MARCO, a macrophage scavenger receptor highly expressed in rodents, mediates dalcetrapib-induced uptake of lipids by rat and mouse macrophages
Introduction
Despite the well-known benefits of statins in lowering levels of low-density lipoprotein cholesterol (LDL-C) and preventing cardiovascular disease, a residual risk of coronary heart disease remains in approximately 60% of patients taking statins (Grundy et al., 2004). Increasing levels of high-density lipoprotein cholesterol (HDL-C) may be an alternative therapeutic strategy.
Dalcetrapib (RO4607381/JTT-705), a novel agent that acts upon cholesteryl ester transfer protein (CETP), is being investigated for the treatment of dyslipidemia and the improvement of cardiovascular disease outcomes. CETP mediates the exchange of cholesteryl esters in HDL for triglycerides (TG); inhibiting its activity is therefore expected to increase plasma levels of HDL-C (Rader, 2006). In vitro studies show that dalcetrapib inhibits human plasma CETP activity with IC50 values of ⩽10 μM (Okamoto et al., 2003, Niesor et al., 2008) while Phase II studies have demonstrated the efficacy of dalcetrapib in increasing HDL-C, both as monotherapy and in combination with statins (de Grooth et al., 2002, Kuivenhoven et al., 2005, Stein et al., 2009; unpublished data, Stein, E.A., 2009).
Dalcetrapib is hydrolyzed in the gastrointestinal tract and in plasma to the active metabolite dalcetrapib-thiol (Okamoto et al., 2003). In order to characterize any potential on- or off-target effects of dalcetrapib that may pose safety concerns, a series of standard and exploratory toxicity studies using up to 1000 mg/kg/day dalcetrapib by oral gavage with treatment durations of up to 1 year were performed in rodents and non-rodents, of which hamsters and monkeys were used as CETP proficient species whereas mice and rats are lacking CETP activity. Primarily studies in two non-CETP expressing rodent species, namely, rats and mice, showed increases in the size and weight of mesenteric lymph nodes, which corresponded microscopically to the accumulation of foamy macrophages. Foamy macrophages were also observed in the mucosa of small intestines of rats. Using special staining techniques, the foamy appearance of macrophages was shown to be due to the accumulation of lipids. Electron microscopy revealed an increased number of enlarged lysosomes containing lipid-like particles or amorphous material.
In vitro studies presented here aimed to identify the receptors that likely mediate the increased lipid uptake associated with dalcetrapib, focusing on the characterized macrophage lectin-like oxidized LDL receptor (LOX-1), scavenger receptor type AI (SR-AI) and MAcrophage Receptor with COllagenous domain (MARCO). LOX-1 is expressed in mouse and human macrophages; it mediates the uptake of oxidized LDL but not acetylated LDL (Moriwaki et al., 1998a, Moriwaki et al., 1998b). Almost all macrophages express SR-AI, while only a subset of macrophages expresses MARCO (Mukhopadhyay et al., 2006). MARCO was originally cloned from mouse macrophages and was found to be expressed only in macrophages in the marginal zone of the spleen and in lymph nodes, and was not detected in other macrophage-rich tissues including lung and liver (Elomaa et al., 1995). A human homologue of MARCO was later identified (Kangas et al., 1999). Although SR-AI and MARCO share structural similarities and bind similar ligands, SR-AI and mouse MARCO bind acetylated and oxidized lipoproteins (Elomaa et al., 1995), while human MARCO does not (Elshourbagy et al., 2000). In addition, mouse MARCO binds Gram-negative and Gram-positive bacteria but not yeast, while other scavenger receptors bind all three (Elomaa et al., 1995). Human MARCO has also been shown to bind Gram-negative bacteria (Mukhopadhyay et al., 2006). There are also differences between SR-AI and mouse MARCO in how their expression is regulated. MARCO expression in J774A.1, a mouse macrophage cell line, was shown to be upregulated by Th1 adjuvants including lipopolysaccharide (LPS), CpG-motif containing oligodeoxynucleotide, interleukin-12 and granulocyte macrophage colony stimulating factor, and was downregulated by the Th2-polarizing factors interleukin-4, macrophage colony stimulating factor, and non-CpG-motif containing oligodeoxynucleotide. In the same cells, these factors had an opposite effect on SR-AI expression or had no effect (Józefowski et al., 2005). Uptake of dalcetrapib-treated lipids and expression of candidate receptors were investigated using both rodent and human macrophages.
Section snippets
Cell culture
Thioglycollate-induced peritoneal macrophages were collected by peritoneal lavage from Sprague–Dawley rats (OFA-SD, Iffa Credo) and C57BL/6J mice (RCC). Peritoneal macrophages were allowed to adhere on tissue culture plates and non-adherent cells were washed away. Peritoneal macrophages were cultured in Dulbecco’s Modified Eagle Medium containing 1 g/L d-glucose supplemented with 10 U/mL penicillin, 10 μg/mL streptomycin (Penicillin–Streptomycin, Invitrogen), and 10% fetal bovine serum
Effect of polyinosinic acid on uptake of dalcetrapib-treated CM + VLDL
Polyinosinic acid is an identified macrophage scavenger receptor type A inhibitor (Lysko et al., 1999). It also inhibits the uptake of oxidized LDL by LOX-1 (Moriwaki et al., 1998a, Moriwaki et al., 1998b), a class E scavenger receptor expressed on a number of cell types including macrophages (Moore and Freeman, 2006). Incubation with polyinosinic acid was shown to decrease the dalcetrapib-mediated increase in the uptake of rat CM + VLDL by rat peritoneal macrophages (Fig. 1). This result
Discussion
Dalcetrapib, an agent that targets CETP, is in development for the treatment of dyslipidemia and the prevention of cardiovascular disease events. Preclinical repeat dose toxicity studies had identified an off-target effect manifested by uptake and deposition of lipid loaded chylomicron into the lamina propria of the small intestine and into mesenteric lymph node macrophages associated with increased lymph node size and slow reversibility. However, this preclinical signal was not observed in the
Disclosure and acknowledgements
Anne Perez, Matthew B. Wright, Cyrille Maugeais, Annamaria Braendli-Baiocco, Thomas Singer, Lutz Mueller, and Eric J. Niesor are employees of F. Hoffmann-La Roche Ltd. Hiroshi Okamoto and Akemi Takahashi are employees of Japan Tobacco. Editorial assistance was provided by Prime Medica Inc. during preparation of this report and was funded by F. Hoffmann-La Roche Ltd.
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