identical to apolipoprotein E (apo-E), a major plasma apolipoprotein involved in cholesterol transport. In addition, we show that expression of apo-E by cultured SMC varies according to growth state: while proliferating SMC produced little apo-E and expressed low levels of apo-E mRNA, quiescent SMC produced significantly more apo-E (relative to other proteins) and expressed markedly increased levels of apo-E mRNA. Northern analysis of RNA extracted from aortic tissue revealed that fully differentiated, quiescent SMC contain significant quantities of apo-E mRNA. These data establish aortic SMC as a vascular source for apo-E and suggest new functional roles for this apolipoprotein, possibly unrelated to traditional concepts of lipid metabolism.     

Visualizing caveolin-1 and HDL in cholesterol-loaded aortic endothelial cells

Caveolae are vesicular invaginations of the plasma membranes that regulate signal transduction and transcytosis, as well as cellular cholesterol homeostasis. Our previous studies indicated that the removal of cholesterol from aortic endothelial cells and smooth muscle cells in the presence of HDL is associated with plasmalemmal invaginations and plasmalemmal vesicles. The goal of the present study was to investigate the location and distribution of caveolin-1, the main structural protein component of caveolae, in cholesterol-loaded aortic endothelial cells after HDL incubation. Confocal microscopic analysis demonstrated that the caveolin-1 appeared to colocalize with HDL-fluorescein 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) conjugates on the cell surface. No free HDL-DiI conjugates were revealed in the cytoplasm. Immunoelectron microscopy further demonstrated that caveolin-1 gold (15 nm) conjugates colocalized with HDL gold (10 nm) conjugates in the plasmalemmal invaginations. These morphological results indicated that caveolae are the major membrane domains facilitating the transport of excess cholesterol to HDL on the cell surface of aortic endothelial cells.     

Cholesterol metabolism in pigeon aortic smooth muscle cells lacking a functional low density lipoprotein receptor pathway

Cholesterol metabolism was examined in aortic smooth muscle cells from atherosclerosis-susceptible White Carneau pigeons that have been shown to lack a functional LDL receptor pathway. In cells incubated in the presence of whole serum or low density lipoprotein (LDL) the rate of cholesterol synthesis from [1-14C]acetate or of HMG-CoA reductase activity was 20-100 times greater than for mammalian cells incubated under the same conditions. Unexpectedly, cholesterol synthesis decreased by nearly 50% after preincubation for 24 hr with lipoprotein-deficient serum (LPDS). This occurred without a change in cellular cholesterol content. Neither the high rate of cholesterol synthesis nor the effect of LPDS could be accounted for by differences in cell turnover or state of growth. Cholesterol added in ethanol was ineffective in altering cellular cholesterol synthesis or esterification even though a near doubling in cellular free cholesterol content occurred. Cholesterol synthesis and esterification were, however, able to be regulated with 25-OH cholesterol and mevalonolactone, as indicated by their ability to suppress cholesterol synthesis and to stimulate cholesterol esterification. In spite of the high rate of endogenous cholesterol synthesis, cellular cholesterol content was maintained at a constant level by the efficient efflux of the newly synthesized cholesterol from the cell. Unlike mammalian cells that require a cholesterol acceptor in the medium for efflux to occur, cholesterol efflux from pigeon cells occurred in the absence of a cholesterol acceptor. This suggests either that pigeon cells utilize a different mechanism for cholesterol efflux or that they produce their own cholesterol acceptor. As a result of a lack of a functional LDL receptor pathway, pigeon smooth muscle cells do not maintain cholesterol homeostasis through the controlled uptake of exogenous LDL cholesterol, as do mammalian cells. Rather, pigeon smooth muscle cells would appear to regulate cholesterol concentrations at the level of either cholesterol synthesis or efflux.     

Intracellular cholesterol accumulation is accompanied by enhanced proliferative activity of human aortic intimal cells

Cholesterol accumulation in smooth muscle cells of unaffected human aortic intimal tissue occurred in the following conditions: (1) incubation of cells with atherogenic blood serum from patients with coronary heart disease (CHD), (2) cultivation of cells in the presence of insoluble associates containing low density lipoprotein (LDL). Preincubation of cells with blood serum from CHD patients resulted in a 2-5-fold increase in intracellular cholesterol and in 1.5-3-fold increase in cellular [3H]thymidine uptake. Blood serum collected from healthy donors had no significant effect on cultivated smooth muscle cells. When intimal cells were preincubated with insoluble associates containing LDL and components of fibrous extracellular matrix, the level of intracellular cholesterol increased from 2-4 times and uptake of [3H]thymidine increased 1.5-2.5-fold. Thus, a strong correlation was found between [3H]thymidine incorporation and intracellular cholesterol accumulation. The current study suggests that intracellular lipid accumulation may stimulate the proliferative activity of human aortic intimal cells from uninvolved tissue.     

Tweaking the cholesterol efflux capacity of reconstituted HDL

Mechanisms to increase plasma high-density lipoprotein (HDL) or to promote egress of cholesterol from cholesterol-loaded cells (e.g., foam cells from atherosclerotic lesions) remain an important target to regress heart disease. Reconstituted HDL (rHDL) serves as a valuable vehicle to promote cellular cholesterol efflux in vitro and in vivo. rHDL were prepared with wild type apolipoprotein (apo) A-I and the rare variant, apoA-I Milano (M), and each apolipoprotein was reconstituted with phosphatidylcholine (PC) or sphingomyelin (SM). The four distinct rHDL generated were incubated with CHO cells, J774 macrophages, and BHK cells in cellular cholesterol efflux assays. In each cell type, apoA-I(M) SM-rHDL promoted the greatest cholesterol efflux. In BHK cells, the cholesterol efflux capacities of all four distinct rHDL were greatly enhanced by increased expression of ABCG1. Efflux to PC-containing rHDL was stimulated by transfection of a nonfunctional ABCA1 mutant (W590S), suggesting that binding to ABCA1 represents a competing interaction. This interpretation was confirmed by binding experiments. The data show that cholesterol efflux activity is dependent upon the apoA-I protein employed, as well as the phospholipid constituent of the rHDL. Future studies designed to optimize the efflux capacity of therapeutic rHDL may improve the value of this emerging intervention strategy.