ABC transporters and scavenger receptor BI : important mediators of lipid metabolism and atherosclerosis Meurs, I. Citation Meurs, I. (2011, June 7). ABC transporters and scavenger receptor BI : important mediators of lipid metabolism and atherosclerosis. Retrieved from https://hdl.handle.net/1887/17686 Version: Corrected Publisher’s Version Licence agreement concerning inclusion of doctoral License: thesis in the Institutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/17686 Note: To cite this publication please use the final published version (if applicable). Chapter 6 Transcriptional profiling of ABC transporters in murine foam cells and atherosclerotic lesions identifies putative novel targets for improving macrophage lipid homeostasis Illiana Meurs, Martine Bot, Bart Lammers, Theo J.C. Van Berkel, and Miranda Van Eck Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands Manuscript in preparation Chapter 6 ABSTRACT Objective- Excessive accumulation of cholesterol by macrophages leading to their transformation into foam cells is the earliest pathological hallmark of atherosclerosis. Key regulators of macrophage cholesterol and phospholipid efflux are the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1. The objective of this study was to identify new ABC transporters that are differentially expressed during macrophage foam cell formation induced by the commonly used pro-atherogenic lipoproteins βVLDL and oxLDL and during atherosclerotic lesion development. Methods and Results- Using a collar-induced carotid artery atherosclerosis model, transcriptional profiling of ABC transporters was performed in atherosclerotic lesions in the carotid artery of LDLr KO mice after 2 weeks of Western-type diet (WTD) feeding. The expression levels of 46 ABC transporters were investigated of which 6 transporters were significantly regulated during lesion development, including ABCB1b, ABCB4, ABCC3, ABCC9, ABCD3, and ABCG1, of which ABCB1b, ABCC3, and ABCG1 displayed the highest upregulation. Next, ABC transporter expression was determined in peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) loaded with βVLDL or oxLDL. In total 12 ABC transporters were differentially expressed in the macrophage foam cells compared to non-foamy cells, including ABCA3, ABCB1b, ABCB2, ABCB4, ABCB6, ABCB7, ABCC3, ABCC5, ABCC10, ABCD3, ABCF2, and ABCG1. Notably, ABCB4 showed a remarkable 8-fold upregulation in BMDM specifically loaded with oxLDL. Conclusion- This study indentified several new ABC transporters which are regulated during foam cell formation and atherosclerotic lesion development. Of particular interest are the ABC transporters ABCB1b, ABCB2, ABCB4, and ABCB6 as they are significantly regulated in both peritoneal and bone marrow-derived macrophages upon cellular lipid loading and showed a 1.2- 8.2 fold-change. In agreement, ABCB1b and ABCB4 were also significantly induced in atherosclerotic lesions in the carotid arteries. These ABC transporters might form new targets to modulate foam cell formation and the extend of atherosclerotic lesion development. INTRODUCTION Maintenance of cholesterol homeostasis in macrophages is essential to prevent foam cell formation and the initiation of atherosclerotic lesion development. Macrophages, however, are incapable of limiting the uptake of lipoproteins via scavenger receptors. Therefore, mechanisms by which macrophages export cellular cholesterol are of particular importance. The principal molecules involved in efflux of cholesterol from macrophage foam cells are the ATP-binding cassette (ABC) transporters ABCA11 and ABCG12 and scavenger receptor BI (SR-BI)3, 4. The ABC transporter genes are evolutionary highly conserved and represent one of the largest family of transmembrane (TM) proteins. ABC transporters utilize the energy of ATP hydrolysis to pump a wide variety of substrates, including sugars, amino acids, metal ions, peptides, proteins, and a large number of hydrophobic compounds and 166 Transcriptional profiling of ABC transporters metabolites across extra- and intracellular membranes.5 To date, 52 members of the ABC transporter family have been identified in humans, which are divided into seven distinct subfamilies (ABCA-G), based on organization of domains and amino acid homology.6 ABC transporters are organized as either full or as half transporters. Full transporters contain two identical TM domains and two nucleotide binding folds (NBFs), while half transporters contain one of each domain and must form either homodimers or heterodimers to become functional. The TM domains contain 6 membrane-spanning α-helices, which determine the substrate specificity and provide the pathway for the translocation of the substrate across the membrane. The NBFs contain characteristic Walker A and B motifs and a signature (C) motif and act as an energy source by binding of ATP.6-8 As ABC transporters play a role in lipid transport and a vast majority is highly expressed in macrophages9, 10, it was evident that more ABC transporters are involved in macrophage lipid homeostasis and play critical roles in foam cell formation and atherogenesis. The aim of this study was to identify new ABC transporters that are differentially expressed during macrophage foam cell formation and atherosclerotic lesion development. Microarray analyses were performed to assess the regulation of ABC transporters in atherosclerotic lesions in carotid arteries in vivo and in lipid-loaded macrophages in vitro. MATERIALS AND METHODS Chapter 6 Chapter Animals Microarray analysis was performed on atherosclerotic lesions induced in the carotid artery of LDL receptor knockout (LDLr KO, 12 weeks of age) by perivascular collar placement. Briefly, after 2 weeks of run-in Western-type diet (WTD) feeding, atherosclerosis was induced by bilateral perivascular collar placement (2mm long, diameter 0.3 mm) around both carotid arteries in female LDLr KO mice. Subsequently, these LDLr KO mice were fed WTD for 2 weeks to induce the development of atherosclerotic lesions. Control arteries were isolated from LDLr KO mice which only received 2 weeks of run-in WTD feeding. After the indicated feeding period, mice were anaesthetized, perfused with PBS via the left ventricle of the heart, and the carotid arteries were prepared free of fat tissue and snap frozen in liquid nitrogen and stored at -80oC until total RNA (tRNA) analysis. Furthermore, for microarray analysis of cultured macrophages, 7 female C57Bl/6 mice, 10 weeks of age and maintained on sterilized regular chow diet containing 4.3% (w/w) fat and no cholesterol (RM3, Special Diet Services, Witham, UK), were used. Animal experiments were performed at the Gorlaeus Laboratories of the Leiden/Amsterdam Center for Drug Research in accordance with the National Laws. All experimental protocols were approved by the Ethics Committee for Animal Experiments of Leiden University. Isolation of lipoproteins Beta-migrating very-low-density lipoprotein (βVLDL) was obtained from rats fed a RMH-B diet, containing 2% cholesterol, 5% olive oil, and 0.5% cholic acid for 2 weeks (Abdiets). The rats were fasted overnight and anesthetized after which blood was collected by puncture of 167 Chapter 6 the abdominal aorta. Serum was centrifuged at 40,000 rpm in a discontinuous KBr gradient for 18 hours as reported earlier.11 βVLDL (density <1.019 g/mL) was collected and dialysed against phosphate buffered saline, containing 1 mM EDTA (PBS/1mM EDTA). Isolated βVLDL was characterized as described previously.12 Furthermore, low-density lipoprotein (LDL) (density 1.063 to 1.019 g/mL) was isolated from plasma of healthy human volunteers by ultracentrifugation in a KBr discontinuous gradient and dialysed against PBS/1mM EDTA according to Redgrave et al.11. For generation of oxidized-LDL (oxLDL), LDL was oxidatively modified by incubation of 200 µg/mL of LDL with 10 µM CuSO4 (subscript) at 37°C for 20 h. Oxidation was terminated by dialysis against PBS containing 0.5 mM EDTA for at least 24h. Macrophage culture Peritoneal macrophages (PM) and bone marrow-derived macrophage (BMDM) were used for analysis. PM were harvested by lavage of the peritoneal cavity of C57Bl/6 mice with 10ml of PBS at 5 days after intraperitoneal injection of 1mL of 3% Brewer thioglycollate medium (Difco, Detroit, MI). After three washing steps, the cells were plated in multi well culture dishes with DMEM containing 10% fetal calf serum (FCS). After 4 hours the non adherent cells were removed by washing and the adherent macrophage were cultured overnight in DMEM containing 10% FCS until start of the experiment. For generation of BMDM, bone marrow cells, isolated from female C57/Bl6 mice, were cultured for 7 days in complete RPMI medium supplemented with 20% FCS and 30% L929 cell-conditioned medium, as the source of macrophage colony-stimulating factor (M-CSF). After 7 days of culture, the BMDM were harvested using 4mM EDTA, washed three times and plated in multi well culture dishes with DMEM containing 10% FCS until start of the experiment. Macrophage lipid loading PM or BMDM were incubated with βVLDL (50 µg/mL), or oxLDL (20 µg/mL) in DMEM containing 0.2% BSA for 48 hours at 37°C. Subsequently, the cells were washed three times with PBS, lysed in lysis buffer (Qiagen, Chatsworth, CA) and snap frozen in liquid nitrogen and stored at -80oC until tRNA isolation. Microarray protocol tRNA was isolated from PM or BMDM