Inhibition of acyl coenzyme A:cholesterol acyl transferase in J774 macrophages enhances down-regulation of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase and prevents low density lipoprotein-induced cholesterol accumulation

Cholesteryl ester accumulation in arterial wall macrophages (foam cells) is a prominent feature of atherosclerotic lesions. We have previously shown that J774 macrophages accumulate large amounts of cholesteryl ester when incubated with unmodified low density lipoprotein (LDL) and that this is related to sluggish down-regulation of the J774 LDL receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. To further explore intracellular cholesterol metabolism and regulatory events in J774 macrophages, we studied the effect of inhibitors of acyl-CoA:cholesterol acyl transferase (ACAT) on the cells' ability to accumulate cholesterol and to down-regulate receptor and reductase. Treatment of J774 cells with LDL in the presence of ACAT inhibitor 58-035 (Sandoz) prevented both cholesteryl ester and total cholesterol accumulation. Furthermore, 58-035 markedly enhanced down-regulation of the J774 LDL receptor and 3-hydroxy-3-methylglutaryl-CoA reductase in the presence of LDL. In dose-response studies, down-regulation of the receptor by 58-035 paralleled its inhibition of ACAT activity. Compound 58-035 also increased the down-regulation of the J774 LDL receptor in the presence of 25-hydroxycholesterol and acetyl-LDL but not in the presence of cholesteryl hemisuccinate, which is not an ACAT substrate. The ability of 58-035 to enhance LDL receptor down-regulation was negated when cells were simultaneously incubated with recombinant high density lipoprotein3 discs, which promote cellular cholesterol efflux. In contrast to the findings with J774 macrophages, down-regulation of the human fibroblast LDL receptor was not enhanced by 58-035. These data suggest that in J774 macrophages, but not in fibroblasts, ACAT competes for a regulatory pool of intracellular cholesterol, contributing to diminished receptor and reductase down-regulation, LDL-cholesterol accumulation, and foam cell formation.     

Acyl coenzyme A:cholesterol acyl transferase in macrophages utilizes a cellular pool of cholesterol oxidase-accessible cholesterol as substrate

Cholesterol esterification by acyl CoA:cholesterol acyl transferase (ACAT) in macrophages is a key process in atheroma foam cell formation. However, the process of cholesterol substrate delivery to ACAT is not well defined. In this study, J774 macrophages, which form foam cells with native low density lipoprotein (LDL), were labeled with [3H]cholesterol-containing liposomes. Most (80-90%) of the cholesterol label could be converted by cholesterol oxidase to cholestenone, suggesting plasma membrane localization; only 0.6% of the label was in cholesteryl ester (CE). In cells chased for 6 h in medium lacking LDL, the distribution of label was essentially unchanged, whereas in cells chased with LDL, 28% of the label was incorporated into CE concomitant with a decrease in cholestenone label to 50%. [3H]Cholesterol-labeled mouse peritoneal macrophages incubated with acetyl-LDL, and both J774 and mouse peritoneal macrophages incubated with 25-hydroxy-cholesterol, also showed a shift of label from cholestenone to CE. Similar results were found when cellular cholesterol was biosynthetically labeled with [3H]mevalonate. The percentage of cholesterol substrate for ACAT in LDL-treated J774 macrophages which originates from endogenous cellular pools (versus that originating from LDL itself) is approximately 50%. We conclude that upon activation of ACAT in macrophages, there is a novel process whereby a cholesterol oxidase-accessible pool of cellular cholesterol, presumably plasma membrane cholesterol, is translocated to ACAT in the endoplasmic reticulum.     

Apoprotein E mediates the interaction of beta-VLDL with macrophages

beta-Very low density lipoproteins (beta-VLDL) isolated from cholesterol-fed rhesus monkeys stimulated cholesteryl ester synthesis and accumulation in mouse peritoneal macrophages. The apoprotein specificity and requirement for the cell surface uptake of beta-VLDL was investigated by treating the beta-VLDL with trypsin (beta-VLDL (T], incubating the beta-VLDL (T) with other lipoproteins or apoproteins, reisolating the beta-VLDL (T) and measuring its biological activity which, for this study, is defined as the ability of the lipoprotein to stimulate cholesterol esterification in the macrophages. Trypsin treatment of beta-VLDL abolished its biological activity. Apoprotein analysis of the beta-VLDL (T) demonstrated the absence of intact apoproteins B-100, B-48, and E. The J774 macrophage-like cell line and mouse peritoneal macrophages responded similarly with respect to cholesterol esterification following incubation with inactive and treated beta-VLDL. The J774 macrophage-like cell line was used to establish the conditions necessary for the restoration of biologic activity to the trypsinized beta-VLDL. The loss of biological activity of beta-VLDL (T) could be reversed by restoring apoprotein E-containing LDL from hyperlipemic monkeys or purified apoprotein E. Apoprotein A-I had no such effect. The restored biological activity of the beta-VLDL (T) was proportional to the amount of apoprotein E acquired by the lipoprotein. beta-VLDL particles composed of apoprotein E and either intact or degraded apoprotein B-100 had comparable biological activity. Thus, intact apoprotein E, without intact apoprotein B, is a sufficient mediator for the biological activity and metabolism of beta-VLDL by macrophages and plays a major role in receptor-lipoprotein interaction.     

Protein synthesis inhibition in mouse peritoneal macrophages results in increased acyl coenzyme A:cholesterol acyl transferase activity and cholesteryl ester accumulation in the presence of native low density lipoprotein

Cholesteryl ester (CE) accumulation in arterial wall macrophages (foam cells), mediated by the intracellular enzyme acyl coenzyme A:cholesterol acyl transferase (ACAT), is a prominent feature of atherosclerotic lesions. However, native low density lipoprotein (LDL) does not cause activation of ACAT or CE accumulation in cultured mouse peritoneal macrophages despite both substantial LDL uptake and degradation and the presence of ACAT in these cells. We now report that when protein synthesis is inhibited in mouse peritoneal macrophages by treatment with cycloheximide, puromycin, or actinomycin D, native LDL-induced whole-cell ACAT activity and CE accumulation is 10-fold higher than that seen in LDL-treated control cells. The enhancement of ACAT activity was seen 4 h after the addition of cycloheximide, and ACAT activity returned to control values 4 h after the withdrawal of cycloheximide. Postnuclear supernatants and microsomes from cycloheximide-treated mouse peritoneal macrophages also had higher ACAT activity than microsomes from control cells, but the relative enhancement (maximum 3.3-fold) was less than that seen when ACAT was assayed in the intact cell. In contrast to the situation with mouse peritoneal macrophages, cycloheximide treatment of J774 macrophages, which under normal conditions display high ACAT activity and CE accumulation in the presence of native LDL, did not result in further enhancement of either ACAT activity or LDL-induced CE accumulation. From these data we postulate that mouse peritoneal macrophages have a short-lived protein that inhibits ACAT-mediated cholesterol esterification which is responsible for their lack of ACAT response and CE accumulation in the presence of native LDL. The explanation for high ACAT activity and LDL-induced CE accumulation in J774 macrophages may be that these cells lack the putative mouse peritoneal macrophage cholesterol esterification inhibitor.     

Dose-dependent dual effects of cholesterol and desmosterol on J774 macrophage proliferation

We addressed the ability of native, oxidized and acetylated low-density lipoproteins (nLDL, oxLDL and acLDL, respectively) and desmosterol to act as sources of sterol for the proliferation of J774A.1 macrophages. Treatment with 0.5 μM lovastatin and lipoprotein-deficient serum suppressed cell proliferation. This inhibition was effectively prevented by nLDL, but only to a lesser extent by oxLDL. AcLDL, despite its ability to deliver a higher amount of cholesterol to J774 macrophages than the other LDLs, was dependent on mevalonate supply to sustain cell proliferation. Similarly, exogenous desmosterol, which is not converted into cholesterol in J774 cells, required the simultaneous addition of mevalonate to support optimal cell growth. Expression of hydroxymethyl glutaryl coenzyme A reductase mRNA was potently down-regulated by acLDL and exogenous desmosterol, but the effect was weaker with other sterol sources. We conclude that nLDL is more efficient than modified LDL in sustaining macrophage proliferation. Despite the requirement of cholesterol or desmosterol for J774 cell proliferation, excessive provision of either sterol limits mevalonate availability, thus suppressing cell proliferation.     

Expression of scavenger receptor BI facilitates sterol movement between the plasma membrane and the endoplasmic reticulum in macrophages

Scavenger receptor BI influences multiple aspects of cellular sterol metabolism. In this series of studies, we evaluated the effect of scavenger receptor BI expression on the distribution and movement of sterol between the plasma membrane and the endoplasmic reticulum in macrophages, by comparing control J774 cells to J774 cells in which SR-BI expression was constitutively increased 3-fold. J774 cells with increased expression of SR-BI (J774-SRBI cells) esterified plasma membrane cholesterol more rapidly as compared to control cells. The esterification of endogenously synthesized cholesterol was also more rapid in cells with increased SR-BI expression; this could be partially suppressed by removing cholesterol from the plasma membrane. The increased plasma membrane sterol esterification in J774-SRBI cells was not due to increased acyl-coA:cholesterol acyltransferase activity and was observed even though J774-SRBI cells manifested a smaller free cholesterol pool in the endoplasmic reticulum. Cholesterol ester hydrolysis was also more rapid in J774-SRBI cells. Increased expression of SR-BI also facilitated the clearance of cellular cholesterol ester to HDL(3). This latter observation, combined with the measurement of the smaller ER free cholesterol pool in J774-SRBI cells, suggests that the free cholesterol derived from the hydrolysis of cholesterol ester was rapidly transported back to the plasma membrane. It is concluded that expression of SR-BI in macrophages increases the rate of free cholesterol transport, and modulates free cholesterol distribution between the plasma membrane and the internal membrane compartments in macrophages.     

Evidence that the gap junction protein connexin-43 is the ATP-induced pore of mouse macrophages.

Extracellular ATP4- opens pores in the plasma membrane of mouse macrophages and the J774 macrophage-like cell line that allow molecules as large as fura-2 (831 daltons) to enter the cytoplasmic matrix of the cells. The functional similarity of the ATP-induced pores to gap junctions led us to examine whether these pores were related to members of the connexin family of gap junction proteins. Under conditions of high stringency, RNA isolated from J774 cells hybridized with cDNA for connexin-43 but not with cDNA for connexin-32, -26, or -46. RNA isolated from several variant J774 cell lines that do not permeabilize in response to extracellular ATP (ATPR cells) did not hybridize with connexin-43 cDNA. Immunoblots demonstrated that J774 cells, but not the variant ATPR B2 cell line, expressed connexin-43 protein. These studies demonstrate that mouse macrophages express the connexin-43 gap junction mRNA and protein and strongly suggest that in these cells connexin-43 forms "half-gap junctions" in response to extracellular ATP4-.     

High-density lipoprotein particle uptake and selective uptake of high-density lipoprotein-associated cholesteryl esters by J774 macrophages

High-density lipoprotein (HDL) cholesteryl esters are taken up by fibroblasts via HDL particle uptake and via selective uptake, i.e., cholesteryl ester uptake independent of HDL particle uptake. In the present study we investigated HDL selective uptake and HDL particle uptake by J774 macrophages. HDL3 (d = 1.125-1.21 g/ml) was labeled with intracellularly trapped tracers: 125I-labeled N-methyltyramine-cellobiose-apo A-I (125I-NMTC-apo A-I) to trace apolipoprotein A-I (apo A-I) and [3H]cholesteryl oleyl ether to trace cholesteryl esters. J774 macrophages, incubated at 37 degrees C in medium containing doubly labeled HDL3, took up 125I-NMTC-apo A-I, indicating HDL3 particle uptake (102.7 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein). Apparent HDL3 uptake according to the uptake of [3H]cholesteryl oleyl ether (470.4 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein) was in significant excess on 125I-NMTC-apo A-I uptake, i.e., J774 macrophages demonstrated selective uptake of HDL3 cholesteryl esters. To investigate regulation of HDL3 uptake, cell cholesterol was modified by preincubation with low-density lipoprotein (LDL) or acetylated LDL (acetyl-LDL). Afterwards, uptake of doubly labeled HDL3, LDL (apo B,E) receptor activity or cholesterol mass were determined. Preincubation with LDL or acetyl-LDL increased cell cholesterol up to approx. 3.5-fold over basal levels. Increased cell cholesterol had no effect on HDL3 particle uptake. In contrast, LDL- and acetyl-LDL-loading decreased selective uptake (apparent uptake 606 vs. 366 ng HDL3 protein/mg cell protein per 4 h in unloaded versus acetyl-LDL-loaded cells at 20 micrograms HDL3 protein/ml). In parallel with decreased selective uptake, specific 125I-LDL degradation was down-regulated. Using heparin as well as excess unlabeled LDL, it was shown that HDL3 uptake is independent of LDL (apo B,E) receptors. In summary, J774 macrophages take up HDL3 particles. In addition, J774 cells also selectively take up HDL3-associated cholesteryl esters. HDL3 selective uptake, but not HDL3 particle uptake, can be regulated.     

Oxidized LDL increase free cholesterol and fail to stimulate cholesterol esterification in murine macrophages

Oxidatively modified low density lipoproteins (Ox-LDL) may be involved in determining the formation of foam cells by inducing cellular cholesteryl ester accumulation. We studied the effect of copper oxidized LDL (Ox-LDL) on cholesterol accumulation and esterification in murine macrophages. Ox-LDL (44 micrograms/ml of lipoprotein cholesterol) increased the total cholesterol content of the cells from 29 to 69 micrograms/mg cell protein. Free cholesterol accounted for 85% of this increase. Acetyl LDL (Ac-LDL) (38 micrograms/ml of lipoprotein cholesterol), raised total cellular cholesterol content to a similar extent (76 micrograms/mg cell protein), however only 25% of the accumulated cholesterol was unesterified. When ACAT activity was determined after incubation of J774 cell with Ox- or Ac-LDL, Ox-LDL were 12 times less effective than Ac-LDL in stimulating cholesteryl ester formation. This was not due to an inhibition of ACAT by Ox-LDL since these lipoproteins failed to inhibit pre activated enzyme in cholesteryl ester-loaded macrophages. The uptake of 125I-Ox-LDL: was 175% that of 125I-Ac-LDL, while degradation was only 20%. All together these data suggest an altered intracellular processing of Ox-LDL, which may be responsible for free cholesterol accumulation.     

Pomegranate juice inhibits oxidized LDL uptake and cholesterol biosynthesis in macrophages

Macrophage cholesterol accumulation and foam cell formation are the hallmarks of early atherogenesis. Pomegranate juice (PJ) was shown to inhibit macrophage foam cell formation and development of atherosclerotic lesions. The aim of this study was to elucidate possible mechanisms by which PJ reduces cholesterol accumulation in macrophages. J774.A1 macrophages were preincubated with PJ followed by analysis of cholesterol influx [evaluated as LDL or as oxidized LDL (Ox-LDL) cellular degradation], cholesterol efflux and cholesterol biosynthesis. Preincubation of macrophages with PJ resulted in a significant reduction (P<.01) in Ox-LDL degradation by 40%. On the contrary, PJ had no effect on macrophage degradation of native LDL or on macrophage cholesterol efflux. Macrophage cholesterol biosynthesis was inhibited by 50% (P<.01) after cell incubation with PJ. This inhibition, however, was not mediated at the 3-hydroxy-3 methylglutaryl coenzyme A reductase level along the biosynthetic pathway. We conclude that PJ-mediated suppression of Ox-LDL degradation and of cholesterol biosynthesis in macrophages can lead to reduced cellular cholesterol accumulation and foam cell formation.     

Modification of low density lipoprotein by lipoprotein lipase or hepatic lipase induces enhanced uptake and cholesterol accumulation in cells

Incubation of low density lipoprotein(s) (LDL) with either lipoprotein lipase or hepatic lipase led to modification of the core lipid composition of LDL. Both lipases modified LDL by substantially reducing core triglyceride content without producing marked differences in size, charge, or lipid peroxide content in comparison to native LDL. The triglyceride-depleted forms of LDL that result from treatment with these two enzymes were degraded at approximately twice the rate of native LDL by human monocyte-derived macrophages (HMDM). Lipase-modified LDL degradation was inhibited by chloroquine, suggesting lysosomal involvement in LDL cellular processing. The increased degradation by macrophages of the LDL modified by these lipases was accompanied by enhanced cholesterol esterification rates, as well as by an increase in cellular free and esterified cholesterol content. In a patient with hepatic triglyceride lipase deficiency, degradation of the triglyceride-rich LDL by HMDM was approximately half that of normal LDL. Following in vitro incubation of LDL from this patient with either lipoprotein or hepatic lipase, lipoprotein degradation increased to normal. Several lines of evidence indicate that LDL modified by both lipases were taken up by the LDL receptor and not by the scavenger receptor. 1) The degradation of lipase-modified LDL in nonphagocytic cells (human skin fibroblast and arterial smooth muscle cells) as well as in phagocytic cells (HMDM, J-774, HL-60, and U-937 cell lines) could be dissociated from that of acetylated LDL and was always higher than that of native LDL. A similar pattern was found for cellular cholesterol esterification and cholesterol mass. 2) LDL receptor-negative fibroblasts did not degrade lipase-modified LDL. 3) A monoclonal antibody to the LDL receptor inhibited macrophage degradation of the lipase-modified LDL. 4) Excess amounts of unlabeled LDL competed substantially with 125I-labeled lipase-modified LDL for degradation by both macrophages and fibroblasts. Thus, lipase-modified LDL can cause significant cholesterol accumulation in macrophages even though it is taken up by LDL and not by the scavenger receptor. This effect could possibly be related to the reduced triglyceride content in the core of LDL, which may alter presentation of the LDL receptor-binding domain of apolipoprotein B on the particle surface, thereby leading to increased recognition and cellular uptake via the LDL receptor pathway.     

Nitric oxide production from a macrophage cell line: Interaction with autologous and allogeneic lymphocytes

The indirect stimulation of macrophages to produce nitrite was examined by using the macrophage cell line J774. J774 spontaneously produced nitrite, when cultured at high concentration. J774 cultured in low concentration ( < 10⁴ cells in 100 μl) barely produced nitrite. J774 cultured in low concentration produced a large amount of nitrite by the co-culture of nonadherent spleen cells or nonadherent peritoneal exudate cells, which were stimulated with con A, anti-CD3, or staphylococcal enterotoxin A. J774 (BALB/c derived: H-2^d) cultured with either syngeneic (BALB/c) or allogeneic (B6; H-2^b B10BR; H-2^k) nonadherent lymphocytes, which were stimulated with conA or anti-CD3, produced nitric oxide. However, J774 produced nitric oxide by stimulation with SEA only when co-cultured with SEA-reactive T lymphocytes. Peritoneal exudate cells from mice, which did not proliferate by the stimulation of conA or anti-CD3, proliferated well by the addition of L-arginine homologue, N^G-monomethyl-L-arginine. The proliferation of nonadherent peritoneal exudate cells stimulated with conA or anti-CD3 was suppressed by the addition of peritoneal macrophages. This suppression was abolished by the addition of N^G-monomethyl-L-arginine.     

Involvement of the mannose receptor in infection of macrophages by influenza virus

Influenza viruses A/PR/8/34 (PR8; H1N1), A/Aichi/68 X-31 (HKx31; H3N2), and A/Beijing/89 X-109 (BJx109; H3N2) show marked differences in their ability to infect murine macrophages, including resident alveolar and peritoneal macrophages as well as the macrophage-derived cell line J774. The hierarchy in infectivity of the viruses (PR8 < HKx31 < BJx109) resembles that of their reactivity with mannose-binding lectins of the collectin family. Since the macrophage mannose receptor recognizes the same spectrum of monosaccharides as the collectins do, we investigated the possible involvement of this receptor in infection of macrophages by influenza virus. In competitive binding studies, the binding of (125)I-labeled mannosylated bovine serum albumin to macrophages was inhibited by the purified hemagglutinin and neuraminidase (HANA) glycoproteins of influenza virus but not by HANA that had been treated with periodate to oxidize its oligosaccharide side chains. The inhibitory activity of HANA from the three strains of virus differed markedly and correlated with the infectivity of each virus for macrophages. Infection of macrophages, but not MDCK cells, by influenza virus was inhibited by yeast mannan. A variant line of J774 cells, J774E, which expresses elevated levels of the mannose receptor, was more readily infected than J774, and the sensitivity of J774E cells to infection was greatly reduced by culture in the presence of D-mannose, which down-modulated mannose receptor expression. Together, the data implicate the mannose receptor as a major endocytic receptor in the infectious entry of influenza virus, and perhaps other enveloped viruses, into murine macrophages.     

Rapid turnover of low-density lipoprotein receptor by a non-lysosomal pathway in mouse macrophage J774 cells and inhibitory effect of brefeldin A

The low-density lipoprotein (LDL) receptor of molecular mass 155 kDa was expressed on the cell surface of cultured mouse macrophage J774 cells. The conversion rate of precursor to mature form of LDL receptor in J774 cells was comparable to that in mouse fibroblast L cells. The half-life of the LDL receptor of J774 cells was about 2 h, that of L cells was about 11 h. The rapid degradation of LDL receptor was not significantly inhibited by the lysosomotropic agents, chloroquine and NH4Cl, nor by the thiol-protease inhibitors leupeptin and E-64. By contrast, incubation at 18 degrees C retarded the degradation of LDL receptor. Treatment of J774 cells with brefeldin A, an inhibitor of membrane transport between the endoplasmic reticulum and the Golgi apparatus, inhibited the rapid turnover of the LDL receptor. Even after a 9-h chase in the presence of brefeldin A, LDL receptor 5-10 kDa smaller than the normal mature form was found to be stable. Rapid turnover of the LDL receptor in the macrophages appeared to occur after exit from the Golgi apparatus, possibly during transport of the LDL receptor to the plasma membrane.     

Reduced uptake of cholesterol esterase-modified low density lipoprotein by macrophages.

Changes in low density lipoprotein (LDL) lipid composition were shown to alter its interaction with the LDL receptor, thus affecting its cellular uptake. Upon incubation of LDL with 5 units/ml cholesterol esterase (CEase) for 1 h at 37 degrees C, there was a 33% reduction in lipoprotein cholesteryl ester content, paralleled by an increment in its unesterified cholesterol. CEase-LDL, in comparison to native LDL, was smaller in size, possessed fewer free lysine amino groups (by 14%), and demonstrated reduced binding to heparin (by 83%) and reduced immunoreactivity against monoclonal antibodies directed toward epitopes along the LDL apoB-100. Incubation of CEase-LDL with the J-774 macrophage-like cell line resulted in about a 30% reduction in lipoprotein binding and degradation in comparison to native LDL, and this was associated with a 20% reduction in macrophage cholesterol mass. Similarly, CEase-LDL degradation by mouse peritoneal macrophages, human monocyte-derived macrophages, and human skin fibroblasts was reduced by 20-44% in comparison to native LDL. CEase-LDL uptake by macrophages was mediated via the LDL receptor and not the scavenger receptor. CEase activity toward LDL was demonstrated in plasma and in cells of the arterial wall such as macrophages and endothelial cells. Thus, CEase modification of LDL may take place in vivo, and this phenomenon may have a role in atherosclerosis.     

Caveolin-1 and caveolin-2 expression in mouse macrophages. High density lipoprotein 3-stimulated secretion and a lack of significant subcellular co-localization

Evidence for caveolin expression in macrophages is scarce and conflicting. We therefore examined caveolin-1 and caveolin-2 expression in resident and thioglycollate-elicited mouse peritoneal macrophages (tg-MPM) and in the J774 mouse macrophage cell line by RT-PCR, ribonuclease protection assay, immunoblotting, and immunofluorescence. We found that relative to 3T3 cells, resident MPM and tg-MPM express low amounts of caveolin-1 (45 and 15% of those in 3T3 fibroblasts, respectively), while J774.A1 cells do not express any. Caveolin-2, on the other hand, is expressed in all cells examined, with highest expression in tg-MPM and the lowest in J774 cells. The relative levels of caveolin expression in the various cells correspond well with their respective mRNA levels, as measured by ribonuclease protection assay. Caveolin-1, present primarily on the cell surface, does not co-localize significantly with caveolin-2, which is present primarily in the Golgi compartment in all macrophages studied. Loading of tg-MPM with cholesterol or variations in unesterified cholesterol content appear to have little effect on the level of caveolin-1 or -2 expression or their distribution. Stimulation of cholesterol efflux by HDL(3) leads to caveolin-1 and caveolin-2 secretion to the cell culture medium, a process not detected in the absence of HDL(3). The lack of significant co-localization of the two caveolin isoforms in primary macrophages and their secretion in the presence of HDL(3) provides an interesting and physiologically relevant model system to study additional aspects of caveolin function.     

Macrophage foam cell formation with native low density lipoprotein

This investigation has elucidated a mechanism for development of macrophage foam cells when macrophages are incubated with native low density lipoprotein (LDL). LDL is believed to be the main source of cholesterol that accumulates in monocyte-derived macrophages within atherosclerotic plaques, but native LDL has not previously been shown to cause substantial cholesterol accumulation when incubated with macrophages. We have found that activation of human monocyte-derived macrophages with phorbol 12-myristate 13-acetate (PMA) stimulates LDL uptake and degradation and acyl-CoA:cholesterol acyltransferase-mediated esterification of LDL-derived cholesterol, resulting in massive macrophage cholesterol accumulation that could exceed 400 nmol/mg of cell protein. Cholesterol accumulation showed a biphasic linear LDL concentration dependence with LDL levels as high as 4 mg/ml, similar to LDL levels in artery intima. Protein kinase C mediated the PMA-stimulated macrophage uptake of LDL because the protein kinase C inhibitors, G?6983 and GF109203X, inhibited cholesterol accumulation. LDL receptors did not mediate macrophage cholesterol accumulation because accumulation occurred with reductively methylated LDL and in the presence of an anti-LDL receptor-blocking monoclonal antibody. LDL-induced cholesterol accumulation was not inhibited by antioxidants, was not accompanied by increased LDL binding to macrophages, did not depend on the apoB component of LDL, and was not down-regulated by prior cholesterol enrichment of macrophages. We have shown that the mechanism of LDL uptake by macrophages was PMA-stimulated endocytosis of LDL taken up as part of the bulk phase fluid (i.e. fluid phase endocytosis). The amount of LDL taken up with the bulk phase fluid was measured with [(3)H]sucrose and accounted for a minimum of 83% of the LDL cholesterol delivery and accumulation in PMA-activated macrophages. This novel mechanism of macrophage cholesterol accumulation shows that modification of LDL is not necessary for foam cell formation to occur. In addition, the findings direct attention to macrophage fluid phase endocytosis as a relevant pathway to target for modulating macrophage cholesterol accumulation in atherosclerosis.     

Essential involvement of 12-lipoxygenase in regiospecific andstereospecific oxidation of low density lipoprotein by macrophages.

To establish a role of the 12-lipoxygenase on the generation of oxidized low density lipoprotein (LDL) in macrophages that leads to foam cell formation in atherosclerosis, we overexpressed 12-lipoxygenases in a macrophage-like cell line, J774A.1, that does not show intrinsic enzyme activity. When the 12-lipoxygenase-expressing cells were incubated with 400 microg.mL-1 LDL in Dulbecco's modified Eagle's medium at 37 degrees C for 12 h, LDL oxidation, as determined by thiobarbituric acid reactive substance, was markedly increased compared with the mock-transfected cells. Oxygenated products in the modified LDL were examined by HPLC before and after alkaline hydrolysis. Most of the oxygenated derivatives were of an esterified form, and the major product was identified as 13S-hydroxyoctadeca-9Z,11E-dienoic acid. These results clearly demonstrate that esterified fatty acids in LDL are oxygenated by the 12-lipoxygenases expressed in the J774A.1 cells. Furthermore, the oxidized LDL generated by intracellular 12-lipoxygenases was recognized by a scavenger receptor as assessed by macrophage degradation assay.     

Effect of fatty acid supplementation on cholesterol and retinol esterification in J774 macrophages

J774 macrophages exposed to medium containing cholesterol-rich phospholipid dispersions accumulate cholesteryl ester. Supplementing this medium with 100 micrograms oleate/ml increased cellular cholesteryl ester contents 3-fold. Cell retinyl ester contents increased 8-fold when medium containing retinol dispersed in dimethyl sulfoxide was supplemented with oleate. These increases were not the result of increases in total lipid uptake by the cells but rather of redistribution of cholesterol and retinol into their respective ester pools. Effective oleate concentration of 15-30 micrograms/ml increased cellular retinyl and cholesteryl ester contents. The effective oleate concentration was reduced to 5 micrograms/ml when the fatty acid/albumin molar ratio was increased. The oleate-stimulated increase in cholesterol esterification was blocked by incubating cells with Sandoz 58-035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), indicating that the effect of fatty acid exposure is mediated through changes in ACAT activity. When cholesterol or retinol was added to cells which had been exposed to oleate for 24 h to provide a triacylglycerol store, the cellular contents of cholesteryl or retinyl ester were also significantly increased compared to cells not previously exposed to oleate. The oleate-stimulated increase in the esterification of cholesterol and/or retinol was also observed in P388D1 macrophages, human (HepG2) and rat (Fu5AH) hepatomas, human fibroblasts, rabbit aortic smooth muscle cells and MCF-7 breast carcinoma cells. In addition to oleate, a number of other fatty acids increased retinol esterification in J774 macrophages; however, cellular cholesterol esterification in these cells was increased only by unsaturated fatty acids and was inhibited in the presence of saturated fatty acids. Although the cellular uptake of radiolabeled oleate and palmitate was similar, a significant difference in the distribution of these fatty acids among the lipid classes was observed. These data demonstrate that exogenous fatty acids are one factor that regulate cellular cholesteryl and retinyl ester contents in cultured cells.