Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Kaohsiung 807, Taiwan, Republic of China
http://www.100md.com
《毒物学科学杂志》
Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei, Taiwan, ROC
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC
ABSTRACT
Arsenic exposure is associated with an increased risk of vascular disorders, and results in increased oxidative stress in endothelial cells and vascular smooth muscle cells (VSMCs). Since oxidative stress is involved in regulating the expression of genes related to atherogenesis, we investigated its involvement in the enhanced expression of three atherosclerosis-related genes coding for heme oxygenase-1 (HO-1), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in VSMCs treated with inorganic sodium arsenite (iAs). In human VSMCs (hVSMCs) and rat VSMCs (rVSMCs), HO-1, MCP-1, and IL-6 mRNA levels were significantly increased by iAs treatment. An increase in HO-1 protein levels in hVSMCs was confirmed by Western blotting technique, while increased MCP-1 and IL-6 secretion by hVSMCs was demonstrated by enzyme-linked immunosorbent assay. Although modulators of oxidative stress inhibited this iAs-induced increase in the expression of these three genes, different modulators had differential effects. In iAs-treated rVSMCs, catalase, dimethylsulfoxide, and L--nitro-L-arginine significantly inhibited the increase in expression of all three genes, allopurinol inhibited the increase in MCP-1 and IL-6 expression, but had no effect on HO-1 expression, while superoxide dismutase had no significant effect on HO-1 expression, but had an inhibitory effect on IL-6 expression and a stimulatory effect on MCP-1 expression. Therefore, iAs may enhance the expression of HO-1, MCP-1, and IL-6 in VSMCs via different reactive oxygen molecules. Furthermore, using tin protoporphyrin IX (SnPP) and anti-MCP-1 antibody to abolish iAs-induced HO-1 and MCP-1 activity, respectively, shows that HO-1 has protective effect against iAs-induced injury in VSMCs and MCP-1 is chemoattractive to human monocytes, THP-1.
Key Words: sodium arsenite; heme oxygenase-1; monocyte chemoattractant protein-1; interleukin-6; oxidative stress; vascular smooth muscle cells.
INTRODUCTION
Drinking arsenic-contaminated groundwater is the main route of human exposure (Sheehy and Jones, 1993). Epidemiological evidence has shown that long-term chronic arsenic exposure is associated with increased risks of skin, bladder, lung, and liver cancers (Chen et al., 1992; IARC, 1987). Arsenic exposure also results in an increased prevalence of diabetes (Rahman et al., 1998; Tseng et al., 2000) and cardiovascular disorders, such as atherosclerosis (Wang et al., 2002), ischemic heart disease (Hsueh et al., 1998), cerebral infraction (Chiou et al., 1997), and hypertension (Chen et al., 1995; Rahman et al., 1999) in humans. However, the mechanisms by which arsenic induces the pathological changes in blood vessels leading to vascular disorders remain to be delineated.
When the blood vessel is injured, a variety of chemokines and growth factors are secreted by various cell types, including endothelial cells (ECs), platelets, immune cells, and vascular smooth muscle cells (VSMCs), and stimulate VSMCs to migrate into the intima layer and expand (Ross, 1999). The migration of VSMCs into the intima and their subsequent proliferation (intimal hyperplasia) is an early pathological process in atherosclerosis. Because of the involvement of a variety of cytokines, chemokines, and immune cells in the development of atherosclerotic lesions, chronic inflammation is suggested to be involved in the progression of atherosclerosis (Libby, 2002).
The generation of reactive oxidants is a general manifestation of an inflammatory reaction. Various atherosclerosis-related factors, such as platelet-derived growth factor (PDGF), angiotensin II (Ang II), thrombin, and oxidized low-density lipoproteins, increase intracellular reactive oxidants or reactive oxygen species (ROS) production in VSMCs (Hsieh et al., 2001; Seshiah et al., 2002; Sundaresan et al., 1995) and stimulate VSMC proliferation (Gosgnach et al., 2000; Griendling et al., 1994; Sundaresan et al., 1995). Mechanisms involved in enhanced VSMC proliferation by generation of ROS are mediated through activation of MAP kinases and Akt and their downstream transcription factors, such as NF-B or AP-1, which are well-documented to regulate the expression of atherogenic related genes (Griendling et al., 2000; Irani, 2000). ROS are therefore implicated in the pathological processes of atherosclerosis (Ross, 1999).
The generation of reactive oxidants during arsenic metabolism has been shown to play an important role in arsenic-induced injury (Gurr et al., 2003; Liu et al., 2001). The involvement of reactive oxidants in iAs-induced DNA and chromosomal damage (Ho et al., 2000; Lynn et al., 2000), DNA repair inhibition (Mei et al., 2002), and apoptosis (Nakagawa et al., 2002) is well documented. Recent epidemiological studies have established a relationship between chronic arsenic exposure and increased levels of reactive oxidants in the blood of human populations drinking arsenic-contaminated water (Pi et al., 2002; Wu et al., 2001). ROS are therefore considered as a possible etiologic factor of atherogenicity (Wu et al., 2001, 2003).
In our previous study (Wu et al., 2003), the expression of several cytokine-related genes is increased in human subjects with increased arsenic exposure, including heme oxygenase-1 (HO-1), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6). Particularly, our study demonstrated an association for MCP-1 plasma protein level with blood arsenic in 65 study subjects of varying arsenic exposure level (Wu et al., 2003). HO-1, a stress response gene with a cytoprotective and inflammation regulation function, MCP-1, a monocyte-recruiting chemokine, and IL-6, a pleiopotent inflammatory cytokine, are regulated by ROS and atherogenic factors and involved in modulating the biological functions of VSMCs (Browatzki et al., 2000; Cushing et al., 1990; Duckers et al., 2001; Durante et al., 1999; Morse and Choi, 2002). Furthermore, the expression of these three genes has been detected in atherosclerotic lesion (Nelken et al., 1991; Seino et al., 1994; Wang et al., 1998). These three genes are possibly involved in the development of atherosclerosis. Since VSMCs are a major cell type in vascular vessels, we therefore examined whether treatment with inorganic trivalent sodium arsenite (iAs, NaAsO2) resulted in the generation of ROS and subsequent enhancement of the expression of these three genes in VSMCs.
MATERIALS AND METHODS
Cell culture.
Rat aorta VSMCs (rVSMCs, ATCC CRL-1476) and human aorta VSMCs (hVSMCs, ATCC CRL-1999) were obtained from the American Type Culture Collection (Manassas, VA) rVSMCs were routinely cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), while hVSMCs were maintained in Kaighn's modified F-12 Ham medium (HAM's F-12K) supplemented with 10% FBS, 10 μg/ml of insulin, 10 μg/ml of transferrin, 10 ng/ml of sodium selenite, and 20 μg/ml of endothelial cell growth supplement. To obtain resting cells, rVSMCs were switched to DMEM containing 1% FBS for 24 h, while hVSMCs were switched to HAM's F-12K containing 0.1% FBS for 48 h. The human fibroblast strain, HFW, derived from human newborn foreskin and kindly provided by Professor W. N. Wen (National Taiwan University), was grown in DMEM supplemented with 10% FBS as described previously (Yih and Lee, 2000). The human monocyte cell line, THP-1 (ATCC, TIB-202), was cultured in RPMI 1640 medium containing 10% FBS.
Measurement of mRNA levels by quantitative RT-PCR.
Total cellular RNA was extracted using the TRI-reagent according to the manufacturer's instructions (Molecular Research Center, Inc., Cincinnati, OH). The quantitative reverse transcriptase-polymerase chain reaction (quantitative RT-PCR) was used to analyze individual mRNA levels as described by Morrison et al. (1988). Each analysis was performed in triplicate. The relative mRNA levels for the genes of interest were calculated from the difference between the Ct value (number of cycles required to reach the detection threshold for the double-strand DNA product) for the gene of interest and that for an internal control gene, glyceraldehyde-3-phosphate dehydrogenase as described previously (Wu et al., 2003). The Ct values were determined by using Sequence Detector System v1.7 software provided by the Applied Biosystems (Foster City, CA).
Measurement of protein levels.
Intracellular HO-1 protein levels were analyzed by Western blotting as described previously (Yih et al., 1997). In brief, rVSMCs or hVSMCs treated with iAs for different time periods were lysed, and the proteins were electrophoretically separated and transferred to a positively charged PVDF membrane. The PVDF membrane was immunoblotted with anti-HO-1 antibody or anti-actin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). HO-1 protein and actin were visualized by the enhanced chemoluminescence method as described previously (Yih et al., 1997). Levels of secreted MCP-1 and IL-6 in the culture medium from iAs-treated VSMCs were determined using enzyme-linked immunosorbent assay (ELISA) kits (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions.
Determination of ROS production.
2',7'-dichlorofluorescein diacetate (DCF-DA) was used to measure ROS production in VSMCs (Huang et al., 1993). In brief, proliferating rVSMCs were treated with iAs for various time periods, DCF-DA (at a final concentration of 20 μM) being added to the culture medium 30 min before the end of iAs treatment. The rVSMCs were then trypsinized and resuspended in Hank's balanced salt solution. The fluorescence of the DCF formed by oxidation of DCF-DA by cellular oxidants was measured using a flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.
Survival assay.
To determine the protective effects of HO-1 on iAs-induced cell injury, the proliferation rates of VSMCs were measured by counting the cell number after treatment with iAs or iAs plus SnPP. In brief, 3 x 104 rVSMCs were seeded onto a 60 mm dish and cultivated for 24 h. The cells were then treated with various concentrations of iAs (0, 1, 2, 5 μM) with vehicle (0.25% DMSO) or 50 μM SnPP (dissolved in DMSO) for 24 h. Afterward, the media were replaced with fresh medium without drugs. After further cultivating for 72 h, the cells were trypsinized and counted with a cell counter (hematology analyzer, EXCELL 300, Metertech).
Chemotaxis assay.
MCP-1-induced monocyte chemotaxis was measured using 12-well Transwell polystyrene trays (Corning Costar Corp., Cambridge, MA) and a 5 μm pore polycarbonate membrane (McQuibban et al., 2002). Conditioned medium from resting hVSMCs incubated with or without 5 μM iAs was harvested and a 600 μl aliquot loaded into the lower chamber, while 100 μl of the human monocytic leukemia cell line, THP-1, at a density of 5 x 106 cells/ml in HAM's F-12K medium containing 0.1% FBS were loaded into the upper chamber. After incubation in a 37°C incubator for 2 h, the THP-1 cells on the lower chamber-side of the membrane were fixed with methanol, stained with DAPI. In each treatment, the average cell number of three randomly selected fields (using 10x eyepieces and 40x objective) was scored under a fluorescence microscopy. In control group, 100 to 140 migrating cells were obtained in each field. The relative migration index was calculated by dividing the number of THP-1 cells that migrated in response to conditioned medium from iAs-treated cultures by the number that migrated in response to conditioned medium from untreated controls supplemented with 5 μM iAs prior to assay. Pilot studies showed that addition of 5 μM iAs to conditioned medium from untreated cultures did not affect the migration index of THP-1 cells. To confirm the observed chemotaxis was due to secreted MCP-1, anti-human MCP-1 antibody (R&D Systems, Inc., Minneapolis, MN) at a final concentration of 0.83 μg/ml was added to the conditioned medium in the lower chamber 10 min before loading the cells into the upper chamber.
Treatment with ROS modulators.
Catalase (CAT) was added to the culture medium for 4 h. To avoid the interference with iAs, the medium was replaced with fresh medium before treatment with iAs (5 μM for 4 h). Superoxide dismutase (SOD) was added to the culture medium 4 h before iAs treatment, while allopurinol (AP), dimethylsulfoxide (DMSO), and L--nitro-L-arginine (L-NNA) were added 30 min before iAs treatment; these reagents were then left in the medium during the subsequent iAs treatment (5 μM for 4 h).
Statistical analysis.
The results are expressed as the mean ± SD. Statistical analyses were performed using Student's two-tailed unpaired t-test. p values < 0.05 were considered statistically significant.
RESULTS
Arsenite Induces Increased Expression of HO-1, MCP-1, and IL-6 in Vascular Smooth Muscle Cells
In a pilot experiment, treatment of resting hVSMC with iAs at concentrations up to 5 μM for 8 h did not affect [3H]thymidine incorporation when they were returned to complete medium containing 10% FBS and growth factor supplements. A concentration of 5 μM iAs was therefore used to investigate stimulatory effects on gene expression in VSMCs. Quantitative RT-PCR showed that treatment of resting hVSMC with 5 μM iAs for 0 to 8 h resulted in an increase in HO-1, MCP-1, and IL-6 mRNA levels, which peaked at 4 h of treatment (Fig. 1A). The appearance of large amounts of HO-1, an intracellular protein, in iAs-treated hVSMCs was confirmed by Western blotting (Fig. 1B). Levels of HO-1 protein were negligible in untreated hVSMCs, became visible after 5 h of iAs treatment, and were markedly increased at 9 h. Low, but significant, levels of MCP-1 (1.7-fold) and IL-6 (1.6-fold) were detected in the culture medium from iAs-treated hVSMCs by ELISA (Figs. 1C and 1D). Allowing for the lag in protein synthesis, the levels of HO-1, MCP-1, and IL-6 protein correlated well with the mRNA levels determined by quantitative RT-PCR. Similar induction profiles of HO-1, MCP-1, and IL-6 were seen in iAs-treated resting rVSMCs (Fig. 2A). Since proliferation of VSMCs is crucial for the development of atherosclerosis, we also examined the effect of iAs treatment on the expression of these three genes in proliferating rVSMCs, with similar results (Fig. 2B). By measuring the cell proliferating activity, 5 μM iAs, under our experimental conditions, was not cytotoxic for either resting or proliferation rVSMCs. Furthermore, HO-1, MCP-1, and IL-6 mRNA levels in proliferating rVSMCs treated with iAs for 4 h were increased in a concentration-dependent manner (Fig. 2C). Treatment of the HFW, cultured human fibroblast strain, with 5 μM iAs for different periods of time (0–8 h) resulted in a marked increase in HO-1 mRNA levels, but a marked decrease in MCP-1 and IL-6 mRNA levels (Fig. 2D). These results are consistent to several other reports (Foresti et al., 2001; Morse and Choi, 2002) showing that increased HO-1 expression is a general response of cells to iAs insult, whereas regulation of MCP-1 and IL-6 expression by iAs differs in different cell types.
Induction of ROS by Arsenite Treatment in VSMCs
To confirm that HO-1, MCP-1, and IL-6 genes were oxidative stress response genes, proliferating rVSMCs were exposed to 200 μM H2O2 for 0 to 8 h. As shown in Figure 3A, significant expression of all three genes was induced, being maximal after 2 h of H2O2 treatment. The generation of intracellular ROS in VSMCs by iAs was confirmed using a redox-sensitive fluorescence dye, DCF-DA, that is known to detect H2O2 and a variety of organic hydroperoxides, nitric oxide and peroxynitrite. As shown in Figure 3B, the fluorescence intensity of the oxidized product, DCF, was increased 1.6-fold in proliferating rVSMCs treated with 5 μM iAs for 4 h.
Roles of Arsenite-Increased Oxidative Stress in Arsenite-Induced HO-1, MCP-1, and IL-6 Expression
The effects of several ROS modulators were examined to explore the roles of oxidative stress in iAs-induced HO-1, MCP-1, and IL-6 expression in VSMCs. CAT catalyzes the degradation of H2O2 to H2O and O2. In proliferating rVSMCs, CAT pretreatment significantly inhibited the iAs-induced increase in HO-1, MCP-1, and IL-6 mRNA expression (Fig. 4A), and a similar effect was seen in iAs-treated resting hVSMCs (data not shown). DMSO, a hydroxyl radical scavenger, and L-NNA, an NO synthase inhibitor, were used to investigate the roles of hydroxyl radicals and NO in iAs-induced HO-1, MCP-1, and IL-6 mRNA expression. As shown in Figures 4B and 4C, DMSO (0.1 % v/v) or L-NNA (100 μM) had a similar effect to CAT on iAs-induced HO-1, MCP-1, and IL-6 mRNA expression in proliferating rVSMCs. These results indicate that H2O2, hydroxyl radicals, and NO are probably involved in the induction of HO-1, MCP-1, and IL-6 expression in iAs-treated VSMCs. In contrast, when proliferating rVSMCs were treated with AP, an inhibitor of the superoxide anion-producing enzyme, xanthine oxidase, or SOD, a scavenger of superoxide anions, before and during iAs treatment, only a moderate, not statistically significant inhibitory effect on HO-1 mRNA levels was seen, whereas the iAs-induced increase in IL-6 mRNA levels was completely blocked (Figs. 5A and 5B). In addition, AP completely inhibited, while SOD enhanced, the iAs-induced increase in MCP-1 mRNA levels in rVSMCs. These results indicated that superoxide anions play a role in the effect of iAs on IL-6 mRNA levels, but are less important in the effect on HO-1 mRNA levels and play a complicated role in MCP-1 induction.
Protective Role of HO-1 on iAs Cytotoxicty in VSMCs
In our previous report, iAs-induced HO-1 has cytoprotective effect on iAs-treated HFW (Ho et al., 2000). Here, we used tin protoporphyrin IX (SnPP), a well-documented HO-1 inhibitor, to examine the protective roles of HO-1 against iAs-induced cytotoxicity. By treatment of rVSMCs with various concentrations of iAs for 24 h, the proliferation of rVSMC was reduced when iAs was at the concentration of 5 μM (Fig. 6A). However, HO-1 protein became remarkable even when iAs at the concentration of 1 μM (Fig. 6B). Co-treatment of rVSMC with 50 μM SnPP and various concentrations of iAs significantly potentiated iAs-resulted inhibition of rVSMC proliferation (Fig. 6A). These results, consistent to previous observation in HFW (Ho et al., 2000), revealed that HO-1 plays a cytoprotective role against iAs-induced cytotoxic effects.
Response of Cultured Monocytes to MCP-1 Secreted by Arsenite-Treated Vascular Smooth Muscle Cells
MCP-1 is a well-documented chemoattractant. The human monocyte chemotactic activity of conditioned medium harvested from cultures of iAs-treated resting hVSMCs was determined using the Transwell migration assay. As shown in Figure 6, conditioned medium from iAs-treated (5 μM, 9 h) resting hVSMCs showed significantly higher chemotactic activity than conditioned medium from untreated cells supplemented with 5 μM iAs prior to assay. Furthermore, an anti-human MCP-1 monoclonal antibody, which neutralizes the bioactivity of secreted MCP-1, reduced the chemotactic activity of conditioned medium from either untreated or iAs-treated resting hVSMCs to a similar level (Fig. 7). These results show that MCP-1 is a functional chemotaxis factor in conditioned medium with or without iAs treatment and that its levels are increased on iAs treatment. Since 0.1% FBS was supplemented in conditioned medium with or without iAs treatment, residual MCP-1 activity is likely presented in conditioned medium without iAs treatment. We also could not rule out the possibility that a small amount of MCP-1 was produced by VSMCs without iAs treatment.
DISCUSSION
Using quantitative RT-PCR and Western blotting, our present study demonstrated that iAs, as well as H2O2, enhanced the expression of the HO-1, MCP-1, and IL-6 genes in cultured human and rat VSMCs. This effect was suppressed by most ROS/NO modulators used in this study, suggesting that ROS/NO were involved. However, AP and SOD had differential effects on the expression of these three genes in iAs-treated VSMCs. Since neither AP nor SOD significantly suppressed the iAs-induced increase in HO-1 expression, superoxide anion itself was not an active inducer of HO-1 expression. However, superoxide anions can react with NO to form the peroxynitrite anion, which is a potent inducer of HO-1 (Foresti et al., 1997). In the present study, the iAs-induced increase in MCP-1 expression was suppressed by AP, but enhanced by SOD. Since increased SOD activity could accelerate the dismutation of superoxide anions to H2O2, we inferred that a rapid increase in H2O2 was crucial for the increase in MCP-1 expression caused by iAs treatment. Similarly, iAs-induced Nrf2 nuclear accumulation is enhanced by copper (II) 3,5-diisopropyl salicylate hydrate, a cell-permeable SOD (Pi et al., 2003). Since all the ROS/NO modulators tested inhibited the iAs-induced increase in IL-6 expression, induction of the IL-6 gene can probably be caused by different kinds of ROS/NO molecules. Thus, different pro-oxidants are probably involved in the enhancement of HO-1, MCP-1, and IL-6 expression seen in iAs-treated VSMCs.
Several reports have shown that iAs treatment results in increased ROS and NO production in a variety of cells in culture (Barchowsky et al., 1999; Lee and Ho, 1995; Lynn et al., 2000), and in the blood of human populations drinking arsenic-contaminated water (Pi et al., 2002; Wu et al., 2001). Precisely how iAs results in an increase in ROS/NO is unclear. However, iAs is reported to increase superoxide anion production by stimulating NADH oxidase in hVSMCs and porcine aortic endothelial cells (Lynn et al., 2000; Smith et al., 2001). Alternatively, iAs may interact with sulfhydryl groups of biomolecules and GSH, resulting in a decreased capacity for scavenging ROS (Nakagawa et al., 2002; Wang et al., 1996). Downregulation of antioxidant enzymes, such as phospholipid hydroperoxide glutathione peroxidase, is another mechanism for increasing intracellular peroxide levels (Huang et al., 2002). The cellular signaling cascade activated by iAs-induced oxidative stress has been reviewed. Emerging evidence has shown that iAs-induced oxidative stress could activate MAP kinases, such as JNK and p38 kinase, and subsequently activate their downstream transcription factors, AP-1 and NF-B (Bernstam and Nriagu, 2000; Qian et al., 2003). In this study, we demonstrated that iAs-treatment resulted in increasing intracellular ROS production in VSMCs. Thus iAs is likely through similar signaling pathway to stimulate the expression of HO-1, MCP-1, and IL-6.
MCP-1, a C-C family chemokine, is expressed in endothelial cells, foam cells, and VSMCs of atherosclerotic lesions (Nelken et al., 1991), and has strong chemotactic activity for immune cells, such as monocytes (Rollins, 1997). The recruitment of circulating monocytes is a crucial step in the initiation of atherosclerosis (Ross, 1993). Numerous reports have shown that MCP-1 is induced in VSMCs by a variety of stimuli, including Ang II (Funakoshi et al., 2001), thrombin (Wenzel et al., 1995), uric acid (Kanellis et al., 2003), Fas ligand plus cycloheximide (Schaub et al., 2000), and activated platelets (Massberg et al., 2003). Expression of MCP-1 may result in the accumulation of inflammatory cells, such as macrophages, in atherosclerotic lesions. Our results showed that the enhanced chemotactic activity for human monocytes seen in conditioned medium from iAs-treated VSMCs could be suppressed by antibody against MCP-1. Since VSMCs are the major cell type in the media of arteries, MCP-1 released from VSMCs by iAs treatment may be involved in the recruitment of circulating monocytes and thus in initiating atherogenesis. Our current results also support our previous finding that plasma levels of MCP-1 show a good correlation with blood arsenic levels in human populations exposed to arsenic in drinking water (Wu et al., 2003).
IL-6, a proinflammatory cytokine and a major inducer of C-reactive protein (CRP) (Heinrich et al., 1990), is also present in atherosclerotic lesions (Seino et al., 1994). Both IL-6 and CRP are considered as strong risk markers of cardiovascular diseases (Blake and Ridker, 2001). Several vasoactive substances, such as PDGF, Ang II, thrombin, and endothelin, are reported to induce IL-6 expression in VSMCs (Browatzki et al., 2000; Funakoshi et al., 1999; Roth et al., 1995; Tokunou et al., 2001). It has been reported that IL-6 at levels of ng/ml range could stimulate VSMCs proliferation and migration (Ikeda et al., 1991; Liu et al., 2004), which are important manifestation of atherogenesis. Since the concentrations of secreted IL-6 in medium of iAs-treated cultures were ranged at pg/ml levels, anti-IL-6 antibody was unable to inhibit the proliferation or migration ability in iAs-treated rVSMCs (data not shown). This result may be explained not only by the low level of IL-6 secretion but also the presence of other mitogens or migration factors in culture medium of rVSMC. IL-6 also stimulates the expression of adhesion molecules and increases cell-cell permeability in ECs (Maruo et al., 1992; Watson et al., 1996). Increased levels of adhesion molecules and increased ECs permeability could promote the attachment and penetration of circulating immune cells, such as monocytes. Although the exact physiological role of iAs-induced IL-6 in VSMCs still requires further investigation, the enhanced expression of IL-6 seen in iAs-treated VSMCs may act as a risk factor of iAs-induced cardiovascular disorders.
In addition to cytokines and chemokines, the heme degradation enzyme, HO-1, is also expressed in atherosclerotic lesions (Wang et al., 1998). The products of heme degradation are iron, carbon monoxide, and biliverdin. Carbon monoxide acts as a vasodilator (Duckers et al., 2001), while bilirubin, produced by reduction of biliverdin, is a potent antioxidant (Stocker et al., 1987). Because of this, HO-1 has been proposed to have roles in cytoprotection and in the regulation of inflammation (Morse and Choi, 2002). In this study, SnPP, a competitive inhibitor of HO-1, was shown to significantly reduce cell survival of proliferating rVSMCs, indicating that iAs-induced HO-1 plays a cytoprotective role in iAs-damaged VSMCs. In fact, the growth promoting properties of HO-1 have been demonstrated in many cell types and in transgenic animal models and humans (Durante, 2003). Recently, HO-1 was further reported to play an important role in angiogenesis (Bussolati et al., 2004). Therefore, the roles of HO-1 on atherogenesis require further clarification.
Since arsenic is a well-documented potent inducer of HO-1 in many cell types (Lee and Ho, 1995; Taketani et al., 1989), HO-1 is also considered as a good marker of exposure to arsenic and oxidative stress. In the present study, iAs treatment of HFW resulted in increased HO-1 gene expression, but suppression of MCP-1 and IL-6 expression. This finding is consistent with the results of our previous study using cDNA microarrays to study the effect of iAs treatment on the gene expression profile of HFW cells (Yih et al., 2002). Inhibition of IL-6 expression by iAs is also observed in human intestinal epithelial cells (Hershko et al., 2002). These results indicated that although HO-1, MCP-1, and IL-6 are oxidative response genes, their response to iAs-enhanced oxidative stress is cell type dependent. The cell type specificity of the induction of MCP-1 and IL-6 requires further study.
Our present study provides evidence that exposure to iAs increases the generation of ROS/NO and thus enhances the expression of the HO-1, MCP-1, and IL-6 genes in VSMCs. Expression of MCP-1 and IL-6 may promote the initiation of atherogenesis by increasing the attachment, penetration, and migration of monocytes. These three gene products are also involved in regulation of VSMC proliferation. The findings presented here suggest a possible mechanism for atherogenesis in arsenic-exposed humans. A better understanding of the atherogenic mechanisms of arsenic should provide the basis for improved interventional approaches for both treatment and prevention.
ACKNOWLEDGMENTS
This work was supported by the Academia Sinica and grants from the National Science Council, Republic of China (NSC91-2320-B010-055 and NSC92-2320-B-010-017).
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Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC
ABSTRACT
Arsenic exposure is associated with an increased risk of vascular disorders, and results in increased oxidative stress in endothelial cells and vascular smooth muscle cells (VSMCs). Since oxidative stress is involved in regulating the expression of genes related to atherogenesis, we investigated its involvement in the enhanced expression of three atherosclerosis-related genes coding for heme oxygenase-1 (HO-1), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in VSMCs treated with inorganic sodium arsenite (iAs). In human VSMCs (hVSMCs) and rat VSMCs (rVSMCs), HO-1, MCP-1, and IL-6 mRNA levels were significantly increased by iAs treatment. An increase in HO-1 protein levels in hVSMCs was confirmed by Western blotting technique, while increased MCP-1 and IL-6 secretion by hVSMCs was demonstrated by enzyme-linked immunosorbent assay. Although modulators of oxidative stress inhibited this iAs-induced increase in the expression of these three genes, different modulators had differential effects. In iAs-treated rVSMCs, catalase, dimethylsulfoxide, and L--nitro-L-arginine significantly inhibited the increase in expression of all three genes, allopurinol inhibited the increase in MCP-1 and IL-6 expression, but had no effect on HO-1 expression, while superoxide dismutase had no significant effect on HO-1 expression, but had an inhibitory effect on IL-6 expression and a stimulatory effect on MCP-1 expression. Therefore, iAs may enhance the expression of HO-1, MCP-1, and IL-6 in VSMCs via different reactive oxygen molecules. Furthermore, using tin protoporphyrin IX (SnPP) and anti-MCP-1 antibody to abolish iAs-induced HO-1 and MCP-1 activity, respectively, shows that HO-1 has protective effect against iAs-induced injury in VSMCs and MCP-1 is chemoattractive to human monocytes, THP-1.
Key Words: sodium arsenite; heme oxygenase-1; monocyte chemoattractant protein-1; interleukin-6; oxidative stress; vascular smooth muscle cells.
INTRODUCTION
Drinking arsenic-contaminated groundwater is the main route of human exposure (Sheehy and Jones, 1993). Epidemiological evidence has shown that long-term chronic arsenic exposure is associated with increased risks of skin, bladder, lung, and liver cancers (Chen et al., 1992; IARC, 1987). Arsenic exposure also results in an increased prevalence of diabetes (Rahman et al., 1998; Tseng et al., 2000) and cardiovascular disorders, such as atherosclerosis (Wang et al., 2002), ischemic heart disease (Hsueh et al., 1998), cerebral infraction (Chiou et al., 1997), and hypertension (Chen et al., 1995; Rahman et al., 1999) in humans. However, the mechanisms by which arsenic induces the pathological changes in blood vessels leading to vascular disorders remain to be delineated.
When the blood vessel is injured, a variety of chemokines and growth factors are secreted by various cell types, including endothelial cells (ECs), platelets, immune cells, and vascular smooth muscle cells (VSMCs), and stimulate VSMCs to migrate into the intima layer and expand (Ross, 1999). The migration of VSMCs into the intima and their subsequent proliferation (intimal hyperplasia) is an early pathological process in atherosclerosis. Because of the involvement of a variety of cytokines, chemokines, and immune cells in the development of atherosclerotic lesions, chronic inflammation is suggested to be involved in the progression of atherosclerosis (Libby, 2002).
The generation of reactive oxidants is a general manifestation of an inflammatory reaction. Various atherosclerosis-related factors, such as platelet-derived growth factor (PDGF), angiotensin II (Ang II), thrombin, and oxidized low-density lipoproteins, increase intracellular reactive oxidants or reactive oxygen species (ROS) production in VSMCs (Hsieh et al., 2001; Seshiah et al., 2002; Sundaresan et al., 1995) and stimulate VSMC proliferation (Gosgnach et al., 2000; Griendling et al., 1994; Sundaresan et al., 1995). Mechanisms involved in enhanced VSMC proliferation by generation of ROS are mediated through activation of MAP kinases and Akt and their downstream transcription factors, such as NF-B or AP-1, which are well-documented to regulate the expression of atherogenic related genes (Griendling et al., 2000; Irani, 2000). ROS are therefore implicated in the pathological processes of atherosclerosis (Ross, 1999).
The generation of reactive oxidants during arsenic metabolism has been shown to play an important role in arsenic-induced injury (Gurr et al., 2003; Liu et al., 2001). The involvement of reactive oxidants in iAs-induced DNA and chromosomal damage (Ho et al., 2000; Lynn et al., 2000), DNA repair inhibition (Mei et al., 2002), and apoptosis (Nakagawa et al., 2002) is well documented. Recent epidemiological studies have established a relationship between chronic arsenic exposure and increased levels of reactive oxidants in the blood of human populations drinking arsenic-contaminated water (Pi et al., 2002; Wu et al., 2001). ROS are therefore considered as a possible etiologic factor of atherogenicity (Wu et al., 2001, 2003).
In our previous study (Wu et al., 2003), the expression of several cytokine-related genes is increased in human subjects with increased arsenic exposure, including heme oxygenase-1 (HO-1), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6). Particularly, our study demonstrated an association for MCP-1 plasma protein level with blood arsenic in 65 study subjects of varying arsenic exposure level (Wu et al., 2003). HO-1, a stress response gene with a cytoprotective and inflammation regulation function, MCP-1, a monocyte-recruiting chemokine, and IL-6, a pleiopotent inflammatory cytokine, are regulated by ROS and atherogenic factors and involved in modulating the biological functions of VSMCs (Browatzki et al., 2000; Cushing et al., 1990; Duckers et al., 2001; Durante et al., 1999; Morse and Choi, 2002). Furthermore, the expression of these three genes has been detected in atherosclerotic lesion (Nelken et al., 1991; Seino et al., 1994; Wang et al., 1998). These three genes are possibly involved in the development of atherosclerosis. Since VSMCs are a major cell type in vascular vessels, we therefore examined whether treatment with inorganic trivalent sodium arsenite (iAs, NaAsO2) resulted in the generation of ROS and subsequent enhancement of the expression of these three genes in VSMCs.
MATERIALS AND METHODS
Cell culture.
Rat aorta VSMCs (rVSMCs, ATCC CRL-1476) and human aorta VSMCs (hVSMCs, ATCC CRL-1999) were obtained from the American Type Culture Collection (Manassas, VA) rVSMCs were routinely cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), while hVSMCs were maintained in Kaighn's modified F-12 Ham medium (HAM's F-12K) supplemented with 10% FBS, 10 μg/ml of insulin, 10 μg/ml of transferrin, 10 ng/ml of sodium selenite, and 20 μg/ml of endothelial cell growth supplement. To obtain resting cells, rVSMCs were switched to DMEM containing 1% FBS for 24 h, while hVSMCs were switched to HAM's F-12K containing 0.1% FBS for 48 h. The human fibroblast strain, HFW, derived from human newborn foreskin and kindly provided by Professor W. N. Wen (National Taiwan University), was grown in DMEM supplemented with 10% FBS as described previously (Yih and Lee, 2000). The human monocyte cell line, THP-1 (ATCC, TIB-202), was cultured in RPMI 1640 medium containing 10% FBS.
Measurement of mRNA levels by quantitative RT-PCR.
Total cellular RNA was extracted using the TRI-reagent according to the manufacturer's instructions (Molecular Research Center, Inc., Cincinnati, OH). The quantitative reverse transcriptase-polymerase chain reaction (quantitative RT-PCR) was used to analyze individual mRNA levels as described by Morrison et al. (1988). Each analysis was performed in triplicate. The relative mRNA levels for the genes of interest were calculated from the difference between the Ct value (number of cycles required to reach the detection threshold for the double-strand DNA product) for the gene of interest and that for an internal control gene, glyceraldehyde-3-phosphate dehydrogenase as described previously (Wu et al., 2003). The Ct values were determined by using Sequence Detector System v1.7 software provided by the Applied Biosystems (Foster City, CA).
Measurement of protein levels.
Intracellular HO-1 protein levels were analyzed by Western blotting as described previously (Yih et al., 1997). In brief, rVSMCs or hVSMCs treated with iAs for different time periods were lysed, and the proteins were electrophoretically separated and transferred to a positively charged PVDF membrane. The PVDF membrane was immunoblotted with anti-HO-1 antibody or anti-actin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). HO-1 protein and actin were visualized by the enhanced chemoluminescence method as described previously (Yih et al., 1997). Levels of secreted MCP-1 and IL-6 in the culture medium from iAs-treated VSMCs were determined using enzyme-linked immunosorbent assay (ELISA) kits (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions.
Determination of ROS production.
2',7'-dichlorofluorescein diacetate (DCF-DA) was used to measure ROS production in VSMCs (Huang et al., 1993). In brief, proliferating rVSMCs were treated with iAs for various time periods, DCF-DA (at a final concentration of 20 μM) being added to the culture medium 30 min before the end of iAs treatment. The rVSMCs were then trypsinized and resuspended in Hank's balanced salt solution. The fluorescence of the DCF formed by oxidation of DCF-DA by cellular oxidants was measured using a flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.
Survival assay.
To determine the protective effects of HO-1 on iAs-induced cell injury, the proliferation rates of VSMCs were measured by counting the cell number after treatment with iAs or iAs plus SnPP. In brief, 3 x 104 rVSMCs were seeded onto a 60 mm dish and cultivated for 24 h. The cells were then treated with various concentrations of iAs (0, 1, 2, 5 μM) with vehicle (0.25% DMSO) or 50 μM SnPP (dissolved in DMSO) for 24 h. Afterward, the media were replaced with fresh medium without drugs. After further cultivating for 72 h, the cells were trypsinized and counted with a cell counter (hematology analyzer, EXCELL 300, Metertech).
Chemotaxis assay.
MCP-1-induced monocyte chemotaxis was measured using 12-well Transwell polystyrene trays (Corning Costar Corp., Cambridge, MA) and a 5 μm pore polycarbonate membrane (McQuibban et al., 2002). Conditioned medium from resting hVSMCs incubated with or without 5 μM iAs was harvested and a 600 μl aliquot loaded into the lower chamber, while 100 μl of the human monocytic leukemia cell line, THP-1, at a density of 5 x 106 cells/ml in HAM's F-12K medium containing 0.1% FBS were loaded into the upper chamber. After incubation in a 37°C incubator for 2 h, the THP-1 cells on the lower chamber-side of the membrane were fixed with methanol, stained with DAPI. In each treatment, the average cell number of three randomly selected fields (using 10x eyepieces and 40x objective) was scored under a fluorescence microscopy. In control group, 100 to 140 migrating cells were obtained in each field. The relative migration index was calculated by dividing the number of THP-1 cells that migrated in response to conditioned medium from iAs-treated cultures by the number that migrated in response to conditioned medium from untreated controls supplemented with 5 μM iAs prior to assay. Pilot studies showed that addition of 5 μM iAs to conditioned medium from untreated cultures did not affect the migration index of THP-1 cells. To confirm the observed chemotaxis was due to secreted MCP-1, anti-human MCP-1 antibody (R&D Systems, Inc., Minneapolis, MN) at a final concentration of 0.83 μg/ml was added to the conditioned medium in the lower chamber 10 min before loading the cells into the upper chamber.
Treatment with ROS modulators.
Catalase (CAT) was added to the culture medium for 4 h. To avoid the interference with iAs, the medium was replaced with fresh medium before treatment with iAs (5 μM for 4 h). Superoxide dismutase (SOD) was added to the culture medium 4 h before iAs treatment, while allopurinol (AP), dimethylsulfoxide (DMSO), and L--nitro-L-arginine (L-NNA) were added 30 min before iAs treatment; these reagents were then left in the medium during the subsequent iAs treatment (5 μM for 4 h).
Statistical analysis.
The results are expressed as the mean ± SD. Statistical analyses were performed using Student's two-tailed unpaired t-test. p values < 0.05 were considered statistically significant.
RESULTS
Arsenite Induces Increased Expression of HO-1, MCP-1, and IL-6 in Vascular Smooth Muscle Cells
In a pilot experiment, treatment of resting hVSMC with iAs at concentrations up to 5 μM for 8 h did not affect [3H]thymidine incorporation when they were returned to complete medium containing 10% FBS and growth factor supplements. A concentration of 5 μM iAs was therefore used to investigate stimulatory effects on gene expression in VSMCs. Quantitative RT-PCR showed that treatment of resting hVSMC with 5 μM iAs for 0 to 8 h resulted in an increase in HO-1, MCP-1, and IL-6 mRNA levels, which peaked at 4 h of treatment (Fig. 1A). The appearance of large amounts of HO-1, an intracellular protein, in iAs-treated hVSMCs was confirmed by Western blotting (Fig. 1B). Levels of HO-1 protein were negligible in untreated hVSMCs, became visible after 5 h of iAs treatment, and were markedly increased at 9 h. Low, but significant, levels of MCP-1 (1.7-fold) and IL-6 (1.6-fold) were detected in the culture medium from iAs-treated hVSMCs by ELISA (Figs. 1C and 1D). Allowing for the lag in protein synthesis, the levels of HO-1, MCP-1, and IL-6 protein correlated well with the mRNA levels determined by quantitative RT-PCR. Similar induction profiles of HO-1, MCP-1, and IL-6 were seen in iAs-treated resting rVSMCs (Fig. 2A). Since proliferation of VSMCs is crucial for the development of atherosclerosis, we also examined the effect of iAs treatment on the expression of these three genes in proliferating rVSMCs, with similar results (Fig. 2B). By measuring the cell proliferating activity, 5 μM iAs, under our experimental conditions, was not cytotoxic for either resting or proliferation rVSMCs. Furthermore, HO-1, MCP-1, and IL-6 mRNA levels in proliferating rVSMCs treated with iAs for 4 h were increased in a concentration-dependent manner (Fig. 2C). Treatment of the HFW, cultured human fibroblast strain, with 5 μM iAs for different periods of time (0–8 h) resulted in a marked increase in HO-1 mRNA levels, but a marked decrease in MCP-1 and IL-6 mRNA levels (Fig. 2D). These results are consistent to several other reports (Foresti et al., 2001; Morse and Choi, 2002) showing that increased HO-1 expression is a general response of cells to iAs insult, whereas regulation of MCP-1 and IL-6 expression by iAs differs in different cell types.
Induction of ROS by Arsenite Treatment in VSMCs
To confirm that HO-1, MCP-1, and IL-6 genes were oxidative stress response genes, proliferating rVSMCs were exposed to 200 μM H2O2 for 0 to 8 h. As shown in Figure 3A, significant expression of all three genes was induced, being maximal after 2 h of H2O2 treatment. The generation of intracellular ROS in VSMCs by iAs was confirmed using a redox-sensitive fluorescence dye, DCF-DA, that is known to detect H2O2 and a variety of organic hydroperoxides, nitric oxide and peroxynitrite. As shown in Figure 3B, the fluorescence intensity of the oxidized product, DCF, was increased 1.6-fold in proliferating rVSMCs treated with 5 μM iAs for 4 h.
Roles of Arsenite-Increased Oxidative Stress in Arsenite-Induced HO-1, MCP-1, and IL-6 Expression
The effects of several ROS modulators were examined to explore the roles of oxidative stress in iAs-induced HO-1, MCP-1, and IL-6 expression in VSMCs. CAT catalyzes the degradation of H2O2 to H2O and O2. In proliferating rVSMCs, CAT pretreatment significantly inhibited the iAs-induced increase in HO-1, MCP-1, and IL-6 mRNA expression (Fig. 4A), and a similar effect was seen in iAs-treated resting hVSMCs (data not shown). DMSO, a hydroxyl radical scavenger, and L-NNA, an NO synthase inhibitor, were used to investigate the roles of hydroxyl radicals and NO in iAs-induced HO-1, MCP-1, and IL-6 mRNA expression. As shown in Figures 4B and 4C, DMSO (0.1 % v/v) or L-NNA (100 μM) had a similar effect to CAT on iAs-induced HO-1, MCP-1, and IL-6 mRNA expression in proliferating rVSMCs. These results indicate that H2O2, hydroxyl radicals, and NO are probably involved in the induction of HO-1, MCP-1, and IL-6 expression in iAs-treated VSMCs. In contrast, when proliferating rVSMCs were treated with AP, an inhibitor of the superoxide anion-producing enzyme, xanthine oxidase, or SOD, a scavenger of superoxide anions, before and during iAs treatment, only a moderate, not statistically significant inhibitory effect on HO-1 mRNA levels was seen, whereas the iAs-induced increase in IL-6 mRNA levels was completely blocked (Figs. 5A and 5B). In addition, AP completely inhibited, while SOD enhanced, the iAs-induced increase in MCP-1 mRNA levels in rVSMCs. These results indicated that superoxide anions play a role in the effect of iAs on IL-6 mRNA levels, but are less important in the effect on HO-1 mRNA levels and play a complicated role in MCP-1 induction.
Protective Role of HO-1 on iAs Cytotoxicty in VSMCs
In our previous report, iAs-induced HO-1 has cytoprotective effect on iAs-treated HFW (Ho et al., 2000). Here, we used tin protoporphyrin IX (SnPP), a well-documented HO-1 inhibitor, to examine the protective roles of HO-1 against iAs-induced cytotoxicity. By treatment of rVSMCs with various concentrations of iAs for 24 h, the proliferation of rVSMC was reduced when iAs was at the concentration of 5 μM (Fig. 6A). However, HO-1 protein became remarkable even when iAs at the concentration of 1 μM (Fig. 6B). Co-treatment of rVSMC with 50 μM SnPP and various concentrations of iAs significantly potentiated iAs-resulted inhibition of rVSMC proliferation (Fig. 6A). These results, consistent to previous observation in HFW (Ho et al., 2000), revealed that HO-1 plays a cytoprotective role against iAs-induced cytotoxic effects.
Response of Cultured Monocytes to MCP-1 Secreted by Arsenite-Treated Vascular Smooth Muscle Cells
MCP-1 is a well-documented chemoattractant. The human monocyte chemotactic activity of conditioned medium harvested from cultures of iAs-treated resting hVSMCs was determined using the Transwell migration assay. As shown in Figure 6, conditioned medium from iAs-treated (5 μM, 9 h) resting hVSMCs showed significantly higher chemotactic activity than conditioned medium from untreated cells supplemented with 5 μM iAs prior to assay. Furthermore, an anti-human MCP-1 monoclonal antibody, which neutralizes the bioactivity of secreted MCP-1, reduced the chemotactic activity of conditioned medium from either untreated or iAs-treated resting hVSMCs to a similar level (Fig. 7). These results show that MCP-1 is a functional chemotaxis factor in conditioned medium with or without iAs treatment and that its levels are increased on iAs treatment. Since 0.1% FBS was supplemented in conditioned medium with or without iAs treatment, residual MCP-1 activity is likely presented in conditioned medium without iAs treatment. We also could not rule out the possibility that a small amount of MCP-1 was produced by VSMCs without iAs treatment.
DISCUSSION
Using quantitative RT-PCR and Western blotting, our present study demonstrated that iAs, as well as H2O2, enhanced the expression of the HO-1, MCP-1, and IL-6 genes in cultured human and rat VSMCs. This effect was suppressed by most ROS/NO modulators used in this study, suggesting that ROS/NO were involved. However, AP and SOD had differential effects on the expression of these three genes in iAs-treated VSMCs. Since neither AP nor SOD significantly suppressed the iAs-induced increase in HO-1 expression, superoxide anion itself was not an active inducer of HO-1 expression. However, superoxide anions can react with NO to form the peroxynitrite anion, which is a potent inducer of HO-1 (Foresti et al., 1997). In the present study, the iAs-induced increase in MCP-1 expression was suppressed by AP, but enhanced by SOD. Since increased SOD activity could accelerate the dismutation of superoxide anions to H2O2, we inferred that a rapid increase in H2O2 was crucial for the increase in MCP-1 expression caused by iAs treatment. Similarly, iAs-induced Nrf2 nuclear accumulation is enhanced by copper (II) 3,5-diisopropyl salicylate hydrate, a cell-permeable SOD (Pi et al., 2003). Since all the ROS/NO modulators tested inhibited the iAs-induced increase in IL-6 expression, induction of the IL-6 gene can probably be caused by different kinds of ROS/NO molecules. Thus, different pro-oxidants are probably involved in the enhancement of HO-1, MCP-1, and IL-6 expression seen in iAs-treated VSMCs.
Several reports have shown that iAs treatment results in increased ROS and NO production in a variety of cells in culture (Barchowsky et al., 1999; Lee and Ho, 1995; Lynn et al., 2000), and in the blood of human populations drinking arsenic-contaminated water (Pi et al., 2002; Wu et al., 2001). Precisely how iAs results in an increase in ROS/NO is unclear. However, iAs is reported to increase superoxide anion production by stimulating NADH oxidase in hVSMCs and porcine aortic endothelial cells (Lynn et al., 2000; Smith et al., 2001). Alternatively, iAs may interact with sulfhydryl groups of biomolecules and GSH, resulting in a decreased capacity for scavenging ROS (Nakagawa et al., 2002; Wang et al., 1996). Downregulation of antioxidant enzymes, such as phospholipid hydroperoxide glutathione peroxidase, is another mechanism for increasing intracellular peroxide levels (Huang et al., 2002). The cellular signaling cascade activated by iAs-induced oxidative stress has been reviewed. Emerging evidence has shown that iAs-induced oxidative stress could activate MAP kinases, such as JNK and p38 kinase, and subsequently activate their downstream transcription factors, AP-1 and NF-B (Bernstam and Nriagu, 2000; Qian et al., 2003). In this study, we demonstrated that iAs-treatment resulted in increasing intracellular ROS production in VSMCs. Thus iAs is likely through similar signaling pathway to stimulate the expression of HO-1, MCP-1, and IL-6.
MCP-1, a C-C family chemokine, is expressed in endothelial cells, foam cells, and VSMCs of atherosclerotic lesions (Nelken et al., 1991), and has strong chemotactic activity for immune cells, such as monocytes (Rollins, 1997). The recruitment of circulating monocytes is a crucial step in the initiation of atherosclerosis (Ross, 1993). Numerous reports have shown that MCP-1 is induced in VSMCs by a variety of stimuli, including Ang II (Funakoshi et al., 2001), thrombin (Wenzel et al., 1995), uric acid (Kanellis et al., 2003), Fas ligand plus cycloheximide (Schaub et al., 2000), and activated platelets (Massberg et al., 2003). Expression of MCP-1 may result in the accumulation of inflammatory cells, such as macrophages, in atherosclerotic lesions. Our results showed that the enhanced chemotactic activity for human monocytes seen in conditioned medium from iAs-treated VSMCs could be suppressed by antibody against MCP-1. Since VSMCs are the major cell type in the media of arteries, MCP-1 released from VSMCs by iAs treatment may be involved in the recruitment of circulating monocytes and thus in initiating atherogenesis. Our current results also support our previous finding that plasma levels of MCP-1 show a good correlation with blood arsenic levels in human populations exposed to arsenic in drinking water (Wu et al., 2003).
IL-6, a proinflammatory cytokine and a major inducer of C-reactive protein (CRP) (Heinrich et al., 1990), is also present in atherosclerotic lesions (Seino et al., 1994). Both IL-6 and CRP are considered as strong risk markers of cardiovascular diseases (Blake and Ridker, 2001). Several vasoactive substances, such as PDGF, Ang II, thrombin, and endothelin, are reported to induce IL-6 expression in VSMCs (Browatzki et al., 2000; Funakoshi et al., 1999; Roth et al., 1995; Tokunou et al., 2001). It has been reported that IL-6 at levels of ng/ml range could stimulate VSMCs proliferation and migration (Ikeda et al., 1991; Liu et al., 2004), which are important manifestation of atherogenesis. Since the concentrations of secreted IL-6 in medium of iAs-treated cultures were ranged at pg/ml levels, anti-IL-6 antibody was unable to inhibit the proliferation or migration ability in iAs-treated rVSMCs (data not shown). This result may be explained not only by the low level of IL-6 secretion but also the presence of other mitogens or migration factors in culture medium of rVSMC. IL-6 also stimulates the expression of adhesion molecules and increases cell-cell permeability in ECs (Maruo et al., 1992; Watson et al., 1996). Increased levels of adhesion molecules and increased ECs permeability could promote the attachment and penetration of circulating immune cells, such as monocytes. Although the exact physiological role of iAs-induced IL-6 in VSMCs still requires further investigation, the enhanced expression of IL-6 seen in iAs-treated VSMCs may act as a risk factor of iAs-induced cardiovascular disorders.
In addition to cytokines and chemokines, the heme degradation enzyme, HO-1, is also expressed in atherosclerotic lesions (Wang et al., 1998). The products of heme degradation are iron, carbon monoxide, and biliverdin. Carbon monoxide acts as a vasodilator (Duckers et al., 2001), while bilirubin, produced by reduction of biliverdin, is a potent antioxidant (Stocker et al., 1987). Because of this, HO-1 has been proposed to have roles in cytoprotection and in the regulation of inflammation (Morse and Choi, 2002). In this study, SnPP, a competitive inhibitor of HO-1, was shown to significantly reduce cell survival of proliferating rVSMCs, indicating that iAs-induced HO-1 plays a cytoprotective role in iAs-damaged VSMCs. In fact, the growth promoting properties of HO-1 have been demonstrated in many cell types and in transgenic animal models and humans (Durante, 2003). Recently, HO-1 was further reported to play an important role in angiogenesis (Bussolati et al., 2004). Therefore, the roles of HO-1 on atherogenesis require further clarification.
Since arsenic is a well-documented potent inducer of HO-1 in many cell types (Lee and Ho, 1995; Taketani et al., 1989), HO-1 is also considered as a good marker of exposure to arsenic and oxidative stress. In the present study, iAs treatment of HFW resulted in increased HO-1 gene expression, but suppression of MCP-1 and IL-6 expression. This finding is consistent with the results of our previous study using cDNA microarrays to study the effect of iAs treatment on the gene expression profile of HFW cells (Yih et al., 2002). Inhibition of IL-6 expression by iAs is also observed in human intestinal epithelial cells (Hershko et al., 2002). These results indicated that although HO-1, MCP-1, and IL-6 are oxidative response genes, their response to iAs-enhanced oxidative stress is cell type dependent. The cell type specificity of the induction of MCP-1 and IL-6 requires further study.
Our present study provides evidence that exposure to iAs increases the generation of ROS/NO and thus enhances the expression of the HO-1, MCP-1, and IL-6 genes in VSMCs. Expression of MCP-1 and IL-6 may promote the initiation of atherogenesis by increasing the attachment, penetration, and migration of monocytes. These three gene products are also involved in regulation of VSMC proliferation. The findings presented here suggest a possible mechanism for atherogenesis in arsenic-exposed humans. A better understanding of the atherogenic mechanisms of arsenic should provide the basis for improved interventional approaches for both treatment and prevention.
ACKNOWLEDGMENTS
This work was supported by the Academia Sinica and grants from the National Science Council, Republic of China (NSC91-2320-B010-055 and NSC92-2320-B-010-017).
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