Nox4 NAD(P)H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. We investigated the role of the NAD(P)H oxidase Nox4 in generation of reactive oxygen species (ROS), hypertrophy, and fibronectin expression in a rat model of type 1 diabetes induced by streptozotocin. Phosphorothioated antisense (AS) or sense oligonucleotides for Nox4 were administered for 2 weeks with an osmotic minipump 72 h after streptozotocin treatment. Nox4 protein expression was increased in diabetic kidney cortex compared with non-diabetic controls and was down-regulated in AS-treated animals. AS oligonucleotides inhibited NADPH-dependent ROS generation in renal cortical and glomerular homogenates. ROS generation by intact isolated glomeruli from diabetic animals was increased compared with glomeruli isolated from AS-treated animals. AS treatment reduced whole kidney and glomerular hypertrophy. Moreover, the increased expression of fibronectin protein was markedly reduced in renal cortex including glomeruli of AS-treated diabetic rats. Akt/protein kinase B and ERK1/2, two protein kinases critical for cell growth and hypertrophy, were activated in diabetes, and AS treatment almost abolished their activation. In cultured mesangial cells, high glucose increased NADPH oxidase activity and fibronectin expression, effects that were prevented in cells transfected with AS oligonucleotides. These data establish a role for Nox4 as the major source of ROS in the kidneys during early stages of diabetes and establish that Nox4-derived ROS mediate renal hypertrophy and increased fibronectin expression.     

Aggravated Cardiac Remodeling post Aortocaval Fistula in Unilateral Nephrectomized Rats

Background

Aortocaval fistula (AV) in rat is a unique model of volume-overload congestive heart failure and cardiac hypertrophy. Living donor kidney transplantation is regarded as beneficial to allograft recipients and not particularly detrimental to the donors. Impact of AV on animals with mild renal dysfunction is not fully understood. In this study, we explored the effects of AV in unilateral nephrectomized (UNX) rats.

Methods

Adult male Sprague-Dawley (SD) rats were divided into Sham (n = 10), UNX (right kidney remove, n = 10), AV (AV established between the levels of renal arteries and iliac bifurcation, n = 18) and UNX+AV (AV at one week after UNX, n = 22), respectively. Renal outcome was measured by glomerular filtration rate, effective renal plasma flow, fractional excretion of sodium, albuminuria, plasma creatinine, and cystatin C. Focal glomerulosclerosis (FGS) incidence was evaluated by renal histology. Cardiac function was measured by echocardiography and hemodynamic measurements.

Results

UNX alone induced compensatory left kidney enlargement, increased plasma creatinine and cystatin C levels, and slightly reduced glomerular filtration rate and increased FGS. AV induced significant cardiac enlargement and hypertrophy and reduced cardiac function and increased FGS, these changes were aggravated in UNX+AV rats.

Conclusions

Although UNX only induces minor renal dysfunction, additional chronic volume overload placement during the adaptation phase of the remaining kidney is associated with aggravated cardiac dysfunction and remodeling in UNX rats, suggesting special medical care is required for UNX or congenital monokidney subjects in case of chronic volume overload as in the case of pregnancy and hyperthyroidism to prevent further adverse cardiorenal events in these individuals.     

Surgical procedures suppress autophagic flux in the kidney

Many surgical models are used to study kidney and other diseases in mice, yet the effects of the surgical procedure itself on the kidney and other tissues have not been elucidated. In the present study, we found that both sham surgery and unilateral nephrectomy (UNX), which is used as a model of renal compensatory hypertrophy, in mice resulted in increased mammalian target of rapamycin complex 1/2 (mTORC1/2) in the remaining kidney. mTORC1 is known to regulate lysosomal biogenesis and autophagy. Genes associated with lysosomal biogenesis and function were decreased in sham surgery and UNX kidneys. In both sham surgery and UNX, there was suppressed autophagic flux in the kidney as indicated by the lack of an increase in LC3-II or autophagosomes seen on immunoblot, IF and EM after bafilomycin A1 administration and a concomitant increase in p62, a marker of autophagic cargo. There was a massive increase in pro-inflammatory cytokines, which are known to activate ERK1/2, in the serum after sham surgery and UNX. There was a large increase in ERK1/2 in sham surgery and UNX kidneys, which was blocked by the MEK1/2 inhibitor, trametinib. Trametinib also resulted in a significant decrease in p62. In summary, there was an intense systemic inflammatory response, an ERK-mediated increase in p62 and suppressed autophagic flux in the kidney after sham surgery and UNX. It is important that researchers are aware that changes in systemic pro-inflammatory cytokines, ERK1/2 and autophagy can be caused by sham surgery as well as the kidney injury/disease itself.Subject terms: Autophagy, Kidney     

Organ specific optical imaging of mitochondrial redox state in a rodent model of hereditary hemorrhagic telangiectasia‐1

Hereditary Hemorrhagic Telangiectasia‐1 (HHT‐1) is a vascular disease caused by mutations in the endoglin (Eng)/CD105 gene. The objective of this study was to quantify the oxidative state of a rodent model of HHT‐1 using an optical imaging technique. We used a cryofluorescence imaging instrument to quantitatively assess tissue metabolism in this model. Mitochondrial redox ratio (FAD/NADH), FAD RR, was used as a quantitative marker of the metabolic status and was examined in the kidneys, and eyes of wild‐type and Eng +/– mice. Kidneys and eyes from wild‐type P21, 6W, and 10M old mice showed, respectively, a 9% (±2), 24% (±0.4), 15% (±1), and 23% (±4), 33% (±0.6), and 30% (±2) change in the mean FAD RR compared to Eng +/– mice at the same age. Thus, endoglin haploinsufficiency is associated with less oxidative stress in various organs and mitigation of angiogenesis. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Nox2 B-loop peptide, Nox2ds, specifically inhibits the NADPH oxidase Nox2

In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1, Nox2, and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibition of these isoforms is an area of intense current investigation. In this study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B-loop of Nox2, would inhibit ROS production by the Nox2-, but not the Nox1- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2ds was assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1-, or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibited superoxide (O₂^•−) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance confirmed that Nox2ds inhibits O₂^•− production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production by either Nox1- or Nox4-oxidase. These findings demonstrate that Nox2ds is a selective inhibitor of Nox2-oxidase and support its utility to elucidate the role of Nox2 in organ pathophysiology and its potential as a therapeutic agent.     

Structural Insights into Nox4 and Nox2: Motifs Involved in Function and Cellular Localization

Regulated generation of reactive oxygen species (ROS) is primarily accomplished by NADPH oxidases (Nox). Nox1 to Nox4 form a membrane-associated heterodimer with p22^phox, creating the docking site for assembly of the activated oxidase. Signaling specificity is achieved by interaction with a complex network of cytosolic components. Nox4, an oxidase linked to cardiovascular disease, carcinogenesis, and pulmonary fibrosis, deviates from this model by displaying constitutive H₂O₂ production without requiring known regulators. Extensive Nox4/Nox2 chimera screening was initiated to pinpoint structural motifs essential for ROS generation and Nox subcellular localization. In summary, a matching B loop was crucial for catalytic activity of both Nox enzymes. Substitution of the carboxyl terminus was sufficient for converting Nox4 into a phorbol myristate acetate (PMA)-inducible phenotype, while Nox2-based chimeras never gained constitutive activity. Changing the Nox2 but not the Nox4 amino terminus abolished ROS generation. The unique heterodimerization of a functional Nox4/p22^phox Y121H complex was dependent on the D loop. Nox4, Nox2, and functional Nox chimeras translocated to the plasma membrane. Cell surface localization of Nox4 or PMA-inducible Nox4 did not correlate with O₂⁻ generation. In contrast, Nox4 released H₂O₂ and promoted cell migration. Our work provides insights into Nox structure, regulation, and ROS output that will aid inhibitor design.The family of NADPH oxidases consists of seven members termed Nox/Duox that differ in their tissue expression profiles, modes of activation, reactive oxygen species (ROS) outputs, and physiological functions. Understanding their distinguishing features is a prerequisite for rational inhibitor design and thus targeted intervention in ROS-mediated pathophysiologies (4). The coexpression of different Nox isoforms, each with potentially distinct functional profiles, in the same cell type necessitates a more discriminating approach than application of pan-Nox inhibitors. Detailed structure-function studies are necessary to identify unique regions and their impact with respect to catalytic function or localization of the enzyme. All Nox/Duox enzymes share a Nox backbone with six predicted transmembrane domains and an intracellular carboxyl-terminal domain which harbors FAD and NADPH binding sites. Nox5 and Duox1/2 enzymes contain additional structural elements such as amino terminal EF-hand motifs, a hallmark of their regulation by the intracellular calcium concentration (13, 30).The founding member of the NADPH oxidase family, the phagocyte oxidase, consists of membrane-bound Nox2 in a complex with the smaller subunit p22^phox (3). Heterodimerization of these two proteins is required for maturation and translocation of the enzyme complex to the plasma membrane or to intracellular vesicles. The Nox family members Nox1, Nox3, and Nox4 follow this paradigm (1, 14, 21, 25, 31). Heterodimer formation and association of the Nox/p22^phox complex at particular cellular membranes is essential for catalytic activity, i.e., for ROS generation. Nox2, and to a lesser degree Nox1 and Nox3, remain dormant under resting conditions and rely on stimulus-dependent translocation and assembly of oxidase components such as p47^phox and p67^phox, or NoxO1 and NoxA1 in the case of Nox1 and Nox3 (16). These steps, together with activation and translocation of the GTPase Rac, ultimately lead to the assembled, catalytically active oxidase and to ROS generation.Nox4 differs from the usual theme of multimeric assembly of active NADPH oxidases found in Nox1 to Nox3 (21, 22, 28, 32). Constitutive H₂O₂ production by Nox4 localized at perinuclear vesicles has been reported (1, 21, 28). Since NADPH oxidases catalyze the one-electron reduction of molecular oxygen to superoxide anion, the current dogma suggests that Nox4 generates intracellular superoxide. The superoxide produced will then dismutate rapidly to H₂O₂, diffusing from the cell into the extracellular milieu. Cytosolic proteins, which regulate the activity of Nox1 to Nox3 by binding to the carboxyl-terminal domains of Nox1 to Nox3, seem to be irrelevant for Nox4 function. The membrane-bound subunit p22^phox is to date the only known protein associated with Nox1 to Nox4. Heterodimerization, translocation, and enzymatic function of these oxidases require p22^phox. Recent structure-function analyses of complexes between Nox2 or Nox4 and the subunit p22^phox documented specific regions and amino acid residues in p22^phox necessary for complex formation and oxidase activity (35, 37). Interestingly, a p22^phox mutant (p22^phox Y121H) is capable of distinguishing between Nox1 to Nox3 and Nox4 by forming a functional complex only with Nox4, further suggesting unique structural features in Nox4 (35).In this study, we expand structure-function analysis of the oxidase complex by comparing Nox4/Nox2 chimeric enzymes with respect to NADPH oxidase activity, type of reactive oxygen species produced, requirement for additional oxidase components, and detailed subcellular localization.     

Hydrogen sulfide alleviates uranium‐induced rat hepatocyte cytotoxicity via inhibiting Nox4/ROS/p38 MAPK pathway

As a gasotransmitter, hydrogen sulfide (H₂S) plays a crucial role in regulating the signaling pathway mediated by oxidative stress. The purpose of this study was to investigate the protective effects of H ₂S on uranium‐induced rat hepatocyte cytotoxicity. Primary hepatocytes were isolated and cultured from Sprague Dawley rat liver tissues. After pretreating with sodium hydrosulfide (an H ₂S donor) for 1 hour (or GKT‐136901 for 30 minutes), hepatocytes were treated by uranyl acetate for 24 hours. Cell viability, reactive oxygen species (ROS), malondialdehyde (MDA), NADPH oxidase 4 (Nox4), and p38 mitogen‐activated protein kinase (p38 MAPK) phosphorylation were respectively determined. The effects of direct inhibition of Nox4 expression by GKT‐136901 (a Nox4 inhibitor) on ROS and phospho‐p38 MAPK levels were examined in uranium‐treated hepatocytes. The results implicate that H ₂S can afford protection of rat hepatocytes against uranium‐induced adverse effects through attenuating oxidative stress via prohibiting Nox4/ROS/p38 MAPK signaling.     

The reductive half-reaction of two bifurcating electron-transferring flavoproteins: Evidence for changes in flavin reduction potentials mediated by specific conformational changes

The EtfAB components of two bifurcating flavoprotein systems, the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from the bacterium Megasphaera elsdenii and the menaquinone-dependent NADH:ferredoxin oxidoreductase from the archaeon Pyrobaculum aerophilum, have been investigated. With both proteins, we find that removal of the electron-transferring flavin adenine dinucleotide (FAD) moiety from both proteins results in an uncrossing of the reduction potentials of the remaining bifurcating FAD; this significantly stabilizes the otherwise very unstable semiquinone state, which accumulates over the course of reductive titrations with sodium dithionite. Furthermore, reduction of both EtfABs depleted of their electron-transferring FAD by NADH was monophasic with a hyperbolic dependence of reaction rate on the concentration of NADH. On the other hand, NADH reduction of the replete proteins containing the electron-transferring FAD was multiphasic, consisting of a fast phase comparable to that seen with the depleted proteins followed by an intermediate phase that involves significant accumulation of FAD⋅⁻, again reflecting uncrossing of the half-potentials of the bifurcating FAD. This is then followed by a slow phase that represents the slow reduction of the electron-transferring FAD to FADH⁻, with reduction of the now fully reoxidized bifurcating FAD by a second equivalent of NADH. We suggest that the crossing and uncrossing of the reduction half-potentials of the bifurcating FAD is due to specific conformational changes that have been structurally characterized.     

Increased renal vascular reactivity to ANG II after unilateral nephrectomy in the rat involves 20-HETE

This study examined the role of intrarenal ANG II in the renal vascular reactivity changes occurring in the remaining kidney undergoing adaptation following contralateral nephrectomy. Renal blood flow responses to intrarenal injections of ANG II (0.25 to 5 ng) were measured in anesthetized euvolemic male Wistar rats 1, 4, 12, and 24 wk after uninephrectomy (UNX) or sham procedure (SHAM). At week 4, renal vasoconstriction induced by 2 ng ANG II was greater in UNX (69 +/- 5%) than in SHAM rats (50 +/- 3%; P < 0.01). This response was inhibited, by 50 and 66%, and by 20 and 25%, in SHAM and UNX rats, after combined injections of ANG II and losartan, or PD-123319 (P < 0.05), respectively. Characteristics of ANG II receptor binding in isolated preglomerular resistance vessels were similar in the two groups. After prostanoid inhibition with indomethacin, renal vasoconstriction was enhanced by 42 +/- 8% (P < 0.05), only in SHAM rats, whereas after 20-HETE inhibition with HET0016, it was reduced by 53 +/- 16% (P < 0.05), only in UNX rats. These differences vanished after concomitant prostanoid and 20-HETE inhibition in the two groups. After UNX, renal cortical protein expression of cytochrome P-450 2c23 isoform (CYP2c23) and cyclooxygenase-1 (COX-1) was unaltered, but it was decreased for CYP4a and increased for COX-2. In conclusion, renal vascular reactivity to ANG II was significantly increased in the postuninephrectomy adapted kidney, independently of protein expression, but presumably involving interactions between 20-HETE and COX in the renal microvasculature and changes in the paracrine activity of ANG II and 20-HETE.     

Rutaecarpine prevents hypertensive cardiac hypertrophy involving the inhibition of Nox4‐ROS‐ADAM17 pathway

Rutaecarpine attenuates hypertensive cardiac hypertrophy in the rats with abdominal artery constriction (AAC); however, its mechanism of action remains largely unknown. Our previous study indicated that NADPH oxidase 4 (Nox4) promotes angiotensin II (Ang II)‐induced cardiac hypertrophy through the pathway between reactive oxygen species (ROS) and a disintegrin and metalloproteinase‐17 (ADAM17) in primary cardiomyocytes. This research aimed to determine whether the Nox4‐ROS‐ADAM17 pathway is involved in the protective action of rutaecarpine against hypertensive cardiac hypertrophy. AAC‐induced hypertensive rats were adopted to evaluate the role of rutaecarpine in hypertensive cardiac hypertrophy. Western blotting and real‐time PCR were used to detect gene expression. Rutaecarpine inhibited hypertensive cardiac hypertrophy in AAC‐induced hypertensive rats. These findings were confirmed by the results of in vitro experiments that rutaecarpine significantly inhibited Ang II‐induced cardiac hypertrophy in primary cardiomyocytes. Likewise, rutaecarpine significantly suppressed the Nox4‐ROS‐ADAM17 pathway and over‐activation of extracellular signal‐regulated kinase (ERK) 1/2 pathway in the left ventricle of AAC‐induced hypertensive rats and primary cardiomyocytes stimulated with Ang II. The inhibition of Nox4‐ROS‐ADAM17 pathway and over‐activation of ERK1/2 might be associated with the beneficial role of rutaecarpine in hypertensive cardiac hypertrophy, thus providing additional evidence for preventing hypertensive cardiac hypertrophy with rutaecarpine.     

Transforming growth factor‐β1 requires NADPH oxidase 4 for angiogenesis in vitro and in vivo

Angiogenesis, the formation of new blood vessels, is a key physiological event in organ development and tissue responses to hypoxia but is also involved in pathophysiologies such as tumour growth and retinopathies. Understanding the molecular mechanisms involved is important to design strategies for therapeutic intervention. One important regulator of angiogenesis is transforming growth factor‐β1 (TGF‐β1). In addition, reactive oxygen species (ROS) and the ROS‐forming NADPH oxidase type 4 (Nox4) have been implicated as additional regulators such as during hypoxia. Here, we show that both processes are indeed mechanistically linked. TGF‐β1‐stimulated Nox4 expression and ROS formation in endothelial cells. In cells from Nox4‐deficient mice, TGF‐β1‐induced cell proliferation, migration and tube formation were abolished. In vivo, TGF‐β1 stimulated growth of blood vessels into sponges implanted subcutaneously, and this angiogenesis was markedly reduced in Nox4 knockout mice. Thus, endothelial cells are regulated by a TGF‐β1 signalling pathway involving Nox4‐derived ROS to promote angiogenesis. In order to abrogate pathological angiogenesis triggered by a multitude of factors, such as TGF‐β1 and hypoxia, Nox4 may thus be an ideal therapeutic target.     

Adrenaline stimulates H2O2 generation in liver via NADPH oxidase

It is known that adrenaline promotes hydroxyl radical generation in isolated rat hepatocytes. The aim of this work was to investigate a potential role of NADPH oxidase (Nox) isoforms for an oxidative stress signal in response to adrenaline in hepatocytes. Enriched plasma membranes from isolated rat liver cells were prepared for this purpose. These membranes showed catalytic activity of Nox isoforms, probably Nox 2 based on its complete inhibition with specific antibodies. NADPH was oxidized to convert O₂ into superoxide radical, later transformed into H₂O₂. This enzymatic activity requires previous activation with either 3 mM Mn²⁺ or guanosine 5′-0-(3-thiotriphosphate) (GTPγS) plus adrenaline. Experimental conditions for activation and catalytic steps were set up: ATP was not required; S_0.5 for NADPH was 44 μM; S_0.5 for FAD was 8 μM; NADH up to 1 mM was not substrate, and diphenyleneiodonium was inhibitory. Activation with GTPγS plus adrenaline was dose- and Ca²⁺-dependent and proceeded through α₁-adrenergic receptors (AR), whereas β-AR stimulation resulted in inhibition of Nox activity. These results lead us to propose H₂O₂ as additional transduction signal for adrenaline response in hepatic cells.     

NADPH oxidases contribute to autophagy regulation

Reactive oxygen species (ROS) are emerging as regulators of autophagy in various cellular contexts. There are many cellular sources of ROS in eukaryotic cells. In phagocytes, the critical immune cells for host defense, the Nox2 NADPH oxidase generates ROS during phagocytosis and plays a central role in microbial killing. Toll-like receptors (TLRs) are important membrane microbial sensing receptors, which can activate Nox2,¹ and were recently demonstrated to signal autophagy targeting of phagosomes to promote their maturation.² Our recent study reveals that Nox2 activity and its generated ROS are key signals that induce TLR-activated autophagy of phagosomes. Our results provide the first evidence that ROS from the Nox2 NADPH oxidase can contribute to regulating autophagy in host defense against bacteria. The association of TLR, Nox2 and autophagy with inflammatory bowel disease (IBD) suggests a significant role of this antibacterial pathway in these diseases.     

Bone marrow derived cells and reactive oxygen species in hypertrophy of contralateral kidney of transient unilateral renal ischemia-induced mouse

Abstract

Renal mass reduction, such as unilateral nephrectomy induces a compensatory hypertrophy of remaining renal mass in response to overload induced by reduction of functional renal parenchyma. In our recent study, we observed that the recovery of ischemic injured kidney following transient unilateral renal ischemia took longer time than that following transient bilateral renal ischemia, indicating that non-damaged kidney may affect the damaged kidney and vice versa. Here, we investigated whether transient and partial renal parenchymal injury by transient unilateral renal ischemia (UI) results in the hypertrophy of its contralateral kidney (CLK) and reactive oxygen species is associated with the hypertrophy. Thirty minutes of UI resulted in gradual increase in CLK weight over time. UI increased superoxide formation, but not lipid peroxidation in the CLK. After UI, a significant increase in the number of NADPH oxidase 2 (Nox2)-expressing cells and the level of Nox2 expression in the CLK was observed. In parallel with the increases in Nox2-expressing cells in CLKs, infiltration of bone marrow-derived cells (BMDC) increased in CLK. Treatments with Mn(III) Tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP, a superoxide dismutase (SOD) mimetic) and apocynin (a putative NADPH oxidase inhibitor) inhibited UI-induced hypertrophy of CLK along with reduction in Nox2-positive cell, BMDC, amount of Nox2 expression and superoxide formation. In conclusion, transient and partial renal mass reduction by UI resulted in the hypertrophy of CLK through increased ROS formation by infiltrated cells into the interstitium of CLK.     

Hypoxia-inducible factor 1 mediates increased expression of NADPH oxidase-2 in response to intermittent hypoxia

Sleep‐disordered breathing with recurrent apnea is associated with intermittent hypoxia (IH). Cardiovascular morbidities caused by IH are triggered by increased generation of reactive oxygen species (ROS) by pro‐oxidant enzymes, especially NADPH oxidase‐2 (Nox2). Previous studies showed that (i) IH activates hypoxia‐inducible factor 1 (HIF‐1) in a ROS‐dependent manner and (ii) HIF‐1 is required for IH‐induced ROS generation, indicating the existence of a feed‐forward mechanism. In the present study, using multiple pharmacological and genetic approaches, we investigated whether IH‐induced expression of Nox2 is mediated by HIF‐1 in the central and peripheral nervous system of mice as well as in cultured cells. IH increased Nox2 mRNA, protein, and enzyme activity in PC12 pheochromocytoma cells as well as in wild‐type mouse embryonic fibroblasts (MEFs). This effect was abolished or attenuated by blocking HIF‐1 activity through RNA interference or pharmacologic inhibition (digoxin or YC‐1) or by genetic knockout of HIF‐1α in MEFs. Increasing HIF‐1α expression by treating PC 12 cells with the iron chelator deferoxamine for 20 h or by transfecting them with HIF‐1alpha expression vector increased Nox2 expression and enzyme activity. Exposure of wild‐type mice to IH (8 h/day for 10 days) up‐regulated Nox2 mRNA expression in brain cortex, brain stem, and carotid body but not in cerebellum. IH did not induce Nox2 expression in cortex, brainstem, carotid body, or cerebellum of Hif1a^+/? mice, which do not manifest increased ROS or cardiovascular morbidities in response to IH. These results establish a pathogenic mechanism linking HIF‐1, ROS generation, and cardiovascular pathology in response to IH. J. Cell. Physiol. 226: 2925–2933, 2011. © 2011 Wiley‐Liss, Inc.     

Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells

Nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the subunit Nox4 is the major Nox isoform in primary human endothelial cells; we also found high levels of NADPH oxidase subunit p22^phox expression. Nox4 was localized by laser scanning confocal microscopy within the cytoplasm of endothelial cells. Endothelial Nox4 overexpression enhanced superoxide anion formation and phosphorylation of p38 MAPK. Nox4 down-regulation by shRNA has in contrast to TGF-β no effect on p38 MAPK phosphorylation. We conclude that Nox4 is the major Nox isoform in human endothelial cells, and forms an active complex with p22^phox. The Nox4-containing complex mediates formation of reactive oxygen species and p38 MAPK activation. This is a novel mechanism of redox-sensitive signaling in human endothelial cells.     

Nox4 modulates collagen production stimulated by transforming growth factor β1 in vivo and in vitro

The synthesis of extracellular matrix including collagen during wound healing responses involves signaling via reactive oxygen species (ROS). We hypothesized that NADPH oxidase isoform Nox4 facilitates the stimulatory effects of the profibrotic cytokine transforming growth factor (TGF) β₁ on collagen production in vitro and in vivo. TGFβ₁ stimulated collagen synthesis and hydrogen peroxide generation in mouse cardiac fibroblasts, and both responses were attenuated by a scavenger of superoxide and hydrogen peroxide (EUK-134). Furthermore, by expressing a dominant negative form of Nox4 (Adv-Nox4^ΔNADPH) in fibroblasts, TGFβ₁-induced hydrogen peroxide production and collagen production were abrogated, suggesting that Nox4-dependent ROS are important for TGFβ₁ signaling in collagen production. This was confirmed by the inhibitory effect of an adenovirus carrying siRNA targeting Nox4 (Adv-Nox4i) on TGFβ₁-induced collagen synthesis and expression of activated myofibroblasts marker smooth muscle alpha actin. Finally we used a mouse model of subcutaneous sponge implant to examine the role of Nox4 in the local stimulatory effects of TGFβ₁ on collagen accumulation in vivo. TGFβ₁-induced collagen accumulation was significantly reduced when the sponges were instilled with Adv-Nox4^ΔNADPH. In conclusion, Nox4 acts as an intermediary in the signaling of TGFβ₁ to facilitate collagen synthesis.