Oxidative Stress Mediates Sodium Arsenite-Induced Expression of Heme Oxygenase-1, Monocyte Chemoattractant Protein-1, and Interleukin-6 in Vascular Smooth Muscle Cells

Pei-Chung Lee 1 2 I-Ching Ho 0 1 Te-Chang Lee 0 1 2 0 Institute of Biomedical Sciences , Academia Sinica, Taipei, Taiwan, ROC 1 Biopharmaceutical Science, School of Life Sciences, National Yang-Ming University , Pei-Tou, Taipei , Taiwan 112. Fax: 2 Institute of Biopharmaceutical Science, National Yang-Ming University , Taipei, Taiwan, ROC Toxicological Sciences vol. 85 no. 1 The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: - 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-v-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 iAsinduced 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. 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 atherosclerosisrelated 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-jB 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 arsenicinduced 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 monocyterecruiting 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 (rV (...truncated)