Upon reaction with electrons, oxygen is transformed into reactive oxygen species (ROS). It has long been known that ROS can destroy bacteria and destroy human cells, but research in recent decades has highlighted new roles for ROS in health and disease. Indeed, while prolonged exposure to high ROS concentrations may lead to non-specific damage to proteins, lipids, and nucleic acids, low to intermediate ROS concentrations exert their effects rather through regulation of cell signalling cascades. Biological specificity is achieved through the amount, duration, and localisation of ROS production. ROS have crucial roles in normal physiological processes, such as through redox regulation of protein phosphorylation, ion channels, and transcription factors. ROS are also required for biosynthetic processes, including thyroid hormone production and crosslinking of extracellular matrix. There are multiple sources of ROS, including NADPH oxidase enzymes; similarly, there are a large number of ROS-degrading systems. ROS-related disease can be either due to a lack of ROS (e.g., chronic granulomatous disease, certain autoimmune disorders) or a surplus of ROS (e.g., cardiovascular and neurodegenerative diseases). For diseases caused by a surplus of ROS, antioxidant supplementation has proven largely ineffective in clinical studies, most probably because their action is too late, too little, and too non-specific. Specific inhibition of ROS-producing enzymes is an approach more promising of clinical efficacy.

The endothelium is a source of reactive oxygen species in short-term models of hypercholesterolemia and atherosclerosis. We examined a chronic model of atherosclerosis for increased vascular production of superoxide (O2-.) and determined whether endothelial overexpression of superoxide dismutase (SOD) would improve endothelium-dependent relaxation. Superoxide generation was 3 times higher in isolated aortas from Watanabe heritable hyperlipidemic (WHHL) rabbits (2 to 4 years old) compared with aortas from New Zealand White (NZ) rabbits (43+/-10 versus 14+/-2 relative light units x min(-1) x mm(-2), n=9, P<0.05). After in vitro transduction with adenovirus containing the gene for CuZn-SOD (AdCMVCuZn-SOD) or extracellular SOD (AdCMVEC-SOD), endothelial O2-. levels in WHHL aortas were significantly reduced. Gene transfer of SOD to WHHL aortas, however, failed to improve the impaired relaxation to acetylcholine or calcium ionophore. By use of the oxidative fluorescent dye hydroethidine, an in situ assay indicated markedly increased generation of O2-. throughout the wall of WHHL aorta, especially within layers of smooth muscle. This finding was confirmed by demonstrating increased O2-. levels in smooth muscle cells cultured from WHHL aorta. We conclude that elevated O2-. levels in atherosclerotic vessels are not confined to the endothelium but occur throughout the vascular wall, including smooth muscle cells. Reduction in endothelial O2-. levels is not sufficient to improve endothelium-dependent relaxation. Generation of reactive oxygen species within the media may contribute to vasomotor dysfunction in atherosclerosis.

Chronic inflammatory diseases are associated with accelerated atherosclerosis and increased risk of cardiovascular diseases (CVD). As the pathogenesis of atherosclerosis is increasingly recognized as an inflammatory process, similarities between atherosclerosis and systemic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel diseases, lupus, psoriasis, spondyloarthritis and others have become a topic of interest. Endothelial dysfunction represents a key step in the initiation and maintenance of atherosclerosis and may serve as a marker for future risk of cardiovascular events. Patients with chronic inflammatory diseases manifest endothelial dysfunction, often early in the course of the disease. Therefore, mechanisms linking systemic inflammatory diseases and atherosclerosis may be best understood at the level of the endothelium. Multiple factors, including circulating inflammatory cytokines, TNF-α (tumor necrosis factor-α), reactive oxygen species, oxidized LDL (low density lipoprotein), autoantibodies and traditional risk factors directly and indirectly activate endothelial cells, leading to impaired vascular relaxation, increased leukocyte adhesion, increased endothelial permeability and generation of a pro-thrombotic state. Pharmacologic agents directed against TNF-α-mediated inflammation may decrease the risk of endothelial dysfunction and cardiovascular disease in these patients. Understanding the precise mechanisms driving endothelial dysfunction in patients with systemic inflammatory diseases may help elucidate the pathogenesis of atherosclerosis in the general population.

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...

Background-Recent reports demonstrate that multiple forms of cardiovascular stress, including pressure overload, chronic ischemia, and infarction-reperfusion injury, provoke an increase in autophagic activity in cardiomyocytes. However, nothing is known regarding molecular events that stimulate autophagic activity in stressed myocardium. Because autophagy is a highly conserved process through which damaged proteins and organelles can be degraded, we hypothesized that stress-induced protein aggregation is a proximal trigger of cardiomyocyte autophagy. Methods and Results-Here, we report that pressure overload promotes accumulation of ubiquitinated protein aggregates in the left ventricle, development of aggresome-like structures, and a corresponding induction of autophagy. To test for causal links, we induced protein accumulation in cultured cardiomyocytes by inhibiting proteasome activity, finding that aggregation of polyubiquitinated proteins was sufficient to induce cardiomyocyte autophagy. Furthermore, attenuation of autophagic activity dramatically enhanced both aggresome size and abundance, consistent with a role for autophagic activity in protein aggregate clearance. Conclusions-We conclude that protein aggregation is a proximal trigger of cardiomyocyte autophagy and that autophagic activity functions to attenuate aggregate/aggresome formation in heart. Findings reported here are the first to demonstrate that protein aggregation occurs in response to hemodynamic stress, situating pressure-overload heart disease in the category of proteinopathies. (Circulation. 2008;117:3070-3078.)

Non-phagocytic NAD(P)H oxidases have been impliinduced stimulation of NAD(P)H oxidase activity in nonphagocytic cell types could represent a mechanism by which oxidant-mediated injury is amplified in vivo and identify these NAD(P)H oxidase enzymes as therapeutic targets for the amelioration of the biological effects of chronic oxidant stress. EXPERIMENTAL PROCEDURESCell Culture-SMC were prepared from 250 to 300-g male HarlanSprague Dawley rats as previously described (10, 11). Aortic adventitial fibroblasts were prepared from C57 BL/6 and from gp91 phox knock-out mice (Jackson Lab, Bar Harbor, ME) as described (12, 13). The cells were grown in minimum essential media supplemented with 10% fetal bovine serum and penicillin (100 IU/ml) and streptomycin (100 g/ml) in a humidified atmosphere containing 5% CO 2 at 37°C. Stocks were subcultured at subconfluence by trypsinization. All experiments were performed on cells between passages 8 and 20 grown to 70% to 95% confluence in 24-well plates, 35-or 60-mm dishes, or 4-well chamber slides.Superoxide Measurement by Enhanced Chemiluminescence-Lucigenin luminescence measurements were carried out using 5 M lucigenin, a concentration which is unlikely to induce redox cycling (14). Cells were grown to subconfluence in 35-mm dishes and growth arrested in serum-free medium for 24 h. After exposure to H 2 O 2 at the indicated concentrations and times, cells were washed twice with PBS, and 5 M lucigenin was added. Immediately afterward, NADPH (0.1 mM) was added, and after dark adaptation, luminescence was measured every 15 s for 5 min in a luminometer (Zylux Corp). Changes in luminescence were expressed as relative luminescent units per 10 5 cells per minute. No changes in luminescence were observed in the absence of NADPH, and addition of H 2 O 2 to lucigenin in PBS in the absence of cells did not increase chemiluminescence. Some experiments were also performed with coelenterazine (5 M), a structurally distinct luminescent probe which reportedly is not capable of redox cycling via autoxidation (15). Data were analyzed by ANOVA followed by Bonferroni t testing.Intracellular O 2 . Determination by Confocal Microscopy-SMC grown on chamber slides were treated as indicated under "Results" and in the figure legends, washed, and incubated with dihydroethidium (HE, 5

Background— Angiotensin II (Ang II) contributes to vascular pathology in part by stimulating NADPH oxidase activity, leading to increased formation of superoxide (O 2 − ). We reported that O 2 − levels, NADPH oxidase activity, and expression of the p47 phox subunit of NADPH oxidase are increased in human abdominal aortic aneurysms (AAAs). Here, we tested the hypothesis that deletion of p47 phox will attenuate oxidative stress and AAA formation in Ang II–infused apoE −/− mice. Methods and Results— Male apoE −/− and apoE −/− p47 phox −/− mice received saline or Ang II (1000 ng · kg −1 · min −1 ) infusion for 28 days, after which abdominal aortic weight and maximal diameter were determined. Aortic tissues and blood were examined for parameters of aneurysmal disease and oxidative stress. Ang II infusion induced AAAs in 90% of apoE −/− versus 16% of apo −/− p47 phox −/− mice ( P <0.05). Abdominal aortic weight (14.1±3.2 versus 35.6±9.0 mg), maximal aortic diameter (1.5±0.2 versus 2.4±0.4 mm), aortic NADPH oxidase activity, and parameters of oxidative stress were reduced in apoE −/− p47 phox −/− mice compared with apoE −/− mice ( P <0.05). In addition, aortic macrophage infiltration and matrix metalloproteinase-2 activity were reduced in apoE −/− p47 phox −/− mice compared with apoE −/− mice. Deletion of p47 phox attenuated the pressor response to Ang II; however, coinfusion of phenylephrine with Ang II, which restored the Ang II pressor response, did not alter the protective effects of p47 phox deletion on AAA formation. Conclusions— Deletion of p47 phox attenuates Ang II–induced AAA formation in apoE −/− mice, suggesting that NADPH oxidase plays a critical role in AAA formation in this model.

BackgroundThe broad applicability of RNA aptamers as cell-specific delivery tools for therapeutic reagents depends on the ability to identify aptamer sequences that selectively access the cytoplasm of distinct cell types. Towards this end, we have developed a novel approach that combines a cell-based selection method (cell-internalization SELEX) with high-throughput sequencing (HTS) and bioinformatics analyses to rapidly identify cell-specific, internalization-competent RNA aptamers.Methodology/Principal FindingsWe demonstrate the utility of this approach by enriching for RNA aptamers capable of selective internalization into vascular smooth muscle cells (VSMCs). Several rounds of positive (VSMCs) and negative (endothelial cells; ECs) selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1) metrics of selection enrichment; and (2) pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs.Conclusions and SignificanceWe describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies.