Genetic deletion of LXRα prevents arsenic-enhanced atherosclerosis, but not arsenic-altered plaque composition.

TOXICOLOGICAL SCIENCES, 142(2), 2014, 477–488 doi: 10.1093/toxsci/kfu197 Advance Access Publication Date: September 30, 2014

Maryse Lemaire*,†, Catherine A. Lemarie´†,‡, Manuel Flores Molina†, Cynthia Guilbert†, Ste´phanie Lehoux†,‡, and Koren K. Mann*,1,† *Department of Oncology, †Lady Davis Institute for Medical Research, and ‡Department of Medicine, McGill University, Montre´al, Canada H3T 1E2 1

To whom correspondence should be addressed at Lady Davis Institute for Medical Research, McGill University, 3755 Cote Sainte Catherine Road, Montre´al, Que´bec, Canada H3T 1E2. Fax: (514) 340-8717. E-mail: [email protected].

ABSTRACT Arsenic exposure has been linked to an increased incidence of atherosclerosis. Previously, we have shown in vitro and in vivo that arsenic inhibits transcriptional activation of the liver X receptors (LXRs), key regulators of lipid homeostasis. Therefore, we evaluated the role of LXRa in arsenic-induced atherosclerosis using the apoE/ mouse model. Indeed, deletion of LXRa protected apoE/ mice against the proatherogenic effects of arsenic. We have previously shown that arsenic changes the plaque composition in apoE/ mice. Arsenic decreased collagen content in the apoE/ model, and we have observed the same diminution in LXRa/apoE/ mice. However, the collagen-producing smooth muscle cells (SMCs) were decreased in apoE/, but increased in LXRa/apoE/. Although transcriptional activation of collagen remained the same in SMC from both genotypes, arsenic-exposed LXRa/apoE/ plaques had increased matrix metalloproteinase activity compared with both control LXRa/apoE/ and apoE/, which could be responsible for both the decrease in plaque collagen and the SMC invasion. In addition, arsenic increased plaque lipid accumulation in both genotypes. However, macrophages, the cells known to retain lipid within the plaque, were unchanged in arsenic-exposed apoE/ mice, but decreased in LXRa/apoE/. We confirmed in vitro that these cells retained more lipid following arsenic exposure and are more sensitive to apoptosis than apoE/. Mice lacking LXRa are resistant to arsenic-enhanced atherosclerosis, but arsenic-exposed LXRa/apoE/ mice still present a different plaque composition pattern than the arsenic-exposed apoE/ mice. Key words: arsenic; atherosclerosis; LXRa; MMP-9; macrophages; smooth muscle cells

Arsenic is a widespread environmental pollutant to which people are exposed worldwide, mostly through ingestion of contaminated drinking water (Nordstrom, 2002). Although well known as a human carcinogen (Abernathy et al., 2003), recent epidemiologic data link chronic arsenic exposure to a significant increased risk of cardiovascular disease, including atherosclerosis (Engel and Smith, 1994; Medrano et al., 2010; Moon et al., 2013). In addition to these epidemiologic studies, we and others have demonstrated that arsenic enhances plaque formation in atherosclerosis-prone mouse models (Bunderson et al.,

2004; Lemaire et al., 2011; Srivastava et al., 2009). We observed that arsenic, at concentrations found widely in the environment, increases atherosclerotic plaque size by 3-fold in the aortic valves of apoE/ mouse, even though animals were not fed a hyperlipidemic diet (Lemaire et al., 2011). Similarly, transplacental exposure induced atherosclerosis in apoE/ mouse offspring without a high-fat diet (Srivastava et al., 2007). Interestingly, we showed that arsenic exposure altered the composition of the plaque, characterized by an increase in lipid and a decrease in collagen within the plaque (Lemaire et al., 2011).

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Genetic Deletion of LXRa Prevents Arsenic-Enhanced Atherosclerosis, But Not Arsenic-Altered Plaque Composition

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MATERIALS AND METHODS Animals exposure protocol. B6.129P2-Apoetm1Unc/J (apoE/) male mice were obtained from Jackson laboratory (Bar Harbor, Maine). LXRa/ mice (C57BL/6 background) were kindly provided by Peter Tontonoz (Howard Hughes Medical Institute, UCLA School of Medicine, Los Angeles, California) (Bradley et al., 2007). LXRa/apoE/ were generated in our facility. Purchased mice were acclimatized to housing conditions under a 12-h light/12-h dark cycle for at least 2 weeks before experiments. All mice were fed ad libitum. The experimental protocol was approved by the McGill animal use committee and animals were handled in accordance with institutional guidelines. Prior to arsenic exposure, all mice were maintained on tap water. Then, 5-week-old mice (n ¼ 10 animals per group) were either maintained on tap water or on tap water-containing 200 ppb of m-sodium arsenite (0.35 mg/l NaAsO2; Sigma-Aldrich, Ontario, Canada) for 13 weeks. This concentration corresponds to 200 ppb of elemental arsenic, and was selected because it was the most proatherogenic concentration in our previous studies (Lemaire et al., 2011). Solutions containing arsenic were refreshed every 2–3 days to minimize oxidation. The mice were fed with standard diet containing 5% fat (by weight) with no cholesterol for all the experiments (2018; Harlan Laboratories Inc, Wisconsin). Minimal levels of arsenic were detected both in the tap water (0.75 ppb) and in the regular chow (1.90 ppb) 6 SD 5%, with detection limit of 0.65 ppb (Lemaire et al., 2011). Plasma analyses. Plasma analyses were performed as previously described (Lemaire et al., 2011). Briefly, blood (0.6 ml) was withdrawn by cardiac puncture and serum was obtained using collection tubes (BD Vacutainer SST). Plasma cholesterol, highdensity lipoproteins (HDLs) and low-density lipoproteins (LDLs), plasma triglycerides, and liver enzymes (aspartate aminotransferase and alanine aminotransferase) were assessed by the Charles River Research Animal Diagnostic Services (Massachusetts) (n 5 animals per group; Table 1). Atherosclerotic lesion characterization. The characterization of the atherosclerotic lesions was performed as previously described (Lemaire et al., 2011). Briefly, the aorta was removed, rinsed with phosphate-buffered saline, and fixed in 4% paraformaldehyde. The vessel was then cut longitudinally and stained en face with oil red O (Electronic Microscopy Sciences, Pennsylvania). Images were acquired using Infinity Capture software and camera (Lumenera, Canada). Percentage of lesion area of the aortic arch, as defined as the region from the first intercostal arteries to the ascending arch, was evaluated with the Image J software (the National Institutes of Health [NIH]; USA). The atherosclerotic lesions were also evaluated within the aortic sinus from 7 animals. Rinsed, fixed, and embedded frozen

TABLE 1. Effect of Arsenic on Plasma Cholesterol Levels, HDL/LDL Ratio, and Mice Weight Mice genotype þ/ arsenic /

apoE apoE/ þ arsenic LXRa/ apoE/ LXRa/apoE/ þ arsenic

CHOL (mg/dl)

HDL/LDL

Weight (g)

356 6 19 357 6 17 390 6 23 299 6 57

0.25 0.27 0.29 0.28

28.8 6 3.6 31.9 6 1.9 33.3 6 4.9 33.8 6 3.3

CHOL, plasma cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein. Data showed correspond to the mean values 6 SD.


It suggests that arsenic modifies the atherosclerotic lesion to an advanced phenotype (Stary et al., 1995), by making them less stable and rendering them potentially more dangerous with an increased risk of rupture (Rao et al., 2005). As a result, lesion fragments can lodge in vessels, leading to myocardial infarction or stroke. However, the mechanisms by which arsenic increases atherosclerosis are unclear. We have shown that arsenic impairs cellular lipid homeostasis by inhibiting the liver X receptors (LXRa and b), key nuclear receptors that regulate transport and efflux of cholesterol (Lemaire et al., 2011; Padovani et al., 2010). Normally tightly regulated, reverse cholesterol efflux removes excess cholesterol from macrophages to prevent lipid accumulation and formation of foam cells within the arterial wall. LXR isoforms heterodimerize with the retinoid X receptor (RXR), and upon activation with endogenous oxysterol ligands, LXR/RXR releases co-repressors molecules and activates gene transcription (Padovani et al., 2010; Wagner et al., 2003). These genes include several cholesterol transport and lipid metabolism genes, such as the ATP-binding cassette A1 (ABCA1) (Baranowski, 2008), the phospholipid transfer protein (Mak et al., 2002), the adipocyte fatty acid-binding protein (aP2) (Liu et al., 2007), and the sterol response element-binding protein 1 (Baranowski, 2008; Bradley et al., 2007; Tontonoz and Mangelsdorf, 2003). In addition, LXRs can act as repressors of gene transcription by antagonizing inflammation-induced transcriptional programs. For example, matrix metalloproteinase-9 (MMP-9) can be inhibited by LXRmediated repression of the transcription factor nuclear factorjB (NFjB) (Castrillo et al., 2003; Tontonoz and Mangelsdorf, 2003). NFjB plays an important role in atherosclerosis, participating in the inflammatory processes and contributing to plaque instability through MMPs. Thus, LXRs are antiatherogenic by promoting cholesterol efflux and by inhibiting inflammation. Our previous results demonstrated that arsenic exposure inhibits the transcriptional activity of LXRa and LXRb in vitro and in vivo, and correlates with decreased LXR target gene expression, increased macrophage lipid accumulation, and increased atherosclerotic plaque in the apoE/ mouse model (Lemaire et al., 2011; Padovani et al., 2010). Interestingly, arsenic inhibits the transactivation of genes involved in lipid homeostasis, but not the transrepression of inflammatory genes (Padovani et al., 2010). Deletion of either LXRa or LXRb disrupts lipid homeostasis slightly, but only double LXRa/b-knockout mice develop atherosclerosis on normal diet (Quinet et al., 2006; Schuster et al., 2002). In double LXRa/b-knockout mice, increased atherosclerosis is associated with increased circulating lipids and foam cell accumulation within arterial plaques. Deletion of LXRa in either the apoE/ or LDL-R/ mouse background exacerbates atherosclerosis (Bischoff et al., 2010; Bradley et al., 2007). However, deletion of LXRb in LDL-R/ mice did not significantly enhance atherosclerosis (Bischoff et al., 2010), indicating that LXRa and b have non-overlapping functions and that LXRa perhaps plays a more significant antiatherogenic role. Based on the important protective role of LXRa and our previous data showing that arsenic inhibits LXRa function, we have investigated whether arsenic exacerbated plaque formation in an LXRa/apoE/ mouse model. As predicted, deletion of LXRa prevented arsenic-enhanced plaque formation. In addition, arsenic-exposed, LXRa/apoE/ mice had a different plaque composition from arsenic-exposed, apoE/ mice. Interestingly, our observations suggest that arsenic may increase atherosclerosis through LXRa inhibition, but changes in plaque composition involve LXRa-independent mechanisms.

LEMAIRE ET AL.

hearts were processed as previously described (Lemaire et al., 2011). Consecutive cryosections of 6 -lm thickness were sliced from the aortic base throughout the aortic sinus, where 5–7 valve sections per animal were stained with oil red O to visualize the plaque areas and analyzed for their lipid content. Aortic valves were also stained and analyzed for their collagen content (types I and III) using picrosirius red (Polysciences, Pennsylvania) (Lemaire et al., 2011).

Thioglycollate-elicited macrophages. In order to study macrophages exposed to arsenic in vivo, 1.5 ml of 4% thioglycollate solution (Criterion, California) was injected intraperitoneally in mice exposed to arsenic for 13 weeks or tap water. Seventy-two hours after the injection, the ascitic fluid containing elicited macrophages was collected (Lemaire et al., 2011; Padovani et al., 2010) and gene expression analysis was performed (n ¼ 3 animals from each group). Alternatively, in order to obtain sufficient macrophages for ex vivo cultures, thioglycollate solution was injected in 12-week-old male apoE/ and LXRa–/– apoE/ mice (n 3), and the macrophages from the ascitic fluid were cultured in vitro overnight and exposed to arsenic (0, 10, 50, or 200 ppb) for 24 h. These concentrations correspond to the amount of elemental arsenic, and represent non-cytotoxic concentrations based on our previous studies in murine macrophages (Padovani et al., 2010). Additionally, apoE/ mice were also exposed or not to geranylgeranyl pyrophosphate ammonium salt (GGPP; 10 lM; Sigma-Aldrich), an LXRa inhibitor. Cells were then stained for either total lipid accumulation with oil red O or with anti-active caspase-3 antibody (Abcam). SMCs isolation and culture. SMCs were isolated from the thoracic aorta of 12-week-old apoE/ and LXRa/apoE/ male mice (Ray et al., 2001). After removal of the adventitia and the endothelial cell layer, the tunica media was digested with 3 mg of collagenase (Worthington Biochemical Corporation, New Jersey) and 3 mg of elastase (Sigma-Aldrich) for 0.5 h at 37 C. Residual SMCs were cultured in DMEM þ 10% fetal bovine serum for 2 weeks prior to experiment.

479

Analysis of gene expression by real-time polymerase chain reaction. Total RNA was isolated from thioglycollate-elicited macrophages collected from mice exposed to arsenic or tap water for 13 weeks. In addition, RNA was isolated from the primary SMC exposed to noncytotoxic concentrations of arsenic (0, 10, or 50 ppb) in vitro for 24 h. In both cases, total RNA was isolated using TRIzol reagent (Invitrogen), followed by chloroform extraction and nucleic acid precipitation in isopropanol. RNA was quantified and cDNA was synthesized from 5 mg RNA using random primers and Superscript II reverse transcriptase (Invitrogen). Quantitative real-time polymerase chain reaction (qPCR) amplification was performed using a 7500 Fast PCR thermocycler (Applied Biosystems, California) and SYBR green chemistry with specific primers spanning different exons from murine acidic ribosomal phosphoprotein P0 (36B4), fatty acid synthase (FASN), and collagen type 1, a1 (Col1a1) (Table 2). Results were analyzed using the DDCt method where calibrator samples were control animals and results were normalized to the 36B4 housekeeping gene. In situ zymography. Aortic sinus vessel sections were incubated at 37 C for 7 h with a fluorogenic gelatin substrate for metalloproteinase (DQ gelatin, Molecular Probes) dissolved to 25 lg/ml in zymography buffer, as provided by the manufacturer. Upon enzymatic activation, the fluorescein-labeled gelatin was digested and became fluorescent. The MMP activity was detected under fluorescence and images were acquired using Infinity Capture software and camera (Lumenera). Percentage positive gelatinolytic activity relative to plaque areas was evaluated with the Image J software (NIH). Apoptosis and proliferation assessment. Immunofluorescent assay using antiactive caspase 3 antibody (Abcam) was performed to visualize apoptosis both in vitro on arsenic-exposed thioglycollate-elicited macrophages collected from apoE/ and LXRa/apoE/ mice and in situ on aortic sinus tissue. In addition, apoptosis was evaluated in arsenic-exposed thioglycollateelicited macrophages using an Apo-BrdU kit according to the manufacturer (BD Pharmingen), also known as the TUNEL assay. Briefly, 1 106 paraformaldehyde-fixed cells were stained for 15 h with DNA-labeling solution that contained Br-dUTP compound and the deoxynucleotidyl transferase enzyme, which catalyzes addition to the 30 -OH end of double- and singlestranded DNA. Cells were washed and stained with anti-BrdU fluorescein-labeled and propidium iodide to detect DNA breaks using a BD Biosciences FACSCalibur flow cytometer. Cell apoptosis was measured as the percentage of fluoroscein-labeled cells. Similarly, Br-dUTPs were utilized to determine primary SMC proliferation by detecting newly synthesized DNA by cells (FITC-BrdU Flow, BD Pharmingen). Briefly, 1 106 fixed SMC were was washed, permeabilized, and stained according to the manufacturer’s protocol. DNAse was then added to expose incorporated BrdU, which was subsequently stained with antiBrdU fluorescein-labeled-specific antibodies. Cell proliferation

TABLE 2. Oligonucleotide Sequences Target Gene (GenBank Accession Number) 36B4 (NM_007475) bactin (NM_007393.3) FASN (NM_007988.3) Col1a1 (NM_007742) LXRb (EU869275.2j)

mRNA Sense Primer 0

mRNA Antisense Primer 0

5 -GGCACCGAGGCAACAGTT-3 50 -GACGGCCAGGTCATCACTAT-30 50 -GCTGCGGAAACTTCAGGAAAT-30 50 -GCTCCTCTTAGGGGCCACT-30 50 -TGCATTCTGTCTCGTCCTTCT-30

50 -TCATCCAGCAGGTGTTTGACA-30 50 -CCTCTGCATCCTGTCAGCAA-30 50 -AGAGACGTGTCACTCCTGGACTT-30 50 -CCACGTCTCACCATTGGGG-30 50 -AAGCAGGTGCCAGGGTTCT-30


In situ immunofluorescence analyses. Smooth muscle cell (SMC), macrophage, and active caspase-3 protein content were assessed within the entire plaque area, as previously described (Lemaire et al., 2011). Briefly, aortic sinus sections were rinsed and blocked with 3% bovine albumin serum (Sigma-Aldrich), incubated with primary antibody (1:100 for monoclonal antia-SMC actin [clone 1A4], 1:100 for anti-monocyte and macrophage [moma-2], or 1:200 for anti-active caspase-3 [Abcam, Massachusetts]), rinsed and incubated with fluorescently labeled secondary antibodies (1:500) (Invitrogen, Ontario, Canada). The presence of the immunofluorescent marker from at least 5 sections per animal was quantified using Image J software (NIH) and expressed as percentage of the total lesion area.

|

480

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was measured on a BD Biosciences FACS Calibur flow cytometer, and the percentage of fluoroscein-labeled cells was assessed. Statistical considerations. For statistical analysis, the one-way ANOVA was performed and the P value was evaluated with a Tukey’s post hoc test using the GraphPad Instat software (San Diego, California). A P < 0.05 indicated statistical significance. The data correspond to the mean values 6 SD.

RESULTS

Arsenic Induces Collagen Depletion and SMC Invasion in LXRa2/2apoE2/2 Atherosclerotic Lesions In addition to enhancing atherosclerosis, we previously published that arsenic exposure alters plaque composition toward a less stable phenotype (Lemaire et al., 2011). Collagen and SMC content were reduced and lipids were increased following arsenic exposure. Here, we studied the aortic sinus plaque composition from apoE/ and LXRa/apoE/ mice exposed to arsenic or maintained on tap water for 13 weeks, in order to determine which of these changes was dependent upon arsenic inhibiting LXRa. First, we measured plaque collagen content using picrosirius red staining of the aortic sinus. As previously observed in the

Arsenic Enhances Lipid Accumulation and Apoptosis in Macrophages from LXRa2/2apoE2/2 Mice In addition to collagen and SMC content, we determined whether arsenic influences the total plaque lipid accumulation. We stained aortic sinus from apoE/ and LXRa/apoE/ mice exposed to arsenic or maintained on tap water with oil red O,


Deletion of LXRa Protected apoE2/2 Mice Against Arsenic-Enhanced Plaque Formation In order to investigate the role of LXRa in arsenic-induced atherosclerosis formation, we exposed LXRa/apoE/ mice to a moderate arsenic concentration (200 ppb), which is proatherogenic in apoE/ mice (Lemaire et al., 2011). After 13 weeks, we assessed the atherosclerotic lesion formation in the aortic arch and in the aortic sinus. The aortic arch en face staining showed that the LXRa/apoE/ control mice developed more lesions than the apoE/ control mice (Figs. 1A and 1B; P < 0.01), as previously reported in literature that established an antiatherogenic effect for LXRa (Bradley et al., 2007). In addition, we have also shown that, arsenic exposure increases plaque size in apoE/ mice (Lemaire et al., 2011). Interestingly, arsenic exposure failed to enhance plaque formation in the LXRa/apoE/ mice (Figs. 1A and 1B; P < 0.001). In the aortic sinus, deletion of LXRa itself promoted plaque formation to a level comparable with arsenic-exposed, apoE/ mice (Fig. 1C). Again, addition of arsenic to LXRa/apoE/ mice did not enhance plaque formation (Figs. 1C and 1D). These observations suggest that arsenic enhances atherosclerosis formation through inhibition of LXRa. Arsenic exposure did not alter circulating plasma cholesterol levels, HDL/LDL ratio, or body weight, as we and others have previously observed (Lemaire et al., 2011; Srivastava et al., 2009) (Table 1). Arsenic exposure significantly decreased plasma triglyceride levels in apoE/ mice, but arsenic did not further decrease the already lower triglyceride levels in LXRa/apoE/ mice (Fig. 2A). FASN is a LXR target gene that encodes the enzyme that catalyzes the elongation of long-chain fatty acids, which are then converted into triglycerides (Paulauskis and Sul, 1989; Schuster et al., 2006). FASN mRNA expression was decreased in thioglycollate-elicited macrophages from arsenicexposed apoE/ mice, when compared with control apoE/. In addition, LXRa/apoE/ control mice had significantly lower levels of FASN mRNA (P < 0.001), which was unchanged by arsenic (Fig. 2B), indicating that arsenic decreases triglyceride levels through LXRa-dependent mechanisms.

arsenic-exposed apoE/ mice, collagen content significantly decreased within the plaque compared with the apoE/ control group (Fig. 3A; P < 0.05) (Lemaire et al., 2011). LXRa/apoE/ mice had significantly more collagen per plaque area than apoE/ mice (Fig. 3A; P < 0.01), but it was still decreased following arsenic exposure (Figs. 3A and 3B; P < 0.01). Because collagen diminution can be a consequence of decreased production, we assessed collagen-producing SMC content within the plaque. Arsenic decreased the percentage of a-actin positive-stained cells per plaque area in apoE/ mice, when compared with tap water-exposed apoE/ mice. However, we surprisingly found that arsenic significantly increased plaque SMC content in LXRa/apoE/ mice (Figs. 3C and 3D; P < 0.01). This finding cannot be explained by changes in the proliferative capacity of the SMC (Fig. 4A). We cultured primary apoE/ and LXRa/apoE/ SMC ex vivo. Although LXRa/apoE deletion increased baseline SMC proliferation significantly compared with apoE/, addition of arsenic did not alter SMC proliferation in either genotype. When we tested whether arsenic might decrease SMC collagen mRNA levels, we did not observe arsenic-induced changes in Col1a1 gene expression from these cells, although LXRa/apoE/ SMC have significantly decreased Col1a1 gene expression (Fig. 4B). Because collagen depletion can also result from increased degradation, we tested the hypothesis that plaque microenvironment MMP activity might be involved in its reduction (Pasterkamp et al., 2000). Some MMPs are known to possess collagenase activity (Bigg et al., 2007). We measured their activity in situ within the atherosclerotic plaque by assessing their digestion of a gelatin substrate. This assay measures MMP-2 and MMP-9 activity, both enzymes having gelatinase and collagenase activity (Bigg et al., 2007). Deletion of LXRa itself slightly enhanced MMP-2/9 activity, although it was not significant (Fig. 4C). The addition of arsenic further increased this MMP-2/9 activity within the plaque of LXRa/apoE/ mice (Figs. 4C and 4D; P < 0.01), which suggests regulation beyond LXRa inhibition. The observed increased MMP activity might explain the reduction of collagen within the plaque of arsenic-exposed, LXRa/apoE/ mice (Fig. 3A), but might also favor tissue remodeling in the plaque microenvironment that could be more amenable to SMC migration and invasion (Pasterkamp et al., 2000) (Fig. 3B). As mentioned, the gelatinase assay assessed both MMP-2 and MMP-9 gelatinase activity, but recent observations suggest that MMP-2 participates in the cellular activation of MMP-9, which will then degrade collagen types I and III (Bigg et al., 2007; Veidal et al., 2010). Therefore, we further assessed MMP-9 mRNA expression from SMC. However, MMP-9 mRNA transcript level in SMC is unchanged following arsenic exposure (Fig. 4E). However, we also investigated whether macrophages, which are important contributors to atherosclerosis, could be the source of MMP-9 observed in vivo. Indeed, arsenic induced MMP-9 expression from both genotypes (Fig. 4F) in thioglycollate-elicited macrophages. This increased MMP-9 production by macrophages may favor plaque matrix breakdown, which may contribute to the impaired plaque integrity observed following arsenic exposure (Lemaire et al., 2011).

LEMAIRE ET AL.

B

30

** ***

apoE-/-

20

ns

LXRα-/apoE-/-

10

0

control

arsenic

apoE -/-

control

arsenic

LXR α -/- apoE -/-

D

C Lesion area (%) / total aortic sinus area

481

30

* * ns

20

LXRα-/apoE-/-

10

0

control

arsenic

apoE

-/-

control

arsenic -/-

LXR α apoE -/-

FIG. 1. Moderate arsenic exposure does not increase atherosclerotic plaque formation in LXRa/apoE/ mice. Five-week-old apoE/ and LXRa/apoE/ mice were exposed to arsenic (200 ppb) for 13 weeks or maintained on tap water. The aorta was cut with the luminal surface facing up, and the inner aortic surface was stained with oil red O (A and B). Percent of lesion area of the aortic arch from the heart to the first intercostal artery was evaluated with the Infinity Analyze software 5.0 (Lumenera) (A). In C and D, sections of the aortic sinus were stained with oil red O, and % of the lesion area was evaluated relative to the total aortic sinus area. Representative pictures from the experiments are shown (B and D). Values are expressed as mean 6 SD; ns: not significant; *P < 0.05; **P < 0.05; ***P < 0.001.

and measured the total lipid content within the entire plaque area. Surprisingly, deletion of LXRa did not increase lipid levels within the plaque (Fig. 5A). However, arsenic significantly increased lipids in both genotypes compared with tap water controls (Figs. 5A and 5B; P < 0.05). Because macrophages are key players in lipid homeostasis, accumulating lipid and forming foam cells in the arterial walls, we evaluated macrophage composition within the plaque. We observed that the macrophage content was diminished in the arsenic-exposed, LXRa/ apoE/ animals compared with all other groups (Figs. 5C

and 5D; P < 0.01). These data suggest that, in the lesions from the arsenic-exposed LXRa/apoE/, there are fewer macrophages that retain more lipids. This could result from impaired macrophage lipid turnover within the plaque or a higher percentage of macrophage cell death. To study these possibilities, we assessed lipid accumulation and the apoptotic profile of apoE/ and LXRa/apoE/, thioglycollate-elicited macrophages exposed in vitro to arsenic. Although deletion of LXRa increased lipid accumulation within the cells, arsenic exposure accentuated lipid accumulation (Figs. 5E and 5F),


Lesion area (%) / total aortic arch area

A

|

|


Plasma triglycerides (mg/dl)

A

500

***

400

B

***

FASN mRNA expression / 36B4

482

300

ns 200

100

control

arsenic -/-

apoE

control

arsenic

LXRα-/-apoE-/-

*** ** 1.0

0.5

ns

0.0

control

arsenic

apoE-/-

control

arsenic

LXRα -/- apoE-/-

FIG. 2. Effect of arsenic exposure on plasma triglyceride levels and macrophage fatty acid synthase (FASN) expression. Five-week-old apoE/ and LXRa/apoE/ mice were exposed to arsenic (200 ppb) for 13 weeks or maintained on tap water. Plasma triglyceride levels (mg/dl) were measured at the end of the experiment (A). In B, total RNA from thioglycollate-elicited macrophages from mice was isolated and gene expression was assessed by qPCR of FASN mRNA. Each sample (n ¼ 3 mice per group) was analyzed in triplicate (technical replicate) and expressed relative to the 36B4 housekeeping gene (arbitrary units). For both graphs, values are expressed as mean 6 SD; ns: not significant; **P < 0.01; ***P < 0.001.

as observed in vivo (Fig. 5A). Furthermore, chemical inhibition of LXRa with GGPP in apoE/ mice confirmed these data (Fig. 5E). Additionally, besides affecting their lipid turnover, we tested whether arsenic exposure could result in increased macrophage death, which would explain the reduction in plaque macrophages (Fig. 5C). In fact, markedly more antiactivated caspase-3 staining could be detected within the arsenic-exposed, LXRa/apoE/ plaque compared with all other groups (Fig. 6A; P < 0.001). To confirm these findings in isolated cells, thioglycollate-elicited macrophages were stained for apoptotic markers and arsenic-exposed, LXRa/apoE/ macrophages exhibited more apoptosis thethan all the other groups (50 ppb; Figs. 6C and 6D; P < 0.001), suggesting increased sensitivity to arsenicinduced cell death. Together, these data indicate that arsenicexposed LXRa/apoE/ macrophages accumulate more lipids and have a higher rate of apoptosis.

DISCUSSION Exposure to arsenic is linked to an increased risk of atherosclerosis (Engel and Smith, 1994; Medrano et al., 2010; Moon et al., 2013). The underlying molecular mechanisms involved in arsenic-induced cardiovascular toxicity remain to be established. Previously, we showed that arsenic inhibits LXRa nuclear receptor transcriptional activity in macrophages, and leads to impaired cholesterol efflux and subsequent lipid accumulation (Lemaire et al., 2011; Padovani et al., 2010). To confirm our data in vivo, we utilized a model of arsenic-enhanced atherosclerosis in apoE/ mice that were also deficient in LXRa. We hypothesized that if arsenic enhances atherosclerosis through nonLXRa-dependent mechanisms, increased plaque size would still be observed. In fact, we observed no additional plaque formation in the arsenic-exposed LXRa/apoE/ mice, compared with the control LXRa/apoE/ animals. This is in contrast to high-fat diet that increased plaque size in the LXRa/apoE/ mice (Bradley et al., 2007). We interpret these data to mean that the effects of arsenic are mediated, in part, through LXRa. Furthermore, our previous data suggest that arsenic acts as an inhibitor of LXRs, not an activator (Lemaire et al., 2011; Padovani

et al., 2010). Thus, we believe that the atheroprotective effects of LXRa are abrogated by arsenic exposure, significantly contributing to the enhanced atherosclerosis. However, inhibition of LXRs is likely not the only means by which arsenic increases atherosclerosis. Several other mechanisms are proposed to add to the proatherogenic effects of arsenic, including induction of various inflammatory molecules (Bunderson et al., 2004; Srivastava et al., 2009; Straub et al., 2009), production of oxidative stress (Flora, 2011; Srivastava et al., 2009), dysfunction of vascular endothelial cells (Hossain et al., 2013), and modulation of various cellular signal transduction, including activation of NFjB (Barchowsky et al., 1996). Furthermore, the multicellular landscape of the atherosclerotic plaque makes it difficult to determine which mechanisms are contributing in each cell type. The use of bone marrow transplantation models and tissue-specific knock-outs may aid in this dissection. We and others have previously reported that arsenic exposure significantly decreases plasma triglyceride levels in apoE/ mice (Lemaire et al., 2011; Srivastava et al., 2009). Earlier, Bradley et al. (2007) showed that apoE/ mice, but not LXRa/apoE/, have increased plasma triglyceride levels when exposed to the synthetic LXR ligand GW3965, indicating that triglyceride regulation is LXRa dependent. We extend our results here by showing that arsenic does not further reduce the already low triglyceride levels in LXRa/apoE/ mice. This indicates that arsenic lowers triglyceride levels through inhibition of LXRa. In addition, we investigated the expression of the LXR target gene, FASN, which is involved in the production of fatty acids that are converted into triglycerides. We observed that FASN mRNA expression correlated with triglyceride levels. Arsenic decreased FASN mRNA in the thioglycollate-elicited macrophages from apoE/, but did not change the lower levels observed in LXRa/apoE/ mice. Evidence from the literature suggests indeed that triglyceride levels might have little influence on plaque formation (Braz et al., 2007). Nevertheless, since high rather than low triglycerides are more likely to be associated with atherosclerosis (Talayero and Sacks, 2011), this effect of arsenic is unlikely to have an important impact on plaque formation.


0

1.5

LEMAIRE ET AL.

Collagen content (%) / lesion area

A

**

75

|

483

B **

* LXRα-/apoE-/-

50

25

control

arsenic apoE-/-

control

arsenic -/-

LXRα apoE-/-

D Smooth muscle cells (%) / lesion area

C

75

50

**

LXRα-/apoE-/-

**

25

0

control

arsenic

apoE -/-

control

arsenic

LXR α-/-apoE -/-

FIG. 3. Arsenic exposure alters plaque collagen and SMC composition differently in apoE/ and LXRa/apoE/ mice. Five-week-old apoE/ and LXRa/apoE/ mice were exposed to arsenic (200 ppb) for 13 weeks or maintained on tap water. Sections of the aortic sinus were stained for collagen (A) with picrosirius red. The collagen content was evaluated relative to the total lesion area. In C, sections of the aortic sinus were stained for SMCs within the entire plaque. Representative pictures from the experiments are shown for collagen staining (B) and for SMC staining (D). Values are expressed as mean 6 SD; *P < 0.05; **P < 0.01 relative to their own control. Circles and arrows indicate example of atherosclerotic lesions.

We previously reported that arsenic exposure alters plaque composition toward a less stable phenotype (Lemaire et al., 2011). In the apoE/ model, arsenic decreased the presence of collagen-producing SMC, correlating with decreased collagen. However, in LXRa/apoE/ mice, arsenic significantly increased SMC, despite decreased collagen content. Thus, arsenic has opposite effects on SMC content within the plaque depending on the LXRa expression. These contradictory observations could not be attributed to arsenic-induced changes in collagen synthesis by SMC or SMC proliferation. Aortic wall fibroblasts, which also produce collagen, could be affected by arsenic exposure, although we have not investigated that possibility here. An alternative hypothesis is that arsenic alters plaque remodeling, which could explain SMC migration or invasion. Furthermore, our data suggest a role for MMPs in this remodeling process. Mice lacking MMP-9 gene expression have significantly decreased SMC migration and proliferation capacities (Cho and Reidy, 2002). In addition, induction of the active form of MMP-9

has been linked with plaque disruption in apoE/ mice (Gough et al., 2006), and epidemiologic data indicate that MMP-9 expression highly correlates with human clinical manifestations of atherosclerosis (Loftus et al., 2000). Furthermore, arsenic exposure correlates with circulating MMP-9 plasma levels in humans (Burgess et al., 2013). LXRs inhibit basal activity of MMPs, including MMP-9, through repression of NFjB-induced transactivation (Castrillo et al., 2003). Interestingly, we showed that arsenic significantly enhances in situ MMP activity in LXRa/apoE/, but only slightly increases MMP activity in apoE/ mice. The enhanced MMP activity correlated with an arsenic-induced increase in plaque-resident SMCs, suggesting some threshold of MMP activity is needed to affect remodeling for SMC invasion. Our previous data show that arsenic does not affect the transrepressive properties of LXR, consistent with the lack of MMP-9 mRNA induction in apoE/ mice. However, when LXRa is deleted, this transrepression is absent and thus, we postulate that arsenic can activate MMPs through NFjB activation. Alternatively, arsenic may post-translationally increase


0


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arsenic

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FIG. 4. Arsenic favors SMC invasion within the plaque by enhancing MMPs activity. Primary SMC cultures were exposed to DMSO vehicle control or arsenic trioxide (10 or 50 ppb) for 24 h. In A, Br-dUTPs incorporation was used to determine cell proliferation. In B, total RNA from the primary SMC exposed or not to arsenic trioxide was isolated and converted to cDNA followed by the analysis of Col1a1 gene expression by qPCR. In C, sections of the aortic sinus were assessed for MMP activity with a fluorogenic gelatin substrate for metalloproteinase. Detected-fluorescent MMP activity was evaluated relative to the total lesion area. Representative pictures from this experiment are shown (D). Primary SMC (E) or thioglycollate-elicited macrophages (F) MMP-9 gene expression was evaluated by qPCR. Data are relative to their own control. For all graphs, values are expressed as mean 6 SD; ns: not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

proteolytic activation of MMPs. Interestingly, arsenic-induced MMP-9 activation is also linked with cellular transformation and it is proposed to facilitate tumor cell invasion (Achanzar et al., 2002; Baruch et al., 2001). Thus, activation of tissue remodeling metalloproteinases may be a common effect of arsenic exposure across pathologies. LXRs are key regulators of lipid metabolism, including cholesterol efflux from plaque-resident macrophages. Our in vitro

work indicated that arsenic could inhibit LXRs transcriptional activation and LXR ligand-induced cholesterol efflux in RAW264.7 cells (Padovani et al., 2010). Thus, we predicted arsenic would not increase lipid accumulation in the absence of LXRa. Surprisingly, arsenic exposure increased plaque lipid accumulation in both mouse models, regardless of LXRa expression. Furthermore, arsenic increased lipid accumulation in macrophages from both apoE/ and LXR/apoE/.


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LXR α -/- apoE -/-

FIG. 5. Effects of arsenic exposure on lipid accumulation and macrophages in plaques from apoE/ and LXRa/apoE/ mice. Five-week-old apoE/ and LXRa/ apoE/ mice were exposed to arsenic (200 ppb) for 13 weeks or maintained on tap water. Sections of the aortic sinus were stained for total lipids with oil red O (A and B). Sections of the aortic sinus were stained for macrophages with moma-2 antibody. The content was evaluated relative to the total lesion area (C). Representative pictures from the experiments are shown for lipids staining (B) and for macrophages (D) and arrows indicate example of atherosclerotic lesions. In E, primary thioglycollate-elicited macrophages were exposed to DMSO vehicle control, arsenic trioxide (10 or 50 ppb), or GGPP for 24 h. Total lipid accumulation was evaluated with oil red O staining and the number of positive cells relative to total cell amount per field was quantified. Representative pictures from this experiment are shown (F). Values are expressed as mean 6 SD; *P < 0.05; **P < 0.01; ***P < 0.001.


control

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arsenic (ppb) FIG. 6. Arsenic exposure effects on lipid accumulation and apoptosis from apoE/ and LXRa/apoE/ thioglycollate-elicited macrophages. Five-week-old apoE/ and LXRa/apoE/ mice were exposed to arsenic (200 ppb) for 13 weeks or maintained on tap water. Sections of the aortic sinus were stained for active caspase-3 activity and quantify within the entire plaque (A). Representative pictures from this experiment are shown in B. In addition, primary thioglycollate-elicited macrophages were exposed to DMSO vehicle control, arsenic trioxide (10 or 50 ppb) for 24 h. Apoptotic primary macrophages were detected by TUNEL assay (C) or for active caspase-3 (D). Representative pictures are shown. For all graphs, values are expressed as mean 6 SD; ***P < 0.001.

Together, these data indicate that arsenic can result in increased lipid accumulation within macrophages in an LXRaindependent manner. Coupled with the increased apoptosis observed in vitro and in vivo, our data further indicate that, while each macrophage from LXRa/apoE/ mice accumulates more lipid following arsenic exposure, there are fewer macrophages, resulting in no net gain of plaque lipid compared with arsenicexposed apoE/ mice. One possibility may be that LXRb compensates for LXRa deletion, as suggested by data showing LXRa/ mice do not increase atherosclerosis formation (Bradley et al., 2007; Quinet et al., 2006), while LXR double-knockout mice displayed foam cells accumulation (Schuster et al., 2002). However, LXRb mRNA expression is not increased in liver or intestine from LXRa/ apoE/ mice (Bradley et al., 2007) or in thioglycollate-elicited, LXRa/apoE/ macrophages (Supplementary Fig. S1). Furthermore, our in vitro data showed that LXRb is also inhibited by arsenic (Padovani et al., 2010), making this hypothesis less likely.

In this study, we show that arsenic enhances atherosclerosis in vivo through LXRa inhibition. In contrast, LXRa-independent mechanisms may contribute to the plaque instability observed following arsenic exposure. A better understanding of cellular and molecular targets of arsenic leading to atherosclerosis might enable targeted interventions to prevent or reverse the disease.

SUPPLEMENTARY DATA Supplementary data are available online at http://toxsci.oxford journals.org/.

FUNDING Funding for this work was provided by the Canadian Institutes of Health Research [MOP-115000 to K.K.M.; MOP102498 to S.L.], and a fellowship from the Canadian Institutes of Health Research [MFE-120914] to M.L.


Capsase-3 positive cells / plaque area (%)

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ACKNOWLEDGMENTS The authors would like to thank Dr Talin Ebrahimian and Stefania Simeone at the Lady Davis Institute for Medical Research (McGill University, Montreal, Canada) for kindly providing their help with the proliferation and the zymography assays. The authors have no conflicts of interest to declare.

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Genetic deletion or TWEAK blocking antibody administration reduce atherosclerosis and enhance plaque stability in mice.

Genetic deletion of AEG-1 prevents hepatocarcinogenesis.

Asymmetry in prevalence of femoral but not carotid atherosclerosis.

TTC39B deficiency stabilizes LXR reducing both atherosclerosis and steatohepatitis.

deletion polymorphisms are associated with atherosclerosis in young black but not young white males. Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group.

Pappa2 deletion in mice affects male but not female fertility.

Serum antibody prevents lethal murine influenza pneumonitis but not tracheitis.

Plague (tuberculosis) and plaque (atherosclerosis)

Genetic Deletion of the Transcriptional Repressor NFIL3 Enhances Axon Growth In Vitro but Not Axonal Repair In Vivo.

Reduced cocaine-induced serotonin, but not dopamine and noradrenaline, release in rats with a genetic deletion of serotonin transporters.

Dietary phytosterol does not accumulate in the arterial wall and prevents atherosclerosis of LDLr-KO mice.

Change in Emiliania huxleyi Virus Assemblage Diversity but Not in Host Genetic Composition during an Ocean Acidification Mesocosm Experiment.

Carotid artery plaque morphology and composition in relation to incident cardiovascular events: the Multi-Ethnic Study of Atherosclerosis (MESA).

Deletion of the cytoplasmic domain of N-cadherin reduces, but does not eliminate, traction force-transmission.

Local but Not Systemic Administration of Uridine Prevents Development of Antigen-Induced Arthritis.

The macrophage LBP gene is an LXR target that promotes macrophage survival and atherosclerosis.

Metabolically healthy obese subjects are at risk of fatty liver but not of pre-clinical atherosclerosis.

Herbal composition of Cinnamomum cassia, Pinus densiflora, Curcuma longa and Glycyrrhiza glabra prevents atherosclerosis by upregulating p27 (Kip1) expression.

Deletion of the titin N2B region accelerates myofibrillar force development but does not alter relaxation kinetics.

Loss of c-REL but not NF-κB2 prevents autoimmune disease driven by FasL mutation.

Dexamethasone prevents autoimmune nephritis and reduces renal expression of Ia but not costimulatory signals.

Triclosan produces statistically significant reduction in plaque, gingivitis and caries but not clinically important benefit.

Imaging of coronary atherosclerosis and identification of the vulnerable plaque.

Antibodies to periodontal pathogens are associated with coronary plaque remodeling but not with vulnerability or burden.