PPARα activation promotes macrophage reverse cholesterol transport through an LXR-dependent pathway

Abstract

Objective

Peroxisome proliferator-activate receptorα (PPARα) activation has been shown in vitro to increase macrophage cholesterol efflux, the initial step in reverse cholesterol transport (RCT). However, it remains unclear whether PPARα activation promotes macrophage RCT in vivo.

Methods and Results

We demonstrated that a specific potent PPARα agonist GW7647 inhibited atherosclerosis and promoted macrophage RCT in hypercholesterolemic mice expressing the human apoA-I gene. We compared the effect of GW7647 on RCT in human apoA-I transgenic (hA-ITg) mice with wild-type (WT) mice and showed that the PPARα agonist promoted RCT in hA-ITg mice to a much greater extent than in WT mice, indicating that human apoA-I expression is important for PPARα-induced RCT. We further investigated the dependence of the macrophage PPARα-LXR pathway on the promotion of RCT by GW7647. Primary murine macrophages lacking PPARα or LXR abolished the ability of GW7647 to promote RCT in hA-ITg mice. In concert, the PPARα agonist promoted cholesterol efflux and ABCA1/ABCG1 expression in primary macrophages and this was also by the PPARα-LXR pathway.

Conclusion

Our observations demonstrate that a potent PPARα agonist promotes macrophage RCT in vivo in a manner that is enhanced by human apoA-I expression and dependent on both macrophage PPARα and LXR expression.

Reverse cholesterol transport (RCT) is believed to be a primary atheroprotective property of high-density lipoprotein (HDL) and its major protein apolipoprotein A-I (apoA-I), which promote efflux of excess cholesterol from macrophages in atherosclerotic lesions and then transport it back to the liver for excretion into bile and eventually the feces.¹ Cholesterol efflux is the initial step in RCT and plays a pivotal role in maintaining intracellular cholesterol levels and preventing the formation of macrophage-derived foam cells in atherosclerotic plaques. ATP binding cassette transporter A1 (ABCA1) has been shown to play an important role in apoA-I-mediated cholesterol efflux from peripheral cells and macrophages, whereas ABCG1 promotes cholesterol efflux from macrophages to HDL particles but not to lipid-poor apoA-I.^2–3 ABCA1 and ABCG1 in macrophages act in synergy against atherosclerosis.⁴ Moreover, we recently developed an assay that specifically traces RCT from the macrophages to the feces in vivo,⁵ and reported that both ABCA1 and ABCG1 in macrophage play critical roles in promoting macrophage RCT in vivo.⁶

Peroxisome proliferator-activated receptors (PPARs) have been reported to regulate the expression of genes that control lipid metabolism by binding as heterodimers with retinoid X receptors to PPAR response element (PPRE) in the promoter or enhancer regions of these genes.⁷ PPARα is a member of this nuclear receptor superfamily that regulates gene expression in response to the binding of fatty acids and their metabolites,^8–9 and is expressed in the major cell types found in the atherosclerotic lesion, including macrophages, endothelial, and smooth muscle cells.^10–12 PPARα activation has been demonstrated in vitro to stimulate ABCA1 gene transcription, which in turn enhances cholesterol efflux.^13–14 Moreover, these events have been suggested to involve upregulation of the liver X receptor (LXR),^13–14 one of the main regulators of ABCA1 gene transcription,¹⁵ although specific mechanisms for these cascades and their relevance to the in vivo setting are yet to be clarified.

In addition, studies in human apoA-I transgenic mice (hA-ITg)¹⁶ and in human subjects¹⁷ have established that PPARα agonists promote human apoA-I gene transcription and its production. We reported that overexpression of human apoA-I promoted macrophage RCT in vivo.¹⁸ In concert with these findings, Duez et al¹⁹ demonstrated that human apoA-I expression is important for PPARα agonist-mediated reduction in atherosclerosis using apoE-KO mice with the human apoA-I transgene.

In the present study, we examined whether a potent PPARα agonist promoted macrophage RCT in vivo in mice, and whether its effect was conditioned by expression of human apoA-I and the expression of macrophage PPARα and LXR.

Methods

An expanded Methods section is available in the Online Data Supplement.

Human apoA-I transgenic (hA-ITg) and PPARα-knockout mice were obtained from the Jackson Laboratory, and LXRα/β double-knockout mice were obtained from Taconic. LDLR/apobec-1 double-knockout mice and hA-ITg mice were crossed to generate LDLR/Apobec-1 double-knockout/human ApoA-I transgenic (LAA) mice. Lipids from fasted plasma and fast performance liquid chromatography (FPLC) samples were quantified using commercially available kits. Primary bone marrow derived macrophages were isolated from femurs and tibias of mice and cultured in DMEM supplemented with 30% L929 conditioned medium. Cholesterol efflux and in vivo RCT studies were performed using established methods. All studies were approved by the University of Pennsylvania Institutional Animal Care and Use Committee.

Results

The PPARα Agonist GW7647 Inhibits the Development of Atherosclerosis in Hypercholesterolemic Mice Expressing Human ApoA-I

LDLR/Apobec-1 double knockout mice have elevated LDL cholesterol and apoB-100 levels and develop extensive atherosclerosis on chow diet,²⁰ thus more closely resembling human atherosclerotic pathophysiology. To investigate potential effects of PPARα activation on the development of atherosclerosis in this hypercholesterolemic murine model in the setting of human apoA-I expression driven by the PPARα-responsive human apoA-I promoter, we generated LDLR/Apobec-1 double-knockout/human ApoA-I transgenic (LAA) mice and performed intervention studies using the potent and specific PPARα agonist GW7647. GW7647 has a median effective concentration of 1nM for the murine PPARα, compared to 2.9 µM and 1.3 µM for murine PPARβ and PPARγ, respectively.²¹ LAA mice were fed chow diet with or without GW7647 for 24 weeks and no adverse health effects were noted throughout the study. GW7647 treatment brought about a 2.3 fold increase in hepatic mRNA levels of acyl-CoA oxidase (AOX), a canonical PPARα target gene, compared with control treatment in the mice (Supplemental Figure I).

GW7647 treatment significantly increased plasma HDL-cholesterol and human apoA-I levels by 45% and 46%, respectively (Table 1). Total cholesterol levels trended higher and triglyceride levels trended lower in mice treated with GW7647 compared with control-treated mice, but neither were statistically significant. Analysis of lipoprotein profiles using fast-performance liquid chromatography (FPLC) revealed a substantial increase in the HDL fraction, but little change in the VLDL and LDL fractions, in the GW7647-treated group compared with the control group (Supplemental Figure IIA).

Table 1.

Effect of the PPARα Agonist on Plasma Lipid Levels in LAA Mice

	Total cholesterol (mg/dL)	HDL Cholesterol (mg/dL)	Triglycerides (mg/dL)	ApoA-I (mg/dL)
Control	314 ± 82	53 ± 20	125 ± 38	126 ± 40
GW7647	386 ± 81	95 ± 24*	100 ± 36	181 ± 36*

The results for 10 mice each group are presented as mean ± SD. Value were determined in plasma samples from fasting animals.

^*P< 0.05.

The extent of atherosclerosis was determined in en face preparations of the aorta as well as in sections of the aortic root. Representative examples of en face Oil Red O-stained aortas from LAA mice treated with GW7647 or control are shown in Figure 1A. When the extent of atherosclerotic lesion was quantified, GW7647 resulted in a significant decrease in atherosclerosis in the aorta by 35% (Figure 1B). We also quantified total lesion area in the aortic root. Representative sections are shown in Figure 1C. The mice treated with GW7647 demonstrated a significant 31% decrease in aortic root lesions compared with control mice (Figure 1D).

Figure 1. The PPARα Agonist Inhibits the Development of Atherosclerosis and Promotes Macrophage RCT in LDLR/Apobec-1 double-knockout/human ApoA-I transgenic (LAA) Mice.

LAA mice (n = 10/each group) were maintained with chow diet containing either the specific PPARα agonist GW7647 (2.5 mg/kg/day) or control for 24 weeks, and then atherosclerosis study was performed as described in Methods. (A) Oil red O-stained en face preparations of aortas. (B) Quantitative analysis of atherosclerotic surface area in the entire aorta. Data are expressed as percent of total aortic area. (C) Oil red O staining of representative sections through the aortic root at the level of the aortic valves. The micrographs were taken of sections at a similar distance from the aortic root. (D) Quantitative analysis of lesion areas in the aortic root.

LAA mice that had been treated with either GW7647 (2.5 mg/kg/day) or control for 2 weeks were intraperitoneally injected with ³H-cholesterol-labeled and acLDL-loaded J774 macrophages. (E) Time course of ³H-cholesterol distribution in plasma. Blood samples obtained from mice at 6, 24, and 48 hours after injection were subjected to ³H-tracer analysis. (F) Fecal ³H-tracer levels. Feces collected continuously from 0 to 48 hours were prepared as described in Methods, and then subjected to ³H-tracer analysis. Data are expressed as percent counts relative to total injected tracer; mean ± SD; n=8 for each group. * P<0.05, ** P<0.01 vs. control.

PPARα Agonism Promotes Macrophage RCT in Hypercholesterolemic Mice Expressing Human ApoA-I

The enhancing effects of PPARα on human apoA-I and HDL in LAA mice led us to hypothesize that its activation inhibited atherogenesis by promoting RCT. We therefore used methods developed in our laboratory⁵ in order to investigate the impact of PPARα agonism on macrophage RCT in LAA mice. Cholesterol-loaded and ³H-cholesterol-labeled J774 macrophages were intraperitoneally injected into LAA mice that had been treated with GW7647 or control for 2 weeks. We then followed the ³H-tracer levels in plasma at 6, 24, and 48 hours and in liver at 48 hours after injection. We also integrated the ³H-tracer levels in feces collected through the 0- to 48- hour period. J774 macrophages were found to express PPARα as detected by RT-PCR (data not shown). The appearance in plasma of macrophage-derived ³H-tracer levels in LAA mice treated with GW7647 showed a non-significant trend toward an increase compared with control-treated mice (Figure 1E). Fractionation of lipoproteins at 48 hours revealed a substantially greater number of ³H-tracer counts in the HDL fraction of GW7647-treated mice than in control mice (Supplemental Figure IIB). Most importantly, GW7647 treatment significantly increased the levels of macrophage-derived ³H-tracer excreted into feces by 98% (Figure 1F). These data indicate that PPARα activation with GW7647 promotes macrophage RCT in LAA mice.

Human ApoA-I Expression Plays an Important Role in the Ability of Systemic PPARα Activation to Promote Macrophage RCT

Next, we investigated whether transgenic human apoA-I expression contributed to PPARα-mediated macrophage RCT in vivo. We compared the effect of the PPARα agonist on macrophage RCT in human apoA-I transgenic (hA-ITg) mice with that in wild-type (WT) mice to specifically evaluate the contribution of human apoA-I. GW7647 significantly increased AOX mRNA levels in the liver from both WT and hA-ITg mice (Supplemental Figure IIIA and IIIB), indicating that GW7647 efficiently activated PPARα in both mice. As expected, in hA-ITg mice GW7647 induced plasma human apoA-I levels and liver human apoA-I gene expression (Supplemental Figure IIIC and IIID), resulting in a significant increase in plasma HDL-C levels (Supplemental Figure IIIF). GW7647 treatment also increased plasma HDL-C levels in WT mice (Supplemental Figure IIIE), but to a lesser extent than in hA-ITg mice.

We performed a macrophage RCT assay in both WT and hA-ITg mice treated with GW7647 or control. The appearance in plasma of macrophage-derived ³H-tracer levels in WT mice treated with GW7647 were modeslty but significantly increased at 24 hours after injection (Figure 2A). GW7647 also modestly, but significantly, induced ³H-tracer levels in feces by 30% in WT mice (Figure 2C). In contrast, hA-ITg mice treated with GW7647 showed substantially greater ³H-tracer levels in plasma at 24 and 48 hours after injection (Figure 2B). Moreover, the fecal ³H-tracer excretion from GW7647-treated hA-ITg mice was increased by 108% (Figure 2D). Taken together, these observations suggest that PPARα-induced human apoA-I expression confers substantial enhancement of macrophage RCT in vivo induced by a PPARα agonist.

Figure 2. Human ApoA-I Expression is Important for PPARα-mediated Macrophage RCT in vivo.

The RCT experiments were performed as described in Figure 1 using wild-type (WT) mice and human apoA-I transgenic (hA-ITg) mice. ³H-cholesterol-labeled and acLDL-loaded J774 macrophages were intraperitoneally injected into WT or hA-ITg mice treated with or without GW7647 (2.5 mg/kg/day). (A and B) Time course of ³H-cholesterol distribution in plasma from WT (A) and hA-ITg (B) mice. (C and D) Fecal ³H-tracer levels from WT (C) and hA-ITg (D) mice. Data are expressed as percent counts relative to total injected tracer; mean ± SD; n=8 for each group. * P<0.05, ** P<0.01 vs. control.

Macrophage PPARα is Required for the Ability of Systemic PPARα Activation to Promote Macrophage RCT

To further explore the mechanism of PPARα-mediated macrophage RCT, we tested whether systemic PPARα activation required macrophage PPARα to promote RCT using primary bone marrow derived macrophages (BMMs) from PPARα-KO and WT mice. We confirmed that PPARα was expressed in WT BMMs but not in PPARα-KO BMMs (data not shown). Human A-ITg mice were treated with GW7647 or control for 2 weeks before injection of labeled BMMs from either PPARα-KO or WT mice and continued to be treated during the 48-hour experiment. The ³H-tracer levels in plasma were significantly increased by GW7647 treatment in the mice injected with WT BMMs (Figure 3A). In contrast, GW7647 had no effect in the plasma ³H-tracer levels in the mice injected with PPARα-KO BMMs (Figure 3B). GW7647 treatment also significantly increased fecal excretion of ³H-tracer in WT BMMs-injected mice by up to 60%, but no effect in the mice with PPARα-KO BMMs (Figure 3C and 3D). These data confirm that macrophage PPARα is required for the promotion of RCT by PPARα.

Figure 3. Macrophage PPARα is Critical for Systemic PPARα Activation-induced Macrophage RCT.

HA-ITg mice that had been treated with either GW7647 (2.5 mg/kg/day) or control for 2 weeks were intraperitoneally injected with ³H-cholesterol-labeled and acLDL-loaded BMMs from WT (PPARα^+/+) or PPARα-KO (PPARα^−/−) mice prepared as described in Methods. (A and B) Time course of ³H-cholesterol distribution in plasma. Blood samples obtained from the mice injected with BMMs from PPARα^+/+ (A) or PPARα^−/− (B) mice at 6, 24, and 48 hours after injection were subjected to ³H-tracer analysis. (C and D) Fecal ³H-tracer levels. Feces collected continuously from the mice injected with BMMs from PPARα^+/+ (C) or PPARα^−/− (D) mice were subjected to ³H-tracer analysis. Data are expressed as percent counts relative to total injected tracer; mean ± SD; n=8 for each group. * P<0.05 vs. control.

Next, in order to elucidate the molecular mechanisms underlying the enhanced macrophage RCT, we determined the impact of PPARα activation on cholesterol efflux and expression of genes involved in the pathway in WT and PPARα-KO BMMs ex vivo. In WT BMMs GW7647 treatment increased apoA-I- and HDL3-mediated cholesterol efflux by 42% and 18%, respectively, whereas in PPARα-KO BMMs this treatment had no effect on cholesterol efflux (Figure 4A and 4B). Supporting the change in cholesterol efflux, GW7647 significantly increased expression of ABCA1/G1 mRNA (Figure 4C, 4D) and protein (Supplemental Figure IV) levels in WT BMMs, whereas the treatment did not change their expression in PPARα-KO BMMs (Figure 4C, 4D). Similar to the changes in ABCA1 and ABCG1, LXRα and AOX mRNA levels were also significantly increased by GW7647 treatment in WT BMMs, whereas these effects were cancelled in PPARα-KO BMMs (Figure 4E and 4F). GW7647 did not significantly change expression of macrophage scavenger receptor CD36, an established PPARγ target gene, or SR-BI (data not shown). Taken together, these findings suggest that PPARα activation enhances cholesterol efflux by increasing ABCA1/G1 expression in primary murine macrophages, which in turn promotes overall RCT in vivo, in a macrophage PPARα-dependent manner.

Figure 4. The PPARα Agonist Enhances Cholesterol Efflux and ABCA1/ABCG1/LXRα/AOX Expression in a PPARα-dependent Manner.

(A and B) BMMs from WT (PPARα^+/+) or PPARα-KO (PPARα^−/−) mice were labeled with ³H-cholesterol and loaded with acLDL as described in Methods. Cells were equilibrated for 18 hours in the presence or absence of 1 µM GW7647. Cholesterol efflux was determined in the presence of 10 µg/mL of human apoA-I (A) or 25 µg/mL of human HDL3 (B) for 4 hours as described in Methods. The results for 3 samples are presented as mean ± SD. (C, D, E, and F) Quantitative analysis of mRNA expression of mouse ABCA1 (C), ABCG1 (D), LXRα (E), and AOX (F) in BMMs from PPARα^+/+ and PPARα^−/− mice by quantitative RT-PCR. Total RNA was extracted as described in Methods from BMMs that were treated with GW7647 (1 µM) or control for 24 hours. The mRNA levels of each gene are standardized for mouse β-actin levels. The results are expressed relative to the controls and presented as mean ± SD. * P<0.05 vs. control.

Macrophage LXR is Required for the Ability of Systemic PPARα Activation to Promote Macrophage RCT

Finally, we investigated whether macrophage LXR is essential for the ability of PPARα to promote RCT using BMMs from LXRα/β double-knockout (LXR-DKO) mice. We performed macrophage RCT studies in hA-ITg mice with WT and LXR-DKO BMMs. GW7647 treatment significantly increased plasma ³H-tracer levels at 24 and 48 hours in the mice injected with WT BMMs, but there was no effect in the mice injected with LXR-DKO BMMs (Figure 5A and 5B). The fecal excretion of ³H-tracer was also increased by GW7647 in WT BMMs-injected mice by up to 188%, but not in LXR-DKO BMMs-injected mice (Figure 5C and 5D). In ex vivo experiments, GW7647 significantly increased apoA-I- and HDL3-mediated cholesterol efflux as well as ABCA1/G1 expression in WT BMMs, whereas this treatment had no effect in LXR-DKO BMMs (Figure 6A–6D). These observations indicate that the macrophage PPARα-LXR pathway plays a critical role in PPARα-induced macrophage RCT.

Figure 5. Macrophage LXR is Critical for Systemic PPARα Activation-induced Macrophage RCT.

The RCT experiments were performed as described in Figure 3 with BMMs from WT (LXR^+/+) or LXR-DKO (LXR^−/−) mice. (A and B) Time coarse of ³H-cholesterol distribution in plasma from the mice injected with BMMs from LXR^+/+ (A) or LXR^−/− (B) mice. (C and D) Fecal ³H-tracer levels from the mice injected with BMMs from LXR^+/+ (C) or LXR^−/− (D) mice. Data are expressed as percent counts relative to total injected tracer; mean ± SD; n=8 for each group. * P<0.05 vs. control.

Figure 6. The PPARα Agonist Enhances Cholesterol Efflux and ABCA1/G1 Expression in a LXR-dependent Manner.

(A and B) Cholesterol efflux assay were performed as described in Figure 4 using BMMs from WT (LXR^+/+) and LXR-DKO (LXR^−/−) mice. Cholesterol efflux was determined in the presence of 10 µg/mL of human apoA-I (A) or 25 µg/mL of human HDL3 (B) for 4 hours. The results for 3 samples are presented as mean ± SD. (C and D) Quantitative RT-PCR and total RNA extraction were performed as described in Figure 4 using BMMs from LXR^+/+ or LXR^−/− mice to determine ABCA1 (C) and ABCG1 (D) mRNA levels. The mRNA levels of each gene are standardized for mouse β-actin levels. The results are expressed relative to the controls and presented as mean ± SD. * P<0.05 vs. control.

Discussion

In the present study, we demonstrated that PPARα activation with the specific and potent agonist GW7647 inhibited the development of atherosclerosis and promoted macrophage RCT in LAA mice, a humanized hypercholesterolemic murine model expressing the human apoA-I gene. Next, we showed that GW7647 brought about much higher rate of macrophage RCT as well as increased plasma human apoA-I levels in hA-ITg mice than in WT mice, suggesting that human apoA-I expression is important for the promotion of macrophage RCT by PPARα. We further determined the impact of the macrophage PPARα-LXR pathway on PPARα-mediated overall RCT. Interestingly, GW7647 significantly promoted macrophage RCT in hA-ITg mice injected with WT primary macrophages but not in the mice injected with macrophages lacking PPARα or LXR. In ex vivo experiments, GW7647 also enhanced cholesterol efflux and ABCA1/G1 expression in primary macrophages by the PPARα-LXR pathway. Taken together, these observations suggest that PPARα activation promotes macrophage RCT in a macrophage PPARα- and LXR-dependent fashion.

The stimulatory effect of PPARα activation on human apoA-I expression has been well documented. Vu-Dac et al²² reported that PPARα agonist increased human apoA-I expression but not rodent apoA-I due to sequence divergences in the respective apoA-I promoters. We also demonstrated that PPARα agonist upregulated apoA-I production in human subjects by kinetic studies.¹⁷ Moreover, Duez et al¹⁹ reported that a PPARα agonist had minimal anti-atherogenic effects in apoE-KO mice but exerted a more pronounced effect in apoE-KO mice carrying the human apoA-I transgene, suggesting that human apoA-I expression is crucial for the reduction in atherosclerosis by PPARα. In the present study, we therefore used LDLR/Apobec-1 double-knockout/human ApoA-I transgenic (LAA) mice and human apoA-I transgenic mice (hA-ITg) mice in order to investigate the effect of PPARα activation on atherosclerosis and RCT in mouse models with the human apoA-I gene, driven by the human apoA-I promoter which has PPARα-responsive elements.

Consistent with the previous observations in humans,^{16, 23–25} the PPARα agonist strongly increased human apoA-I and HDL-cholesterol levels in LAA mice. Therefore, we hypothesized that PPARα inhibited atherogenesis by promoting macrophage RCT. As expected, in concert with its effect on atherosclerosis, GW7647 significantly promoted the fecal excretion of HDL-derived cholesterol and thus the overall pathway of macrophage RCT in LAA mice. Recently, Rotllan N et al²⁶ also reported that a well-known PPARα agonist fenofibrate promoted macrophage RCT, supporting that the effect of GW7647 on RCT is not an off-target effect.

ApoA-I is also present free in the circulation and interstitial space under varying degrees of lipidation.²⁷ It is therefore plausible that apoA-I associated with HDL may have direct effects on HDL anti-atherogenic function. Abundant data from studies in animals have indicated that enhancement of human apoA-I expression inhibited atherosclerosis.^28–29 We also reported that overexpression of human apoA-I promoted macrophage RCT in vivo, which is thought to contribute at least in part to its anti-atherogenic properties.¹⁸ Therefore, we investigated whether PPARα-induced human apoA-I expression results in the promotion of macrophage RCT. We compared the effect of GW7647 on macrophage RCT in hA-ITg mice with WT mice to specifically evaluate the contribution of human apoA-I and found that GW7647 treatment had a much higher rate of RCT in hA-ITg mice. These studies provide the first in vivo evidence of the substantial contribution of human apoA-I expression to the enhancement of macrophage RCT by PPARα, consistent with the previous study indicating the importance of human apoA-I in the inhibition of atherosclerosis by PPARα.¹⁹ These observations also support the concept that the promotion of macrophage RCT may be one mechanism by which PPARα activation functions to inhibit the development of atherosclerosis in the hypercholesterolemic mice.

It has been shown that PPARα promotes cholesterol efflux from macrophages in vitro and that this may be related to upregulation of LXR expression. LXR, especially LXRα, has been reported to be activated by oxysterols and its primary function is to maintain cellular cholesterol homeostasis. It is therefore relevant that activation of LXRα leads to induction of the genes required for cholesterol efflux, such as those encoding ABCA1 and ABCG1 through the LXR response elements in their proximal promoters.¹⁵ Moreover, Laffitte et al³⁰ reported that LXR agonist-induced ABCA1 and ABCG1 expression in macrophages were completely abolished by deletion of both LXRα and LXRβ. In fact, it was reported that PPARγ directly induces LXRα expression through a response element upstream of the LXR promoter region and leads to the induction of ABCA1, and then regulates a pathway of cholesterol efflux from macrophages.³¹ PPARα agonists also have been shown to induce ABCA1 expression and cholesterol efflux in macrophages in an LXRα-dependent manner.^13–14 In addition, it was recently reported that activation of LXRα enhanced not only ABCA1 but also PPARα gene transcription,³² indicating that the cellular regulation of cholesterol transport is via a complex pathway involving PPARα and LXR.

A previous study using LDLR-KO mice reconstituted with bone marrow from PPARα-KO mice demonstrated that macrophage PPARα conferred anti-atherogenic effects via modulation of macrophage cholesterol metabolism.³³ Moreover, our present data suggested that an important factor in the promotion of overall RCT by the PPARα agonist was likely to be the enhancement of cholesterol efflux from the injected macrophages, which was reflected in the increased plasma tracer levels. We therefore examined whether macrophage PPARα-LXR pathway plays a crucial role in the promotion of RCT by PPARα using primary bone marrow macrophages lacking PPARα or LXR. Interestingly, primary macrophages lacking PPARα or LXR completely abolished PPARα-induced overall RCT in vivo, indicating the critical role of macrophage PPARα and LXR in RCT. In ex vivo experiments, we also demonstrated that PPARα activation enhanced cholesterol efflux and ABCA1/G1 gene expression through the PPARα-LXR pathway.

Since we previously reported that macrophage ABCA1 and ABCG1 promoted RCT in vivo and were additive in their effects,⁶ our in vivo and ex vivo findings are consistent with the concept that PPARα activation enhances cholesterol efflux in association with increased expression of ABCA1/G1 in macrophages, resulting in promotion of overall RCT, by the PPARα-LXR pathway. The above observations also suggest that modest promotion of RCT by PPARα in WT mice might be mainly due to enhanced cholesterol efflux from macrophages. Moreover, since macrophage PPARα and LXR were required for the promotion of RCT even in the condition with increased human apoA-I expression, the ability of human apoA-I to promote RCT may require the interaction with the macrophage PPARα-LXR pathway. Thus further investigation is needed to elucidate the exact mechanisms.

On the other hand, several recent studies have provided conflicting results on the roles of PPARα in ABCA1 and ABCG1 expression in macrophages. Li et al³⁴ showed that PPARα agonist inhibited foam-cell formation in cholesterol-loaded murine peritoneal macrophages in a LXR-dependent manner, but did not affect ABCA1 expression. Babaev et al³³ also reported that PPARα agonist treatment had a trend for increases in ABCA1 and ABCG1 expression in WT peritoneal macrophages, but these were not significant. The discrepancies between these studies and our results might be partly ascribed to differences between peritoneal macrophages, which are stimulated by thioglycollate, and BMMs. Moreover, PPARα agonists have been reported to inhibit pro-inflammatory pathways by antagonizing NF-κB or activator protein-1.^35–36 Clearly, macrophage cholesterol efflux and RCT constitute one factor involved in the overall impact of PPARα on atherosclerosis, and other factors including inhibition of inflammation can also have effects on atherosclerosis besides those of RCT.

In summary, we demonstrate that PPARα activation with a specific agonist inhibits the development of atherosclerosis and promotes macrophage RCT in humanized hypercholesterolemic mice expressing the human apoA-I gene. We also show that human apoA-I expression is important for PPARα-mediated macrophage RCT. More interestingly, macrophage PPARα and LXR expression are critical for the ability of PPARα to promote cholesterol efflux and overall RCT. These results indicate that PPARα exerts an anti-atherogenic property by promoting RCT through the macrophage PPARα-LXR pathway, and are consistent with the concept that specific and potent PPARα activation might be expected to enhance macrophage RCT in humans.