Toxicology and Applied Pharmacology 273 (2013) 651–658

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Arsenic augments the uptake of oxidized LDL by upregulating the expression of lectin-like oxidized LDL receptor in mouse aortic endothelial cells Ekhtear Hossain a, Akinobu Ota a,⁎, Sivasundaram Karnan a, Lkhagvasuren Damdindorj a, Miyuki Takahashi a,b, Yuko Konishi a, Hiroyuki Konishi a, Yoshitaka Hosokawa a a b

Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan Division of Hematology, Department of Internal Medicine, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan

a r t i c l e

i n f o

Article history: Received 8 July 2013 Revised 25 September 2013 Accepted 4 October 2013 Available online 19 October 2013 Keyword: Arsenic exposure Atherosclerosis LOX-1 NF-κB Reactive oxygen species (ROS) Molecular biology

a b s t r a c t Although chronic arsenic exposure is a well-known risk factor for cardiovascular diseases, including atherosclerosis, the molecular mechanism underlying arsenic-induced atherosclerosis remains obscure. Therefore, this study aimed to elucidate this molecular mechanism. We examined changes in the mRNA level of the lectin-like oxidized LDL (oxLDL) receptor (LOX-1) in a mouse aortic endothelial cell line, END-D, after sodium arsenite (SA) treatment. SA treatment significantly upregulated LOX-1 mRNA expression; this finding was also verified at the protein expression level. Flow cytometry and fluorescence microscopy analyses showed that the cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with SA treatment. In addition, an anti-LOX-1 antibody completely abrogated the augmented uptake of Dil-oxLDL. We observed that SA increased the levels of the phosphorylated forms of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB)/p65. SA-induced upregulation of LOX-1 protein expression was clearly prevented by treatment with an antioxidant, N-acetylcysteine (NAC), or an NF-κB inhibitor, caffeic acid phenethylester (CAPE). Furthermore, SA-augmented uptake of Dil-oxLDL was also prevented by treatment with NAC or CAPE. Taken together, our results indicate that arsenic upregulates LOX-1 expression through the reactive oxygen species-mediated NF-κB signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of the aberrant LOX-1 signaling pathway in the pathogenesis of arsenic-induced atherosclerosis. © 2013 Elsevier Inc. All rights reserved.

Introduction Arsenic is a naturally occurring toxic element that is present in air, food, soil, and water in several countries, including Bangladesh, India, Taiwan, Chile, Argentina, and the USA (Argos et al., 2010; Chen et al., 2011; Fendorf et al., 2010; Oremland and Stolz, 2003). Epidemiologic evidence indicates that chronic arsenic exposure enhances the risk of cardiovascular diseases, including acute myocardial infarctions (Yuan Abbreviations: ABCA1, ATP-binding cassette sub-family A member 1; AP-1, activator protein 1; CAPE, caffeic acid phenethylester; DAPI, 4′,6-diamidine-2′-phenylindole dihydrochloride; DCFH-DA, 2,7-dichlorofluorescin diacetate; Dil, 1,1′-Dioctadecyl3,3,3′,3′-tetramethylindocarbocyanine perchlorate; ECs, endothelial cells; eNOS, endothelial nitric oxide synthetase; Erk1/2, extracellular signal-regulated protein kinases 1 and 2; FACS, fluorescence-activated cell sorting; GAPDH, glyceraldehyde3-phosphate dehydrogenase; JNK, C-Jun N-terminal kinases; ICAM-1, intercellular adhesion molecule 1; LOX-1, lectin-like oxidized low-density lipoprotein receptor; LXR, liver X receptor; MAPK, mitogen-activated protein kinase; NAC, N-acetylcysteine; NF-κB, nuclear factor of kappa light polypeptide gene enhancer in B cells; oxLDL, oxidized low-density lipoprotein; ROS, reactive oxygen species; SA, sodium arsenite; SR-A, scavenger receptor A; VCAM-1, vascular cell adhesion molecule; VSMCS, vascular smooth muscle cells. ⁎ Corresponding author at: Department of Biochemistry, Aichi Medical University School of Medicine, 1-1 Yazako karimata, Building #2, Room 362, Nagakute, Aichi 480-1195, Japan. Fax: +81 561 61 056. E-mail address: [email protected] (A. Ota). 0041-008X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2013.10.012

et al., 2007), ischemic heart disease (Chen et al., 1996; Hsueh et al., 1996), hypertension (Abhyankar et al., 2012; Chen et al., 1995; Rahman et al., 1999), and carotid atherosclerosis (Wang et al., 2002). Recently, Srivastava et al. (2009) and Lemaire et al. (2011) independently reported that a low concentration of arsenic exacerbates atherosclerotic lesion formation and increases the production of pro-inflammatory cytokines in apolipoprotein E-knockout (ApoE−/−) mice, accompanied by lipid accumulation in the arterial walls. Arsenic-induced oxidative stress has been reported to mediate abnormal expression of inflammatory genes and impair nitric oxide (NO) homeostasis (Simeonova and Luster, 2004). These events are thought to lead subsequently to endothelial dysfunction, which is recognized as one of the initial mechanisms for the development and progression of atherosclerosis (Kumagai and Pi, 2004). However, detailed information regarding the mechanism underlying arsenic-induced atherosclerosis is not available. Atherosclerosis is characterized by the accumulation of lipids and inflammatory cells in the arterial wall. Oxidized low-density lipoproteins (oxLDLs) play a number of pro-atherogenic roles in endothelial dysfunction, increased endothelial apoptosis, and increased vascular smooth muscle cells (VSMCs) proliferation (Goyal et al., 2012). Lectinlike oxidized low-density lipoprotein receptor-1 (LOX-1) was initially identified as a major receptor for oxLDL, and the oxLDL uptake through LOX-1 induces vascular ROS generation. ROS generation

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inhibits the expression of endothelial nitric oxide synthetase (eNOS), whose function is known to prevent a number of events associated with atherosclerosis (Chen et al., 2002; Sawamura et al., 1997). LOX-1 knockout mice fed by a high-cholesterol diet have been reported to show reduced binding of oxLDL to the aortic endothelium, which preserves endothelial function (Hu et al., 2008), whereas transgenic ApoE−/−mice overexpressing LOX-1 have been reported to show the increases in atheroma-like lesions (Inoue et al., 2005). Thus, it is probable that LOX-1 plays an important role in the development and progression of atherosclerosis. Given the association between arsenic exposure and the increased risk for atherosclerosis, it is of particular interest to examine whether arsenic affects the LOX-1 expression and uptake of oxLDL in endothelial cells (ECs). To our knowledge, this study is the first to demonstrate that sodium arsenite (SA) augments uptake of oxLDL through LOX-1 upregulation in a mouse aortic endothelial cell line, END-D. We have also discussed a possible molecular mechanism underlying SA-induced LOX1 upregulation.

Table 1 Primers applied for quantitative RT-PCR.

Materials and methods

Cellular uptake of Dil-OxLDL. Cellular uptake of oxLDL was measured using both confocal microscopy and fluorescence-activated cell sorting (FACS). For confocal microscopy, END-D cells were seeded on a poly-LLysine-coated culture cover glass (Matsunami Glass Ind., Ltd., Osaka, Japan). After the cells were incubated overnight, they were next incubated in a medium containing 1 μmol/L of SA for 6 h and then treated with 1 μg/mL of Dil-labeled oxLDL (Dil-oxLDL) for another 3 h in the presence of SA. For the neutralizing assay, cells were incubated with 10 μg/mL of either anti-LOX-1 antibody or normal goat IgG for 1 h prior to SA treatment. Then, the cells were washed 3 times with PBS and fixed with 1% ice-cold paraformaldehyde in PBS. For counterstaining, cell nuclei were stained with 4′,6-diamidine-2′phenylindole dihydrochloride (DAPI, l μg/mL) for 10 min at room temperature. The cells were then analyzed using LSM710 confocal microscopy (Carl Zeiss, Oberkochen, Germany). Cellular uptake of DiloxLDL was confirmed by competition with an excess amount (50 μg/ mL) of unlabeled human oxLDL (Biomedical Technologies). For FACS analysis, cells (1 × 105 cells/well) were seeded in a 6-well plate. The cells were treated with the indicated concentration (0.01, 0.1, 1, 2, and 5 μmol/L) of SA as described above for microscopy analysis. Following SA treatment, the cells were detached, washed, resuspended in PBS, and then examined using a FACSCantoII system (BD Bioscience, Tokyo, Japan), in which a total of 10,000 events (determined by forward and side scatter) were analyzed. The data provided represent the mean fluorescence intensity of triplicate determinations ± standard error (SE).

Reagents. Dulbecco's minimal essential medium (DMEM), penicillin-streptomycin solution, SP600125 (JNK inhibitor), and SB203580 (p38 inhibitor) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Caffeic acid phenethylester (CAPE, NFκB inhibitor), sodium arsenite (SA), 2,7-dichlorofluorescin diacetate (DCFH-DA), and N-acetylcysteine (NAC) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Goat anti-mouse LOX-1/ OLR1 antibody was obtained from R&D Systems (Minneapolis, MN, USA). Phycoerythrin (PE)-labeled anti-mouse CD31 antibody and PE-labeled isotype control antibody were obtained from BioLegend (San Diego, CA, USA). All other primary and secondary antibodies for western blot analysis were obtained from Cell Signaling Technology Japan, K.K. (Tokyo, Japan). 1,1′-Dioctadecyl-3,3,3′,3′tetramethylindocarbocyanine perchlorate (Dil)-labeled human oxidized LDL (Dil-oxLDL) was obtained from KALEN BioMedical, LLC (Montgomery Village, MD, USA). Human unlabeled oxLDL was obtained from Biomedical Technologies, Inc. (Stoughton MA, USA). The arsenic concentrations (0.01, 0.1, 1, 2, and 5 μmol/L) used in this study are within the range found in arsenic-contaminated well water in Bangladesh (Chen et al., 2011). Cell culture. END-D cells (mouse aortic endothelial cell line), which were kindly provided by Dr. Takashi Yokochi (Aichi Medical University School of Medicine, Aichi, Japan), were maintained in DMEM containing 10% heat-inactivated fetal bovine serum and penicillin at 37°C in 5% CO2 humidified air. END-D cells are positive for vascular cell adhesion molecule (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) and negative for E-selectin (Morikawa et al., 2000), and are suitable for the analysis of vascular development and vascular diseases (Morita et al., 1990). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. END-D cells (1×105 cells/well) were seeded in 6-well plates and incubated for 24 h. Cells were incubated in a medium containing 1 μmol/L of SA for the indicated time points (0, 1, 2, 4, 6, 8, 12, 24, and 36 h). qRT-PCR analysis using SYBR Green I was performed as described previously (Takahashi et al., 2013). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The sequences of the primers used in this study have been provided in Table 1. Western blot analysis. END-D cells (1×105 cells/well) were incubated with SA as described above. The cells were washed with ice-cold phosphate buffer saline (PBS) and lysed in loading buffer (125 mmol/L of Tris [pH 6.8], 4% sodium dodecyl sulfate [SDS], 10% βmercaptoethanol, 20% glycerol, and 0.02% bromophenol blue). Western

Gene symbol

Sequences (5′-)

Product size (bp)

LOX-1

5′- ACA AGA TGA AGC CTG CGA AT 5′- GCT GAG TAA GGT TCG CTT GG 5′- TCA CTG GAT GCA ATC TCC AA 5′- ACG TGC GCT TGT TCT TCT TT 5′- TGC TGG AGC TGT TAT TGG TG 5′- TGG GTT TTG CAC ATC AAA GA 5′- GAT GAC ATC AAG AAG GTG GTG A 5′- TGC TGT AGC CGT ATT CAT TGT C

213

SR-A CD36 GAPDH

Sense Antisense Sense Antisense Sense Antisense Sense Antisense

194 190 199

blot analysis was performed as described previously (Ota et al., 2009). Immune complexes were detected with ImmunoStar LD (Wako) by using the LAS-4000 image analyzer (GE Healthcare, Tokyo, Japan). Band intensity was measured using the ImageQuant TL software (GE Healthcare). The relative protein levels were calculated after normalization to an internal control, β-actin.

Detection of reactive oxygen species (ROS). ROS generation was detected using DCFH-DA — an uncharged cell-permeant fluorescent probe. Inside the cells, DCFH-DA is cleaved by nonspecific esterases to form DCFH, the nonfluorescent form, which in turn is oxidized to the fluorescent compound 2′,7′-dichlorofluorescein (DCF) in the presence of ROS. END-D cells were treated with or without the antioxidant NAC (10 mmol/L). Following 1 h of NAC treatment, cells were incubated with SA (1 μmol/L) in the presence of NAC. Cells were additionally incubated with DCFH-DA fluorescent probe in the presence of SA for 0.5 h. After incubation for the indicated times (0, 1, 2, 4, and 8 h), cells were detached using 0.05% trypsin, washed, and fixed in ice-cold PBS containing 1% paraformaldehyde for 15 min. Following fixation, cells were again re-suspended in PBS and then examined using FACSCantoII, which analyzed a total of 10,000 events (determined by forward and side scatter). Data are presented as mean fluorescence intensity of triplicate determinations ± SE. Statistics. We performed at least 3 independent experiments and 3 replicates per experiment. The results have been expressed as mean ± SE values. Statistical significance between groups was determined using

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one-way ANOVA and Dunnett's comparison. Statistical analyses were performed using the SPSS 15.0 program (SPSS Inc., Chicago, Illinois). Results Upregulation of LOX-1 expression by arsenic exposure in END-D cells LOX-1, an oxLDL receptor, is highly expressed in ECs, whereas the other oxLDL receptors, macrophage scavenger receptor 1 (SR-A) and CD36, are expressed in minimal amounts (Goyal et al., 2012). Therefore, we first investigated the basal mRNA expression of these receptors in END-D cells. RT-PCR analysis showed that LOX-1 mRNA expression was readily detected in END-D cells, whereas neither SR-A nor CD36 mRNA expression was detected (Supplementary Fig. S1A). Using FACS analysis, we also confirmed that CD31, a cell surface marker for ECs, is expressed in END-D cells (Supplementary Fig. S1B), indicating that END-D cells are suitable for endothelial biology studies. We next performed qRT-PCR analysis to examine whether SA affects LOX-1 mRNA expression. Notably, LOX-1 mRNA expression was found to significantly increase at 1, 2, and 4 h after SA treatment (p b 0.05, Fig. 1A), whereas no significant changes were detected in CD36 and SR-A mRNA expression (data not shown). We also confirmed that SA augments LOX-1 protein expression. The total LOX-1 protein level increased following 6 or 12 h of SA treatment (Fig. 1B). Augmentation of cellular Dil-oxLDL uptake by arsenic exposure Many studies have reported the association between upregulation of LOX-1 and atherosclerosis, in which the uptake of oxLDL mediated by LOX-1 promotes endothelial dysfunctions, regarded as an initial event before atheroma formation (Chen et al., 2002; Hu et al., 2008; Sawamura et al., 1997). The results of these studies prompted us to investigate whether SA affects the cellular uptake of oxLDL. As evidenced by the results of FACS analysis (Fig. 2A), the uptake of Dil-oxLDL was significantly augmented following 6 h of SA treatment in a dose-dependent manner, compared with that in untreated cells. Cellular uptake of Dil-oxLDL was confirmed by competition with an excess amount of unlabeled oxLDL (data not shown). We further examined the involvement of LOX-1 in the uptake of oxLDL. DiloxLDL uptake, which was augmented by SA, was completely blocked by treatment with an anti-LOX-1 antibody but not with a control antibody (Fig. 2B). To further obtain experimental evidence for the involvement of LOX-1, we visualized the uptake of Dil-oxLDL by using confocal fluorescence microscopy. The intracellular fluorescence signals of Dil-oxLDL clearly increased after SA treatment; the increases were again blocked by treatment with an anti-LOX-1 antibody (Fig. 3). Taken together, these results indicate that SA augments the cellular uptake of oxLDL through upregulating LOX-1 expression. A possible molecular mechanism underlying SA-induced LOX-1 expression Arsenic exposure activates nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and/or a series of mitogen-activated protein kinases (MAPKs), which eventually leads to the activation of the downstream transcriptional factor, activator protein 1 (AP-1) (Shi et al., 2004). Therefore, we examined the effect of SA on the activation of NF-κB and 3 MAPKs (p38, extracellular signal-regulated kinase [Erk1/2], and c-jun N-terminal kinase [JNK]). The level of the phosphorylated form of NF-κB/p65 was found to have significantly increased following SA treatment (Fig. 4A, p b 0.05), whereas no significant changes were detected in the phosphorylation levels of p38, ERK (p44/42), and JNK (Fig. 4B). To investigate the involvement of NFκB on SA-induced LOX-1 expression, we further examined the effects of CAPE (specific NF-κB inhibitor), SP600125 (JNK inhibitor), and SB203580 (p38 inhibitor) on SA-induced LOX-1 protein expression by using western blot analysis. Unlike SP600125 and SB203580, CAPE

Fig. 1. Upregulation of LOX-1 expression by arsenic in END-D cells. (A) OX-1 mRNA expression was examined using quantitative real-time PCR analysis. END-D cells were treated with 1 μmol/L of SA for the indicated times. The relative LOX-1 mRNA expression level is shown after normalization to GAPDH mRNA expression. Data are expressed relative to the mRNA levels found in the control (at 0 h after SA treatment), which was arbitrarily defined as 1. (B) LOX-1 protein expression was examined using western blot analysis. END-D cells were treated with SA (1 μmol/L) for the indicated times. A total of 1 μg of the protein was subjected to western blot analysis by using a goat anti-LOX-1 polyclonal antibody. After normalization to β-actin protein, data were expressed relative to the protein levels found in the control (at 0 h after SA treatment), which were arbitrarily defined as 1. The values shown represent the mean ± SD of 3 separate experiments. An asterisk (⁎⁎ or ⁎) indicates a significant difference with a p-value of less than 0.005 or 0.05, respectively (n = 3).

significantly inhibited the SA-induced LOX-1 protein expression (Fig. 4C, p b 0.05). Furthermore, the SA-augmented uptake of Dil-oxLDL was significantly prevented by CAPE (Fig. 4D, p b 0.05), strongly indicating that the activation of NF-κB may play a pivotal role in LOX-1 expression. Arsenic, a strong oxidant, has been reported to induce ROS generation (Shi et al., 2004). Arsenic-induced ROS generation is known to be critical in the activation of transcription factors, including NF-κB and AP-1 (Shi et al., 2004). Therefore, we examined the effect of SA on ROS generation using FACS analysis. FACS analysis revealed that SA increases the levels of ROS following 1, 2, 4, and 8h of SA exposure; these increases were clearly prevented by an antioxidant NAC (Fig. 5A). We further examined the effect of NAC on LOX-1 mRNA expression. qRT-PCR analysis showed that SA-induced LOX-1 mRNA expression was inhibited by NAC (Fig. 5B). Similarly, western blot analysis showed that SA-induced LOX-1 protein expression was prevented by NAC (Fig. 5C). In addition, SA-induced phosphorylation of NF-κB/p65 was found to be abrogated by NAC (Fig. 5D). Finally, we examined the effect of NAC on uptake of Dil-oxLDL. The SA-augmented uptake of Dil-oxLDL was significantly prevented by NAC treatment (Fig. 5E and Supplementary Fig. S2).

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Taken together, these results indicated that the ROS-mediated NF-κB activation plays an important role in SA-augmented LOX-1 expression and the subsequent uptake of oxLDL (Fig. 6). Discussion

Fig. 2. Arsenic augments the uptake of Dil-labeled oxLDL. (A) A flow cytometry analysis for Dil-oxLDL uptake in END-D cells after SA treatment. END-D cells were treated with the indicated concentration of SA for 6 h and then incubated with Dil-oxLDL (1 μg/mL) for 3 h. Data are expressed relative to the mean fluorescence intensity found in the untreated cells, which was arbitrarily defined as 100% (n = 3). (B) Effect of an anti-LOX-1 antibody on Dil-oxLDL uptake following SA treatment. END-D cells were incubated either with 10 μg/mL of an anti-LOX-1 IgG or a control goat IgG for 1 h prior to SA treatment (1 μmol/L) and then a similar experiment was performed as in A. An asterisk (⁎⁎ or ⁎) indicates a significant difference with a p-value of less than 0.005 or 0.05, respectively (n = 3).

There is a growing body of evidence supporting an enhanced risk of cardiovascular disease, including atherosclerosis, due to arsenic exposure. However, the molecular basis underlying arsenic-induced atherosclerosis remains obscure. To our knowledge, this study is the first to demonstrate that arsenic upregulates LOX-1 expression through ROS-mediated activation of the NF-κB signaling pathway, thereby enhancing the cellular uptake of oxLDL in END-D mouse aortic endothelial cells. LOX-1, identified as a receptor for oxLDL, has been shown to mediate vascular dysfunction, accumulate foam cells, and promote atheroma formation. Recent studies have demonstrated the association between elevated LOX-1 expression and pathological conditions, including diabetes mellitus (Tan et al., 2008), hypertension (Sankaralingam et al., 2009), myocardial ischemia (Nagase et al., 1997), and atherosclerosis (Morawietz, 2010). In this study, we found that SA treatment causes a significant increase in both LOX-1 mRNA and protein levels, whereas no significant increases were observed in CD36 or SR-A mRNA expression. To date, LOX-1 has been reported to be upregulated by proinflammatory molecules (Kume et al., 1998; Nagase et al., 1998), phorbol ester (Kume et al., 1998), angiotensin II (Morawietz et al., 1999), oxLDL (Li and Mehta, 2000a), and fluid shear stress (Murase et al., 1998). In addition, arsenic has been reported to activate AP-1 and NF-κB, known as stress response transcription factors, in various types of cells (Iwama et al., 2001; Lynn et al., 2000). Low arsenite concentrations (0.5–5 μmol/L) have been reported to promote DNA synthesis and to activate NF-κB in aortic endothelial cells (Barchowsky et al., 1996). Cavigelli et al. (1998) previously reported that a sublethal dose of arsenic resulted in the activation of 3 major MAPKs: ERK, JNK, and p38. We observed that SA enhances the phosphorylated forms of NF-κB. Our observation that SA-induced LOX-1 protein expression was inhibited by the NF-κB inhibitor

Fig. 3. Arsenic exposure augments intracellular Dil-oxLDL signals. END-D cells were treated with 1 μmol/L of SA as described in the legend for Fig. 2B. The cells were then fixed with 1% formaldehyde and visualized using fluorescence microscopy. An excess amount (50 μg/mL) of unlabeled oxLDL was used as a competitor to confirm specific binding of DiI-oxLDL. The Dil-oxLDL signals are shown as red. DAPI (blue) was used to counterstain the cell nuclei. None, cells incubated without Dil-oxLDL; magnification, ×400.

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Fig. 4. Involvement of NF-κB in SA-induced LOX-1 expression and Dil-oxLDL uptake. (A) Effect of SA on the phosphorylation levels of NF-κB (p65). END-D cells were treated with SA (1 μmol/L) for the indicated times. A total of 1 μg of the protein was subjected to western blot analysis to detect phospho-p65, p65, and β-actin by using their specific antibodies. βActin protein was used as an internal control. After normalization to the β-actin protein, the bar graph values are calculated as described in Fig. 1B. * p b 0.05 versus control (0 h). (B) Effect of SA on the phosphorylation levels of MAPKs (p38, ERK, and JNK). END-D cells were treated as described in A. A total of 1 μg of the protein was subjected to western blot analysis to detect phospho-p38, p38, phospho-ERK, ERK, and β-actin with their specific antibodies. A total of 5 μg of the protein was used to detect phospho-JNK and JNK. (C) Effect of CAPE or MAPK inhibitors on SA-induced LOX-1 protein expression. END-D cells were treated with CAPE (NF-κB inhibitor, 5 μmol/L), SP600125 (JNK inhibitor, 10 μmol/L), or SB203580 (p38 inhibitor, 10 μmol/L) for 1 h and then incubated with SA (1 μmol/L) for 12 h in the presence of CAPE, SP600125, or SB203580. Western blot analysis was performed as described in Fig. 1B. βActin protein was used as an internal control. After normalization to the β-actin protein, the bar graph values are calculated as described in Fig. 1B. An asterisk (⁎) indicates a significant difference with a p-value of less than 0.05 (n = 3). (D) Effect of CAPE on Dil-oxLDL uptake following SA treatment. END-D cells were treated with CAPE (5 μmol/L) for 1 h and incubated with SA (1 μmol/L) for 6 h in the presence of CAPE. The cells were then incubated with Dil-oxLDL (1 μg/mL) for 3 h in the presence of both NAC and SA. Data are expressed relative to the mean fluorescence intensity found in the untreated cells, which was arbitrarily defined as 100% (n = 3). An asterisk (⁎⁎) indicates a statistically significant difference (p b 0.005).

(CAPE) suggests that NF-κB signaling cascade may contribute to the upregulation of LOX-1 protein expression. Our results are supported by those of a previous study that reported that an NF-κB-responsive element is located in the promoter of the LOX-1 gene (Nagase et al., 1998). Therefore, it will be of particular interest to analyze SAregulated LOX-1 promoter activity in detail.

LOX-1-mediated uptake of oxLDL can induce endothelial apoptosis, the expression of adhesion and inflammatory molecules, and the downregulation of eNOS activity, eventually leading to the pathophysiological condition of atherosclerosis (Li and Mehta, 2000a,b; Morawietz, 2010). We found that SA significantly augments the cellular uptake of Dil-oxLDL and that an anti-LOX-1 antibody blocked the

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E. Hossain et al. / Toxicology and Applied Pharmacology 273 (2013) 651–658

Fig. 6. A possible molecular mechanism by which arsenic enhances uptake of oxLDL through upregulation of LOX-1 expression. Arsenic exposure induces ROS generation, which results in oxidative stress. ROS generation-mediated oxidative stress then causes an increase in phosphorylation of NF-κB. NF-κB, one of the redox sensitive transcriptional factors, may act as a transcriptional activator for arsenic exposure-induced LOX-1 gene expression. Arsenic-induced LOX-1 expression can promote the cellular uptake of oxLDL by modulating an aberrant LOX-1 signaling pathway, thus contributing to the development of atherosclerosis.

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the uptake of oxidized LDL through upregulating the expression of LOX-1 in ECs add another molecular mechanism underlying arsenicinduced atherosclerosis. Many studies have reported the involvement of LOX-1 in the molecular pathogenesis of atherosclerosis in both humans and mice (Morawietz, 2010; Nagase et al., 1997; Sankaralingam et al., 2009; Tan et al., 2008). Recently, Karim et al. (2013) have reported that arsenic exposure is associated with the elevation of plasma oxLDL levels with the concomitant reduction of HDL levels. In this study, we performed a series of experiments using a single cell line, END-D. Thus, it will be of particular interest to use human ECs to examine whether SA upregulates LOX-1 expression and/or enhances the cellular uptake of oxLDL. In conclusion, to our knowledge, this study is the first to demonstrate that SA upregulates LOX-1 expression through ROS-mediated activation of NF-κB, thereby enhancing the cellular uptake of oxLDL. Our novel findings raise the possibility that arsenic exposure can promote the pathogenic actions of oxLDL by evoking an aberrant LOX-1 signaling pathway, thus implicating the pathophysiology of cardiovascular diseases. Further studies, including in vivo experiments, are certainly warranted and may contribute to a better understanding of the molecular basis underlying the increasing risk of atherosclerosis associated with arsenic exposure. Conflict of interest We declare that we have no conflict of interest. Acknowledgments

uptake of Dil-oxLDL. These results strongly suggest that SA may augment the uptake of oxLDL through upregulation of LOX-1 expression. Arsenic exposure has been reported to generate ROS in human aortic vascular ECs (Barchowsky et al., 1999; Shi et al., 2004). We observed that SA-induced ROS generation following 1 h of SA exposure in ENDD cells; this ROS generation was clearly prevented by an antioxidant, NAC. ROS generation subsequently leads to the activation of stress response transcriptional factors, such as AP-1 and NF-κB. In this study, LOX-1 expression, activation of NF-κB, and the uptake of Dil-oxLDL were found to be suppressed by an antioxidant, NAC. Thus, our results strongly suggest that ROS-mediated activation of the NF-κB signaling pathway may contribute to the upregulation of LOX-1 expression, thereby augmenting the uptake of oxLDL. There are a number of studies linking arsenic with atherogenesis in ECs, VSMCs, or macrophages. Arsenic activates NF-κB, which results in upregulation of tumor necrosis factor α and interleukin 8 productions in ECs (Tsai et al., 2002). Arsenic increases ROS generation, expression of monocyte chemoattractant protein 1 and interleukin 6 in VSMCs (Lee et al., 2005). Padovani et al. (2010) have reported that arsenic decreases liver X receptor (LXR) ligands-induced gene expression of ATPbinding cassette, sub-family A, member 1 (ABCA1) and inhibits reverse cholesterol efflux in macrophages. Our findings that arsenic augments

This work was partly supported by a grant from the Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) [S1101027 to S. K., H. K., and Y. H.]; and the AIKEIKAI Foundation [to A. O.]. We would like to thank Dr. Takashi Yokochi at Aichi Medical University (Aichi, Japan) for kindly providing the END-D mouse aortic vascular endothelial cell line. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.taap.2013.10.012. References Abhyankar, L.N., Jones, M.R., Guallar, E., Navas-Acien, A., 2012. Arsenic exposure and hypertension: a systematic review. Environ. Health Perspect. 120, 494–500. Argos, M., Kalra, T., Rathouz, P.J., Chen, Y., Pierce, B., Parvez, F., Islam, T., Ahmed, A., Rakibuz-Zaman, M., Hasan, R., Sarwar, G., Slavkovich, V., van Geen, A., Graziano, J., Ahsan, H., 2010. Arsenic exposure from drinking water, and all-cause and chronicdisease mortalities in Bangladesh (HEALS): a prospective cohort study. Lancet 376, 252–258. Barchowsky, A., Dudek, E.J., Treadwell, M.D., Wetterhahn, K.E., 1996. Arsenic induces oxidant stress and NF-kappa B activation in cultured aortic endothelial cells. Free Radic. Biol. Med. 21, 783–790. Barchowsky, A., Klei, L.R., Dudek, E.J., Swartz, H.M., James, P.E., 1999. Stimulation of reactive oxygen, but not reactive nitrogen species, in vascular endothelial cells exposed to low levels of arsenite. Free Radic. Biol. Med. 27, 1405–1412.

Fig. 5. Involvement of ROS generation in SA-induced LOX-1 expression and Dil-oxLDL uptake. (A) Effect of arsenic exposure on generation of ROS. END-D cells were treated with or without the antioxidant NAC (10 mmol/L) for 1 h and then incubated with SA (1 μmol/L) for 1, 2, 4, 8 h in the presence or absence of NAC. Data are expressed relatively to the mean fluorescence intensity of untreated cells, arbitrarily defined as 100%. An asterisk (⁎⁎) or (⁎) indicates statistically significant differences from control of p b 0.005 or 0.05, respectively (n = 3). Black bar, cells treated with SA; gray bar, cells treated with SA in the presence of NAC. (B) Effect of NAC on SA-induced LOX-1 mRNA expression. END-D cells were treated with the antioxidant NAC (10 mmol/L) for 1 h and then incubated with SA (1 μmol/L) for 2 h in the presence of NAC. Quantitative real-time PCR analysis was performed as described in the legend for Fig. 1A. An asterisk (⁎⁎) indicates a statistically significant difference of p b 0.005 (n = 3), compared to only SA-treated cells. (C) Effect of NAC on SA-induced LOX-1 protein expression. END-D cells were incubated with SA (1 μmol/L) for 12 h, as described in A. Western blot analysis was performed as described in Fig. 1B. After normalization to the β-actin protein, the bar graph values are calculated as described in Fig. 1B. * p b 0.05 versus control (None). (D) Effect of NAC on SA-induced phosphorylation of NF-κB (p65). END-D cells were treated as described in A. A total of 1 μg of the protein was subjected to western blot analysis to detect phospho-p65, p65, and β-actin with their specific antibodies. β-Actin protein was used as an internal control. After normalization to the β-actin protein, the bar graph values are calculated as described in Fig. 1B. * p b 0.05 versus control (None). (E) Effect of NAC on Dil-oxLDL uptake following SA treatment. END-D cells were treated with NAC (10 mmol/L) for 1 h and incubated with SA (1 μmol/L) for 4 h in the presence of NAC. The cells were then incubated with Dil-oxLDL (1 μg/mL) for 3 h in the presence of both NAC and SA. Data are expressed relative to the mean fluorescence intensity found in the untreated cells, which was arbitrarily defined as 100% (n = 3). An asterisk (**) indicates a statistically significant difference (p b 0.005).

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