Abstract-Reactive oxygen species (ROS) are mediators of intracellular signals for a myriad of normal and pathologic cellular events, including differentiation, hypertrophy, proliferation, and apoptosis. NADPH oxidases are important sources of ROS that are present in diverse tissues throughout the body and activate many redox-sensitive signal transduction and gene expression pathways. To avoid toxicity and provide specificity of signaling, ROS production and metabolism necessitate tight regulation that likely includes subcellular compartmentalization. However, the constituent elements of NADPH oxidase-dependent cell signaling are not known. To address this issue, we examined cytokine generation of ROS and subsequent activation of the transcription factor nuclear factor B in vascular smooth muscle cells (SMCs). Tumor necrosis factor-␣ and interleukin (IL)-1␤ stimulation of SMCs resulted in diphenylene iodonium-sensitive ROS production within intracellular vesicles. Nox1 and p22 phox , integral membrane subunits of NADPH oxidase, coimmunoprecipitated with early endosomal markers in SMCs. ClC-3, an anion transporter that is primarily found in intracellular vesicles, also colocalized with Nox1 in early endosomes and was necessary for tumor necrosis factor-␣ and interleukin-1␤ generation of ROS. Cytokine activation of nuclear factor B in SMCs required both Nox1 and ClC-3. We conclude that in response to tumor necrosis factor-␣ and interleukin-1␤, NADPH oxidase generates ROS within early endosomes and that Nox1 cannot produce sufficient ROS for cell signaling in the absence of ClC-3. These data best support a model whereby ClC-3 is required for charge neutralization of the electron flow generated by Nox1 across the membrane of signaling endosomes. Key Words: smooth muscle cells Ⅲ NAPDH oxidase Ⅲ cell signaling Ⅲ ion channels I n response to diverse extracellular stimuli, intracellular signaling is dependent on generation of reactive oxygen species (ROS) by NADPH oxidase. The catalytic core of NADPH oxidase consists of a membrane-bound flavocytochrome b558 composed of a Nox (NADPH oxidase) (reviewed elsewhere 1 ) subunit and p22phox . The prototypical model of NAPDH oxidase is found in phagosomes, where the orientation and biochemical properties of this heterodimer obligate reduction of oxygen to superoxide on the side of the membrane opposite from where NADPH and the cytosolic subunits of the oxidase bind. 2 Based on this orientation, activation of NADPH oxidase in nonphagocytes should generate superoxide into the extracellular space or, following endocytosis, into intracellular vesicles. We hypothesized that extracellular stimuli activating the Nox1-based NADPH oxidase would produce superoxide in endocytotic vesicles.The phagocyte NADPH oxidase is electrogenic, moving electrons from cytoplasmic NADPH through the enzyme into the phagosome to reduce oxygen to superoxide. Without charge compensation, this electron flux rapidly depolarizes the membrane (the voltage in the cytoplasm becomes positive relative to the phagoso...

Objective Examine the contribution of Nox1 NADPH oxidase to atherogenesis. Methods and Results Male apolipoprotein E deficient mice (ApoE−/−) and male mice deficient in both apolipoprotein E and Nox1 (ApoE−/− Nox1−/y) received an atherogenic diet for 18 weeks. Mean blood pressures, body weights, and serum cholesterol levels were similar between the two groups of mice. Deficiency of Nox1 decreased superoxide levels and reduced lesion area in the aortic arch from 43% (ApoE−/−) to 28% (ApoE−/− Nox1−/y). The reduction in lesion size at the level of the aortic valve in ApoE−/−/Nox1−/y was accompanied by a decrease in macrophage infiltration as compared to ApoE−/− mice. Carotid artery ligation in ApoE−/− mice induced accelerated intimal hyperplasia with decreased cellular proliferation and increased collagen content in the neointima of vessels deficient in Nox1. Conclusions Nox1-derived ROS modify lesion composition and contribute to lesion size in a murine model of atherosclerosis.

Internalization of activated receptors to a compartment enriched with NAPDH oxidase and associated signaling molecules is expected to facilitate regulation of redox-mediated signal transduction. The aim of this study was to test the hypothesis that endocytosis is necessary for generation of reactive oxygen species (ROS) by Nox1 and for redox-dependent signaling in smooth muscle cells (SMCs). Within minutes of treatment with tumor necrosis factor (TNF)-a or thrombin, SMCs increased cellular levels of ROS that was inhibited by shRNA to Nox1. Treatment of SMC with TNF-a induced a dynamin-dependent endosomal generation of ROS, whereas thrombinmediated ROS production did not occur within endosomes and was not prevented by dominant-negative dynamin (dn-dynamin), but instead required transactivation of the epidermal growth factor receptor (EGFR). Activation of the phosphatidylinositol 3-kinase (PI3K)-Akt-activating transcription factor-1 (ATF-1) pathway by TNF-a and thrombin were both Nox1-and dynamin-dependent. In conclusion, we show that formation of specific ligand-receptor complexes results in spatially distinct mechanisms of Nox1 activation and generation of ROS. These findings provide novel insights into the role of compartmentalization for integrating redoxdependent cell signaling. Antioxid. Redox Signal. 12, 583-593.

Objective-We have shown that the chloride-proton antiporter chloride channel-3 (ClC-3) is required for endosome-dependent signaling by the Nox1 NADPH oxidase in SMCs.In this study, we tested the hypothesis that ClC-3 is necessary for proliferation of smooth muscle cells (SMCs) and contributes to neointimal hyperplasia following vascular injury. Methods and Results-Studies were performed in SMCs isolated from the aorta of ClC-3-null and littermate control (wild-type [WT]) mice. Thrombin and tumor necrosis factor-␣ (TNF-␣) each caused activation of both mitogen activated protein kinase extracellular signal-regulated kinases 1 and 2 and the matrix-degrading enzyme matrix metalloproteinase-9 and cell proliferation of WT SMCs. Whereas responses to thrombin were preserved in ClC-3-null SMCs, the responses to TNF-␣ were markedly impaired. These defects normalized following gene transfer of ClC-3. Carotid injury increased vascular ClC-3 expression, and compared with WT mice, ClC-3-null mice exhibited a reduction in neointimal area of the carotid artery 28 days after injury. Conclusion-ClC-3 is necessary for the activation of SMCs by TNF-␣ but not thrombin. Deficiency of ClC-3 markedly reduces neointimal hyperplasia following vascular injury. In view of our previous findings, this observation is consistent with a role for ClC-3 in endosomal Nox1-dependent signaling. These findings identify ClC-3 as a novel target for the prevention of inflammatory and proliferative vascular diseases.

Objective-To examine the effect of an oxidized extracellular oxidation-reduction (redox) state (E h ) on the expression of NADPH oxidases in vascular cells. Methods and Results-The generation of reactive oxygen species by NADPH oxidase (Nox)-based NADPH oxidases activates redox-dependent signaling pathways and contributes to the development of "oxidative stress" in vascular disease. An oxidized plasma redox state is associated with cardiovascular disease in humans; however, the cellular mechanisms by which the extracellular redox state may cause disease are not known. Aortic segments and cultured aortic smooth muscle cells were exposed to E h between Ϫ150 mV (reduced) and 0 mV (oxidized) by altering the concentration of cysteine and its disulfide, cystine, the predominant redox couple in plasma. A more oxidized E h increased the expression of Nox1 and resulted in Nox1-dependent proliferation of smooth muscle cells. Oxidized E h rapidly induced epidermal growth factor receptor phosphorylation via shedding of epidermal growth factor-like ligands from the plasma membrane and caused extracellular signal-regulated kinase 1/2-dependent phosphorylation of the transcription factors activating transcription factor-1 and cAMP-response element-binding protein. Inhibition of epidermal growth factor receptor or extracellular signal-regulated kinase 1/2 activation, or addition of small interference RNA to activating transcription factor-1, prevented the increase in Nox1 expression. Conclusion-Our results identify a novel mechanism by which extracellular oxidative stress increases expression and activity of Nox1 NADPH oxidase and contributes to vascular disease. Key Words: oxidative stress Ⅲ atherosclerosis Ⅲ NADPH oxidase Ⅲ epidermal growth factor receptor R eactive oxygen species (ROS) contribute to the pathogenesis of cardiovascular diseases, including hypertension, atherosclerosis, cardiac hypertrophy, heart failure, and restenosis. [1][2][3] An increase in cellular ROS contributes to the pathogenesis of vascular disease by altering endothelial cell function, enhancing vascular smooth muscle cell (SMC) growth and proliferation, stimulating the expression of proinflammatory genes, and modulating reconstruction of the extracellular matrix. 1,4 Although an excessive amount of ROS is considered harmful and contributes to the pathogenesis of disease, ROS normally participate in the regulation of important cellular processes, including cell signaling, gene expression, cellular death and senescence, regulation of growth, oxygen sensing, activation of matrix metalloproteinases (MMPs), and angiogenesis. [5][6][7] Although several cellular sources of ROS have been identified, the primary source of ROS in vascular cells is NADPH oxidases. 3,8 Of the Nox isoforms, Nox1, Nox2, Nox4, and Nox5 are expressed in vascular cells, with expression patterns showing cell specificity. For example, only Nox1 and Nox4 are expressed in rodent aortic SMCs. 9 The activity of the vascular NADPH oxidases is regulated by cytokines, hormones, and mecha...

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ABSTRACT:The effects of clinoptilolite in pig's diets were examined on sixty Landrace × Yorkshire crossbred pigs of both sexes divided into two groups: 1. control group, with basal diets; 2. treatment, with basal diets supplemented with 5 g clinoptilolite per 1 kg diet (Cp group). Feed and water were available ad libitum. Individual live weights were recorded on days 45, 90 and 135 of the experiment. Feed consumptions were recorded weekly. Blood was drawn from the anterior vena cava of each pig at day 135 for the determination of serum biochemical parameters. During the first 90 days of the experiment pigs from the Cp group had higher body weight gain (BWG) compared with the control group (+2.5% and +7.0%, respectively) but in the finishing phase of growing their growth parameters were significantly lower (-4.8%). Blood serum biochemical parameters from all experimental pigs were generally within the normal range. Higher triglyceride concentration, lower total cholesterol concentration and increased activity of AST were recorded in the serum of the Cp group.

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