Introduction: Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) serves as a signaling molecule and promotes endothelial cell proliferation and migration as well as protecting against cell death. However, the role of NOX4 in EPC function is not completely understood. Methods: EPCs were isolated from human saphenous vein and mammary artery discarded during bypass surgery. NOX4 gene and protein expression in EPCs were measured by real time-PCR and Western blot analysis respectively. NOX4 gene expression was inhibited using an adenoviral vector expressing human NOX4 shRNA (Ad-NOX4i). H2O2 production was measured by Amplex red assay. EPC migration was evaluated using a transwell migration assay. EPC proliferation and viability were measured using trypan blue counts. Results: Inhibition of NOX4 using Ad-NOX4i reduced Nox4 gene and protein expression as well as H2O2 formation in EPCs. Inhibition of NOX4-derived H2O2 decreased both proliferation and migration of EPCs. Interestingly, pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) decreased NOX4 expression and reduced survival of EPCs. However, the survival of EPCs was further diminished by TNF-α in NOX4-knockdown cells, suggesting that NOX4 has a protective role in EPCs. Conclusion: These findings suggest that NOX4-type NADPH oxidase is important for proliferation and migration functions of EPCs and protects against pro-inflammatory cytokine induced EPC death. These properties of NOX4 may facilitate the efficient function of EPCs which is vital for successful neovascularization.

Nox5 was the last member of the Nox enzyme family to be identified. Functionally distinct from the other Nox isoforms, our understanding of its physiological significance has been hampered by the absence of Nox5 in mouse and rat genomes. Nox5 is present in the genomes of other species such as the rabbit that have broad utility as models of cardiovascular disease. However, the mRNA sequence, characteristics, and functional analysis of rabbit Nox5 has not been fully defined and were the goals of the current study.

Objective: AIP1 expression is downregulated in human atherosclerotic plaques and global deletion of AIP1 in mice exacerbates atherosclerosis in ApoE-KO mouse models. However, the direct role of AIP1 in endothelium, vascular remodeling and associated vascular diseases has not been determined. Approach and Results: We used endothelial cell (EC)-specific AIP1-deficient (AIP1-ECKO) mice to define the role of AIP1 in vascular remodeling and intima-media thickening in a mouse carotid artery ligation model characterized by both neointimal hyperplasia and inward vessel remodeling. Compared to WT littermates, AIP1-ECKO mice had 2.2-fold larger intima area and 4.4-fold thicker intima as measured by intima/media ratio in arteries with more proliferating vascular smooth muscle cells (VSMCs) at week 2-4 post-injury. Increased reactive oxygen species (ROS) in endothelium at early time points induced inflammation and vessel dysfunction in AIP1-ECKO prior to VSMC accumulations. Moreover, knockdown of AIP1 in human EC enhanced ROS generation which was attenuated by co-silencing of NOX2. Mechanistically, AIP1 via its proline-rich region binds to the SH3 domain of cytosolic subunit p47phox to disrupt formation of an active NOX2 complex, attenuating ROS production. Conclusion: Our study supports that AIP1 regulates vascular remodeling with intima-media thickening by suppressing endothelial NOX2-dependent oxidative stress. Highlights: •In a carotid ligation model, endothelial cell (EC)-specific AIP1-deficient (AIP1-ECKO) mice had much larger media area, thicker vessel wall and augmented neointima formation.•Increased production of reactive oxygen species in vascular EC at early time points concomitant with vessel dysfunction in AIP1-ECKO.•AIP1 via its proline-rich region binds to the SH3 domain of cytosolic subunit p47phox to disrupt formation of an active NOX2 complex, attenuating ROS production.

Oral treatment of apolipoprotein E-knockout (ApoE-KO) mice with the putative sirtuin 1 (SIRT1) activator resveratrol led to a reduction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the heart. In contrast, the SIRT1 inhibitor EX527 enhanced the superoxide production in isolated human polymorphonuclear granulocytes. In human monocytic THP-1 cells, phorbol ester-stimulated superoxide production was enhanced by inhibitors of histone deacetylases (HDACs; including quisinostat, trichostatin A (TSA), PCI34051, and tubastatin A) and decreased by inhibitors of histone acetyltransferases [such as garcinol, curcumin, and histone acetyltransferase (HAT) Inhibitor II]. These results indicate that protein acetylation and deacetylation may represent crucial mechanisms regulating NADPH oxidase-mediated superoxide production. In cell-free systems, incubation of recombinant Rac1 with SIRT1 resulted in decreased Rac1 acetylation. Mass spectrometry analyses identified lysine 166 (K166) in Rac1 as a residue targeted by SIRT1. Deacetylation of Rac1 by SIRT1 markedly reduced the interaction of Rac1 with p67phox in in vitro assays. Computational modeling analyses revealed that K166 deacetylation of Rac1 led to a 5-fold reduction in its binding affinity to guanosine-5'-triphosphate, and a 21-fold decrease in its binding potential to p67phox. The latter is crucial for Rac1-mediated recruitment of p67phox to the membrane and for p67phox activation. In conclusion, both SIRT1 and non-sirtuin deacetylases play a role in regulating NADPH oxidase activity. Rac1 can be directly deacetylated by SIRT1 in a cell-free system, leading to an inhibition of Rac1-p67phox interaction. The downstream targets of non-sirtuin deacetylases are still unknown. The in vivo significance of these findings needs to be investigated in future studies.

Background: Muscle wasting is a debilitating phenotype associated with chronic heart failure (CHF). We have previously demonstrated that angiotensin II (AII) directly induces muscle wasting in mice through the activation of NADPH oxidase (Nox). In this study, we tested the hypothesis that deficiency of NADPH oxidase 4 (Nox4), a major source of oxidative stress, ameliorates AII-induced muscle wasting through the regulation of redox balance. Methods and Results: Nox4 knockout (KO) and wild-type (WT) mice were used. At baseline, there were no differences in physical characteristics between the WT and KO mice. Saline (vehicle, V) or AII was infused via osmotic minipumps for 4 weeks, after which, the WT + AII mice showed significant increases in Nox activity and NOX4 protein compared with the WT + V mice, as well as decreases in body weight, gastrocnemius muscle weight, and myocyte cross-sectional area. These changes were significantly attenuated in the KO + AII mice (27 ± 1 vs. 31 ± 1 g, 385 ± 3 vs. 438 ± 13 mg, and 1,330 ± 30 vs. 2281 ± 150 μm2, respectively, all P < 0.05). The expression levels of phospho-Akt decreased, whereas those of muscle RING Finger-1 (MuRF-1) and MAFbx/atrogin-1 significantly increased in the WT + AII mice compared with the WT + V mice. Furthermore, nuclear factor erythroid-derived 2-like 2 (Nrf2) and the expression levels of Nrf2-regulated genes significantly decreased in the WT + AII mice compared with the WT + V mice. These changes were significantly attenuated in the KO + AII mice (P < 0.05). Conclusion: Nox4 deficiency attenuated AII-induced muscle wasting, partially through the regulation of Nrf2. The Nox4-Nrf2 axis may play an important role in the development of AII-induced muscle wasting.

Elevated concentrations of sphingomyelinase (SMase) have been detected in a variety of diseases. SMase has been shown to increase muscle derived oxidants and decrease skeletal muscle force; however, the sub-cellular site of oxidant production has not been elucidated. Using redox sensitive biosensors targeted to the mitochondria and NADPH oxidase (Nox2), we demonstrate that SMase increased Nox2-dependent ROS and had no effect on mitochondrial ROS in isolated FDB fibers. Pharmacological inhibition and genetic knockdown of Nox2 activity prevented SMase induced ROS production and provided protection against decreased force production in the diaphragm. In contrast, genetic overexpression of superoxide dismutase within the mitochondria did not prevent increased ROS production and offered no protection against decreased diaphragm function in response to SMase. Our study shows that SMase induced ROS production occurs in specific sub-cellular regions of skeletal muscle; however, the increased ROS does not completely account for the decrease in muscle function.

Chronic intermittent hypoxia (CIH) causes upper airway muscle dysfunction. We hypothesized that the superoxide generating NADPH oxidase (NOX) is upregulated in CIH-exposed muscle causing oxidative stress. Adult male Wistar rats were exposed to intermittent hypoxia (5% O2 at the nadir for 90 s followed by 210 s of normoxia), for 8 h per day for 14 days. The effect of CIH exposure on the expression of NOX subunits, total myosin and 4-hydroxynonenal (4-HNE) protein adducts in sternohyoid muscle was determined by western blotting and densitometry. Sternohyoid protein free thiol and carbonyl group contents were determined by 1D electrophoresis using specific fluorophore probes. Aconitase and glutathione reductase activities were measured as indices of oxidative stress. HIF-1α content and key oxidative and glycolytic enzyme activities were determined. Contractile properties of sternohyoid muscle were determined ex vivo in the absence and presence of apocynin (putative NOX inhibitor). We observed an increase in NOX 2 and p47 phox expression in CIH-exposed sternohyoid muscle with decreased aconitase and glutathione reductase activities. There was no evidence, however, of increased lipid peroxidation or protein oxidation in CIH-exposed muscle. CIH exposure did not affect sternohyoid HIF-1α content or aldolase, lactate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase activities. Citrate synthase activity was also unaffected by CIH exposure. Apocynin significantly increased sternohyoid force and power. We conclude that CIH exposure upregulates NOX expression in rat sternohyoid muscle with concomitant modest oxidative stress but it does not result in a HIF-1α-dependent increase in glycolytic enzyme activity. Constitutive NOX activity decreases sternohyoid force and power. Our results implicate NOX-dependent reactive oxygen species in CIH-induced upper airway muscle dysfunction which likely relates to redox modulation of key regulatory proteins in excitation-contraction coupling.

Patients with heart failure (HF) have diaphragm abnormalities that contribute to disease morbidity and mortality. Studies in animals suggest that reactive oxygen species (ROS) cause diaphragm abnormalities in HF. However, the effects of HF on ROS sources, antioxidant enzymes, and protein oxidation in the diaphragm of humans is unknown. NAD(P)H oxidase, especially the Nox2 isoform, is an important source of ROS in the diaphragm. Our main hypothesis was that diaphragm from patients with HF have heightened Nox2 expression and p47phox phosphorylation (marker of enzyme activation) that is associated with elevated protein oxidation. We collected diaphragm biopsies from patients with HF and brain-dead organ donors (controls). Diaphragm mRNA levels of Nox2 subunits were increased 2.5-4.6-fold over controls (p < 0.05). Patients also had increased protein levels of Nox2 subunits (p47phox, p22phox, and p67phox) and total p47phox phosphorylation, while phospho-to-total p47phox levels were unchanged. The antioxidant enzyme catalase was increased in patients, whereas glutathione peroxidase and superoxide dismutases were unchanged. Among markers of protein oxidation, carbonyls were increased by ~40% (p < 0.05) and 4-hydroxynonenal and 3-nitrotyrosines were unchanged in patients with HF. Overall, our findings suggest that Nox2 is an important source of ROS in the diaphragm of patients with HF and increases in levels of antioxidant enzymes are not sufficient to maintain normal redox homeostasis. The net outcome is elevated diaphragm protein oxidation that has been shown to cause weakness in animals.

Kinin B1 receptor (B1R) enhanced superoxide anion ([Formula: see text]) production in the vasculature of diabetic rats. This study investigates the induction and distribution of B1R in diabetic blood vessels and addresses the hypothesis that B1R is co-localized with NADPH oxidase (NOX1 and NOX2) and produces its activation via protein kinase C (PKC). Diabetes was induced in rats with streptozotocin (STZ 65 mg.kg-1, i.p.). Two weeks later, the production of [Formula: see text] was measured in aorta rings in response to the B1R agonist (Sar[D-Phe8]-des-Arg9-BK, 20 μM) by the method of lucigenin-enhanced chemiluminescence. Various inhibitors were added (10 μM) to block PKCtotal (Ro-31-8220), PKCβ1/2 (LY333531), or NADPH oxidase (Diphenyleneiodonium). The cellular localization of B1R was studied in the aorta, popliteal artery, and renal glomerulus/arteries by immunofluorescence and confocal microscopy with markers of endothelial cells (anti-RECA-1), macrophages (anti-CD11), vascular smooth muscle cells (anti-SMA), and NADPH oxidase (anti-NOX1 and NOX2). Although B1R was largely distributed in resistant vessels, it was sparsely expressed in the aorta's endothelium. The greater basal production of [Formula: see text] in STZ-diabetic aorta was significantly enhanced by the B1R agonist (15-45 min). The peak response to the agonist (30 min) was inhibited by all inhibitors. Immunofluorescent staining for B1R, NOX1, and NOX2 was significantly increased in endothelial cells, vascular smooth muscle cells, and macrophages of STZ-diabetic aorta on which they were found co-localized. Data showed that B1R enhanced [Formula: see text] by activating vascular NADPH oxidase through PKCβ1/2. This was substantiated by the cellular co-localization of B1R with NOX1 and NOX2 and opens the possibility that B1R-enhanced oxidative stress is derived from vascular and infiltrating immune cells in diabetes.

Obstructive sleep apnea (OSA), manifested by exposure to chronic intermittent hypoxia (CIH) and excess production of reactive oxygen species (ROS) in the airways, is associated with hyperreactive airway diseases. ROS, particularly when created by NADPH oxidase, are known to sensitize lung vagal C fibers (LVCFs), which may contribute to airway hypersensitivity pathogenesis. We investigated whether CIH augments the reflex and afferent responses of LVCFs to chemical stimulants and the roles of ROS and NADPH oxidase in such airway hypersensitivity. Rats were exposed to room air (RA) or CIH with/without daily treatment with MnTMPyP (a superoxide anion scavenger), apocynin (an NADPH oxidase inhibitor), or vehicle. At 16 h after their last exposure, intravenous capsaicin, adenosine, or α,β-methylene-ATP evoked an augmented apneic response in anesthetized rats with 14-days CIH exposure, compared to anesthetized rats with 14-days RA exposure. The augmented apneic responses to these LVCF stimulants were abolished by bilateral vagotomy or perivagal capsaicin treatment, which block LVCFs neural conduction and were significantly suppressed by treatment with MnTMPyP or apocynin, but not vehicle. Electrophysiological studies revealed that 14-days CIH exposure potentiated the responses of LVCFs to these stimulants. This effect was inhibited by treatment with MnTMPyP or apocynin treatment and was not seen in rats who received 7-days of CIH exposure. Biochemical analysis indicated that 14-days CIH exposure increased both lung lipid peroxidation, which is indicative of oxidative stress, and expression of the p47(phox) subunit in the membrane fraction of lung tissue, which is an index of NADPH oxidase activation. The former was prevented by treatment with either MnTMPyP or apocynin, while the later was prevented by treatment with apocynin only. These results suggest that 14-days CIH exposure sensitizes LVCFs in rats, leading to an exaggerated reflex and afferent responses to stimulants and that this sensitization is mediated via ROS generated by NADPH oxidase.

Reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide, are reported to contribute to the dynamic regulation of contractility in various arterial preparations, however, the situation in pressurized, myogenically active resistance arteries is much less clear. In the present study, we have utilized established pharmacological inhibitors of NADPH oxidase activity to examine the potential contribution of ROS to intrinsic myogenic contractility in adult Sprague-Dawley rat resistance arteries and responses to vasoactive agents acting via the endothelium (i.e., acetylcholine, SKA-31) or smooth muscle (i.e., sodium nitroprusside, phenylephrine). In cannulated and pressurized cremaster skeletal muscle and middle cerebral arteries, the NOX inhibitors 2-acetylphenothiazine (2-APT) and VAS2870, selective for NOX1 and NOX2, respectively, evoked concentration-dependent inhibition of basal myogenic tone in a reversible and irreversible manner, respectively, whereas the non-selective inhibitor apocynin augmented myogenic contractility. The vasodilatory actions of 2-APT and VAS2870 occurred primarily via the vascular endothelium and smooth muscle, respectively. Functional responses to established endothelium-dependent and -independent vasoactive agents were largely unaltered in the presence of either 2-APT or apocynin. In cremaster arteries from Type 2 Diabetic (T2D) Goto-Kakizaki rats with endothelial dysfunction, treatment with either 2-APT or apocynin did not modify stimulus-evoked vasoactive responses, but did affect basal myogenic tone. These same NOX inhibitors produced robust inhibition of total NADPH oxidase activity in aortic tissue homogenates from control and T2D rats, and NOX isozymes 1, 2 and 4, along with superoxide dismutase 1, were detected by qPCR in cremaster arteries and aorta from both species. Based on the diverse effects that we observed for established, chemically distinct NOX inhibitors, the functional contribution of vascular NADPH oxidase activity to stimulus-evoked vasoactive signaling in myogenically active, small resistance arteries remains unclear.

Obstructive sleep apnea, similar to intermittent hypoxia (IH) during sleep, is associated with laryngeal airway hyperreactivity (LAH). IH-induced laryngeal oxidative stress may contribute to LAH, but the underlying mechanism remains unknown. Conscious rats were subjected to repetitive 75 s cycles of IH for 7 or 14 consecutive days. Reflex apneic responses to laryngeal provocations with chemical stimulants were measured to reflect laryngeal reflex reactivity. Compared with control rats, rats exposed to IH for 14 days, but not for 7 days, displayed enhanced apneic response to laryngeal chemical stimulants. The apneic response to chemical stimulants, but not to mechanical stimulation, was totally abolished by perineural capsaicin treatment of superior laryngeal nerves (SLNs) or by the sectioning of the SLNs, suggesting that the reflex was mediated through capsaicin-sensitive SLNs. Daily intraperitoneal administration of N-acetyl-L-cysteine [NAC, a reactive oxygen species (ROS) scavenger], apocynin (an inhibitor of NADPH oxidase) or YC-1 (an inhibitor of HIF-1α), but not their vehicles, largely attenuated this augmented apneic response in 14 days IH rats. Laryngeal lipid peroxidation (an index of oxidative stress) was elevated in 7 days IH rats and 14 days IH rats, and was abolished by any of these three pharmacologic interventions. The protein expression of HIF-1α (an index of HIF-1 activation) and p47phox subunit in the membrane fraction (an index of NADPH oxidase activation) in the laryngeal tissues increased in 14 days IH rats; the former was reduced by NAC, whereas the latter was inhibited by YC-1. These results suggest that 14 days of IH exposure may sensitize capsaicin-sensitive SLNs and result in exaggerated apneic reflex response to laryngeal chemical stimulants. This phenomenon depends on the action of HIF-1α-mediated, NADPH oxidase-derived ROS.

Background and Aims: The angiotensin-(1-7)/angiotensin-converting enzyme 2/Mas receptor axis counter-regulates the detrimental effects of angiotensin II. Beneficial effects of angiotensin-(1-7), including anti-inflammation, oxidative stress reduction, and anti-thrombosis, have been reported. Previous studies documented that ramipril decreased thrombin generation in human hypertension and that the anti-thrombotic effects of captopril and losartan were angiotensin-(1-7)-dependent, suggesting an interaction between thrombin and angiotensin-(1-7). However, it is not clear whether angiotensin-(1-7) can alleviate the endothelial phenotypic changes induced by thrombin. We have previously documented cytoskeleton remodeling, cell adhesion, and cell migration as dominant altered phenotypes in thrombin-stimulated human aortic endothelial cells (HAECs). In this study, we investigated whether angiotensin-(1-7) can modulate thrombin-induced phenotypic changes. Furthermore, we investigated whether NAPDH oxidase 5 (Nox5)-produced reactive oxygen species (ROS) play a significant role in angiotensin-(1-7)-mediated phenotypic changes. Methods: HAECs were pretreated with 100 nM angiotensin-(1-7) for 1 h, followed by stimulation with 2 units/mL thrombin for different times. Immunofluorescent assay, monocyte adhesion assay, wound-healing assay, ROS assay, real-time PCR, Western blotting, and Nox5 siRNA transfection were conducted. HAECs were pretreated with the ROS scavenger N-acetylcysteine (NAC) to determine whether thrombin-induced phenotypic changes depended on ROS production. Results: Angiotensin-(1-7) prevented thrombin-induced actin cytoskeleton derangements, monocyte adhesion, and migratory impairment. Nox5 siRNA transfection confirmed that thrombin-induced Nox5 expression stimulated ROS production and increased HO-1/NQO-1/ICAM-1/VCAM-1 gene expression, all of which were decreased by angiotensin-(1-7). Phenotypic changes induced by thrombin were prevented by NAC pretreatment. Conclusion: Angiotensin-(1-7) prevents actin cytoskeleton derangement, monocyte adhesion, and migration impairment induced by thrombin via downregulation of ROS production. In addition, thrombin-induced Nox5 expression is involved in the production of ROS, and angiotensin-(1-7) decreases ROS through its inhibitory effect on Nox5 expression.

Despite popular usage of medicinal plants, their effects as cardiovascular protective agents have not been totally elucidated. We hypothesized that treatment with aqueous extract from Prosopis strombulifera (AEPs) and Tessaria absinthioides (AETa), Argentinian native plants, produces antioxidant effects on vascular smooth muscle cells (VSMCs) and attenuates atherogenesis on apolipoprotein E-knockout (ApoE-KO) mice. In VSMCs, both extracts (5-40 μg/ml) inhibited 10% fetal calf serum-induced cell proliferation, arrested cell in G2/M phase, reduced angiotensin II-induced reactive oxygen species (ROS) generation, and decreased NADPH oxidase subunit expression. In ApoE-KO mice, extracts significantly reduced triglycerides and lipid peroxidation [plasma thiobarbituric acid reactive substances (TBARS)], increased plasma total antioxidant status (TAS), and improved glutathione peroxidase activity in the liver. Under high-fat diet (HFD), both extracts were able to inhibit O2 - generation in the aortic tissue and caused a significant regression of atheroma plaques (21.4 ± 1.6% HFD group vs. 10.2 ± 1.2%∗ AEPs group and 14.3 ± 1.0%∗ AETa group; ∗ p < 0.01). Consumption of AEPs and AETa produces antioxidant/antimitogenic/anti-atherosclerotic effects, and their use may be beneficial as a complementary strategy regarding cardiovascular disease therapies.

Salusin-β, a multifunctional vasoactive peptide, has a potentially important function in the pathological development of hypertension. However, the exact functional role of salusin-β and the underlying mechanism in this process are still not fully understood. The current study aimed to investigate the effects of silencing salusin-β on vascular function and vascular remodeling, as well as its signaling pathways in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY).

The participation of reactive oxygen species (ROS) generated by NOX1 and NOX2/NADPH oxidase has been documented during inflammatory pain. However, the molecular mechanism involved in their activation is not fully understood. We reported earlier a key role of Cyclin-dependent kinase 5 (Cdk5) during inflammatory pain. In particular, we demonstrated that TNF-α increased p35 expression, a Cdk5 activator, causing Cdk5-mediated TRPV1 phosphorylation followed by an increment in Ca2+ influx in nociceptive neurons and increased pain sensation. Here we evaluated if Cdk5 activation mediated by p35 transfection in HEK293 cells or by TNF-α treatment in primary culture of nociceptive neurons could increase ROS production. By immunofluorescence we detected the expression of catalytic subunit (Nox1 and Nox2) and their cytosolic regulators (NOXO1 and p47phox) of NOX1 and NOX2/NADPH oxidase complexes, and their co-localization with Cdk5/p35 in HEK293 cells and in nociceptive neurons. By using a hydrogen peroxide sensor, we detected a significant increase of ROS production in p35 transfected HEK293 cells as compared with control cells. This effect was significantly blocked by VAS2870 (NADPH oxidase inhibitor) or by roscovitine (Cdk5 activity inhibitor). Also by using another ROS probe named DCFH-DA, we found a significant increase of ROS production in nociceptive neurons treated with TNF-α and this effect was also blocked by VAS2870 or by roscovitine treatment. Interestingly, TNF-α increased immunodetection of p35 protein and NOX1 and NOX2/NADPH oxidase complexes in primary culture of trigeminal ganglia neurons. Finally, the cytosolic regulator NOXO1 was significantly translocated to plasma membrane after TNF-α treatment and roscovitine blocked this effect. Altogether these results suggest that Cdk5 activation is implicated in the ROS production by NOX1 and NOX2/NADPH oxidase complexes during inflammatory pain.

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia with patients dying frequently of stroke. In view of the unclear etiologies of AF and a potential role of oxidative stress, the present study examined cardiac reactive oxygen species production and NADPH oxidase (NOX) expression in AF patients.

Toll-like receptor (TLR) 4 was originally thought to be the sole pattern recognition receptor for lipopolysaccharide (LPS). Transient receptor potential ankyrin 1 (TRPA1), a Ca2+-permeant channel, has been suggested as a non-TLR receptor membrane-bound sensor of LPS. We recently reported that TRPA1 is expressed in lung epithelial cells (LECs) and mediates lung inflammation induced by cigarette smoke. However, the role of TRPA1 in LPS-induced lung inflammation has not been conclusively defined, and its underlying cellular mechanisms remain unclear. In this study, our in vitro results showed that LPS sequentially produced a cascade of events, including the elevation of intracellular Ca2+, the activation of NADPH oxidase, increase in intracellular reactive oxygen species (ROS), the activation of mitogen-activated protein kinase (MAPK)/nuclear factor-kB (NF-κB) signaling, and the induction of IL-8. The increase in intracellular Ca2+ was inhibited by HC030031 (a TRPA1 antagonist) but was unaffected by TAK-242 (a TLR-4 inhibitor). The activation of NADPH oxidase was prevented by its inhibitor apocynin, EGTA (an extracellular Ca2+ chelator), and HC030031. The increase in intracellular ROS was attenuated by apocynin, N-acetyl-cysteine (NAC, a ROS scavenger), EGTA, and HC030031. The activation of the MAPK/NF-κB signaling was halted by NAC, EGTA, and HC030031. IL-8 induction was suppressed by HC030031 and TRPA1 siRNA, and further reduced by the combination of HC030031 and TAK-242. Our in vivo studies showed that trpa1-/- mice exhibited a reduced level of LPS-induced lung inflammation compared with wild-type mice as evidenced by the alleviations of increases in vascular permeability, inflammatory cell infiltration, inflammatory cytokine levels, oxidative stress, and MAPK signaling activation. Thus, in LECs, LPS may activate TRPA1 resulting in an increase in Ca2+ influx. The increased intracellular Ca2+ leads to NADPH oxidase activation, which causes an increase in intracellular ROS. The intracellular ROS activates the MAPK/NF-κB signaling resulting in IL-8 induction. This mechanism may possibly be at work to induce lung inflammation in mice.

The present study examined whether angiotensin II (Ang II) mediates the pressor effect through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS)-mitogen-activated protein kinase (MAPK) signaling in the glutamatergic neurons of the rostral ventrolateral medulla (RVLM) in stress-induced hypertensive rats (SIHR). The SIHR model was established using electric foot-shocks combined with noises for 15 days. We observed that Ang II type 1 receptor (AT1R) and the glutamatergic neurons co-localized in the RVLM of SIHR. Furthermore, glutamate levels in the intermediolateral column of the spinal cord were higher in SIHR than in controls. Microinjection of Ang II into the RVLM of SIHR activated stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), extracellular signal-regulated protein kinase (ERK) 1/2, and p38MAPK. Compared with controls, the activation of SAPK/JNK, ERK1/2, p38MAPK, and ROS in the RVLM were higher in SIHR, an effect that was blocked by an NADPH oxidase inhibitor (apocynin) and an AT1R antagonist (candesartan). RVLM microinjection of apocynin or a SAPK/JNK inhibitor (SP600125), but not an ERK1/2 inhibitor (U0126) or a p38MAPK inhibitor (SB203580), decreased AT1R mRNA and mean arterial blood pressure (MABP) in SIHR. The increase of AT1R protein expression and MABP was inhibited by intracerebroventricular infusion (ICV), for 14 days, of SP600125, but not U0126 or SB203580 in SIHR. We conclude that Ang II modulates the pressor effect through AT1R-dependent ROS-SAPK/JNK signaling in glutamatergic neurons in the RVLM of SIHR.

Cigarette smoking causes chronic lung inflammation that is mainly regulated by redox-sensitive pathways. Our previous studies have demonstrated that cigarette smoke (CS) activates reactive oxygen species (ROS)-sensitive mitogen-activated protein kinases (MAPKs)/nuclear factor-κB (NF-κB) signaling resulting in induction of lung inflammation. Eicosapentaenoic acid (EPA), a major type of omega-3 polyunsaturated fatty acid, is present in significant amounts in marine-based fish and fish oil. EPA has been shown to possess antioxidant and anti-inflammatory properties in vitro and in vivo. However, whether EPA has similar beneficial effects against CS-induced lung inflammation remains unclear. Using a murine model, we show that subchronic CS exposure for 4 weeks caused pulmonary inflammatory infiltration (total cell count in bronchoalveolar lavage fluid (BALF), 11.0-fold increase), increased lung vascular permeability (protein level in BALF, 3.1-fold increase), elevated levels of chemokines (11.4-38.2-fold increase) and malondialdehyde (an oxidative stress biomarker; 2.0-fold increase) in the lungs, as well as lung inflammation; all of these CS-induced events were suppressed by daily supplementation with EPA. Using human bronchial epithelial cells, we further show that CS extract (CSE) sequentially activated NADPH oxidase (NADPH oxidase activity, 1.9-fold increase), increased intracellular levels of ROS (3.0-fold increase), activated both MAPKs and NF-κB, and induced interleukin-8 (IL-8; 8.2-fold increase); all these CSE-induced events were inhibited by pretreatment with EPA. Our findings suggest a novel role for EPA in alleviating the oxidative stress and lung inflammation induced by subchronic CS exposure in vivo and in suppressing the CSE-induced IL-8 in vitro via its antioxidant function and by inhibiting MAPKs/NF-κB signaling.