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
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6
Chapter
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
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
Chapter 6 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 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
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 loaded with βVLDL, or oxLDL for 48h (3 samples per condition) using an RNAesy mini kit (Qiagen, Chatsworth, CA) for microarray analysis.
In addition, for microarray analyses of the atherosclerotic lesions in the carotid artery, tRNA from the carotid arteries was isolated using the Trizol method. Upon receipt of the tRNA samples, ServiceXS (Leiden, The Netherlands) analyzed the concentration and the integrity of the RNA samples using the NanoDrop ND-100 Spectrophotometer and Agilent 2100 Bioanalyzer, respectively. Amplification and labeling of the RNA samples was performed according to the manufacturer’s specifications (Illumina, San Diego, CA). Hereto, the Ambion® Illumina TotalPrep RNA Amplification Kit (Ambion, #IL1791) was used, which generates biotinylated, amplified cRNA. Hybridisation of the labelled RNA samples to the MouseWG-6 v2.0 array was performed according to manufacturer’s specifications (Illumina,
Chapter 6 San Diego, CA). Signal was developed with streptavidin-Cy3 and the BeadChip was scanned
with the Illumina BeadArray Reader (Illumina, San Diego, CA).
Data analyses
Microarray data were acquired and imported in Excel (Microsoft office Excel 2007) for further analysis. In the macrophage in vitro study, the means of triplicates obtained for the individual genes for each loading condition were calculated and expressed relative to control non-foamy cells. Significant differences between the indicated loading condition and control non-foamy cells were calculated by using the two-tailed student’s T-test. In the in vivo atherosclerotic lesion study, the means of triplicates for the genes after WTD feeding were calculated and expressed relative to control (no WTD feeding), which represents a carotid artery free of atherosclerotic lesion. Significant differences in genes expressed in atherosclerotic lesions compared to control mice were calculated by using the two-tailed student’s T-test. Hierarchical clustering of significant genes was performed using TIBCO Spotfire 3.1 (http://spotfire.tibco.com).
Statistical analyses were performed using a two-tailed student’s T-test. P values <0.05 were considered significant.
RESULTS
Transcriptional profiling of ABC transporter gene expression in atherosclerotic lesions
We designed this experiment to identify new ABC transporters that are specifically regulated during atherosclerotic lesion development. Transcriptional differences between carotid arteries of control mice and carotid arteries of mice subjected to collar placement and challenged with WTD were determined by microarray analysis. To provide insight in the degree of lesion formation and the cellular composition of the lesions in the carotid arteries, the mRNA expression levels of CD68 (macrophage specific), α-actin (smooth muscle cell specific), and PECAM (endothelial cell specific) were determined from the microarrays (Fig.
1A). A significant 9-fold (p<0.0001) increase in CD68 mRNA expression was observed, indicative of massive infiltration of macrophages into the carotid arterial wall and the presence of atherosclerotic lesion development. The mRNA expression levels of smooth muscle cell-specific α-actin was downregulated by 20% (p<0.01) upon WTD feeding, while PECAM expression remained unchanged.
In addition, the expression of cholesterol-responsive genes were also measured, including low-density lipoprotein receptor (LDLr), 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR), very low-density lipoprotein receptor (VLDLr), LDL receptor-related protein 1 (LRP1), scavenger receptor class B type I (SR-BI), scavenger receptor class A (SR-A), and CD36 (Fig. 1B). Transcriptional profiling of genes involved in cholesterol homeostasis revealed a significant increase in expression of LRP1 in carotid arteries of LDLr KO mice fed the WTD (1.5-fold, p<0.05). Additionally, expression of the VLDLr was significantly decreased in atherosclerotic lesions (0.3-fold, p<0.05).
Fig. 1. mRNA expression of cellular markers (A) and cholesterol-responsive genes (B) in the athero- sclerotic carotid artery.
The cellular composition of the carotid artery was determined by identification of CD68 (macrophage specific), α-actin (smooth muscle cell specific), and PCEM (endothelial cell specific) expression in the microarray. The ex- pression of cholesterol-responsive genes were also measured, included low-density lipoprotein receptor (LDLr), 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGCR), very low-density lipoprotein receptor (VLDLr), LDL receptor-related protein 1 (LRP1), scavenger receptor class B type I (SR-BI), scavenger receptor class A (SR-A), and CD36.
An unpaired Student t test was applied to test whether the mRNA expression levels were significantly different from the mRNA levels in control carotid artery. Values represent the mean ± SEM of 3 mice per group. Statistically significant difference *p<0.05, **p<0.01 and ***p<0.001.
Although to date, 52 members of the ABC-transporter family have been identified in humans only 46 murine ABC transporters were measured, as the mRNA expression levels of 6 ABC transporters, including ABCA8, ABCA8a, ABCB1a, ABCC12, ABCG2, and ABCG8 were too low in macrophages and atherosclerotic lesions to represent reliable expression levels.
Therefore, these ABC transporters were excluded from the results.
Hierarchical clustering of ABC transporters, other known cholesterol-responsive genes, and cellular markers expressed in atherosclerotic lesions, identified 7 specific gene clusters (C). C1 and C2 represent genes that are markedly reduced during atherosclerotic lesion development, whereas C3 represents genes that are only modestly regulated in atherosclerotic carotid arteries compared to control arteries (Fig. 2). C4, C5, C6, and C7 represent genes that are upregulated in atherosclerotic lesions, of which C6 and C7 contain genes that showed the highest upregulation compared to control arteries.
Identification of the genes present in the specific clusters in the atherosclerotic arteries are listed in Table 1A, of which significantly regulated genes compared to control cells
CD 68
0 5 10 15
***
Relative expression
α-Actin
0.0 0.5 1.0 1.5
**
PECAM
0.0 0.5 1.0 1.5
Control arteries Atherosclerotic arteries
A
B
HMGCR LDLr
VLDLr LRP1
SR-BI SR-A
CD36 0.00
0.25 0.50 0.75 1.00 1.25 1.50 1.75
Relative expression
*
*
Chapter 6 are underscored. Furthermore, the fold-change of these significantly regulated genes are
presented in Table 1B.
In total 6 ABC transporters out of 46 measured ABC transporters were significantly regulated in atherosclerotic carotid arteries compared to control arteries, including ABCB1b, ABCB4, ABCC3, ABCC9, ABCD3, and ABCG1 (Table 1B). ABCD3 expression was significantly reduced during atherosclerotic lesion development compared to carotid arteries of control LDLr KO (0.5-fold, p<0.001), whereas the expression of ABCB1b, ABCB4, ABCC3 ABCC9, and ABCG1 were significantly induced during atherosclerotic lesion development in LDLr KO mice (ABCB1b: 2.1-fold, p<0.01; ABCB4: 1.2-fold, p<0.05; ABCC3: 3.2-fold, p<0.001;
ABCC9: 1.5-fold, p<0.05; ABCG1: 2.3-fold, p<0.01).
Cluster 4
ABCA3 ABCA7 ABCB2 ABCB4 ABCB7 ABCC4
ABCC5 ABCF2
Cluster 1 VLDLr
Cluster 2
ABCA2 ABCB6 ABCB8 ABCB9 ABCD3
Cluster 6
ABCB1b ABCC3 ABCG1
Cluster 5
ABCC9 LRP1
Cluster 3
ABCA1 ABCA4 ABCA5 ABCA6 ABCA8b ABCA9
ABCA12 ABCA13 ABCA14 ABCA15 ABCA16 ABCA17
ABCB5 ABCB10 ABCC1 ABCC2 ABCC6 ABCC8
ABCC10 ABCC12 ABCD1 ABCD2 ABCD4 ABCE1
ABCF3 ABCG2 ABCG3 ABCG4 ABCG5 ABCGF1
CD36 HMGCR LDLr PECAM SR-AI SR-BI
α-actin
Cluster 7 CD68
Underscored genes represent genes which are significantly regulated
Table 1A. Clusters 1-7 of ABC transporters, other known cholesterol-responsive genes, and cellular markers in atherosclerotic carotid arteries of LDLr KO mice fed WTD compared to control carotid arteries
Table 1B . Genes significantly regulated in atherosclerotic carotid arteries of LDLr KO mice fed WTD compared to control carotid arteries
C2
C6 C4 C3 C1
C5
C7
Fig. 2. Hierarchical clustering of relative expression of ABC transporters, known cholesterol-respon- sive genes, and cellular markers in atherosclerotic lesions of LDLr KO mice upon WTD feeding compared to control mice.
The color scale ranges from saturated green to saturated red, indicating low to high expression against the average relative expression in carotid arteries without atherosclerotic lesions, respectively. Values represent the means of 3 mice.
Statistical significance of *p<0.05, **p<0.01, and ***p<0.001 compared to control arteries
Genes Cluster Fold-change
VLDLr 1 0,3*
ABCD3 2 0,5***
α-actin 3 0,8**
ABCB4 4 1,2*
LRP1 5 1,5*
ABCC9 5 1,5*
ABCG1 6 2,3**
ABCC3 6 3,2***
ABCB1b 6 2,1**
CD68 7 8.7***
Chapter 6
Transcriptional profiling of ABC transporter gene expression in lipid loaded PM and BMDM
As massive macrophage infiltration in the carotid arterial wall was observed in vivo, we next determined mRNA expression levels of ABC transporters specifically in macrophages loaded with different lipoproteins in vitro. Peritoneal macrophages (PM) and bone marrow- derived macrophage (BMDM), loaded with βVLDL or oxLDL are commonly used as an in vitro model for macrophage foam cells in atherosclerotic lesions. Therefore both types of macrophages were included in the studies.
Transcriptional profiling of known cholesterol-responsive genes revealed a significant reduction in gene expression of LDLr (0.4-fold, p<0.001 and 0.5-fold, p<0.01 for PM after loading with βVLDL and oxLDL, respectively; 0.4-fold, p<0.001 and 0.6-fold, p>0.05 for BMDM after loading with βVLDL and oxLDL, respectively) and HMGCR (0.7-fold, p<0.01 and 0.7-fold, p<0.01 for PM after loading with βVLDL and oxLDL, respectively; 0.7-fold, p<0.05 and 1.1-fold, p>0.05 for BMDM after loading with βVLDL and oxLDL, respectively) in both types of macrophages after incubation with βVLDL or oxLDL compared to non- foam cells (Fig 3A and B). Previous studies, showed that LDLr and HMGCR gene expression is downregulated upon lipid loading.13, 14 The observed reduction, therefore, might be considered as a sensor to indicate lipid accumulation in the macrophages, leading to the formation of foam cells. Furthermore, a significant reduction in expression of LRP1 in PM loaded with oxLDL (0.8-fold, p<0.05) was observed, whereas loading of BMDM with βVLDL resulted in significantly induced expression of VLDLr (1.4-fold, p<0.01). The expression of SR-BI, SR-A and CD36 were not affected in PM and BMDM upon lipid loading.
A
B
Fig. 3. mRNA expression of cholesterol-responsive genes in PM (A) and BMDM (B) compared to control non-foamy cells.
Values represent the mean ± SEM of 3 mice per loading condition. Statistically significant difference *p<0.05,
**p<0.01 and ***p<0.001.
Fig. 4. Hierarchical clustering of relative expression of ABC transporters and known cholesterol- responsive genes in PM (A) and BMDM (B) compared to control non-foamy cells.
The color scale ranges from saturated green to saturated red, indicating low to high expression against the aver- agerelative expression in non-foamy control cells, respectively. Values represent the means of 3 per loading condi- tion.
Hierarchical clustering of ABC transporter gene expression in PM and BMDM loaded with βVLDL and oxLDL is displayed in Fig. 4A and 4B, respectively. Identification of the genes present in the specific clusters of PM and BMDM are listed in Table 2A and 2B, respectively, of which significantly regulated genes compared to control cells are underscored. Furthermore, the fold-change of these significantly regulated genes are presented in Table 3A and 3B, respectively. All data are values compared to control non-foamy cells.
In the hierarchical clustering of genes expressed in PM, 6 unique clusters were identified (Fig. 4A). C1 and C2 represent genes which were upregulated in PM loaded with βVLDL, while oxLDL loading had differential effects. C3 represent genes which were not or only moderately affected by loading of PM with βVLDL and downregulated in oxLDL-loaded PM, with the exception of 2 genes namely ABCB6 and ABCC4 which were both upregulated upon oxLDL loading. C4, C5 and C6 represent genes which were downregulated in PM loaded with either βVLDL or oxLDL.
In PM loaded with βVLDL or oxLDL, 8 ABC transporters out of 46 measured ABC transporters showed a statistically significant change in expression, including ABCB2, ABCB4, ABCB6, ABCB7, ABCC3, ABCC10, ABCF2, and ABCG1(Table 3A). ABCC10 was slightly reduced under both conditions in PM. However, this change in ABCC10 was only significantly different compared to control cells upon incubation with oxLDL (βVLDL: 0.9-fold, p>0.05;
oxLDL:0.9-fold, p<0.05). Additionally, oxLDL significantly reduced specifically the expression of ABCB2 (0.5-fold, p<0.05), while ABCB4 (1.4-fold, p<0.01), ABCB6 (1.4-fold, p<0.05), and ABCF2 (1.3-fold, p<0.05) were significantly increased. PM foam cell formation induced by
βVLDL oxLDL
C1 C2
C3
C5 C4
C6 ABCB6
ABCC4
A βVLDL oxLDL
C1
C2
C6 C3 C4 C5 B
Chapter 6
βVLDL did result in a significant increase in expression of ABCB7, ABCC3, and ABCG1 expression (1.3-fold, p<0.05; 2.2-fold, p<0.05; and 1.7-fold, p<0.05 compared to control cells, respectively), genes that were not affected upon loading of PM with oxLDL.
In the hierarchical clustering of genes expressed in BMDM, 6 unique clusters were identified (Fig. 4B). C1 and C6 represent genes which were upregulated in BMDM loaded with either βVLDL or oxLDL of which C6 contains genes with the highest upregulation upon loading with βVLDL or oxLDL. C2 represents genes in BMDM which were not or moderately affected by βVLDL or oxLDL. C3 represents genes which were upregulated in BMDM loaded with βVLDL and downregulated by oxLDL loading. C4 represents genes which were upregulated in BMDM loaded with βVLDL but were differentially expressed after loading BMDM with oxLDL, whereas C5 represents genes which were downregulated in BMDM upon loading with either βVLDL or oxLDL.
Loading of BMDM with βVLDL or oxLDL resulted in a significant change in expression
Cluster 4
ABCA2 ABCA3 ABCA7 ABCA14 ABCA17 ABCB8
ABCB9 ABCC10 ABCC5 ABCD4 ABCG3 SR-A
Cluster 1
ABCC3 ABCG1
Cluster 2
ABCA9 ABCB2 ABCB4 ABCB7 ABCD3 ABCF2
Cluster 3
ABCA1 ABCA4 ABCA5 ABCA6 ABCA8 ABCA12
ABCA13 ABCA15 ABCA16 ABCB1b ABCB5 ABCB6
ABCB10 ABCB11 ABCC1 ABCC2 ABCC4 ABCC6
ABCC8 ABCC9 ABCD1 ABCD2 ABCE1 ABCF1
ABCF3 ABCG5 CD36 LRP1 SR-BI VLDLr
Cluster 5 HMGCR
Cluster 6 LDLR
Underscored genes represent genes which are significantly regulated
Table 2A. Clusters 1-6 of ABC transporter genes and genes involved in cholesterol homeostasis in loaded peritoneal macrophages compared to non-loaded control cells
Underscored genes represent genes which are significantly regulated
of 7 out of 46 measured ABC transporters, including ABCA3, ABCB1b, ABCB2, ABCB4, ABCB6, ABCC5, and ABCD3 (Table 3B). βVLDL or oxLDL-loaded BMDM showed reduced expression of ABCB2, which was only significant compared to control cells after incubation with βVLDL (βVLDL: 0.5-fold, p<0.05; oxLDL: 0.8-fold, p>0.05). BMDM loaded with βVLDL or oxLDL showed increased expression of ABCA3, which, however, reached a significant difference compared to control cells only after βVLDL loading (βVLDL: 1.4-fold, p<0.05;
oxLDL: 1.2-fold, p>0.05). In addition, loading of BMDM with βVLDL or oxLDL led to increased expression of ABCB1b and ABCD3, which reached significances compared to control cells only after oxLDL loading (ABCB1b: 1.8-fold, p<0.05; ABCD3: 1.5-fold; p<0.01 for oxLDL and ABCB1b: 1.2-fold, p>0.05; ABCD3: 1.2-fold; p>0.05 for βVLDL). Furthermore, oxLDL- loaded BMDM showed moderately increased expression of ABCC5 (1.2-fold, p<0.05), and, interestingly, a large increase in expression of ABCB4 (8.2-fold, p<0.001, respectively) and ABCB6 (2.1-fold, p<0.05, respectively).
Cluster 4
ABCA2 ABCA3 ABCA7 ABCA14 ABCA17 ABCB8
ABCB9 ABCC10 ABCC5 ABCD4 ABCG3 SR-A
Cluster 1
ABCC3 ABCG1
Cluster 2
ABCA9 ABCB2 ABCB4 ABCB7 ABCD3 ABCF2
Cluster 3
ABCA1 ABCA4 ABCA5 ABCA6 ABCA8 ABCA12
ABCA13 ABCA15 ABCA16 ABCB1b ABCB5 ABCB6
ABCB10 ABCB11 ABCC1 ABCC2 ABCC4 ABCC6
ABCC8 ABCC9 ABCD1 ABCD2 ABCE1 ABCF1
ABCF3 ABCG5 CD36 LRP1 SR-BI VLDLr
Cluster 5 HMGCR
Cluster 6 LDLR
Underscored genes represent genes which are significantly regulated
Table 2B. Clusters 1-6 of ABC transporter genes and genes involved in cholesterol homeostasis in loaded bone marrow-derived macrophages compared to non-loaded control cells
Underscored genes represent genes which are significantly regulated
Chapter 6 Table 3A . Genes significantly regulated in loaded peritoneal macrophages compared to non-foamy
control cells
Table 3B . Genes significantly regulated in loaded bone marrow-derived macrophages compared to non-foamy control cells
Statistical significance of *p<0.05, **p<0.01, and ***p<0.001 compared to control non
Genes Cluster βVLDL
Fold –change oxLDL
Fold -change
ABCA3 1 1,4* 1,2
VLDLR 1 1,4** 1,1
ABCC5 2 1,0 1,2*
HMGCR 3 0,7* 1,1
ABCB1B 4 1,2 1,8*
ABCB6 4 0,9 2,1*
ABCD3 4 1,2 1,5**
ABCB2 5 0,5* 0,8
LDLR 5 0,4** 0,6*
ABCB4
6 1,7 8,2***
-loaded cells
DISCUSSION
In the present study, the effect of foam cell formation and atherosclerotic lesion development on the expression levels of 46 ABC transporters were determined. The ABC transporter genes represent one of the largest family of transmembrane 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 metabolites across extra- and intracellular membranes.5
Out of the 46 ABC transporters analysed 6 transporters were significantly regulated during atherosclerotic lesion development in the carotid arteries of LDLr KO. The mRNA expression levels of ABCB1b, ABCB4, ABCC3 ABCC9, and ABCG1 were significantly
Statistical significance of *p<0.05, **p<0.01, and ***p<0.001 compared to control non-loaded cells
Statistical significance of *p<0.05, **p<0.01, and ***p<0.001 compared to control non-loaded cells
Genes Cluster βVLDL
Fold –change oxLDL
Fold-change
ABCC3 1 2,2* 0,7
ABCG1 1 1,7* 1,3
ABCB2 2 1,2 0,5*
ABCB4 2 1,2 1,4**
ABCB7 2 1,3* 0,8
ABCF2 2 1,2 1,3*
ABCB6 3 1,0 1,4*
LRP1 3 1,0 0,8*
ABCC10 4 0,9 0,9*
HMGCR 5 0,7** 0,7**
LDLR 6 0,4*** 0,5**
upregulated during lesion development, whereas the mRNA expression level of ABCD3 was significantly downregulated.
In addition, an in vitro study was performed using PM and BMDM to assess ABC-transporter expression levels during foam cell formation. Out of the 46 ABC transporters 12 transporters were significantly regulated during foam cell formation compared to non-foamy cells, including ABCA3, ABCB1b, ABCB2, ABCB4, ABCB6, ABCB7, ABCC3, ABCC5, ABCC10, ABCD3, ABCF2, and ABCG1. The transporters which were significantly upregulated upon foam cell formation induced by different lipoproteins are ABCA3, ABCB1b, ABCB4, ABCB6, ABCB7, ABCC3, ABCC5, ABCF2 and ABCG1. On the other hand, foam cell formation resulted in significant downregulation of the transporters ABCB2 and ABCC10. ABC transporters which were similarly regulated in both types of macrophages include ABCB2, ABCB4, and ABCB6, of which ABCB2 and ABCB4 both showed an interesting high fold- change (ABCB2: 0.5-fold, p<0.05 in PM loaded with oxLDL; 0.5-fold, p<0.05 in BMDM loaded with ßVLDL; ABCB4: 1.4-fold, p<0.01 in PM loaded with oxLDL; 8.2-fold, p<0.001 in BMDM loaded with oxLDL). Importantly, PM and BMDM showed different regulatory expression patterns of several ABC transporters upon lipid loading, indicating that the different types of macrophages do exhibit differences in gene regulation. In addition, this study also showed that loading of macrophages with the different pro-atherogenic lipoproteins, βVLDL and oxLDL, also resulted in different expression patterns of several ABC transporters, which might be due to the differences in lipid and protein composition of the pro-atherogenic lipoproteins. In addition, oxidized LDL does not only induce pro-atherogenic effects but also induces pro-inflammatory effects in macrophages, including generation of reactive oxygen species, survival of foam cells, reduced phagocytic capacity toward apoptotic cells, cytoskeletal rearrangements and macropinocytosis, and expression of inflammatory genes.15 The biological function/processes of all significantly regulated ABC transporters, if known, are described below.
The ABCA3 gene encodes a protein which is highly expressed in the lung and has been localized to the limiting membrane of lamellar bodies.16 ABCA3 is thus possibly important for the maturation of lamellar bodies and surfactant production. In addition, ABCA3 deficiency was reported to induces interstitial lung disease.17 Loading of BMDM with βVLDL significantly upregulated ABCA3 expression, suggesting that ABCA3 might play a role in cholesterol homeostasis.
Furthermore, ABCB1 mRNA is expressed in human atherosclerotic specimens18 and it mediates the esterification of plasma membrane cholesterol.19 In addition, ABCB1 (MDR1) expression is regulated in response to the cellular content of cholesterol. Human monocyte- derived macrophages incubated with HDL for cholesterol depletion or with acetylated LDL for cholesterol loading displayed upregulation and downregulation of ABCB1 expression levels, respectively.20 In the present study, ABCCB1b expression was highly induced in atherosclerotic carotid arteries and in BMDM loaded with oxLDL, which suggests a possible role for ABCB1 in foam cell formation and atherogenesis. However, we recently have found that specific disruption of macrophage ABCB1a/b in LDLr KO mice did not affect atherosclerotic lesion development (Meurs et al. unpublished). In addition, ABCB2 (MDR/TAP) is also suggested to play a role in extracellular transport and import.21 Microarray analysis showed that ABCB2 mRNA expression was reduced in PM loaded with oxLDL and in BMDM loaded with ßVLDL,
Chapter 6 suggesting a role for ABCB2 in macrophage cholesterol homeostasis. ABCB4 is a lipid flippase
that transports phosphatidylcholine across the canalicular membrane during bile formation by the liver.22 Previously, we identified an essential role for ABCB4 in macrophage cholesterol homeostasis and prevention of atherosclerotic lesion development.23 In the current study, ABCB4 was highly induced both in foam cells in vitro as in atherosclerotic lesions in vivo, again indicating the importance of ABCB4 in macrophage lipid homeostasis. ABCB6 and ABCB7 are mitochondrial transport proteins, located in the inner and outer mitochondrial membrane where they function in important physiological processes, including multidrug resistance and protection against oxidative stress.24 Both ABCB6 and ABCB7 were induced upon loading of PM and BMDM with ßVLDL or oxLDL, indicating that these transporters are cholesterol responsive. ABCC9 is another transporter that regulate potassium channels and mutations of ABCC9 was suggested to be associated with dilated cardiomyopathy in human.25 ABCC3, ABCC5 and ABCC10 are all anticipated to have an important role in multidrug resistance.26-28 In the present study, the mRNA expression of ABCC3, ABCC5 and ABCC9 were significantly upregulated in atherosclerotic lesions and macrophage foam cells, whereas expression of ABCC10 was significanlty downregulated in PM loaded with oxLDL, indicating that these ABC transporters are cholesterol responsive and may play a role in macrophage cholesterol homeostasis. ABCD3 is a member of the peroxisomal ABC transporter family and plays a role in fatty acid β-oxidation.29 ABCD3 was differently regulated during atherosclerotic lesion development and in BMDM loaded with oxLDL. ABCD3 expression was significantly reduced in atherosclerotic carotid ateries, whereas loading of BMDM with oxLDL resulted in increased ABCD3 mRNA expression. Furthermore, ABCF2 belongs to the ABCF (GCN20) subfamily.It encodes a protein of unknown function. However, ABCF2 mRNA expression was significantly upregulated in PM loaded with oxLDL, suggesting a role for ABCF2 in cholesterol homeostasis. ABCG1 has been extensively studied in relation to its function in cholesterol efflux from macrophages.2 Nevertheless the role of macrophage ABCG1 in the development of atherosclerosis remains uncertain, as independent studies using the technique of bone marrow transplantation have shown that deficiency of ABCG1 in macrophages resulted in either a modest increase30, 31 or a decrease in atherosclerosis32,
33. In the present study, mRNA expression of ABCG1 was significantly upregulated in PM foam cells in vitro and in atherosclerotic lesions, confirming the importace of ABCG1 for maintaining macrophage cholesterol homeostasis.
Comparison of the transcriptional profiling of ABC transporters in macrophage foam cells in vitro and in atherosclerotic carotid arteries in vivo revealed one ABC transporter, namely ABCB4 which was significantly upregulated in PM and BMDM foam cells as well as in atherosclerotic lesions. Furthermore, ABCB1b and ABCD3 were both significantly regulated in BMDM and in atherosclerotic lesions. However, the expression levels of these transporters were not affected in PM foam cells. Previously, we have evaluated the expression patterns of ABC transporters in the liver of C57Bl/6 mice fed regular chow diet or WTD.9 In this study we found that WTD feeding induced the expression of 4 primarily Kupffer cell expressed genes, including ABCA5, ABCB9, ABCD3, and ABCD4. From these four ABC transporters, only ABCD3 expression was significantly downregulated in atherosclerotic lesions of LDLr KO mice, whereas ABCD3 expression was significantly induced in BMDM loaded with oxLDL in the present study. In addition to macrophages, other cell types accumulate in
atherosclerotic lesions, thereby possibly masking the effects on ABC transporter expression in macrophages specifically. Studies in which laser microdissection is used to measure specifically macrophage areas inside the atherosclerotic lesions will be informative to show ABC transporters expression levels of macrophage foam cells inside atherosclerotic lesions.
It is important to note that the expression of ABCA1, a key transporter in cholesterol and phospholipid efflux from macrophages1 that plays an essential protective role during atherogenesis, was not significantly induced during all conditions studied in vitro as well as in vivo. The expression of ABCA1 in macrophages is tightly controlled by intracellular cholesterol levels.34, 35 Its activity is dramatically increased on cholesterol loading of macrophages and the subsequent transformation into foam cells.36 However, as in this study the mRNA expression of ABCA1 was not induced, the importance of other ABC transporters in macrophage cholesterol homeostasis might also be underestimated.
In conclusion, this study identifies several new ABC transporters, including ABCA3, ABCB1b, ABCB2, ABCB4, ABCB6, ABCB7, ABCC3, ABCC9, ABCC10, ABCD3, and ABCF2 which might serve as novel candidates to modulate macrophage foam cell formation and the initiation of atherosclerotic lesion development. Of particular interest are the ABC transporters ABCB1b, ABCB2, ABCB4, and ABCB6 as they are significantly regulated in BM and BMDM loaded with different lipoproteins and showed a several fold-change. In line with these findings, ABCB1b and ABCB4 were also significantly induced in atherosclerotic lesions in the carotid arteries
ACKNOWLEDGEMENTS
This research was supported by The Netherlands Organization for Scientific Research (Grant 917.66.301 (I.M. and M.V.E.)). M.V.E. is an Established Investigator of the Netherlands Heart Foundation (Grant 2007T056).
Chapter 6
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