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EP 80317, a ligand of the CD36 scavenger receptor, protects apolipoprotein E-deficient mice from developing atherosclerotic lesions

Sylvie Marleau^*,1, Diala Harb^*,1, Kim Bujold^*,1, Roberta Avallone^*, Khadija Iken^*, Yanfei Wang^*, Annie Demers^*, Martin G. Sirois, Maria Febbraio, Roy L. Silverstein, André Tremblay and Huy Ong^*,2

^* Faculty of Pharmacy and Department of Pharmacology, Faculty of Medicine, Université de Montréal, Québec, Canada;
Montreal Heart Institute, Montréal, Québec, Canada;
Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA; and
Ste-Justine Hospital Research Center, Québec, Canada

² Correspondence: Faculty of Pharmacy, Université de Montréal, Pavillon Jean-Coutu, P.O. Box 6128, Station Centre-Ville, Montréal, Québec, Canada, H3C 3J7. E-mail: huy.ong{at}umontreal.ca

SPECIFIC AIMS

The original aim of the present study was to assess whether EP 80317, a novel CD36 ligand, may attenuate atherosclerotic disease progression in apoE-deficient (apoE^–/–) mice fed a high fat, high cholesterol (HFHC) diet, using apoE/CD36 double-deficient (apoE^–/–/CD36^–/–) mice as negative controls. We observed a striking, CD36-dependent anti-atherosclerotic effect of EP 80317, which led us to investigate its effects on plasma lipid profile and on the expression of genes associated with cholesterol metabolism and trafficking in macrophages.

PRINCIPAL FINDINGS

1. EP 80317 reduces lesion formation and total plasma cholesterol in apoE deficient, but not in apoE/CD36 double-deficient mice
ApoE^–/– and apoE^–/–/CD36^–/– mice fed a HFHC diet from 6 wk of age were split into vehicle (0.9% NaCl) and EP 80317-treated groups. Eighteen-wk-old vehicle-treated mice showed a significant number of oil red-O-stained lesions as visualized in longitudinal aorta sections and elevated plasma cholesterol levels. Chronic (12 wk) treatment with EP 80317 from 6 to 18 wk of age significantly reduced the percentage of aortic lesion areas and total plasma cholesterol by 51 and 30%, respectively (Fig. 1 A, B, D). Neither HDL cholesterol nor triglyceride levels were significantly modulated. Figure 1C shows lesions and cholesterol deposition protruding into the lumen at lesion-prone sites in 0.9% NaCl-treated mice. A significant reduction (up to 61%) in plaque surface area was observed in EP 80317-treated apoE^–/– mice. The body weights and food intake did not differ significantly between the vehicle and treated groups during the last week of the study (Fig. 1E ). In contrast, EP 80317 did not significantly modify the percentage of aortic lesions in apoE^–/–/CD36^–/– mice fed a HFHC diet, nor did it modulate plasma lipids (data not shown), suggesting that EP 80317 might negatively modulate atherosclerosis in a CD36-dependent manner.

View larger version (26K): [in this window] [in a new window]   Figure 1. Effects of long-term (12 wk) administration of EP 80317 in apoE–/– mice. Representative en face oil red-O-stained aortas from apoE–/– mice fed an atherogenic diet from 6 wk of age, and treated daily with 0.9% NaCl (top) or EP 80317 (300 µg/kg) (bottom) (A) and mean % of aortic lesion areas in apoE–/– mice treated with EP 80317 (n=6) or vehicle (n=4) (B). Representative histochemistry of aortic sections of apoE–/– mice treated with vehicle or EP 80317 (C). Total plasma cholesterol, HDL cholesterol and triglyceride levels in apoE–/– mice treated with vehicle or EP 80317 (D). Mean body weight and food intake during the last week of treatment with vehicle or EP 80317 (E). Results are expressed as mean ± SE. **P < .01; ***P < 0.001 compared with vehicle (0.9% NaCl).

2. EP 80317 shows curative effects that are reversible upon cessation of treatment
We then investigated whether EP 80317 could inhibit the progression of atherosclerotic lesions by delaying treatment initiation by 4–8 wk after the introduction of the HFHC diet. EP 80317 appeared to delay lesion progression in 18-wk-old apoE^–/– mice, inasmuch as the percentage of total aortic lesion areas was reduced by 39% and 31% when the treatment was delayed by 4 and 6 wk, respectively after introducing the HFHC diet (data not shown). A similar trend was observed when EP 80317 treatment was delayed until the mice were 14 wk old (data not shown). In contrast to the hypocholesterolemic effect of EP 80317 observed after chronic administration, total plasma cholesterol levels were modestly reduced or unchanged when drug administration was delayed by 4–8 wk after initiating the HFHC diet. Neither HDL cholesterol nor triglyceride levels were significantly modulated.

3. EP 80317 reduces oxLDL internalization and regulates the PPAR-LXR-ABCA1 signaling pathway in macrophages
To investigate whether EP 80317 treatment leads to reduced intracellular levels of oxLDL, peritoneal macrophages from apoE^–/– mice fed a HFHC diet and treated with EP 80317 for 12 wk were harvested and exposed to DiI-labeled oxidized LDL. Figure 2 A shows that a 2 h incubation of macrophages with 5 µg/mL DiI-oxLDL reduced the percentage of mean fluorescence intensity by 21 ± 3% (P<0.01) in mice treated with EP 80317.

View larger version (29K): [in this window] [in a new window]   Figure 2. Effect of EP 80317 on DiI-oxLDL internalization and gene expression of scavenger receptors, nuclear receptors, and ABC transporters. DiI-oxLDL internalization in peritoneal macrophages harvested from apoE–/– mice treated daily for 12 wk with 0.9% NaCl or EP 80317 (300 µg/kg) (A). The overlay histogram shows FACS plots of peritoneal macrophages harvested from 0.9% NaCl-treated (black line) and EP 80317-treated mice (grey line) (representative from 3 experiments). Bar graph represents the % change (±SEM) in mean fluorescence intensity over the control macrophage population set at 100%. **P < 0.01 compared with macrophages from 0.9% NaCl-treated mice. Representative steady-state mRNA levels of CD36, SR-A, PPAR, and PPAR, LXR, ABCA1, ABCG1, and GAPDH peritoneal macrophages harvested from apoE–/– mice after chronic (12 wk) treatment with EP 80317 receiving (left panel) or not (right panel) an i.p. injection of oxLDL (250 µg) prior to collection of macrophages. ApoE–/– mice were maintained on a HFHC diet and treated daily as in Fig. 1 . Immunoblot analyses of CD36, LXR, PPAR, ABCA1, and ABCG1 expression in macrophages were performed (C). Data are representative of 2–4 independent experiments.

To further elucidate the role of EP 80317 in the regulation of cholesterol efflux within macrophages, we assessed the effects of long-term treatment on the expression of components of the PPAR-LXR-ABC transporters pathway. Our results show that in the absence of exposure to exogenous oxLDL, EP 80317 (300 µg/kg per day) elicits a significant increase in mRNA expression (2-fold) of targeted genes including LXR and ABCG1, whereas PPAR and ABCA1 gene expression were modestly increased (1.4-fold) (Fig. 2B , right panel). However, the combined exposure to both exogenous oxLDL and EP 80317 treatment induced a higher expression of the transcription factors PPAR and LXR (2.5- to 3-fold) and of ABC transporters (2.5- to 2.6-fold) (Fig. 2B , left panel). The modulation of targeted genes appears to be specific inasmuch as there was no significant change in PPAR or SRA mRNA expression. The increase in gene expression was paralleled with an increase in protein expression of PPAR and LXR (1.4- to 2.8-fold) and of ABC transporters (1.4-fold) (Fig. 2C ). Despite an apparent increase in CD36 mRNA levels (1.5- to 1.9-fold), CD36 protein levels were not significantly increased in peritoneal macrophages harvested from EP 80317 pretreated mice, as assessed by Western blot.

CONCLUSIONS AND SIGNIFICANCE

The discovery of a key role for the scavenger receptor CD36 in the early uptake of modified lipoproteins and of its protective role in limiting lesion progression in an atherosclerotic mouse model, as well as our recent findings identifying GHRPs as novel CD36 ligands, led us to investigate the potentially beneficial effects of EP 80317 on atherosclerosis progression in apoE^–/– mice fed an atherogenic diet.

A prolonged (12 wk) administration of the peptide, initiated before the development of aortic lesions in 6-wk-old apoE^–/– mice, prevented lesions formation at 18 wk by 51% and reduced total plasma cholesterol levels by 30%. In contrast, the same therapeutic regimen in apoE^–/–/CD36^–/– mice was without significant effect on total lesion area and plasma lipids. Chronic treatment with the selective GHS-R1a endogenous peptide ghrelin did not exert significant beneficial anti-atherosclerotic effects in hypercholesterolemic mice (Harb et al., unpublished results). Altogether, these results suggest that the anti-atherosclerotic properties of EP 80317 are CD36-dependent.

EP 80317 also exerted curative effects, inducing a 31–39% reduction in total aortic lesion areas when delaying treatment by 4 and 6 wk after initiating the HFHC diet. However, shorter periods of administration of the peptide elicited either modest or had no significant effect on total plasma cholesterol levels, suggesting that additional mechanisms might contribute to the anti-atherosclerotic properties of EP 80317.

The importance of foam cell formation in initiating atherosclerotic lesions and the preponderant role of CD36 in scavenging modified lipoproteins in macrophages led us to investigate the effect of EP 80317 on macrophage foam cell formation and on the expression of the nuclear receptors PPAR and LXR, which behave as transcriptional regulators of genes involved in lipid metabolism and cholesterol trafficking in macrophages. The protective role of LXR ligands in atherogenesis appears to be largely due to an increase in LXR target genes such as ABCA1 and ABCG1. It is thus interesting that EP 80317 treatment, by itself or associated with an exogenous administration of oxLDL, promotes gene and protein expression of LXR and of the ABC transporter gene target ABCG1 and, to a lesser degree in unprimed cells, of ABCA1 (Fig. 2B, C ). It has been reported that PPAR activation is beneficial in the atherosclerotic setting, inasmuch as the cholesterol efflux pathways are stimulated through an increase in LXR, a target PPAR gene, which may offset the potentially adverse effect of PPAR agonists in up-regulating CD36 expression. In the present study, chronic treatment with EP 80317 elicited modest changes in CD36 mRNA expression within macrophages, but total CD36 protein levels were not significantly modulated as assessed by Western blot. As reported previously, it may be possible that LXR stimulation provides an escape pathway from the autoregulatory positive feedback that normally occurs between PPAR and CD36. However, unlike LXR agonists, for which beneficial effects on macrophage efflux pathways may be counteracted by an up-regulation of fatty acid synthesis and lipogenesis, EP 80317 did not modulate triglyceride levels or induce hepatic steatosis.

Our results show that EP 80317 interfered with the intracellular accumulation of oxLDL, as shown by a reduced DiI-oxLDL internalization in macrophages. Hence, the regulation of cholesterol metabolism and trafficking in macrophages might contribute, in addition to its favorable effect on plasma lipid profile, to the CD36-dependent atheroprotective effects of EP 80317. Elucidation of the cellular and molecular effects of this drug at different tissue levels will be needed to fully understand its beneficial potential in atherogenesis.

View larger version (37K): [in this window] [in a new window]   Figure 3. Modulation of cholesterol trafficking in macrophages by EP 80317. Intimal macrophages are involved in oxLDL uptake through their scavenger receptors including CD36, which appears to play a major role. OxLDL internalization and metabolism is associated with activation of the nuclear receptor PPAR, leading to an increase in the expression of CD36 and of the LXR-ABCA1 efflux pathway in macrophages (left panel). EP 80317, upon its binding to CD36, interferes with the internalization of oxLDL and modulates selectively the PPAR-LXR-ABC transporters pathway involved in reverse cholesterol transport in macrophages (right panel).

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3253fje;

¹ These authors contributed equally to the work

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