Upregulation Of NADPH Oxidase Research Articles

Iron sucrose accelerates early atherogenesis by increasing superoxide production and upregulating adhesion molecules in CKD.

High-dose intravenous iron supplementation is associated with adverse cardiovascular outcomes in patients with CKD, but the underlying mechanism is unknown. Our study investigated the causative role of iron sucrose in leukocyte-endothelium interactions, an index of early atherogenesis, and subsequent atherosclerosis in the mouse remnant kidney model. We found that expression levels of intracellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and adhesion of U937 cells increased in iron-treated human aortic endothelial cells through upregulated NADPH oxidase (NOx) and NF-κB signaling. We then measured mononuclear-endothelial adhesion and atherosclerotic lesions of the proximal aorta in male C57BL/6 mice with subtotal nephrectomy, male apolipoprotein E-deficient (ApoE(-/-)) mice with uninephrectomy, and sham-operated mice subjected to saline or parenteral iron loading. Iron sucrose significantly increased tissue superoxide production, expression of tissue cell adhesion molecules, and endothelial adhesiveness in mice with subtotal nephrectomy. Moreover, iron sucrose exacerbated atherosclerosis in the aorta of ApoE(-/-) mice with uninephrectomy. In patients with CKD, intravenous iron sucrose increased circulating mononuclear superoxide production, expression of soluble adhesion molecules, and mononuclear-endothelial adhesion compared with healthy subjects or untreated patients. In summary, iron sucrose aggravated endothelial dysfunction through NOx/NF-κB/CAM signaling, increased mononuclear-endothelial adhesion, and exacerbated atherosclerosis in mice with remnant kidneys. These results suggest a novel causative role for therapeutic iron in cardiovascular complications in patients with CKD.

Effect of ketones on ROS, NADPH oxidases, and monocyte adhesion in endothelial cells (767.8)

Cardiovascular disease (CVD) incidence is higher in Type 1 diabetic (T1D) patients. Along with hyperglycemia, T1D is associated with hyperketonemia, a condition with elevated blood levels of acetoacetate (AA) and 3‐β‐hydroxybutyrate (BHB). It is not known whether hyperketonemia per se contributes to the excess CVD in T1D. This study investigates the hypothesis that ketones upregulate NADPH oxidases (NOX) and ROS and therby increase monocyte‐endothelial adhesion. HUVEC were treated with AA or BHB (0‐8 mM) ± high‐glucose (25 mM) for 24 h. Results show that AA, but not BHB, increased ROS production, NOX activity, and NOX4 expression in HUVEC. Pretreatment with DPI (20 μM , inhibitor of NOX) abolished the effect of AA on ROS production. This suggests that AA effect on ROS production is mediated through the NOX. To further examine the role of NOX, we determined the effect of ketones in NOX4 knockdown (KD) HUVEC. There were significantly lower levels of ROS (65% vs. 20%), ICAM‐1 (42% vs. 23%), and monocyte adhesion (22% vs. 1%) in AA (4 mM) treated NOX4 KD cells compared with control cells. Effect of AA on ROS and ICAM‐1 was higher in cells knocked down for GCLC (that induces GSH deficiency). This suggests that ketone‐induced increase in ICAM‐1 and adhesion is mediated by the upregulation of NOX and ROS production. This study provides evidence for a biochemical mechanism for the role of hyperketonemia in the development of CVD in T1D.Grant Funding Source: Supported by NIH and Malcolm Feist Chair in Diabetes

Increased oxidative stress enhances endocannabinoid tone (671.13)

Cardiovascular disease (CVD) has been characterized as a chronic inflammatory disease that has become prevalent in industrialized societies. NADPH oxidase contributes to atherosclerosis through the activation of macrophages leading to the internalization of oxidized low‐density lipoproteins (oxLDL). Chronic inflammation is caused in part by monocytes invading the vasculature leading to the formation of lipid‐laden macrophage foam cells with increased flux of oxygen/nitrogen radicals and subsequent chemical modification of extracellular LDL. Endogenous cannabinoids (eCB), such as 2‐arachidonoylglycerol (2‐AG), may be a link between oxidative stress and atherosclerosis. We hypothesize that 2‐AG biosynthesis is enhanced following CD36 ligation by oxLDL and subsequent activation of diacylglycerol lipase β (DAGLβ), the key biosynthetic enzyme of 2‐AG, via upregulated NADPH oxidase activity. Treatment of murine J774 macrophages and human THP1 macrophages/monocytes with either extracellular xanthine/xanthine oxidase or phorbol 12‐myristate 13‐acetate (PMA) caused an increase in superoxide (O2•−) levels and enhanced 2‐AG biosynthesis (2.3‐fold and 2.8‐fold, respectively) compared to vehicle controls. These treatments were not cytotoxic. These data suggest a significant positive correlation between oxygen radical flux and 2‐AG biosynthesis in macrophages. Increased 2‐AG biosynthesis may be an adaptive response to elevated oxidative stress because of antioxidant and anti‐inflammatory actions associated with this bioactive lipid. Therefore, the pathogenesis of atherosclerosis within the vessel wall intima may be reduced by enhancing eCB tone.Grant Funding Source: Supported in part by NIH R15ES015348‐02

Upregulation of mitochondrial Nox4 mediates TGF-β-induced apoptosis in cultured mouse podocytes

Injury to podocytes leads to the onset of chronic renal diseases characterized by proteinuria. Elevated transforming growth factor (TGF)-β in kidney tissue is associated with podocyte damage that ultimately results in apoptosis and detachment. We investigated the proapoptotic mechanism of TGF-β in immortalized mouse podocytes. Exogenous TGF-β1-induced podocyte apoptosis through caspase-3 activation, which was related to elevated ROS levels generated by selective upregulation of NADPH oxidase 4 (Nox4). In mouse podocytes, Nox4 was predominantly localized to mitochondria, and Nox4 upregulation by TGF-β1 markedly depolarized mitochondrial membrane potential. TGF-β1-induced ROS production and caspase activation were mitigated by an antioxidant, the Nox inhibitor diphenyleneiodonium, or small interfering RNA for Nox4. A TGF-β receptor I blocker, SB-431542, completely reversed the changes triggered by TGF-β1. Knockdown of either Smad2 or Smad3 prevented the increase of Nox4 expression, ROS generation, loss of mitochondrial membrane potential, and caspase-3 activation by TGF-β1. These results suggest that TGF-β1-induced mitochondrial Nox4 upregulation via the TGF-β receptor-Smad2/3 pathway is responsible for ROS production, mitochondrial dysfunction, and apoptosis, which may at least in part contribute to the development and progression of proteinuric glomerular diseases such as diabetic nephropathy.

Non-Essential Role for TLR2 and Its Signaling Adaptor Mal/TIRAP in Preserving Normal Lung Architecture in Mice

Myeloid differentiation factor 88 (MyD88) and MyD88-adaptor like (Mal)/Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) play a critical role in transducing signals downstream of the Toll-like receptor (TLR) family. While genetic ablation of the TLR4/MyD88 signaling axis in mice leads to pulmonary cell death and oxidative stress culminating in emphysema, the involvement of Mal, as well as TLR2 which like TLR4 also signals via MyD88 and Mal, in the pathogenesis of emphysema has not been studied. By employing an in vivo genetic approach, we reveal here that unlike the spontaneous pulmonary emphysema which developed in Tlr4−/− mice by 6 months of age, the lungs of Tlr2−/− mice showed no physiological or morphological signs of emphysema. A more detailed comparative analysis of the lungs from these mice confirmed that elevated oxidative protein carbonylation levels and increased numbers of alveolar cell apoptosis were only detected in Tlr4−/− mice, along with up-regulation of NADPH oxidase 3 (Nox3) mRNA expression. With respect to Mal, the architecture of the lungs of Mal−/− mice was normal. However, despite normal oxidative protein carbonylation levels in the lungs of emphysema-free Mal−/− mice, these mice displayed increased levels of apoptosis comparable to those observed in emphysematous Tlr4−/− mice. In conclusion, our data provide in vivo evidence for the non-essential role for TLR2, unlike the related TLR4, in maintaining the normal architecture of the lung. In addition, we reveal that Mal differentially facilitates the anti-apoptotic, but not oxidant suppressive, activities of TLR4 in the lung, both of which appear to be essential for TLR4 to prevent the onset of emphysema.

Induction of senescence and identification of differentially expressed genes in tomato in response to monoterpene.

Monoterpenes, which are among the major components of plant essential oils, are known for their ecological roles as well for pharmaceutical properties. Geraniol, an acyclic monoterpene induces cell cycle arrest and apoptosis/senescence in various cancer cells and plants; however, the genes involved in the process and the underlying molecular mechanisms are not well understood. In this study, we demonstrate that treatment of tomato plants with geraniol results in induction of senescence due to a substantial alteration in transcriptome. We have identified several geraniol-responsive protein encoding genes in tomato using suppression subtractive hybridization (SSH) approach. These genes comprise of various components of signal transduction, cellular metabolism, reactive oxygen species (ROS), ethylene signalling, apoptosis and DNA damage response. Upregulation of NADPH oxidase and antioxidant genes, and increase in ROS level after geraniol treatment point towards the involvement of ROS in geraniol-mediated senescence. The delayed onset of seedling death and induced expression of geraniol-responsive genes in geraniol-treated ethylene receptor mutant (Nr) suggest that geraniol-mediated senescence involves both ethylene dependent and independent pathways. Moreover, expression analysis during tomato ripening revealed that geraniol-responsive genes are also associated with the natural organ senescence process.

P47 Hydrogen Sulfide reduces hypertension, proteinuria and renal damage

Background Hypertension and proteinuria are important mediators of renal damage. Despite therapeutic intervention, many renal patients ultimately progress to end stage renal disease. Hydrogen sulfide (H 2 S) is, in addition to nitric oxide and carbon monoxide, acknowledged as the third gasotransmitter sharing many physiological functions with these gases. It has vasodilatory, anti-inflammatory and anti-oxidant properties, making it an attractive candidate for pharmacological use in renal disease. We therefore investigated the protective properties of the H 2 S donor sodium hydrosulfide (NaHS) in Angiotensin II (AngII)-induced renal disease in rats. Methods Male Sprague Dawley rats were infused with AngII (435 ng/kg/min) or saline (control) for three weeks via subcutaneously placed osmotic minipumps. At placement of the pumps, rats were randomized to two different treatment regimens (vehicle and NaHS 5.6 mg/kg/day) and treated with intra-peritoneal injections twice a day during the three weeks of AngII infusion (n = 7/group). Control animals (n = 6) received vehicle injections only. Twenty-four hour urine was collected weekly. After three weeks, intra-aortic blood pressure was measured under anesthesia and subsequently rats were sacrificed. Upon sacrifice, plasma and kidneys were collected. Renal tissue was studied for tubular epithelial damage (Kidney Injury Molecule-1 (KIM-1)), interstitial fibrosis (collagen III), and upregulation of NADPH oxidase (NOX2). In an ex vivo experiment, rat kidneys (n = 4) in an isolated perfused kidney (IPK) setup were pre-contracted with phenylephrine (PE) (1uM) and subjected to 1 mM NaHS for 1 min. Before and after NaHS treatment intra-renal pressure was measured. Results Compared to controls, AngII infusion caused a 48% increase of systolic blood pressure (SBP) (142 vs 212 mmHg, p vs 346 mg/24 h, p vs 1.4 ml/min, p vs 212 mmHg, p vs 346 mg/24 h, p vs 1.4 ml/min, p Conclusion AngII induced hypertension, proteinuria and subsequent renal damage are markedly decreased by treatment with NaHS. Blood pressure regulation is a possible mechanism of renal tissue protection, but anti-proteinuric, anti-inflammatory and anti-oxidant effects may also play a role. Our data show that H 2 S might be a valuable addition to the already existing anti-hypertensive and reno-protective therapies.

CCAAT/enhancer-binding proteins mediate interferon gamma-induced upregulation of NADPH oxidase in human aortic smooth muscle cells

Aim: In atherosclerosis, oxidative stress-induced vascular Smooth Muscle Cells (SMC) dysfunction is partially mediated by upregulated NADPH oxidases (Nox); the mechanisms of enzyme regulation are not entirely defined. CCAAT/Enhancer-Binding Proteins (C/EBP) regulate the expression of inflammatory and immune genes, the differentiation of immigrating and resident cells, eventually influencing atherosclerotic plaque development. We aimed at elucidating the role of C/EBP in the regulation of Nox in SMC exposed to proinflammatory conditions. Methods and results: Human aortic SMC were exposed to interferon gamma (IFNγ) for up to 24-h. Lucigenin-enhanced chemiluminescence, real-time PCR, Western blot, promoter-luciferase reporter analysis, and chromatin immunoprecipitation assays were employed to investigate Nox regulation. The expression and function of various C/EBP isoforms were controlled by pharmacological and molecular interventions. IFNγ dose-dependently induced Nox activity/expression, nuclear translocation of C/EBPα, C/EBPβ, and C/EBPδ, C/EBP protein expression levels. Silencing of C/EBPα, C/EBPβ or C/EBPδ greatly reduced the IFNγ-induced upregulation of Nox activity and gene/protein expression. Pharmacological inhibition of p38MAPK and ERK1/2 diminished significantly the IFNγ-induced C/EBP nuclear translocation and Nox activity/expression. In silico analysis indicated the existence of typical C/EBP within Nox1, Nox4, and Nox5 promoters. Transient overexpression of C/EBPα, C/EBPβ or C/EBPδ upregulated the luciferase level directed by the promoters of all SMC-expressed Nox isoforms. Chromatin immunoprecipitation demonstrated the physical interaction of C/EBPα, C/EBPβ or C/EBPδ proteins with the Nox1-5 promoters. Moreover, transactivation assays demonstrated the existence of a crosstalk among C/EBP, AP1, and STAT1/3 transcription factors. Conclusions: C/EBP transcription factors are important regulators of Nox enzymes by both direct and indirect pathways in IFNγ-exposed SMC. Activation of C/EBP may induce excessive Nox-derived reactive oxygen species formation, further contributing to phenotypic alterations of SMCs in atherosclerosis. Acknowledgments: Work supported by Romanian Ministry of Education, and Research (PNII-TE 65/2010, PNII-TE 26/2011, PNII-IDEI 107/2011).

Resveratrol Protects Vascular Endothelial Cells from High Glucose–Induced Apoptosis through Inhibition of NADPH Oxidase Activation–Driven Oxidative Stress

Hyperglycemia-induced oxidative stress has been implicated in diabetic vascular complications in which NADPH oxidase is a major source of reactive oxygen species (ROS) generation. Resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG)-induced oxidative stress in endothelial cells. We aimed to examine whether HG-induced NADPH oxidase activation and ROS production contribute to glucotoxicity to endothelial cells and the effect of resveratrol on glucotoxicity. Using a murine brain microvascular endothelial cell line bEnd3, we found that NADPH oxidase inhibitor (apocynin) and resveratrol both inhibited HG-induced endothelial cell apoptosis. HG-induced elevation of NADPH oxidase activity and production of ROS were inhibited by apocynin, suggesting that HG induces endothelial cell apoptosis through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunit Nox1 but not Nox2, Nox4, and p22(phox) expression through NF-κB activation, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced endothelial cell apoptosis through inhibiting HG-induced NF-κB activation, NADPH oxidase activity elevation, and ROS production. HG induces endothelial cell apoptosis through NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide new potential therapeutic targets against brain vascular complications of diabetes.

MicroRNA-25-dependent up-regulation of NADPH oxidase 4 (NOX4) mediates hypercholesterolemia-induced oxidative/nitrative stress and subsequent dysfunction in the heart

Diet-induced hypercholesterolemia leads to oxidative/nitrative stress and subsequent myocardial dysfunction. However, the regulatory role of microRNAs in this phenomenon is unknown. We aimed to investigate, whether hypercholesterolemia-induced myocardial microRNA alterations play a role in the development of oxidative/nitrative stress and in subsequent cardiac dysfunction. Male Wistar rats were fed with 2% cholesterol/0.25% cholate-enriched or standard diet for 12weeks. Serum and tissue cholesterol levels were significantly elevated by cholesterol-enriched diet. Left ventricular end-diastolic pressure was significantly increased in cholesterol-fed rats both in vivo and in isolated perfused hearts, indicating diastolic dysfunction. Myocardial expression of microRNAs was affected by cholesterol-enriched diet as assessed by microarray analysis. MicroRNA-25 showed a significant down-regulation as detected by microarray analysis and QRT-PCR. In silico target prediction revealed NADPH oxidase 4 (NOX4) as a putative target of microRNA-25. NOX4 protein showed significant up-regulation in the hearts of cholesterol-fed rats, while NOX1 and NOX2 remained unaffected. Cholesterol-feeding significantly increased myocardial oxidative/nitrative stress as assessed by dihydroethidium staining, protein oxidation assay, and nitro-tyrosine ELISA, respectively. Direct binding of microRNA-25 mimic to the 3′ UTR region of NOX4 was demonstrated using a luciferase reporter assay. Transfection of a microRNA-25 mimic into primary cardiomyocytes decreased superoxide production, while a microRNA-25 inhibitor resulted in an up-regulation of NOX4 protein and an increase in oxidative stress that was attenuated by the NADPH oxidase inhibitor diphenyleneiodonium. Here we demonstrated for the first time that hypercholesterolemia affects myocardial microRNA expression, and by down-regulating microRNA-25 increases NOX4 expression and consequently oxidative/nitrative stress in the heart. We conclude that hypercholesterolemia-induced microRNA alterations play an important role in the regulation of oxidative/nitrative stress and in consequent myocardial dysfunction.

Abstract 5406: Transcriptional upregulation of NADPH Oxidase 1 (NOX1) by Interleukin-4 (IL-4) enhances tumor cell proliferation and reactive oxygen production in human colon cancer cells.

Abstract Reactive oxygen species (ROS) produced by the family of NOX genes play a critical role in cytokine-mediated signal transduction in both normal and tumor tissue. Human colon cancers, compared to adjacent normal tissues, overexpress NOX1; and premalignant, chronic inflammatory conditions of the colon (ulcerative colitis, Crohn's disease) are associated with NOX up-regulation. However, a mechanistic understanding of how cytokines might increase ROS production and NOX1 expression in colon cancer remains unclear. We screened a panel of human colon cancer cells with a series of pro-inflammatory cytokines to evaluate their effects on NOX1 expression and function, as well as cell proliferation. Cytokine IL-4 significantly up-regulated NOX1 expression (but not other members of the NOX family) in HT-29 (as well as other) colon cancer cells while increasing O2• production and growth; decreasing NOX1 expression by 90% in stable, NOX1-shRNA-containing HT-29 clones blocked IL-4-related enhancement of cell growth, NOX1 expression, or ROS production. IL-4 up-regulated and activated the GATA3 transcription factor in addition to the Jak1-Stat6 pathway in HT-29 cells. GATA3 played an important role in the transcriptional up-regulation of NOX1 by IL-4; RNAi-mediated silencing of Jak1, Stat6, or GATA3 expression attenuated IL-4-induced NOX1 up-regulation in HT-29 cells. Transient overexpression of GATA3 cDNA in the presence of IL-4 resulted in a further increase in NOX1 expression with enhanced ROS activity beyond IL-4 treatment alone. Using an 1139 bp segment of upstream sequence from the transcription start site of the human NOX1 promoter as a promoter-reporter assay, we found that IL-4 treatment increased NOX1 reporter gene transcription 4-fold in HT-29 cells. DNA sequence analysis of this upstream promoter suggests the presence of four putative GATA3 binding, cis-active elements that may be sufficient for maximal IL-4-enahanced activity of the NOX1 promoter. Ongoing mutational analysis of the NOX1 promoter will further define the role of GATA3 in the control of NOX1 transcriptional up-regulation by IL-4. In summary, our results support a role for IL-4 in producing a NOX1-dependent pro-oxidant milieu that could increase the growth potential of colon cancer cells. Citation Format: Han Liu, Agnes Juhasz, Guojian Jiang, Smitha Antony, Jennifer L. Meitzler, Yongzhong Wu, Jiamo Lu, Krishnendu Roy, James H. Doroshow. Transcriptional upregulation of NADPH Oxidase 1 (NOX1) by Interleukin-4 (IL-4) enhances tumor cell proliferation and reactive oxygen production in human colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5406. doi:10.1158/1538-7445.AM2013-5406

SIRT1 inhibits NADPH oxidase activation and protects endothelial function in the rat aorta: Implications for vascular aging

Vascular aging is characterized by up-regulation of NADPH oxidase, oxidative stress and endothelial dysfunction. Previous studies demonstrate that the activity of the evolutionarily conserved NAD+-dependent deacetylase SIRT1 declines with age and that pharmacological activators of SIRT1 confer significant anti-aging cardiovascular effects. To determine whether dysregulation of SIRT1 promotes NADPH oxidase-dependent production of reactive oxygen species (ROS) and impairs endothelial function we assessed the effects of three structurally different inhibitors of SIRT1 (nicotinamide, sirtinol, EX527) in aorta segments isolated from young Wistar rats. Inhibition of SIRT1 induced endothelial dysfunction, as shown by the significantly reduced relaxation to the endothelium-dependent vasodilators acetylcholine and the calcium ionophore A23187. Endothelial dysfunction induced by SIRT1 inhibition was prevented by treatment of the vessels with the NADPH oxidase inhibitor apocynin or superoxide dismutase. Inhibition of SIRT1 significantly increased vascular superoxide production, enhanced NADPH oxidase activity, and mRNA expression of its subunits p22phox and NOX4, which were prevented by resveratrol. Peroxisome proliferator-activated receptor-α (PPARα) activation mimicked the effects of resveratrol while PPARα inhibition prevented the effects of this SIRT1 activator. SIRT1 co-precipitated with PPARα and nicotinamide increased the acetylation of the PPARα coactivator PGC-1α, which was suppressed by resveratrol. In conclusion, impaired activity of SIRT1 induces endothelial dysfunction and up-regulates NADPH oxidase-derived ROS production in the vascular wall, mimicking the vascular aging phenotype. Moreover, a new mechanism for controlling endothelial function after SIRT1 activation involves a decreased PGC-1α acetylation and the subsequent PPARα activation, resulting in both decreased NADPH oxidase-driven ROS production and NO inactivation.

Prostacyclin Signalling Promotes Cytoprotection and Angiogenesis Via Up-Regulation of NADPH Oxidase 4

Prostacyclin Signalling Promotes Cytoprotection and Angiogenesis Via Up-Regulation of NADPH Oxidase 4

Oxidized LDL Triggers Pro-Oncogenic Signaling in Human Breast Mammary Epithelial Cells Partly via Stimulation of MiR-21

Dyslipidemia and obesity are primary risk factors for the development of atherosclerosis and are also epidemiologically linked to increased susceptibility to a variety of cancers including breast cancer. One of the prominent features of dyslipidemia is enhanced production of oxidized LDL (ox-LDL), which has been shown to be implicated in key steps of atherogenesis including inflammatory signaling and proliferation of vascular cells. In this study we analyzed the effects of ox-LDL in human mammary epithelial cells (MCF10A). MCF10A cells avidly internalized dil-ox-LDL and exhibited increased proliferative response to ox-LDL within the range of 1–50 µg/ml in a dose-dependent manner. Treatment of cells with 20 µg/ml ox-LDL for 2 and 12 hours was associated with upregulation of LOX-1 and CD36 scavenger receptors while MSR1 and CXLC16 receptors did not change. Ox-LDL-treated cells displayed significant upregulation of NADPH oxidases (subunits P22phox and P47phox), lipoxygenases-12 and -15, and cytoplasmic, but not mitochondrial, SOD. Ox-LDL also triggered phosphorylation of IκBα coupled with nuclear translocation of NF-κB and stimulated p44/42 MAPK, PI3K and Akt while intracellular PTEN (PI3K/Akt pathway inhibitor and target of miR-21) declined. Quantitative PCR revealed increased expression of hsa-miR-21 in ox-LDL treated cells coupled with inhibition of miR-21 target genes. Further, transfection of MCF10A cells with miR-21 inhibitor prevented ox-LDL mediated stimulation of PI3K and Akt. We conclude that, similarly to vascular cells, mammary epithelial cells respond to ox-LDL by upregulation of proliferative and pro-inflammatory signaling. We also report for the first time that part of ox-LDL triggered reactions in MCF10A cells is mediated by oncogenic hsa-miR-21 through inhibition of its target gene PTEN and consequent activation of PI3K/Akt pathway.

Targeting NADPH Oxidase Decreases Oxidative Stress in the Transgenic Sickle Cell Mouse Penis

Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) ), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Relative to hemi mice, SCD increased (P<0.05) protein expression of NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) , 4-HNE-modified proteins, induced eNOS uncoupling, and reduced Gpx1 expression in the penis. Apocynin treatment of sickle mice reversed (P<0.05) the abnormalities in protein expressions of p47(phox) , gp91(phox) (but not p67(phox) ) and 4-HNE, but only slightly (P>0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a potential target for improving vascular function in the SCD mouse penis.

NADPH Oxidase Mediates Depressive Behavior Induced by Chronic Stress in Mice

Stress is a potent risk factor for depression, yet the underlying mechanism is not clearly understood. In the present study, we explored the mechanism of development and maintenance of depression in a stress-induced animal model. Mice restrained for 2 h daily for 14 d showed distinct depressive behavior, and the altered behavior persisted for >3 months in the absence of intervention. Acute restraint induced a surge of oxidative stress in the brain, and stress-induced oxidative stress progressively increased with repetition of stress. In vitro, the stress hormone glucocorticoid generated superoxide via upregulation of NADPH oxidase. Consistently, repeated restraints increased the expression of the key subunits of NADPH oxidase, p47phox and p67phox, in the brain. Moreover, stressed brains markedly upregulated the expression of p47phox to weak restress evoked in the poststress period, and this molecular response was reminiscent of amplified ROS surge to restress. Pharmacological inhibition of NADPH oxidase by the NADPH oxidase inhibitor apocynin during the stress or poststress period completely blocked depressive behavior. Consistently, heterozygous p47phox knock-out mice (p47phox(+/-)) or molecular inhibition of p47phox with Lenti shRNA-p47phox in the hippocampus suppressed depressive behavior. These results suggest that repeated stress promotes depressive behavior through the upregulation of NADPH oxidase and the resultant metabolic oxidative stress, and that the inhibition of NADPH oxidase provides beneficial antidepression effects.

Ethanol Induces Oxidative Stress in Alveolar Macrophages via Upregulation of NADPH Oxidases

Chronic alcohol abuse is a comorbid variable of acute respiratory distress syndrome. Previous studies showed that, in the lung, chronic alcohol consumption increased oxidative stress and impaired alveolar macrophage (AM) function. NADPH oxidases (Noxes) are the main source of reactive oxygen species in AMs. Therefore, we hypothesized that chronic alcohol consumption increases AM oxidant stress through modulation of Nox1, Nox2, and Nox4 expression. AMs were isolated from male C57BL/6J mice, aged 8-10 wk, which were treated with or without ethanol in drinking water (20% w/v, 12 wk). MH-S cells, a mouse AM cell line, were treated with or without ethanol (0.08%, 3 d) for in vitro studies. Selected cells were treated with apocynin (300 μM), a Nox1 and Nox2 complex formation inhibitor, or were transfected with Nox small interfering RNAs (20-35 nM), before ethanol exposure. Human AMs were isolated from alcoholic and control patients' bronchoalveolar lavage fluid. Nox mRNA levels (quantitative RT-PCR), protein levels (Western blot and immunostaining), oxidative stress (2',7'-dichlorofluorescein-diacetate and Amplex Red analysis), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol increased Nox expression and oxidative stress in mouse AMs in vivo and in vitro. Experiments using apocynin and Nox small interfering RNAs demonstrated that ethanol-induced Nox4 expression, oxidative stress, and AM dysfunction were modulated through Nox1 and Nox2 upregulation. Further, Nox1, Nox2, and Nox4 protein levels were augmented in human AMs from alcoholic patients compared with control subjects. Ethanol induces AM oxidative stress initially through upregulation of Nox1 and Nox2 with downstream Nox4 upregulation and subsequent impairment of AM function.

Oxidative stress and vein graft failure: a focus on NADH oxidase, nitric oxide and eicosanoids

Recent interest has focused on superoxide and the upregulation of NADPH oxidase expression in the aetiology of vein graft failure. Implantation of saphenous vein grafts promotes upregulation of NADPH oxidase through a number of distinct interrelated mechanisms: (a) endothelial denudation, (b) factors released by adherent platelets, monocytes and neutrophils, (c) hypoxia and (d) altered prostacyclin (PGI(2)) and enhanced isoprostane formation. These, in turn, impact on neointima (NI) formation (vascular smooth muscle cell [VSMC] replication and migration) and metalloproteinase (MMP) expression, key events in vein graft thickening. NADPH oxidase in the aetiology of vein graft failure will be discussed in this review with particular reference to nitric oxide and eicosanoids and related drugs that inhibit its activity and expression.

Abstract 200: CD40 Signaling Mediates Postischemic Inflammation, Oxidative Stress, And Tissue Injury Following Focal Cerebral Ischemia

Rationale: Although CD40/CD40 ligand (CD40L) signaling has been implicated in clinical and experimental ischemic strokes, the underlying mechanisms are largely unclear. Objective: We investigated how CD40 participates in the cellular and molecular events underlying the postischemic inflammation and oxidative stress that may contribute to the tissue damage during cerebral ischemia. Methods and Results: Wild-type (WT, n=164) and CD40 knockout mice (n=132) were subjected to middle cerebral artery occlusion (MCAO, 60 minutes) followed by reperfusion. We found that ischemia/reperfusion induced CD40 expression in the brain in a time-dependent manner, primarily localized to the microvascular endothelial cells in the early phase (6h) and then to the activated microglia in the later time (24h). The adhesion and infiltration of neutrophils as well as the activation and expansion of microglia induced by ischemia/reperfusion were inhibited in CD40-/- mice, which were time-dependently correlated with suppressing nuclear factor-kB activation and proinflammatory cytokines (IL-1β, TNFα) and adhesion molecules (E- and P-selectin, ICAM-1,MCP-1). Infarct volumes and mortality were reduced in CD40-/- mice at 72h after ischemia/reperfusion. Treatment with an inhibitor of either NADPH oxidase or COX-2, the known enzymes that contributes to the tissue damage, reduced ischemic brain injury in wild-type mice, but not in CD40-/- mice. In contrast, treatment with an inhibitor of inducible nitric oxide synthase (iNOS) further reduced tissue injury in CD40-/- mice. Consistently, ischemia/reperfusion-induced upregulation of NADPH oxidase (Nox2, and Nox4) and COX-2, but not iNOS, were attenuated in CD40-/- mice. Conclusions: The findings unveil an essential role for CD40 in the regulation of early molecular and cellular events leading to postischemic inflammation. Inhibition of CD40 signaling may be a valuable therapeutic approach to counteract the deleterious effects of postischemic inflammation.

C‐reactive protein promotes adhesion of monocytes to endothelial cells via NADPH oxidase‐mediated oxidative stress

Enhanced monocyte adhesion to endothelial cells is an early event in atherogenesis. It has been shown that C-reactive protein (CRP) plays a key role in atherogenesis. Here, we investigated the effects of CRP on monocyte-endothelial cell adhesion and tested the hypothesis that NADPH oxidase (NOX)-mediated oxidative stress might play a key role in CRP-induced monocyte-endothelial cell adhesion. Firstly, 36 patients with carotid intima-media thickness (IMT) incrassation and 34 controls were enrolled in this study. The levels of glucose, lipids, CRP, monocyte chemotractant protein (MCP-1), malondialdehyde (MDA), and protein carbonylation were analyzed. The results showed that carotid IMT was associated with abnormal lipid metabolism, including elevated CRP, triglycerides (TG) (P < 0.01) and decreased high density lipoprotein (HDL) level (P < 0.05). The levels of CRP and MCP-1 in patients with carotid IMT incrassation were increased compared with the controls (P < 0.01). Moreover, patients with carotid IMT incrassation displayed enhanced MDA and protein carbonylation levels (P < 0.01), accompanied by activation and up-regulation of NOX in monocytes (P < 0.05) compared with the controls. The monocytes isolated from five healthy donors were used for in vitro experiments. Reactive oxygen species (ROS) production and NOX expression in monocytes were examined. The results also indicated that CRP could promote the adhesion of monocyte-endothelial cell by up-regulation of MCP-1 expression (P < 0.05). Importantly, NFκ B and p38 MAPK signaling pathways, which were activated by NOX-derived ROS, were involved in CRP-induced monocyte-endothelial cell adhesion and up-regulation of MCP-1 expression. These data suggested that CRP could promote the adhesion of monocytes to endothelial cells via NOX-mediated oxidative stress. J. Cell. Biochem. 113: 857-867, 2012. © 2011 Wiley Periodicals, Inc.