Elsevier

Toxicology in Vitro

Volume 24, Issue 3, April 2010, Pages 745-750
Toxicology in Vitro

MARCO, a macrophage scavenger receptor highly expressed in rodents, mediates dalcetrapib-induced uptake of lipids by rat and mouse macrophages

https://doi.org/10.1016/j.tiv.2010.01.002Get rights and content

Abstract

Dalcetrapib (RO4607381/JTT-705), an agent that targets cholesteryl ester transfer protein, is in development for prevention of cardiovascular events. In vitro studies were performed to identify receptors that mediate an off-target effect of dalcetrapib observed in preclinical models: increased lipid uptake into the lamina propria of the small intestine and into mesenteric lymph node macrophages. Uptake of oxidized low-density lipoprotein (LDL) cholesterol or dalcetrapib-treated chylomicrons was quantitated by triglyceride assay or fluorescent labeling in primary macrophages and the cell lines CHO, J774A.1 (mouse macrophages) and THP-1 (human macrophages). Quantitative reverse-transcriptase polymerase chain reaction and immunoblotting measured candidate receptor expression. Lectin-like oxidized LDL receptor (LOX-1) and scavenger receptor type AI (SR-AI) were excluded as candidate receptors based on lack of association between their expression and uptake of dalcetrapib-treated lipids. In J774A.1 cells, uptake of dalcetrapib-treated chylomicrons was increased by LPS and associated with expression of MAcrophage Receptor with COllagenous domain (MARCO). MARCO was expressed at very low levels in human macrophages and was not inducible by LPS. The MARCO receptor may account for the variable species susceptibility towards dalcetrapib-mediated chylomicron uptake by 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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