Supplementary Figure Legends 1-6 from Overactivation of the MEK/ERK Pathway in Liver Tumor Cells Confers Resistance to TGF-β–Induced Cell Death through Impairing Up-regulation of the NADPH Oxidase NOX4
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Supplementary Figure Legends 1-6 from Overactivation of the MEK/ERK Pathway in Liver Tumor Cells Confers Resistance to TGF-β–Induced Cell Death through Impairing Up-regulation of the NADPH Oxidase NOX4Keywords:
NOX4
BACKGROUND: Many types of hypertension are associated with increased level of angiotensin II and vascular oxidative stress. Previously, we have shown that NOX2, an adventitial NADPH oxidase (NOX) isoform, generates reactive oxygen species in cardiac vascular system. Although NOX4, as another major isoform of NOX, has been reported to generate reactive oxygen species and play a detrimental role, current publications allude to the possibility that NOX4 may play a beneficial role in the blood vessels. The current project examined this aspect by investigating the expression of adventitial NOX4 and reactive oxygen species. OBJECTIVE: The objective of this study is to test our hypothesis that the expression of adventitial NOX4 would not increase vascular ROS in Angiotensin II‐induced hypertension. METHOD: Four C57BL and NOX2 knockout mice, sixteen to eighteen weeks of age, were separated into two groups, a normotensive and Angiotensin II infused group. Angiotensin II infused animals developed hypertension. Thoracic aortas were sterilely removed, embedded in paraffin and then fixed on glass slides as 5µm thick sections. Expression of 3’‐nitrotyrosine, a reactive oxygen species indicator, NOX2, and NOX4 were determined by immunoflorescence method. RESULTS AND CONCLUSION: Similar to NOX2, NOX4 is expressed in both endothelium and adventitia of the aorta. The level of NOX4 in the Angiotensin II‐infused hypertensive is greatly increased about seven times in comparison to normotensive group. 3’‐Nitrotyrosin levels are increased in Angiotensin II infused mouse aorta segments. However, Angiotensin II induced hypertension and NT is not increased in NOX2 knockout mouse while there is significant amount of NOX4 is still expressed. In conclusion, our data indicate that NOX2 but not NOX4 contributes to vascular production of reactive oxygen species and hypertension. Grant Funding Source : Brock University
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Introduction
The characteristic hyperglycaemia of diabetes drives reactive oxygen species (ROS) production in endothelial cells (ECs), leading to microvascular dysfunction and cardiovascular complications. NADPH oxidases are enzymes whose primary function is to generate ROS, and which contribute to development and progression of cardiovascular disease. This study aimed to investigate the role of the NOX4 isoform, which is highly expressed in ECs, under hyperglycaemic conditions, in EC signalling and paracrine communication with fibroblasts, as a major determinant of diabetic cardiovascular remodelling.Methods
Human aortic endothelial cells (HAoECs) were treated with normal (NG, 5.5 mM) or high (HG, 25 mM) glucose for up to 5 days with or without NOX4 siRNA knockdown (KD) prior to assessment of mRNA expression (real-time RT-PCR; relative to β-actin) and superoxide generation (DHE fluorescence). NIH 3 T3 fibroblasts were treated with conditioned media from NOX4-modified HAoECs for 24 hour to interrogate effects on paracrine signalling.Results
HG treatment of HAoECs for 5 days increased mRNA expression of NOX4 (NG 1.01±0.06, HG 1.27±0.06; n=9, p<0.05) and associated antioxidant and proinflammatory genes (e.g. NRF2: 0.91±0.04 vs 1.22±0.04; IL-6: 1.03±0.13 vs 2.59±0.58; n=6, p<0.05). This was associated with increased superoxide production (262±12 vs 338±11 arbitrary units; n=4, p<0.05) at 2 but not 5 days. Interestingly, NOX4 KD under HG conditions increased mRNA expression of endogenous antioxidant enzymes after 2 days (e.g. NRF2: 1.90±0.06 vs 2.21±0.06; n=3, p<0.05) whilst normalising increased superoxide production. Furthermore, increased TGFβ-induced differentiation of NIH 3 T3 fibroblasts observed in the presence of conditioned media from HG-treated HAoECs was ablated by NOX4 KD (e.g. α-SMA: scrambled control 1.31±0.04, NOX4 KD 0.94±0.07; n=3, p<0.05).Conclusions
HG-induced NOX4 signalling regulates ROS production and endogenous antioxidant expression in ECs, driving paracrine stimulation of fibroblast differentiation. It therefore seems likely that EC NOX4 NADPH oxidase signalling contributes significantly to adverse diabetic cardiovascular remodelling.NOX4
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NADPH oxidases are important sources of reactive oxygen species (ROS) in the vasculature. In phagocytic cells, the catalytic subunit of NADPH oxidase is a glycoprotein, gp91phox. However, vascular smooth muscle cells (VSMCs), which show prominent NADPH oxidase activity, lack gp91phox. Hence, we examined the role of Nox4, a gp91phox homologue, in superoxide production in mouse-cultured VSMCs.Incubation of VSMCs with NADPH increased ROS production whether detected by lucigenin-enhanced chemiluminescence or dichlorofluorescein. Superoxide production was inhibited by the NADPH oxidase inhibitors, diphenyleneiodonium and apocynin, but not by inhibitors of other potential sources of superoxide. In unstimulated VSMCs, phosphorothioate antisense oligonucleotides against Nox4 down-regulated mRNA expression of the subunit by 65% and attenuated superoxide production by 41% without affecting Nox1 expression. Interleukin-1beta (IL-beta) thrombin and platelet-derived growth factor (PDGF) also reduced Nox4 mRNA expression after 3 h without affecting Nox1 levels. Of these stimuli, only IL-beta reduced superoxide, but this effect was more rapid (< or =30 min) than its actions on Nox4.Under resting conditions, NADPH oxidase activity in VSMCs is largely dependent upon Nox4 expression. Proinflammatory mediators down-regulated Nox4 but did not affect Nox1 expression, so other factors must compensate to regulate superoxide production.
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Oxidative stress is known to play a role in cardiovascular disease including atherosclerosis. While several ROS may be involved, NADPH oxidase appears to play a key role. Studies have shown VSMC phenotypic modulation is critical to the development of atherosclerotic plaques. KCa3.1 is one mechanism by which increased VSMC proliferation and migration occurs. Our objective was to determine if NADPH oxidase plays a role in KCa3.1 upregulation. In coronary SMCs, bFGF increased superoxide (O2.‐) production; increased KCa3.1 mRNA levels ~2.5 fold, protein expression ~2.5 fold, and channel activity ~4.0 fold; and increased AP‐1 activity ~2.0 fold. Apo inhibited the bFGF increase in O2.‐, KCa3.1 message and activity, and AP‐1 activity. Coronary smooth muscle expressed all four cardiovascular Nox isoforms (Nox1,2,4, and 5) with Nox4 being the predominant isoform. bFGF decreased Nox1,2, and 4 message, while Apo increased message of all four isoforms. Nox2 and Nox4 siRNA did not affect KCa3.1 message response to bFGF or Apo. Our findings provide novel evidence that NADPH oxidase contributes to VSMC phenotypic modulation through AP‐1 transcriptional upregulation of KCa3.1.
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The antioxidant response element (ARE)-mediated antioxidant pathway has an important role in maintaining the redox status of the retina. The expression of ARE-mediated antioxidants, such as heme oxygenase-1 (HO-1), remains unclear in the db/db mice. We evaluated the expression of HO-1 in the retinas of db/db mice and investigated a possible role for NADPH oxidase.Fresh retinas were harvested from 8-, 12-, and 20-week db/db or db/m mice. Reactive oxygen species were detected by dihydroethidium. The expression levels of HO-1, Nox2, and Nox4 were evaluated by immunohistochemistry and Western blotting. In vitro retina explants culture was used to assess the role of NADPH oxidase in high glucose-induced HO-1 expression.The expression of HO-1 was increased in the retinas of 8-week db/db mice, while it was decreased in 20-week db/db mice compared to age-matched controls. Similarly, the activation of Nox4 was increased in the retinas at 8 weeks and returned to basal levels at 20 weeks in db/db mice compared to age-matched controls. The activation of Nox2 was increased in the retinas of 8-, 12-, and 20-week db/db mice compared to age-matched controls. The NADPH oxidase inhibitors apocynin and DPI significantly blocked the HO-1 expression that was induced by high glucose levels in cultured retina explants.The expression patterns of HO-1, Nox2, Nox4 in db/db mouse retinas, and the suppressive effects of NADPH oxidase inhibitors on the expression of HO-1 induced by high glucose levels in cultured retina explants suggest that the expression of HO-1 is, at least partially, mediated by NADPH oxidase in this diabetic animal model.
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The Noxes (NADPH oxidases) are a family of ROS (reactive oxygen species)-generating enzymes. Of the seven family members, four have been identified as important sources of ROS in the vasculature: Nox1, Nox2, Nox4 and Nox5. Although Nox isoforms can be influenced by the same stimulus and co-localize in cellular compartments, their tissue distribution, subcellular regulation, requirement for cofactors and NADPH oxidase subunits and ability to generate specific ROS differ, which may help to understand the multiplicity of biological functions of these oxidases. Nox4 and Nox5 are the newest isoforms identified in the vasculature. Nox4 is the major isoform expressed in renal cells and appear to produce primarily H2O2. The Nox5 isoform produces ROS in response to increased levels of intracellular Ca2+ and does not require the other NADPH oxidase subunits for its activation. The present review focuses on these unique Noxes, Nox4 and Nox5, and provides novel concepts related to the regulation and interaction in the vasculature, and discusses new potential roles for these isoforms in vascular biology.
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AimsOxidative stress is thought to be a risk for cardiovascular disease and NADPH oxidases of the Nox family are important producers of reactive oxygen species. Within the Nox family, the NADPH oxidase Nox4 has a unique position as it is constitutively active and produces H2O2 rather than O2− . Nox4 is therefore incapable of scavenging NO and its low constitutive H2O2 production might even be beneficial. We hypothesized that Nox4 acts as an endogenous anti-atherosclerotic enzyme.
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Mineralocorticoid excess increases superoxide production by activating NADPH oxidase (NOX), and intracerebroventricular infusions of NADPH oxidase inhibitors attenuate aldosterone (Aldo)/salt-induced hypertension. It has been hypothesized that increased reactive oxygen species (ROS) in the brain may be a key mechanism in the development of hypertension. The present study investigated the brain regional specificity of NADPH oxidase and the role of NOX2 and NOX4 NADPH oxidase subunits in the hypothalamic paraventricular nucleus (PVN) in Aldo/salt-induced hypertension. PVN injections of adenoviral vectors expressing small interfering (si)RNA targeting NOX2 (AdsiRNA-NOX2) or NOX4 (AdsiRNA-NOX4) mRNAs were used to knock down NOX2 and NOX4 proteins. Three days later, delivery of Aldo (0.2 mg·kg(-1)·day(-1) sc) via osmotic pump commenced and 1% NaCl was provided in place of water. PVN injections of either AdsiRNA-NOX2 or AdsiRNA-NOX4 significantly attenuated the development of Aldo/NaCl-induced hypertension. In an additional study, Aldo/salt-induced hypertension was also significantly attenuated in NOX2 (genomic) knockout mice compared with wild-type controls. When animals from both functional studies underwent ganglionic blockade, there was a reduced fall in blood pressure in the NOX2 and NOX4 knockdown/knockout mice. Western blot analyses of the PVN of siRNA-NOX2- or siRNA-NOX4-injected mice confirmed a marked reduction in the expression of NOX2 or NOX4 protein. In cultured PVN neurons, silencing either NOX2 or NOX4 protein production by culturing PVN cells with siRNA-NOX2 or siRNA-NOX4 attenuated Aldo-induced ROS. These data indicate that both NOX2 and NOX4 in the PVN contribute to elevated sympathetic activity and the hypertensivogenic actions induced by mineralocorticoid excess.
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Reactive oxygen species (ROS) play an important role in normal cellular physiology. They regulate different biologic processes such as cell defense, hormone synthesis and signaling, activation of G protein-coupled receptors, and ion channels and kinases/phosphatases. ROS are also important regulators of transcription factors and gene expression. On the other hand, in pathologic conditions, a surplus of ROS in tissue results in oxidative stress with various injurious consequences such as inflammation and fibrosis. NADPH oxidases are one of the many sources of ROS in biologic systems, and there are seven isoforms (Nox1-5, Duox1, Duox2). Nox4 is the predominant form in the kidney, although Nox2 is also expressed. Nox4 has been implicated in the basal production of ROS in the kidney and in pathologic conditions such as diabetic nephropathy and CKD; upregulation of Nox4 may be important in renal oxidative stress and kidney injury. Although there is growing evidence indicating the involvement of NADPH oxidase in renal pathology, there is a paucity of information on the role of NADPH oxidase in the regulation of normal renal function. Here we provide an update on the role of NADPH oxidases and ROS in renal physiology and pathology.
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