NADPH Oxidase-derived Reactive Oxygen Species Research Articles

Microphthalmia-associated transcription factor modulates expression of NADPH oxidase type 4: A negative regulator of melanogenesis

How signaling via reactive oxygen species (ROS) influences skin pigmentation is unclear. We have investigated how NADPH oxidase-derived ROS modulates the expression of the key pigment “melanin” synthesizing enzymes in B16 mouse melanoma cells. A melanin inducer α-melanocyte-stimulating hormone (α-MSH) caused ROS generation that was inhibited by the NADPH oxidase inhibitor Diphenyleneiodonium (DPI) and was insensitive to antagonists of other ROS-producing enzyme systems including mitochondrial enzymes, cycloxygenase, and xanthine oxidase. NADPH oxidase 4 (Nox4) was found to be the most abundant isoform expressed in B16 cells, and its gene levels, as well as ROS generation, were enhanced by α-MSH. Interestingly, silencing Nox4 gene expression with Nox4 siRNA augmented melanin formation under basal conditions and after α-MSH stimulation, demonstrating that constitutive or stimulated Nox4-dependent ROS inhibits melanin formation. This process may be mediated by targeting the promoter region of a melanin synthesizing enzyme tyrosinase, because Nox4 siRNA enhanced tyrosinase promoter activity. Moreover, inhibition of tyrosinase mRNA expression in Nox4 siRNA-treated cells by blocking de novo mRNA and protein synthesis with actinomycin D and cycloheximide respectively indicates that Nox4 repression induces melanogenesis by increasing tyrosinase gene expression. We also found that α-MSH activated its downstream signal transducer microphthalmia-associated transcription factor (MITF) to stimulate Nox4 gene expression. We thus identified a novel mechanism by MITF signaling that in turn stimulates Nox4 to drive ROS generation, thereby repressing melanin synthesis. Such sequence of actions appears to act as an internal feedback mechanism to fine-tune melanin synthesis in response to exogenous challenges such as UV radiation.

NADPH Oxidase-derived Reactive Oxygen Species Increases Expression of Monocyte Chemotactic Factor Genes in Cultured Adipocytes

Excess glucose and free fatty acids delivered to adipose tissue causes local inflammation, which contributes to insulin resistance. Glucose and palmitate generate reactive oxygen species (ROS) in adipocytes, leading to monocyte chemotactic factor gene expression. Docosahexaenoate (DHA) has the opposite effect. In this study, we evaluated the potential sources of ROS in the presence of excess nutrients. Differentiated 3T3-L1 adipocytes were exposed to palmitate and DHA (250 μM) in either 5 or 25 mM glucose to evaluate the relative roles of mitochondrial electron transport and NADPH oxidases (NOX) as sources of ROS. Excess glucose and palmitate did not increase mitochondrial oxidative phosphorylation. However, glucose exposure increased glycolysis. Of the NOX family members, only NOX4 was expressed in adipocytes. Moreover, its activity was increased by excess glucose and palmitate and decreased by DHA. Silencing NOX4 inhibited palmitate- and glucose-stimulated ROS generation and monocyte chemotactic factor gene expression. NADPH, a substrate for NOX, and pentose phosphate pathway activity increased with glucose but not palmitate and decreased with DHA exposure. Inhibition of the pentose phosphate pathway by glucose-6-phosphate dehydrogenase inhibitors and siRNA suppressed ROS generation and monocyte chemotactic factor gene expression induced by both glucose and palmitate. Finally, both high glucose and palmitate induced NOX4 translocation into lipid rafts, effects that were blocked by DHA. Excess glucose and palmitate generate ROS via NOX4 rather than by mitochondrial oxidation in cultured adipocytes. NOX4 is regulated by both NADPH generated in the PPP and translocation of NOX4 into lipid rafts, leading to expression of monocyte chemotactic factors.

Depressed Baroreflex Sensitivity in Hypertensive Rats: A Role for Reactive Oxygen Species

One of the key mechanisms in controlling blood pressure in health and disease is the baroreflex. Baroreceptors located in the carotid sinuses and aortic arch detect changes in blood pressure and trigger reflex autonomic adjustments that buffer alterations in blood pressure. In pathological conditions such as hypertension, there is impairment in the autonomic control of blood pressure resulting in changes in baroreflex sensitivity [4-6]. Indeed, compelling evidence show that the baroreflex modulation of heart rate is impaired in spontaneously hypertensive rats and patients with primary hypertension [6,7]. We have recently demonstrated that acute antioxidant therapy improves baroreflex sensitivity in models of primary (essential) and secondary hypertension [5,6]. In a model of renovascular hypertension, the 2-kidney-1-clip (2K1C), we have shown that acute treatment with Vitamin C (150 mg/Kg, i.v.), a well-known antioxidant, restored the depressed baroreflex sensitivity in those hypertensive animals [5]. Furthermore, administration of apocynin (30 µg/Kg, i.v.), a NADPH oxidase inhibitor, improved baroreflex sensitivity in the 2K1C group. In the same study, autonomic blockade with either methylatropine or propranolol reduced the changes in heart rate to the same extent in normotensive and hypertensive groups suggesting that improved baroreflex sensitivity by antioxidants were mediated by improvement in autonomic function. Taken together, those data suggest that NADPH oxidase-derived reactive oxygen species are involved in the blunted baroreflex sensitivity in secondary hypertension and that acute scavenging of superoxide restores baroreflex sensitivity. Regarding primary hypertension, we have also demonstrated in spontaneously

Hypoxia induces Kv channel current inhibition by increased NADPH oxidase-derived reactive oxygen species

There is current discussion whether reactive oxygen species are up- or downregulated in the pulmonary circulation during hypoxia, from which sources (i.e., mitochondria or NADPH oxidases) they are derived, and what the downstream targets of ROS are. We recently showed that the NADPH oxidase homolog NOX4 is upregulated in hypoxia-induced pulmonary hypertension in mice and contributes to the vascular remodeling in pulmonary hypertension. We here tested the hypothesis that NOX4 regulates Kv channels via an increased ROS formation after prolonged hypoxia. We showed that (1) NOX4 is upregulated in hypoxia-induced pulmonary hypertension in rats and isolated rat pulmonary arterial smooth muscle cells (PASMC) after 3days of hypoxia, and (2) that NOX4 is a major contributor to increased reactive oxygen species (ROS) after hypoxia. Our data indicate colocalization of Kv1.5 and NOX4 in isolated PASMC. The NADPH oxidase inhibitor and ROS scavenger apocynin as well as NOX4 siRNA reversed the hypoxia-induced decrease in Kv current density whereas the protein levels of the channels remain unaffected by siNOX4 treatment. Determination of cysteine oxidation revealed increased NOX4-mediated Kv1.5 channel oxidation. We conclude that sustained hypoxia decreases Kv channel currents by a direct effect of a NOX4-derived increase in ROS.

Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

BackgroundADPH oxidase-derived reactive oxygen species (ROS) play important roles in redox homeostasis and signal transduction in endothelial cells (ECs). We previously demonstrated that c-Src plays a key role in VEGF-induced, ROS-dependent selective activation of PI3K-Akt but not PLCγ-1-ERK1/2 signaling pathways. The aim of the present study was to understand how VEGFR-2-c-Src signaling axis ‘senses’ NADPH oxidase-derived ROS levels and couples VEGF activation of c-Src to the redox state of ECs.Methodology/Principal FindingsUsing biotinylated probe that detects oxidation of cysteine thiol (cys-OH) in intracellular proteins, we demonstrate that VEGF induced oxidative modification in c-Src and VEGFR-2, and that reduction in ROS levels using siRNA against p47phox subunit of Rac1-dependent NADPH oxidase inhibited this phenomenon. Co-immunoprecipitation studies using human coronary artery ECs (HCAEC) showed that VEGF-induced ROS-dependent interaction between VEGFR-2 and c-Src correlated with their thiol oxidation status. Immunofluorescence studies using antibodies against internalized VEGFR-2 and c-Src demonstrated that VEGF-induced subcellular co-localization of these tyrosine kinases were also dependent on NADPH oxidsase-derived ROS.Conclusion/SignificanceThese results demonstrate that VEGF induces cysteine oxidation in VEGFR-2 and c-Src in an NADPH oxidase-derived ROS-dependent manner, suggesting that VEGFR-2 and c-Src can ‘sense’ redox levels in ECs. The data also suggest that thiol oxidation status of VEGFR-2 and c-Src correlates with their ability to physically interact with each other and c-Src activation. Taken together, these findings suggest that prior to activating downstream c-Src-PI3K-Akt signaling pathway, VEGFR-2-c-Src axis requires an NADPH oxidase-derived ROS threshold in ECs.

Apocynin attenuates oxidative stress and hypertension in young spontaneously hypertensive rats independent of ADMA/NO pathway

Both NADPH oxidase-derived reactive oxygen species (ROS) and asymmetric dimethylarginine (ADMA) are increased in hypertension. Apocynin, an NADPH oxidase inhibitor, could inhibit ROS, thus we tested whether apocynin can block NADPH oxidase and prevent increases of ADMA and blood pressure (BP) in spontaneously hypertensive rats (SHRs). SHRs and Wistar Kyoto (WKY) rats, aged 4 weeks, were assigned to four groups: untreated SHRs and WKY rats, SHRs and WKY rats that received 2.5 mM apocynin for 8 weeks. BP was significantly higher in SHRs compared to WKY rats, which was attenuated by apocynin. Apocynin prevented p47phox translocation in SHR kidneys, but not the increase of superoxide and H2O2. Additionally, apocynin did not protect SHRs against increased ADMA. Apocynin blocks NADPH oxidase to attenuate hypertension, but has little effect on the ADMA/nitric oxide (NO) pathway in young SHRs. The reduction of ROS and the preservation of NO simultaneously might be a better approach to restoring ROS–NO balance to prevent hypertension.

Reactive oxygen species drive herpes simplex virus (HSV)-1-induced proinflammatory cytokine production by murine microglia

BackgroundProduction of reactive oxygen species (ROS) and proinflammatory cytokines by microglial cells in response to viral brain infection contributes to both pathogen clearance and neuronal damage. In the present study, we examined the effect of herpes simplex virus (HSV)-1-induced, NADPH oxidase-derived ROS in activating mitogen-activated protein kinases (MAPKs) as well as driving cytokine and chemokine expression in primary murine microglia.MethodsOxidation of 2', 7'-dichlorodihydrofluorescin diacetate (H2DCFDA) was used to measure production of intracellular ROS in microglial cell cultures following viral infection. Virus-induced cytokine and chemokine mRNA and protein levels were assessed using real-time RT-PCR and ELISA, respectively. Virus-induced phosphorylation of microglial p38 and p44/42 (ERK1/2) MAPKs was visualized using Western Blot, and levels of phospho-p38 were quantified using Fast Activated Cell-based ELISA (FACE assay). Diphenyleneiodonium (DPI) and apocynin (APO), inhibitors of NADPH oxidases, were used to investigate the role of virus-induced ROS in MAPK activation and cytokine, as well as chemokine, production.ResultsLevels of intracellular ROS were found to be highly elevated in primary murine microglial cells following infection with HSV and the majority of this virus-induced ROS was blocked following DPI and APO treatment. Correspondingly, inhibition of NADPH oxidase also decreased virus-induced proinflammatory cytokine and chemokine production. In addition, microglial p38 and p44/42 MAPKs were found to be phosphorylated in response to viral infection and this activation was also blocked by inhibitors of NADPH oxidase. Finally, inhibition of either of these ROS-induced signaling pathways suppressed cytokine (TNF-α and IL-1β) production, while chemokine (CCL2 and CXCL10) induction pathways were sensitive to inhibition of p38, but not ERK1/2 MAPK.ConclusionsData presented herein demonstrate that HSV infection induces proinflammatory responses in microglia through NADPH oxidase-dependent ROS and the activation of MAPKs.

Angiotensin II induced catabolic effect and muscle atrophy are redox dependent

Angiotensin II (Ang II) causes skeletal muscle wasting via an increase in muscle catabolism. To determine whether the wasting effects of Ang II were related to its ability to increase NADPH oxidase-derived reactive oxygen species (ROS) we infused wild-type C57BL/6J or p47 phox −/− mice with vehicle or Ang II for 7 days. Superoxide production was increased 2.4-fold in the skeletal muscle of Ang II infused mice, and this increase was prevented in p47 phox −/− mice. Apocynin treatment prevented Ang II-induced superoxide production in skeletal muscle, consistent with Ang II increasing NADPH oxidase derived ROS. Ang II induced loss of body and skeletal muscle weight in C57BL/6J mice, whereas the reduction was significantly attenuated in p47 phox −/− animals. The reduction of skeletal muscle weight caused by Ang II was associated with an increase of proteasome activity, and this increase was completely prevented in the skeletal muscle of p47 phox −/− mice. In conclusion, Ang II-induced skeletal muscle wasting is in part dependent on NADPH oxidase derived ROS.

Inhibition of hypoxia-induced retinal neovascularization in mice with short hairpin RNA targeting Rac1, possibly via blockading redox signaling

NADPH oxidase-derived reactive oxygen species are involved in angiogenesis in vitro and regulated by ras-related C3 botulinum toxin substrate 1 (Rac1). This study has employed vector-based short hairpin RNA targeting Rac1 (Rac1–shRNA) to investigate the inhibitory effect on hypoxia-induced retinal neovascularization (RN) in vivo and the underlying mechanism. pSUPER–Rac1–shRNA was intravitreally injected into the mouse model of oxygen-induced retinopathy. RN was evaluated by FITC–dextran angiography and quantitated histologically. Expressions of Rac1, nuclear factor kappa B (NF-κB) subunit p65, hypoxia-inducible factor-1 alpha (HIF-1α), and vascular endothelial growth factor (VEGF) were determined by real-time quantitative RT-PCR and western blotting. After intravitreal administration of pSUPER–Rac1–shRNA, retinal Rac1 gene expression was reduced by 72% at postnatal day 17 (P17). Retinal flat mount and quantification of the neovascular nuclei demonstrated that RN was significantly inhibited. Meanwhile, the expression levels of NF-κB and HIF-1α, the redox-dependent transcription factors, were significantly downregulated. HIF-1α and its downstream gene VEGF were found to be significantly decreased at both transcriptional and translational levels. Our findings not only suggest that Rac1 may be involved in the process of RN in mouse oxygen-induced retinopathy via regulating the redox signaling, but may also provide a novel therapeutic target for hypoxia-induced retinal neovascular diseases.

NAD(P)H oxidase and renal epithelial ion transport

A fundamental requirement for cellular vitality is the maintenance of plasma ion concentration within strict ranges. It is the function of the kidney to match urinary excretion of ions with daily ion intake and nonrenal losses to maintain a stable ionic milieu. NADPH oxidase is a source of reactive oxygen species (ROS) within many cell types, including the transporting renal epithelia. The focus of this review is to describe the role of NADPH oxidase-derived ROS toward local renal tubular ion transport in each nephron segment and to discuss how NADPH oxidase-derived ROS signaling within the nephron may mediate ion homeostasis. In each case, we will attempt to identify the various subunits of NADPH oxidase and reactive oxygen species involved and the ion transporters, which these affect. We will first review the role of NADPH oxidase on renal Na(+) and K(+) transport. Finally, we will review the relationship between tubular H(+) efflux and NADPH oxidase activity.

Nox4 NADPH oxidase-derived reactive oxygen species, via endogenous carbon monoxide, promote survival of brain endothelial cells during TNF-α-induced apoptosis.

We investigated the role of reactive oxygen species (ROS) in promoting cell survival during oxidative stress induced by the inflammatory mediator tumor necrosis factor-α (TNF-α) in cerebral microvascular endothelial cells (CMVEC) from newborn piglets. Nox4 is the major isoform of NADPH oxidase responsible for TNF-α-induced oxidative stress and apoptosis in CMVEC. We present novel data that Nox4 NADPH oxidase-derived ROS also initiate a cell survival mechanism by increasing production of a gaseous antioxidant mediator carbon monoxide (CO) by constitutive heme oxygenase-2 (HO-2). TNF-α rapidly enhanced endogenous CO production in a superoxide- and NADPH oxidase-dependent manner in CMVEC with innate, but not with small interfering RNA (siRNA)-downregulated Nox4 activity. CORM-A1, a CO-releasing compound, inhibited Nox4-mediated ROS production and enhanced cell survival in TNF-α-challenged CMVEC. The ROS-induced CO-mediated survival mechanism requires functional interactions between the protein kinase B/Akt and extracellular signal-related kinase (ERK)/p38 MAPK signaling pathways activated by TNF-α. In Akt siRNA-transfected CMVEC and in cells with pharmacologically inhibited Akt, Erk1/2, and p38 mitogen-activated protein kinase (MAPK) activities, CORM-A1 was no longer capable of blocking Nox4 activation and apoptosis caused by TNF-α. Overall, Nox4 NADPH oxidase-derived ROS initiate both death and survival pathways in TNF-α-challenged CMVEC. The ROS-dependent cell survival pathway is mediated by an endogenous antioxidant CO, which inhibits Nox4 activation via a mechanism that includes Akt, ERK1/2, and p38 MAPK signaling pathways. The ability of CO to inhibit TNF-α-induced ERK1/2 and p38 MAPK activities in an Akt-dependent manner appears to be the key element in ROS-dependent survival of endothelial cells during TNF-α-mediated brain inflammatory disease.

Priming of Neutrophils and Differentiated PLB-985 Cells by Pathophysiological Concentrations of TNF-α Is Partially Oxygen Dependent

Activation of polymorphonuclear leukocytes (PMN) can be modulated to intermediate ‘primed’ states characterized by enhanced responsiveness to subsequent stimuli. We studied priming in response to TNF-α in human PMN and PLB-985 cells, a myeloid cell line differentiated to a neutrophilic phenotype (PLB-D). PMN generated reactive oxygen species (ROS) in response to TNF-α alone, and NADPH oxidase activity increased in response to stimulation with formyl-Met-Leu-Phe after priming. PLB-D cells also demonstrated priming of NADPH oxidase activity. Similar to priming by endotoxin, priming of the respiratory burst by TNF-α was predominantly oxygen dependent, with marked attenuation of ROS generation if primed anaerobically. Both PMN and PLB-D cells displayed significant increases in cell surface CD11b and gp91^phox expression after TNF-α priming and PMN displayed activation of MAPK. In response to TNF-α priming, neither mobilization of intracellular proteins nor activation of MAPK pathways was NADPH oxidase dependent. Priming of PMN and PLB-D cells by low TNF-α concentrations enhanced chemotaxis. These data demonstrate that pathophysiological concentrations of TNF-α elicit NADPH oxidase-derived ROS and prime cells for enhanced surface protein expression, activation of p38 and ERK1/2 MAPK pathways, and increased chemotaxis. Furthermore, PLB-D cells undergo TNF-α priming and provide a genetically modifiable model to study priming mechanisms.

NADPH oxidase 4 mediates TGF-β-induced smooth muscle α-actin via p38MAPK and serum response factor

In contrast to other cell types, vascular smooth muscle cells modify their phenotype in response to external signals. NADPH oxidase 4 (Nox4) is critical for maintenance of smooth muscle gene expression; however, the underlying mechanisms are incompletely characterized. Using smooth muscle α-actin (SMA) as a prototypical smooth muscle gene and transforming growth factor-β (TGF-β) as a differentiating agent, we examined Nox4-dependent signaling. TGF-β increases Nox4 expression and activity in human aortic smooth muscle cells (HASMC). Transfection of HASMC with siRNA against Nox4 (siNox4) abolishes TGF-β-induced SMA expression and stress fiber formation. siNox4 also significantly inhibits TGF-β-stimulated p38MAPK phosphorylation, as well as that of its substrate, mitogen-activated protein kinase-activated protein kinase-2. Moreover, the p38MAPK inhibitor SB-203580 nearly completely blocks the SMA increase induced by TGF-β. Inhibition of either p38MAPK or NADPH oxidase-derived reactive oxygen species impairs the TGF-β-induced phosphorylation of Ser103 on serum response factor (SRF) and reduces its transcriptional activity. Binding of SRF to myocardin-related transcription factor (MRTF) is also necessary, because downregulation of MRTF by siRNA abolishes TGF-β-induced SMA expression. Taken together, these data suggest that Nox4 regulates SMA expression via activation of a p38MAPK/SRF/MRTF pathway in response to TGF-β.

NADPH oxidase-derived reactive oxygen species: involvement in vascular physiology and pathology

Reactive oxygen species (ROS) are essential mediators of normal cell physiology. However, in the last few decades, it has become evident that ROS overproduction and/or alterations of the antioxidant system associated with inflammation and metabolic dysfunction are key pathological triggers of cardiovascular disorders. NADPH oxidases (Nox) represent a class of hetero-oligomeric enzymes whose primary function is the generation of ROS. In the vasculature, Nox-derived ROS contribute to the maintenance of vascular tone and regulate important processes such as cell growth, proliferation, differentiation, apoptosis, cytoskeletal organization, and cell migration. Under pathological conditions, excessive Nox-dependent ROS formation, which is generally associated with the up-regulation of different Nox subtypes, induces dysregulation of the redox control systems and promotes oxidative injury of the cardiovascular cells. The molecular mechanism of Nox-derived ROS generation and the means by which this class of molecule contributes to vascular damage remain debatable issues. This review focuses on the processes of ROS formation, molecular targets, and neutralization in the vasculature and provides an overview of the novel concepts regarding Nox functions, expression, and regulation in vascular health and disease. Because Nox enzymes are the most important sources of ROS in the vasculature, therapeutic perspectives to counteract Nox-dependent oxidative stress in the cardiovascular system are discussed.

AMP-activated Protein Kinase (AMPK) Negatively Regulates Nox4-dependent Activation of p53 and Epithelial Cell Apoptosis in Diabetes

Diabetes and high glucose (HG) increase the generation of NADPH oxidase-derived reactive oxygen species and induce apoptosis of glomerular epithelial cells (podocytes). Loss of podocytes contributes to albuminuria, a major risk factor for progression of kidney disease. Here, we show that HG inactivates AMP-activated protein kinase (AMPK), up-regulates Nox4, enhances NADPH oxidase activity, and induces podocyte apoptosis. Activation of AMPK blocked HG-induced expression of Nox4, NADPH oxidase activity, and apoptosis. We also identified the tumor suppressor protein p53 as a mediator of podocyte apoptosis in cells exposed to HG. Inactivation of AMPK by HG up-regulated the expression and phosphorylation of p53, and p53 acted downstream of Nox4. To investigate the mechanism of podocyte apoptosis in vivo, we used OVE26 mice, a model of type 1 diabetes. Glomeruli isolated from these mice showed decreased phosphorylation of AMPK and enhanced expression of Nox4 and p53. Pharmacologic activation of AMPK by 5-aminoimidazole-4-carboxamide-1-riboside in OVE26 mice attenuated Nox4 and p53 expression. Administration of 5-aminoimidazole-4-carboxamide-1-riboside also prevented renal hypertrophy, glomerular basement thickening, foot process effacement, and podocyte loss, resulting in marked reduction in albuminuria. Our results uncover a novel function of AMPK that integrates metabolic input to Nox4 and provide new insight for activation of p53 to induce podocyte apoptosis. The data indicate the potential therapeutic utility of AMPK activators to block Nox4 and reactive oxygen species generation and to reduce urinary albumin excretion in type 1 diabetes.

FERONIA receptor-like kinase regulates RHO GTPase signaling of root hair development

Plant RHO GTPases (RAC/ROPs) mediate multiple extracellular signals ranging from hormone to stress and regulate diverse cellular processes important for polarized cell growth, differentiation, development, reproduction, and responses to the environment. They shuttle between the GDP-bound inactive state and the GTP-bound activated state and their activation is predominantly mediated by a family of guanine nucleotide exchange factors (GEFs) referred to as ROPGEFs. Using the Arabidopsis ROPGEF1 as bait, we identified members of a receptor-like kinase (RLK) family as potential upstream regulators for RAC/ROP signaling. NADPH oxidase-derived reactive oxygen species (ROS) are emerging as important regulators for growth and development and play a crucial role in mediating RAC/ROP-regulated root hair development, a polarized cell growth process. We therefore screened T-DNA insertion mutants in these RLKs for root hair defects and found that mutations in one of them, At3g51550 encoding the FERONIA (FER) receptor-like kinase, induced severe root hair defects. We show that the fer phenotypes correlated with reduced levels of active RAC/ROPs and NADPH oxidase-dependent, auxin-regulated ROS accumulation in roots and root hairs and that up-regulating RAC/ROP signaling in fer countered the mutant phenotypes. Taken together, these observations strongly support FER as an upstream regulator for the RAC/ROP-signaled pathway that controls ROS-mediated root hair development. Moreover, FER was pulled down by ROP2 GTPase in a guanine nucleotide-regulated manner implying a dynamic signaling complex involving FER, a ROPGEF, and a RAC/ROP.

Acute superoxide scavenging restores depressed baroreflex sensitivity in renovascular hypertensive rats

In some pathological conditions such as hypertension, there is an impairment in the autonomic control of blood pressure resulting in changes in baroreflex sensitivity. In the present study we tested the hypothesis that acute superoxide scavenging would restore the reduced baroreflex sensitivity in renovascular hypertension. Male Wistar rats underwent 2-Kidney-1-Clip (2K1C) or sham surgery and were maintained untouched for six weeks to develop hypertension. After six weeks, animals from the 2K1C group were hypertensive when compared to the sham group (165 ± 9 vs. 108 ± 7 mmHg, P < 0.05). As a proof of principle for the hypertension model adopted, animals from the 2K1C group presented increased non-clipped kidney and cardiac mass index and reduced clipped kidney mass index. Regarding baroreflex, 2K1C rats presented diminished baroreflex sensitivity when compared to the sham group (2K1C + saline: −1.61 ± 0.15 vs. sham + saline: −2.79 ± 0.24 bpm mm Hg −1, p < 0.05). Moreover, acute administration of Vitamin C (150 mg/Kg, i.v.) restored baroreflex sensitivity in 2K1C rats (2K1C + Vit C: −3.08 ± 0.37 vs. 2K1C + saline: −1.61 ± 0.15 bpm mm Hg −1, p < 0.05). Furthermore, administration of apocynin (30 μg/Kg, i.v.), a NADPH oxidase inhibitor, also improved baroreflex sensitivity in the 2K1C group (2K1C + apocynin: −2.81 ± 0.24 vs. 2K1C + saline: −1.61 ± 0.15 bpm mm Hg −1, p < 0.05). In addition, autonomic blockade with either methylatropine or propranolol reduced the changes in heart rate to the same extent in all groups suggesting that improved baroreflex sensitivity by antioxidants were mediated by improvement in autonomic function. Taken together, these data suggest that NADPH oxidase-derived reactive oxygen species are involved in the blunted baroreflex sensitivity in renovascular hypertension and that acute scavenging of superoxide restores baroreflex sensitivity.

Endothelium-Dependent Coronary Vasodilatation Requires NADPH Oxidase-Derived Reactive Oxygen Species

To determine the functional significance of physiological reactive oxygen species (ROS) levels in endothelium-dependent nitric oxide (NO)-mediated coronary vasodilatation. Endothelium-derived NO is important in regulating coronary vascular tone. Excess ROS have been shown to reduce NO bioavailability, resulting in endothelial dysfunction and coronary diseases. NADPH oxidase is a major source of ROS in endothelial cells (ECs). By using lucigenin-based superoxide production and dichlorfluorescein diacetate (DCFH-DA) fluorescence-activated cell sorter assays, we found that mouse heart ECs from NADPH oxidase-knockdown (p47(phox-/-)) animals have reduced NADPH oxidase activity (>40%) and ROS levels (>30%) compared with wild-type mouse heart ECs. Surprisingly, a reduction in ROS did not improve coronary vasomotion; rather, endothelium-dependent vascular endothelial growth factor-mediated coronary vasodilatation was reduced by greater than 50% in p47(phox-/-) animals. Western blots and L-citrulline assays showed a significant reduction in Akt/protein kinase B (PKB) and endothelial NO synthase phosphorylation and NO synthesis, respectively, in p47(phox-/-) coronary vessels and mouse heart ECs. Adenoviral expression of constitutively active endothelial NO synthase restored vascular endothelial growth factor-mediated coronary vasodilatation in p47(phox-/-) animals. Endothelium-dependent vascular endothelial growth factor regulation of coronary vascular tone may require NADPH oxidase-derived ROS to activate phosphatidylinositol 3-kinase-Akt-endothelial NO synthase axis.

Oxidative Stress and Endothelial Dysfunction: Say NO to Cigarette Smoking!

Smoking is a significant independent risk factor for cardiovascular disease and is a leading cause of structural and functional alterations of the cardiovascular system. Increasing evidence supports the hypothesis that oxidative stress and endothelial dysfunction are the fundamental pathophysiological mechanisms linking cigarette smoking to cardiovascular disease. The cardiovascular system is a rich source of NADPH oxidase-derived reactive oxygen species, which under pathological conditions play a fundamental role in vascular damage. Endothelium-derived nitric oxide (NO) plays a major role in the regulation of vascular tone, structure, and function, and endothelial dysfunction could be considered the first step in the pathogenesis of atherosclerosis and cardiovascular disease. Indeed, the bioavailability of NO is modulated by reactive oxygen species that degrade NO, uncouple NO synthase, and inhibit synthesis. Reduced bioavailability of NO and consequent endothelial dysfunction are involved in the initiation, progression and complications of atherosclerosis and also are predictive of future cardiovascular events. Thus, although data from clinical trials exploring the role of antioxidants on cardiovascular risk and disease are equivocal as yet, the role of oxidative stress in cardiovascular disease is an important area of research, which is likely to continue to be fruitful. This review focuses on possible interactions between oxidative stress, endothelial dysfunction and cigarette smoking--favouring the atherosclerotic process and cardiovascular disease--also focusing on the potential role for antioxidants in the prevention of adverse cardiovascular outcomes.

Paroxetine Prevents Loss of Nigrostriatal Dopaminergic Neurons by Inhibiting Brain Inflammation and Oxidative Stress in an Experimental Model of Parkinson’s Disease

The present study examined whether the antidepressant paroxetine promotes the survival of nigrostriatal dopaminergic (DA) neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. MPTP induced degeneration of nigrostriatal DA neurons and glial activation as visualized by tyrosine hydroxylase, macrophage Ag complex-1, and/or glial fibrillary acidic protein immunoreactivity. Real-time PCR, Western blotting, and immunohistochemistry showed upregulation of proinflammatory cytokines, activation of microglial NADPH oxidase and astroglial myeloperoxidase, and subsequent reactive oxygen species production and oxidative DNA damage in the MPTP-treated substantia nigra. Treatment with paroxetine prevented degeneration of nigrostriatal DA neurons, increased striatal dopamine levels, and improved motor function. This neuroprotection afforded by paroxetine was associated with the suppression of astroglial myeloperoxidase expression and/or NADPH oxidase-derived reactive oxygen species production and reduced expression of proinflammatory cytokines, including IL-1beta, TNF-alpha, and inducible NO synthase, by activated microglia. The present findings show that paroxetine may possess anti-inflammatory properties and inhibit glial activation-mediated oxidative stress, suggesting that paroxetine and its analogues may have therapeutic value in the treatment of aspects of Parkinson's disease related to neuroinflammation.