Model combustion-generated particulate matter containing persistent free radicals redox cycle to produce reactive oxygen species

Particulate matter (PM) is emitted during thermal decomposition of waste. During this process, aromatic compounds chemisorb to the surface of metal-oxide-containing PM, forming a surface-stabilized environmentally persistent free radical (EPFR). We hypothesized that EPFR-containing PM redox cycle to produce ROS and that this redox cycle is maintained in biological environments. To test our hypothesis, we incubated model EPFRs with the fluorescent probe dihydrorhodamine (DHR). Marked increases in DHR fluorescence were observed. Using a more specific assay, hydroxyl radicals ( •OH) were also detected, and their level was further increased by cotreatment with thiols or ascorbic acid (AA), known components of epithelial lining fluid. Next, we incubated our model EPFR in bronchoalveolar lavage fluid (BALF) or serum. Detection of EPFRs and •OH verified that PM generate ROS in biological fluids. Moreover, incubation of pulmonary epithelial cells with EPFR-containing PM increased •OH levels compared to those in PM lacking EPFRs. Finally, measurements of oxidant injury in neonatal rats exposed to EPFRs by inhalation suggested that EPFRs induce an oxidant injury within the lung lining fluid and that the lung responds by increasing antioxidant levels. In summary, our EPFR-containing PM redox cycle to produce ROS, and these ROS are maintained in biological fluids and environments. Moreover, these ROS may modulate toxic responses of PM in biological tissues such as the lung. © 2013 American Chemical Society

Radical-containing ultrafine particulate matter initiates epithelial-to-mesenchymal transitions in airway epithelial cells

Environmentally persistent free radicals (EPFRs) in combustion generated particulate matter (PM) are capable of inducing pulmonary pathologies and contributing to the development of environmental asthma. In vivo exposure of infant rats to EPFRs demonstrates their ability to induce airway hyperresponsiveness to methacholine, a hallmark of asthma. However, the mechanisms by which combustion-derived EPFRs elicit in vivo responses remain elusive. In this study, we used a chemically defined EPFR consisting of approximately 0.2 μm amorphrous silica containing 3% cupric oxide with the organic pollutant 1,2-dichlorobenzene (DCB-230). DCB-230 possesses similar radical content to urban-collected EPFRs but offers several advantages, including lack of contaminants and chemical uniformity. DCB-230 was readily taken up by BEAS-2B and at high doses (200 μg/cm2) caused substantial necrosis. At low doses (20 μg/cm2), DCB-230 particles caused lysosomal membrane permeabilization, oxidative stress, and lipid peroxidation within 24 hours of exposure. During this period, BEAS-2B underwent epithelial-to-mesenchymal transition (EMT), including loss of epithelial cell morphology, decreased E-cadherin expression, and increased α-smooth muscle actin (α-SMA) and collagen I production. Similar results were observed in neonatal air-liquid interface culture (i.e., disruption of epithelial integrity and EMT). Acute exposure of infant mice to DCB-230 resulted in EMT, as confirmed by lineage tracing studies and evidenced by coexpression of epithelial E-cadherin and mesenchymal α-SMA proteins in airway cells and increased SNAI1 expression in the lungs. EMT in neonatal mouse lungs after EPFR exposure may provide an explanation for epidemiological evidence supporting PM exposure and increased risk of asthma. Copyright © 2013 by the American Thoracic Society

Essential role of ER-alpha-dependent NO production in resveratrol-mediated inhibition of restenosis

Resveratrol (Resv), a red wine polyphenol, is known to exhibit vascular protective effects and reduce vascular smooth muscle cell mitogenesis. Vascular smooth muscle cell proliferation is a critical factor in the pathogenesis of restenosis, the renarrowing of vessels that often occurs after angioplasty and/or stent implantation. Although Resv has been shown to be an estrogen receptor (ER) modulator, the role of the ER in Resv-mediated protection against restenosis has not yet been elucidated in vivo. Therefore, with the use of a mouse carotid artery injury model, our objective was to determine the role of ER in modulating Resv-mediated effects on neointimal hyperplasia. Female wild-type and ER-α(-/-) mice were administered a high-fat diet ± Resv for 2 wk. A carotid artery endothelial denudation procedure was conducted, and the mice were administered a high-fat diet ± Resv for an additional 2 wk. Resv-treated wild-type mice exhibited a dramatic decrease in restenosis, with an increased arterial nitric oxide (NO) synthase (NOS) activity and NO production. However, in the ER-α(-/-) mice, Resv failed to afford protection and failed to increase NO production, apparently because of a decreased availability of the NOS cofactor tetrahydrobiopterin. To verify the role of NO in Resv-mediated effects, mice were coadministered Resv plus a nonselective NOS inhibitor, N(G)-nitro-l-arginine methyl ester (l-NAME). Cotreatment with l-NAME significantly attenuated the antirestenotic properties of Resv. These data thus suggest that Resv inhibits vascular proliferative responses after injury, predominately through an ER-α-dependent increase in NO production

Resveratrol inhibits rat aortic vascular smooth muscle cell proliferation via estrogen receptor dependent nitric oxide production

Vascular smooth muscle cell (VSMC) proliferation is pivotal in the progression of hypertension, atherosclerosis, and restenosis. Resveratrol is a grape polyphenol that is implicated as an important contributor to red wine\u27s vascular protective effects. Its antimitogenic action on VSMC is attributed to an array of pleiotropic effects, including modulation of the estrogen receptor (ER). To elucidate the mechanisms underlying resveratrol-mediated ER modulation and its inhibition of VSMC proliferation, we treated VSMC with resveratrol with or without the ER antagonist ICI 182,780 and measured cell proliferation and nitric oxide (NO) production. Resveratrol dose-dependently decreased VSMC DNA synthesis, with a half maximal inhibitory concentration (IC50) of 3.73+/-0.57 microM, and dramatically slowed cell growth, but did not induce VSMC apoptosis. Resveratrol-mediated decrease in proliferation was reversed by cotreatment with ICI 182,780, and resveratrol effectively competed with 17beta-estradiol for binding to the ER, exhibiting an IC50 of 8.92+/-0.14 microM. Resveratrol induced a sustained increase in ER-dependent NO production. Further, resveratrol-mediated decrease in VSMC proliferation was blunted by cotreatment with the general nitric oxide synthase (NOS) inhibitor N5-(1-Iminomethyl)-L-ornithine, dihydrochloride or with the inducible NOS (iNOS)-selective inhibitor S,S\u27-1,4-phenylene-bis (1,2-ethanediyl)bis-isothiourea, dihydrobromide, but not with the neuronal NOS-selective inhibitor 7-nitroindazole. Though resveratrol did not alter iNOS protein levels, it dose-dependently increased levels of iNOS activity, of the iNOS cofactor tetrahydrobiopterin (BH4), and of guanosine triphosphate cyclohydrolase I protein, the rate-limiting enzyme in BH4 biosynthesis. In addition, all of these effects were abolished by cotreatment with ICI 182,780. Thus, the antimitogenic effects of resveratrol on VSMC may be mediated by an ER-induced increase in iNOS activity

Antiretrovirals induce direct endothelial dysfunction in vivo

HIV-associated cardiovascular diseases have been widely described, but clinical studies aimed at establishing cause-effect relationships between HIV-associated cardiovascular disease and either the HIV infection or antiretroviral therapy have been problematic. Endothelial dysfunction is a sensitive marker and early event in atherosclerosis, and many have suggested that protease inhibitors promote endothelial dysfunction indirectly by inducing elevations in circulating lipids. To determine whether nucleoside reverse transcriptase inhibitors and/or protease inhibitors induce endothelial dysfunction, and to test whether this effect is dependent upon drug-mediated alteration in plasma lipid concentrations, we treated male Sprague-Dawley rats with pharmacological doses of azidothymidine (AZT), indinavir, or AZT plus indinavir through their drinking water for 1 month and assessed endothelial function in aortic rings using an isometric force measurement. Circulating levels of plasma lipids and endothelin-1, a marker for endothelial injury and/or dysfunction, were also determined. We found that AZT and AZT plus indinavir treatments dramatically reduced endothelium-dependent vessel relaxation. However, AZT treatment did not significantly alter plasma levels of cholesterol or triglyceride. In addition, plasma endothelin-1 levels were elevated in rats treated with AZT plus indinavir. Indinavir treatment alone increased plasma cholesterol levels but had no effect on endothelial function. These findings suggest that in addition to modulating plasma lipid levels, antiretrovirals, particularly AZT and perhaps other nucleoside reverse transcriptase inhibitors, may have direct effects on the vascular endothelium. Together with other increased risk factors for atherosclerosis in HIV patients, AZT-induced endothelial dysfunction may contribute to the cardiovascular diseases associated with HIV antiretroviral therapy

Role of COX-2 in the bioactivation of methylenedianiline and in its proliferative effects in vascular smooth muscle cells

4,4\u27-Methylenedianiline (DAPM) is an aromatic diamine used directly in the production of polyurethane foams and epoxy resins, or as a precursor to MDI in the manufacture of some polyurethanes. In our prior experiments, we showed that chronic, intermittent treatment of female rats with DAPM resulted in vascular medial hyperplasia of pulmonary arteries. In addition, treatment of vascular smooth muscle cells (VSMC) in culture with DAPM increased the rates of proliferation in a manner that was inhibited by co-treatment with N-acetylcysteine but was not associated with oxidative stress. We thus hypothesized that NAC treatment inhibited DAPM toxicity by competing for binding reactive intermediates formed through DAPM metabolism. Because the peroxidase enzyme cyclooxygenase is constitutively expressed in VSMC, and because cyclooxygenase is known to metabolize similar aromatic amines to electrophilic intermediates, we further hypothesized that DAPM-induced VSMC proliferation was dependent upon COX-1/2-mediated bioactivation. To test this hypothesis, we treated VSMC with DAPM and measured cell proliferation, COX-2 expression, COX-1/2 activity, and levels of covalent binding. DAPM treatment resulted in a dose-dependent increase in proliferation that was abolished by co-treatment with the COX-2-selective inhibitor celecoxib. In addition, DAPM exposure increased the rates of proliferation in VSMC isolated from wild-type but not COX-2 (-/-) mice. Paradoxically, treatment with DAPM reduced the cellular production of PGE(2) and PGF(2α), but dose-dependently increased the COX-2 protein levels. Covalent binding of [(14)C]-DAPM to VSMC biomolecules was greater in wild-type than in COX-2 (-/-) cells. However, covalent binding of [(14)C]-DAPM was not altered by co-treatment with a nonselective inhibitor of cytochromes P450. These studies thus suggest that DAPM-induced VSMC proliferation may be due to bioactivation of DAPM, perhaps through the action of cyclooxygenase. The data furthermore suggest that DAPM\u27s mechanism of action may possibly involve inhibition or suicide inactivation of COX-2. In addition, because we observed an increase in DAPM-induced VSMC proliferation in cells isolated from female compared to male rats, further studies into the potential interplay between DAPM, the estrogen receptor, and COX-2 seem warranted