Manganese enhances peroxynitrite and leukotriene E4 formation in bovine aortic endothelial cells exposed to arsenic.

Long-term exposure to arsenic in drinking water has been linked to cancer and other health effects, including cardiovascular disease. Arsenic in the environment is found in combination with a range of metals that could influence its toxicity. Manganese, in particular, is a metal that is typically found in conjunction with arsenic in contaminated groundwater. Peroxynitrite is a powerful oxidant formed from the reaction between nitric oxide and superoxide anion. Arsenic has been shown to increase the formation of peroxynitrite in bovine aortic endothelial cells (BAECs) and promote the formation of 3-nitrotyrosine (3-NY) in the atherosclerotic plaque of ApoE-/-/LDLr-/- mice. Arsenic exposure also increases leukotriene E4 (LTE4) formation in both the mice and BAECs, an effect that is partially reversed by the addition of Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor. In the present study, we investigated the effect of adding nontoxic concentrations of manganese along with arsenic to BAEC cultures. Manganese increased arsenic toxicity and enhanced peroxynitrite, 3-NY, and LTE4 formation in BAECs. Addition of LNAME reduced 3-NY formation induced by arsenic/manganese mixtures, but in contrast to its effect on arsenic alone, L-NAME actually increased LTE4 synthesis in BAECs treated with the arsenic/manganese combination. Overall, these data suggest that manganese may exacerbate the toxic effects of arsenic on the vascular system.

Increased hydrogen peroxide downregulates soluble guanylate cyclase in the lungs of lambs with persistent pulmonary hypertension of the newborn.

Similar to infants born with persistent pulmonary hypertension of the newborn (PPHN), there is an increase in circulating endothelin-1 (ET-1) and decreased cGMP-mediated vasodilation in an ovine model of PPHN. These abnormalities lead to vasoconstriction and vascular remodeling. Our previous studies have demonstrated that reactive oxygen species (ROS) levels are increased in pulmonary arterial smooth muscle cells (PASMC) exposed to ET-1. Thus the initial objective of this study was to determine whether the development of pulmonary hypertension in utero is associated with elevated production of the ROS hydrogen peroxide (H(2)O(2)) and if this is associated with alterations in antioxidant capacity. Second we wished to determine whether chronic exposure of PASMC isolated from fetal lambs to H(2)O(2) would mimic the decrease in soluble guanylate cyclase expression observed in the ovine model of PPHN. Our results indicate that H(2)O(2) levels are significantly elevated in pulmonary arteries isolated from 136-day-old fetal PPHN lambs (P 0.05). In addition, we determined that catalase and glutathione peroxidase expression and activities remain unchanged. Also, we found that the overnight exposure of fetal PASMC to a H(2)O(2)-generating system resulted in significant decreases in soluble guanylate cyclase expression and nitric oxide (NO)-dependent cGMP generation (P 0.05). Finally, we demonstrated that the addition of the ROS scavenger catalase to isolated pulmonary arteries normalized the vasodilator responses to exogenous NO. As these scavengers had no effect on the vasodilator responses in pulmonary arteries isolated from age-matched control lambs this enhancement appears to be unique to PPHN. Overall our data suggest a role for H(2)O(2) in the abnormal vasodilation associated with the pulmonary arteries of PPHN lambs.

Arsenic exposure exacerbates atherosclerotic plaque formation and increases nitrotyrosine and leukotriene biosynthesis.

A correlation between arsenic and cardiovascular disease (CVD) has been established through epidemiological studies, although the mechanisms are unknown. Using a mouse model that develops atherosclerotic lesions on a normal chow diet, we have confirmed a connection between long-term arsenic intake and CVD. Our results reveal a significant increase in the degree of atherosclerotic plaque stenosis within the innominate artery of ApoE-/-/LDLr-/- mice treated with 10 ppm sodium arsenite (133 microM) in drinking water for 18 weeks compared to controls. Immunohistochemistry shows nitrotyrosine formation, a marker of reactive nitrogen species generation, is significantly higher within the atherosclerotic plaque of arsenic-treated mice. In addition, there is a significant increase in the 5-lipoxygenase (5-LO) product, leukotriene E4 (LTE4), in the serum of arsenic-treated mice. This is supported by induction of the 5-LO protein and subsequent increases in LTE4 synthesis in bovine aortic endothelial cells. This increase in LTE4 is partially inhibited by inhibitors of nitric oxide synthase, suggesting a link between reactive nitrogen species and arsenic-induced inflammation. Furthermore, there is a significant increase in prostacyclin (PGI2) in the serum of arsenic-treated mice. We conclude that changes in specific inflammatory mediators such as LTE4 and PGI2 are related to arsenic-induced atherosclerosis. In addition, amplified synthesis of reactive species such as peroxynitrite results in increased protein nitration in response to arsenic exposure. This finding is consistent with the pathology seen in human atherosclerotic plaques.

Important role for Rac1 in regulating reactive oxygen species generation and pulmonary arterial smooth muscle cell growth.

Vascular NADPH oxidases have been shown to be a major source of reactive oxygen species (ROS). Recent studies have also implicated ROS in the proliferation of vascular smooth muscle cells. However, the components required for activation of the NADPH oxidase complex have not been clearly elucidated. Here we demonstrate that ROS generation in ovine pulmonary arterial smooth muscle cells (PASMCs) requires the activation of Rac1, implicating this protein as an important subunit of the NADPH oxidase complex. Our results, using a geranylgeranyl transferase inhibitor (GGTI-287), demonstrated a dose-dependent inhibition of Rac1 activity and ROS production. This was associated with an inhibition of PASMC proliferation with an arrest at G(2)/M. The inhibition of Rac1 by GGTI-287 led us to more specifically target Rac1 to investigate its role in the generation of ROS and cellular proliferation. To accomplish this, we utilized a dominant negative Rac1 (N17Rac1) and a constitutively active Rac1 (V12Rac1). These two forms of Rac1 were transiently expressed in PASMCs using adenovirus-mediated gene transfer. N17Rac1 expression resulted in decreased cellular Rac1 activity, whereas V12Rac1 infection showed increased activity. Compared with controls, the V12Rac1-expressing cells had higher levels of ROS production and increased proliferation, whereas the N17Rac1-expressing cells had decreased ROS generation and proliferation and cell cycle arrest at G(2)/M. However, the inhibition of cell growth produced by N17Rac1 overexpression could be overcome if cells were co-incubated with the Cu,Zn superoxide dismutase inhibitor DETC. These results indicate the importance of Rac1 in ROS generation and proliferation of vascular smooth muscle cells.

Arsenic induces peroxynitrite generation and cyclooxygenase-2 protein expression in aortic endothelial cells: possible role in atherosclerosis.

Epidemiological evidence suggests that exposure to the metalloid arsenic constitutes a risk factor for cardiovascular disease. The purpose of this study was to determine whether arsenic could stimulate generation of factors involved in oxidative stress and inflammation, conditions associated with atherosclerosis, or coronary artery disease. We found that production of peroxynitrite, a strong oxidant formed from the coupling of nitric oxide and superoxide anion, was significantly increased in bovine aortic endothelial (BAE) cells exposed to sodium arsenite at concentrations as low as 0.5 microM. Expression of the inflammatory mediator cyclooxygenase-2 (COX-2) was also upregulated in response to arsenite exposure as demonstrated by Western blot analysis. The increase in COX-2 protein was time dependent with highest levels at 30 min and 48 h. This result was supported by an increase in the generation of prostaglandin E(2) following exposure to arsenic. Nitrotyrosine residues in proteins are indicative of peroxynitrite generation, and extensive nitrotyrosine formation has been detected in atherosclerotic plaques. Therefore, COX-2 protein was immunoprecipitated from BAE cells and submitted to Western blot analysis using an antibody to nitrotyrosine. Nitration of COX-2 was detected in arsenic-treated cells, but not in untreated control cells. The findings in this report suggest an increase in reactive species, notably peroxynitrite, in BAE cells exposed to arsenic. Furthermore, induction of important inflammatory mediators such as COX-2 may exacerbate the inflammatory state typical of atherosclerosis.

Comparative study of isoflavone, quinoxaline and oxindole families of anti-angiogenic agents.

A study designed to compare the effects on VEGF-induced angiogenesis of a number of known anti-angiogenic agents together with some novel derivatives thereof was undertaken. Thus the isoflavone biochanin A 1[structure: see text], indomethacin 2[structure: see text], the 3-arylquinoxaline SU1433 and its derivatives 3-6[structure: see text], the benzoic acid derivative 7[structure: see text], the oxindoles SU5416 8[structure: see text] and SU6668 11[structure: see text], together with their simple N-benzyl derivatives 9, 10, and 12[structure: see text] were selected for study. Using an in vitro assay the compounds were evaluated for their ability to inhibit VEGF-induced angiogenesis in HUVECs, and the cytotoxicity of representative compounds was also studied in tumour cell lines using 24-h exposure. The results indicate that the SU compounds, SU1433, SU 5416 and SU6668, are more potent inhibitors of VEGF-induced angiogenesis than indomethacin or the naturally occurring biochanin A, presumably because they inhibit VEGF receptor signalling. Blocking one of the phenolic OH groups of SU1433 reduced anti-angiogenic activity, as did blocking the NH groups of SU5416 and SU6668. Cytotoxicity studies indicate that none of the compounds examined exhibited cytotoxicity at anti-angiogenic concentrations.