Enhanced Synthesis of the Oxysterol 24(S),25-Epoxycholesterol in Macrophages by Inhibitors of 2,3-Oxidosqualene:Lanosterol Cyclase
A Novel Mechanism for the Attenuation of Foam Cell Formation
Oxysterols are key regulators of lipid metabolism and regulate gene expression by activating the liver X receptor (LXR). LXR plays a vital role in macrophage foam cell formation, a central event in atherosclerosis. It is known that addition of exogenous oxysterols to cultured macrophages activates LXR, leading to increased expression of ABCA1 and cholesterol efflux. In this study, we tested the novel hypothesis that stimulation of endogenous oxysterol synthesis would block foam cell formation induced by atherogenic lipoproteins. Macrophage synthesis of 24(S),25-epoxycholesterol, a potent LXR ligand, increased 60-fold by partial inhibition of 2,3-oxidosqualene:lanosterol cyclase (OSC), a microsomal enzyme in both the cholesterol biosynthetic pathway and the alternative oxysterol synthetic pathway. When macrophages were challenged with human hypertriglyceridemic VLDL (HTG-VLDL), cellular cholesteryl ester accumulation increased 12-fold. This was reduced dramatically, by 65%, after preincubation with an OSC inhibitor (OSCi). The HTG-VLDL–induced accumulation of macrophage TG (70-fold) was unaffected by the OSCi or exogenous 24(S),25-epoxycholesterol, an effect associated with suppression of SREBP-1 processing. By contrast, TO901317, a synthetic LXR agonist, increased cellular TG significantly and markedly increased SREBP-1 processing. OSC inhibition decreased HTG-VLDL uptake through downregulation of LDL-receptor expression, despite substantial inhibition of cholesterol synthesis. Furthermore, OSC inhibition significantly upregulated ABCA1 and ABCG1 expression, which led to enhanced macrophage cholesterol efflux, an effect mediated through LXR activation. Therefore, increased macrophage synthesis of endogenous oxysterols represents a new mechanism for the dual regulation of LXR- and SREBP-responsive genes, an approach that inhibits foam cell formation without detrimental effect on TG synthesis.
The uptake of apolipoprotein (apo) B–containing lipoproteins by macrophages within the arterial wall results in cholesteryl ester (CE) deposition and foam cell formation, a hallmark of early and late atherosclerotic lesions.1 Foam cell formation is the result of an imbalance in processes mediating lipoprotein uptake and cholesterol efflux.2 The importance of cholesterol efflux in the maintenance of macrophage cellular cholesterol homeostasis arose from elucidation of mutations in the ATP binding cassette protein (ABC) A1 in Tangier disease, which is characterized by macrophage cholesterol accumulation (reviewed by Attie et al3). ABCA1 is a transmembrane protein that controls the transfer of cholesterol and phospholipids to apoA1, the initial step in HDL formation and reverse cholesterol transport (reviewed by Tall et al,2 Francis et al,4 and Oram5). The discovery of ABCA1 and its key role in regulating macrophage cholesterol efflux has stimulated development of therapeutic interventions that enhance the removal of cholesterol from arterial cells as a strategy to reduce atherosclerosis.2
Expression of ABCA1 is transcriptionally controlled and increases in response to cellular cholesterol, which is dependent on the ligand-activated nuclear hormone receptors, liver X receptor (LXRα/β).2,3,5,6 LXRs are activated by oxysterols, permitting heterodimerization with the retinoid X receptor (RXR), resulting in coactivator recruitment and binding to specific response elements (LXREs) in target genes.6 LXRs coordinate the regulation of both cholesterol and fatty acid metabolism through induction of genes encoding ABCA1, ABCG1, apoE, lipoprotein lipase (LPL), fatty acid synthase, and the sterol regulatory element binding protein (SREBP)-1c (reviewed by Repa and Mangelsdorf6 and Francis et al7). Furthermore, proteolytic processing of SREBP-1c results in activation of genes involved in fatty acid and TG metabolism.8 Cellular cholesterol loading most likely results in the production of cholesterol-derived oxysterols, including 27-hydroxycholesterol, which are capable of activating both LXR subtypes.9,10 Indeed, LXRs are expressed in activated macrophages,6 and the addition of exogenous oxysterols or synthetic LXR agonists induces macrophage ABCA1 expression and cholesterol efflux independent of cholesterol loading.11 Importantly, the impact of oxysterols on macrophage uptake of atherogenic lipoproteins and CE deposition is unknown.
The naturally occurring oxysterols 24(S),25-epoxycholesterol [24(S),25-epoxy], 24(S)-hydroxycholesterol, and 22(R)-hydroxycholesterol [22(R)-OH] are potent LXR ligands.9,12 The latter two are synthesized from cholesterol in the brain and adrenals, respectively, whereas 24(S),25-epoxy is uniquely derived from a shunt in the cholesterol biosynthetic pathway (Figure 1).9,12 2,3-Oxidosqualene:lanosterol cyclase (OSC) catalyzes conversion of 2,3-monoepoxysqualene (MOS) to lanosterol, the dedicated step in cholesterol biosynthesis. However, OSC also catalyzes cyclizing of 2,3;22,23-diepoxysqualene (DOS) to 24(S),25-epoxylanosterol, which is subsequently transformed into 24(S),25-epoxy.13–15 Synthesis of 24(S),25-epoxy is favored over cholesterol under conditions of partial OSC inhibition.13–15 Because 24(S),25-epoxy is also a repressor of HMG-CoA reductase activity,14 OSC inhibitors can uniquely stimulate endogenous synthesis of oxysterols while simultaneously inhibiting endogenous cholesterol synthesis. This suggests that 24(S),25-epoxy synthesis can be stimulated in the absence of cellular cholesterol loading and regulate LXR-sensitive genes, including ABCA1.
Macrophage foam cell formation can be induced by VLDL and their remnants (REM), isolated from patients with moderate hypertriglyceridemia (HTG) in both their native16,17 and oxidized forms.18 Native HTG-VLDL and VLDL-REM induce macrophage lipid accumulation via a 2-step process.16,17 TG accumulation occurs via interaction of HTG-VLDL with cell-surface LPL, resulting in core TG hydrolysis, cellular free fatty acid uptake, and reesterification. CE accumulation results from CE-rich remnant uptake by LDL receptors (LDL-R), and lipoprotein-derived free cholesterol (FC) is utilized, reesterified, and stored as CE droplets or effluxed from the cell via ABCA1.
In the present study, we tested the hypothesis that partial inhibition of OSC in macrophages would attenuate CE accumulation and foam cell formation induced by HTG-VLDL. Exposure of macrophages to OSC inhibitors dramatically decreased cholesterol synthesis while concomitantly increasing endogenous synthesis of 24(S),25-epoxy. This resulted in significant attenuation of macrophage CE accumulation by two mechanisms: (1) reduced HTG-VLDL uptake through decreased LDL-R expression and (2) enhanced ABCA1 and ABCG1 expression leading to increased cholesterol efflux, mediated by LXR activation. Macrophage TG metabolism was unaltered, an effect associated with suppression of SREBP-1 processing. Thus, this study provides a new mechanism by which cellular oxysterol synthesis can be induced without cholesterol loading to modulate LXR-responsive genes such as ABCA1.
Materials and Methods
Subjects and Lipoprotein Isolation
Subjects were recruited from the Lipid Clinics at the London Health Sciences Center (London, Ontario, Canada). Studies were approved by the University of Western Ontario Health Science Standing Committee on Human Research. VLDL (Sf 20 to 400) and LDL (Sf 0 to 12) were isolated from plasma of type IV hypertriglyceridemic and normal subjects, respectively, and VLDL-REM and acetylated LDL (AcLDL) were prepared.18,19
The following cells were used in this study: J774A.1 (murine macrophages), THP-1 (human macrophages), HepG2 (human hepatocytes), and mouse peritoneal macrophages isolated from LDL-R–null mice and their control C57Bl/6 mice. Cells were cultured as previously outlined.16–19 Several OSC inhibitors (OSCi), designated Ro-48-8071, Ro-61-3479, Ro-71-3852, and Ro-71-4565 (all in DMSO), were tested, which inhibit OSC in rat hepatic microsomes with IC50s of 8.1, 17.2, 4.4, and 28.4 nmol/L, respectively (F. Hoffmann-La Roche AG, Pharmaceuticals Division). Cell viability and protein contents were not affected at any dose tested. In some experiments, cells were incubated with 24(S),25-epoxy, 22(R)-OH, 22(S)-OH (LXR antagonist), the LXR antagonist 5α,6α-epoxycholesterol-3-sulfate20 (ECHS), or the synthetic LXR ligand TO901317.
Cellular and Immunoblot Analyses
Cellular lipid synthesis, cellular lipid mass, LPL activity, LDL-R activity, and mRNA abundances were measured.16–19 Cholesterol efflux was quantified in macrophages that were either non–cholesterol-loaded or cholesterol-loaded (5 μg or 100 μg total cholesterol [TC] of AcLDL/mL, respectively) before efflux studies.19 For immunoblot analyses, a polyclonal antibody for ABCA1 or monoclonal antibody for SREBP-1 was used. SREBP-1 precursor and activated forms were determined in the postnuclear and nuclear fractions, respectively.
Dual Luciferase Reporter Assay
LXR-activated gene expression was determined in HepG2 cells cotransfected with the TK-LXRE3-luc construct containing three copies of the LXRE upstream of a luciferase reporter (prepared by Dr D. Mangelsdorf, University of Texas Southwestern, Dallas, Tex) and the TK promoter–Renilla luciferase construct pRL-TK (a kind gift of D. Ory, Washington School of Medicine, St Louis, Mo21).
Data are expressed as mean±SEM. Statistical analyses were performed using 2-sample t tests and 1-way ANOVA, with significance at P<0.05.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
OSC Inhibitors Decrease Cholesterol Biosynthesis and Enhance Endogenous Synthesis of 24(S),25-Epoxy
Each OSCi tested dose-dependently decreased cholesterol synthesis, with a maximal inhibition of ≈90% (P<0.05) (Figure 2). IC50 for cholesterol synthesis inhibition ranged from 5 to 30 nmol/L. By comparison, 22(R)-OH maximally decreased cholesterol biosynthesis by 40% (P<0.05) (data not shown).
The effects of OSCi on synthesis of MOS, DOS, and 24(S),25-epoxy were determined after TLC separation (Figure 3A). The 24(S),25-epoxy band was identified by comparison with purified standards. Previous studies demonstrated that 24(S),25-epoxy was the only oxysterol synthesized in OSCi-treated hepatocytes.13 Ro-71-4565 dose-dependently increased 24(S),25-epoxy synthesis, with a maximal increase of 64-fold at 1 μmol/L (P<0.05, Figure 3B). Higher OSCi concentrations (>2 μmol/L) decreased 24(S),25-epoxy such that at 10 μmol/L, levels were 20% of those observed with 1 μmol/L. MOS and DOS increased dose-dependently at OSCi concentrations >1 μmol/L to maximum levels at 10 μmol/L (Figure 3B). These results are consistent with the higher affinity of OSC for DOS compared with MOS,15 such that partial inhibition of OSC (<1 μmol/L Ro-71-4565) results in increased conversion of MOS to DOS and enhanced formation, via OSC, of 24(S),25-epoxy (Figure 1). Therefore, 1 μmol/L of Ro-71-4565 was used in subsequent experiments. All OSCis tested were quantitatively similar (data not shown).
OSCi Decreases Macrophage Cellular Lipid Accumulation Induced by HTG-VLDL
J774A.1 macrophages incubated with HTG-VLDL demonstrated substantial accumulation of cellular Oil Red O– stained lipid droplets, indicative of cytoplasmic neutral lipids (Figure 4A). Cells treated with Ro-71-4565 and HTG-VLDL demonstrated a marked reduction in neutral lipid inclusions (Figure 4B). J774A.1 macrophages incubated with HTG-VLDL increased CE accumulation 12-fold, compared with control (P<0.05, Figure 4C). Preincubation with Ro-71-4565, significantly decreased HTG-VLDL–induced CE accumulation in a dose-responsive manner, with maximal inhibition (63%) at 1 μmol/L. Higher doses resulted in marked attenuation of this decrease such that cellular CE accumulation was reduced by only 24% at 10 μmol/L of Ro-71-4565. Similarly, VLDL-REM– and LDL–induced CE accumulation in macrophages was reduced significantly by preincubation with Ro-71-4565 (data not shown). OSC inhibitors had no significant effect on the 70-fold increase in TG accumulation induced by HTG-VLDL except at the 1 μmol/L dose, at which an 11% increase was observed (P<0.05, Figure 4D). By comparison, macrophages incubated with HTG-VLDL and exogenous 24(S),25-epoxy resulted in a dose-dependent decrease in CE (−40% at 4 μmol/L; Figure 4E). TG mass was unaffected (Figure 4F). Macrophages preincubated with either 22(R)-OH or the synthetic LXR agonist TO901317 reduced HTG-VLDL–induced CE mass by 61% and 63%, respectively (P<0.05, Figure 4E). However, TG accumulation was increased 35% with 22(R)-OH and 47% to 52% with TO901317 (all P<0.05, Figure 4F).
Macrophage FC concentrations were unaffected by HTG-VLDL alone (33±3 versus 31±4 μg/mg cell protein); however, FC mass decreased by 14% (P<0.001) in cells preincubated with Ro-71-4565. Preincubation of macrophages with exogenous 24(S),25-epoxy or TO901317 had no effect on cellular FC (data not shown). HTG-VLDL enhanced CE accumulation in mouse peritoneal macrophages isolated from C57Bl/6 mice by 2.2-fold (P<0.05). This was inhibited 60% (P<0.05) by preincubation of cells with Ro-71-4565 (1 μmol/L). In mouse peritoneal macrophages from LDL-R–null mice, HTG-VLDL increased cellular CE by only 10% above baseline. Ro-71-4565 prevented this small increase in CE accumulation; however, the TG increase was unchanged in both cell types (data not shown).
Effect of OSCi on Cholesterol Esterification
No significant effect on oleate incorporation into CE was observed during the first 5-hour incubation of macrophages with Ro-71-4565. However, after 19 hours of preincubation with 1 μmol/L Ro-71-4565, CE synthesis was reduced by 44% (P<0.05, data not shown). This indicates that OSC inhibitors did not directly affect acylCoA:cholesterol acyltransferase activity but reduced cholesterol esterification secondary to cholesterol synthesis inhibition.
OSC Inhibition Does Not Affect TG Synthesis or LPL Activity
Incubation of macrophages with Ro-71-4565 had no effect on oleate incorporation into TG, LPL activity (Figures 5A and 5B), or fatty acid synthesis (data not shown). Exogenous 24(S),25-epoxy also had no significant effect on TG synthesis (Figure 5A). In contrast, 22(R)-OH and TO901317 significantly increased TG synthesis (Figure 5A). Furthermore, 22(R)-OH increased LPL activity, whereas Ro-71-4565 had no significant effect (Figure 5B). LPL activities correlated with the effect of these compounds on TG accumulation (Figure 4F).
SREBP-1, primarily SREBP-1c, is a potent activator of genes involved in fatty acid and TG metabolism.8 Therefore, we examined expression and activation of SREBP-1. SREBP-1 mRNA was significantly increased in macrophages incubated with Ro-71-4565, 24(S),25-epoxy, or TO901317 (Figure 6A). Although our probe did not distinguish between SREBP-1a and SREBP-1c, it is likely that the increased mRNA represents SREBP-1c, because LXR agonists do not affect mouse SREBP-1a expression.22 Immunoblot analyses indicated that the precursor form of SREBP-1 also increased with each treatment (Figure 6B). However, TO901317 markedly increased the active, nuclear form of SREBP-1 (4-fold), whereas Ro-71-4565 and exogenous 24(S),25-epoxy produced only minor increases (<1.4-fold, Figure 6C).
Regulation of ABCA1 and ABCG1 mRNA and ABCA1 Protein by OSC Inhibition
Cholesterol efflux to apoAI is mediated by ABCA1 and ABCG1, both of which can be induced by oxysterol-dependent LXR activation.6,9 Ro-71-4565 significantly increased ABCA1 and ABCG1 mRNA (2- and 2.8-fold, respectively, both P<0.05) (Figures 7A and 7B). Similarly, exogenous 24(S),25-epoxy and TO901317 significantly induced both ABCA1 and ABCG1 mRNA. Immunoblot analyses demonstrated similar increases in ABCA1 protein (Figure 7C).
OSCi Induces Cholesterol Efflux via LXR
The OSCi-induced expression of ABCA1 and ABCG1 and reduction in cellular cholesterol in cells challenged with HTG-VLDL were determined under conditions in which cells were not preloaded with cholesterol. Therefore, we examined the effect of OSCi on [3H]cholesterol efflux in J774A.1 macrophages preincubated with [3H]cholesterol and minimal AcLDL (5 μg TC/mL). Compared with control, apoAI stimulated cholesterol efflux 3.0-fold (Figure 7D). In cells preincubated with Ro-71-4565, cholesterol efflux was enhanced a further 38% (P<0.05). Similarly, exogenous 24(S),25-epoxy increased efflux to apoAI by 53% (P<0.05) compared with apoAI alone. TO901317 and 22(R)-OH also enhanced cholesterol efflux. In addition, incubation of human THP-1 macrophages with Ro-71-4565 (10 nmol/L) significantly increased 24(S),25-epoxy synthesis and enhanced cholesterol efflux to apoAI. Similar values for cholesterol efflux were observed in response to exogenous 24(S),25-epoxy (4 μmol/L; data not shown).
A role for LXR in the induction of cholesterol efflux by 24(S),25-epoxy was demonstrated in J774A.1 macrophages coincubated with either ECHS or 22(S)-OH, two specific LXR antagonists.20,23 Both ECHS and 22(S)-OH completely inhibited the increase in apoAI-mediated cholesterol efflux induced by Ro-71-4565, 24(S),25-epoxy, and TO901317 (Figure 7D and data not shown). To confirm that partial OSC inhibition results in LXR-mediated gene expression, HepG2 cells were transfected with a reporter construct driven by an LXR response element. LXR activity was markedly increased in cells treated with either Ro-71-4565, 24(S),25-epoxy, or TO90317 (Figure 7E). Collectively, these results establish that increased cellular 24(S),25-epoxy, subsequent to OSC inhibition, enhances ABCA1 expression and macrophage cholesterol efflux via LXR-activation.
OSC inhibition had no effect on cholesterol efflux to apoAI in macrophages loaded with high amounts of cholesterol (see online Figure IA in the data supplement, available at http://www.circresaha.org). By contrast, TO901317 significantly increased cholesterol efflux to apoAI (online Figure IA). This inconsistency is explained by the observation that in cholesterol-loaded cells, both cholesterol and 24(S),25-epoxy synthesis were inhibited >90% (online Figures IB and IC). The OSCi further reduced cholesterol synthesis to undetectable levels and importantly, was unable to increase 24(S),25-epoxy synthesis. These results correlated with an absence of ABCA1 mRNA induction by the OSCi in these cells above that observed with cholesterol loading alone (online Figure ID).
Effect of OSC Inhibition on LDL-R mRNA and Activity
Some oxysterols can decrease LDL-R expression through suppression of SREBP activation, a process independent of LXR.8 We therefore examined whether OSCi-induced 24(S),25-epoxy synthesis would decrease LDL-R expression and activity. Ro-71-4565 reduced LDL-R mRNA 42% (P<0.05). By comparison, exogenous 24(S),25-epoxy decreased LDL-R mRNA 50% (P<0.05) (Figure 8A). By contrast, the LXR agonist TO901317 increased LDL-R mRNA 25%. Ro-71-4565 also significantly reduced 125I-LDL specific binding and uptake (−25 to −54%, P<0.05, Figure 8B). Similar reductions were observed with 22(R)-OH treatment.
Oxysterols play an important role in regulating genes involved in lipid metabolism and modulate the development of atherosclerosis.6,7 Oxysterols activate LXR, which increases the expression of genes involved in foam cell formation, including ABCA1, ABCG1, apoE, and SREBP-1c.6 Some oxysterols also suppress SREBP activation.8 SREBP-1c is a potent activator of genes involved in biosynthesis and esterification of unsaturated fatty acids, whereas SREBP-2 primarily activates genes involved in cholesterol metabolism, including the LDL-R and HMG-CoA reductase.8,24,25 In this study, we demonstrate that partial inhibition of OSC enhanced endogenous oxysterol synthesis, resulting in attenuated macrophage foam cell formation induced by the atherogenic lipoprotein HTG-VLDL. Our results indicate a mechanism involving (1) decreased lipoprotein uptake through inhibition of LDL-R expression and activity and (2) increased ABCA1 and ABCG1 expression. These effects were directly correlated with the extent of OSC inhibition and increase in endogenous synthesis of the regulatory oxysterol 24(S),25-epoxy.
Macrophages take up HTG-VLDL, resulting in significant CE and TG deposition, demonstrating the atherogenic potential of these lipoproteins.16 The novel aspect of the present study is that partial OSC inhibition in macrophages increases 24(S),25-epoxy synthesis, leading to significant reductions in HTG-VLDL–induced CE accumulation, an observation supported by marked decreases in Oil Red O–stained cytoplasmic lipid droplets. Similar decreases in CE mass were measured in macrophages treated with exogenous 24(S),25-epoxy, suggesting that the effects of OSC inhibition on cellular CE accumulation are primarily a result of the regulatory activity of endogenous 24(S),25-epoxy. Reductions in HTG-VLDL–induced CE accumulation were observed in macrophages incubated with the synthetic LXR ligand TO901317, indicating that endogenously synthesized 24(S),25-epoxy was acting, in part, through LXR activation.
An important concept emerging from this study is that 24(S),25-epoxy synthesis and inhibition of HTG-VLDL–induced foam cell formation is biphasic. Peak cellular concentrations of 24(S),25-epoxy achieved with Ro-71-4565 correlate exactly with maximal inhibition of macrophage CE accumulation. However, the effect is significantly attenuated as the extent of OSC inhibition increases, whereas cholesterol synthesis remains inhibited. Thus, reduced HTG-VLDL–induced CE accumulation results from increased 24(S),25-epoxy synthesis and not inhibition of cholesterol synthesis. Furthermore, this observation reveals an elegant feedback mechanism for regulation of endogenous synthesis of a potent regulatory oxysterol. Further OSC inhibition blocks the conversion of DOS to 24(S),25-epoxylanosterol, preventing the formation of 24(S),25-epoxy. This is in contrast to the effect of exogenous oxysterols or TO901317, in which no biphasic response was observed.
LDL-R expression and activity were reduced by OSC inhibition and exogenous 24(S)25-epoxy, contributing to the decrease in lipoprotein uptake and cellular CE accumulation. This provides evidence that 24(S),25-epoxy mediates transcriptional regulation of the LDL-R in OSCi-treated cells. LDL-R downregulation occurred despite inhibition of cholesterol synthesis. Depletion of cellular cholesterol would normally increase LDL-R expression; however, this response occurs when oxysterol concentrations are low, thereby stimulating SREBP-2 processing.8 Exogenous 24(S),25-epoxy is known to suppress SREBP-2 processing,26 supporting the concept that OSCi-induced 24(S),25-epoxy synthesis also suppressed SREBP-2 processing, resulting in the downregulation of LDL-R expression.8 The importance of the LDL-R in macrophage foam cell formation is demonstrated in atherosclerosis-susceptible mice, whereby transplantation of irradiated C57Bl/6 mice with LDL-R–null bone marrow significantly decreased diet-induced atherosclerosis.27
Oxysterols regulate transcription of key genes in cellular lipid homeostasis in part through activation of LXR.6,9 24(S),25-Epoxy is a potent LXR ligand,9 known to activate the LXR-responsive genes ABCA110 and ABCG1.6 Cholesterol loading of macrophages induces ABCA1 expression via LXR, and recently it was determined that the oxysterol responsible was 27-hydroxycholesterol, whereas 22(R)-OH and 24(S),25-epoxy, two oxysterols believed to be endogenous ligands for LXR, were undetected.10 We demonstrate clearly that in the absence of cholesterol loading, inhibition of OSC increased 24(S),25-epoxy synthesis, leading to increased ABCA1 and ABCG1 expression, ABCA1 protein, and cholesterol efflux. LXR is implicated in this process, because LXR antagonists completely attenuated the OSCi-induced increase in apoAI-mediated cholesterol efflux, and partial inhibition of OSC resulted in an increase in LXR-mediated gene expression.
In macrophages preincubated with small amounts of cholesterol, OSC inhibition markedly increased cholesterol efflux, similar to that observed with exogenous 24(S),25-epoxy, 22(R)-OH, or TO901317. Therefore, OSCi-induced cholesterol efflux contributed to attenuated CE accumulation in macrophages exposed to HTG-VLDL. However, in macrophages loaded with high concentrations of cholesterol, OSC inhibition did not induce cholesterol efflux, whereas the nonsterol LXR agonist TO901317 was effective. This was because of decreased cholesterol synthesis via feedback inhibition of HMG-CoA reductase by AcLDL-derived cholesterol,8,10 thus preventing Ro-71-4565 from enhancing 24(S)25-epoxy synthesis and subsequent ABCA1 upregulation. Collectively, our experiments suggest that OSCis may be beneficial for enhancing cholesterol efflux and preventing foam cell formation during the lipoprotein uptake phase but may be less effective once macrophages become cholesterol loaded.
A concern with LXR agonists as therapeutic agents is the potential to increase liver and plasma TG concentrations through LXR activation of fatty acid synthase directly28 or via the activation of SREBP-1c, a transcriptional regulator of TG and fatty acid synthesis.8,24,25 OSCi-induced 24(S),25-epoxy synthesis had no appreciable effect on macrophage TG or free fatty acid synthesis, accumulation of cellular TG by HTG-VLDL, or LPL activity. Furthermore, exogenous 24(S),25-epoxy did not influence TG accumulation induced by HTG-VLDL. In contrast, 22(R)-OH or the synthetic LXR agonist TO901317 induced both LPL activity and TG synthesis. The OSCi, exogenous 24(S),25-epoxy and TO901317 significantly increased both SREBP-1 mRNA and precursor protein levels. However, only TO901317 resulted in a marked increase in active, nuclear SREBP-1; the OSCi and exogenous 24(S),25-epoxy appeared to suppress SREBP-1 processing. Although other oxysterols, including 25-hydroxycholesterol, are known to suppress SREBP-1 processing,8,29 this has not been reported for 24(S),25-epoxy. These observations provide a potential mechanism for the lack of effect of OSC inhibition on macrophage TG metabolism. Recently, Joseph et al30 demonstrated in mice that a synthetic LXR agonist decreased atherosclerosis but only transiently increased TGs, suggesting that alteration of TG metabolism may not affect the atheroprotective effect of LXR activation. Whether this applies to humans is unknown. Nevertheless, the lack of effect of OSC inhibition on TG metabolism is a potential therapeutic advantage.
In the present study, we clearly show that partial OSC inhibition decreases macrophage uptake of native lipoproteins and CE accumulation through decreased LDL-R expression and enhanced expression of ABCA1 and ABCG1, leading to increased cholesterol efflux. Therefore, increased macrophage synthesis of endogenous oxysterols represents a new mechanism for dual regulation of LXR- and SREBP-responsive genes, an approach that inhibits foam cell formation without detrimental effect on TG synthesis. Thus, OSC inhibition within vascular wall macrophages represents a potential new strategy for the prevention of atherosclerosis.
This work was supported by a grant from the Canadian Institutes of Health Research (CIHR; MT 8014 to M.W.H.). A.H.R. is the recipient of an Ontario Graduate Scholarship in Science and Technology, and C.A.A. is the recipient of a CIHR Studentship. M.W.H. and R.A.H. are Career Investigators of the Heart and Stroke Foundation of Ontario, and R.A.H. holds a Canada Research Chair (Tier I) in Human Genetics. We thank Allison Kulchycki, Cara Ooi, and Janine van Gils for their expert technical assistance. OSC inhibitors were prepared by J. Aebi and H. Dehmlow, F. Hoffmann-La Roche Ltd.
↵*Both authors contributed equally to this study.
At the time these experiments were conducted, Olivier H. Morand was an employee of F. Hoffmann-La Roche Ltd, Pharmaceuticals Division. Present address: Actelion Pharmaceuticals Ltd, Allschwil, Switzerland.
Original received March 24, 2003; revision received September 15, 2003; accepted September 16, 2003.
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