Nox4 Gene Expression Research Articles

944 NADPH Oxidase NOX2 facilitates retinal neovascularisation in a mouse model of oxygen-induced retinopathy

Background: High blood pressure and diabetes co-exist in clinical practice. Both conditions increase the risk of small vessel damage in the kidney and eye and diabetic retinopathy remains a leading cause of vision impairment. The advanced stage of proliferative diabetic retinopathy is associated with pathological neovascularisation subsequent to an initial phase of pre-existing vessel loss or damage. Although current ophthalmic therapy is often effective, many patients continue to lose vision in spite of best care. Therefore, an understanding of the underlying mechanism may provide better treatment strategies. Study Design: Accumulating evidence implicates an oxidative stress-related enzyme NADPH oxidase in retinal neovascularisation, so we examined its contribution to neovascularisation in a mouse model of oxygen-induced retinopathy (OIR). Because Nox2 is the major isoform expressed in endothelial cells and involved in vessel growth, we compared the degree of retinal neovascularisation between Nox2-deficient (KO) and wildtype (WT) mice in OIR. Neonatal pups aged 7 days (P7) were placed in a hyperoxic chamber (75%O2) for 5 days to obliterate developing blood vessels, after which they were returned to room air for 5 days (P17). The avascular retina becomes hypoxic, which triggers neovascularisation. Results: Nox2 and VEGF gene expression was gradually elevated during the phase of neovascularisation and both responses were significantly reduced in Nox2 KO animals (see below figure).Conclusions: Nox2 is an important signalling mediator involved in retinal neovascularisation in OIR, possibly via VEGF. Our findings suggest an additional avenue for intervention to suppress neovascularisation associated with proliferative retinopathy.

105 NOX2 NADPH—oxidase a novel target to prevent insulin resistance related endothelial cell dysfunction

IntroductionInsulin resistance, a central pathophysiological feature of type 2 diabetes is characterised by a deleterious change in endothelial cell phenotype, a hallmark of which is increased generation of reactive oxygen...

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.

Chronic C-Type Natriuretic Peptide Infusion Attenuates Angiotensin II-Induced Myocardial Superoxide Production and Cardiac Remodeling

Myocardial oxidative stress and inflammation are key mechanisms in cardiovascular remodeling. C-type natriuretic peptide (CNP) is an endothelium-derived cardioprotective factor, although its effect on cardiac superoxide generation has not been investigated in vivo. This study tested the hypothesis that suppression of superoxide production contributes to the cardioprotective action of CNP. Angiotensin II (Ang II) or saline was continuously infused subcutaneously into mice using an osmotic minipump. Simultaneously with the initiation of Ang II treatment, mice were infused with CNP (0.05 μg/kg/min) or vehicle for 2 weeks. The heart weight to tibial length ratio was significantly increased by Ang II in vehicle-treated mice. Treatment with CNP decreased Ang II-induced cardiac hypertrophy without affecting systolic blood pressure. Echocardiography showed that CNP attenuated Ang II-induced increase in wall thickness, left ventricular dilatation, and decrease in fractional shortening. CNP reduced Ang II-induced increases in cardiomyocyte size and interstitial fibrosis and suppressed hypertrophic- and fibrosis-related gene expression. Finally, CNP decreased Ang II-induced cardiac superoxide production. These changes were accompanied by suppression of NOX4 gene expression. Our data indicate that treatment with CNP attenuated Ang II-induced cardiac hypertrophy, fibrosis, and contractile dysfunction which were accompanied by reduced cardiac superoxide production.

Abstract P032: Nanoparticle Mediated Nox2-siRNA Therapy for Preventing Cardiac Dysfunction Following Myocardial Infarction

Myocardial infarction (MI) is the most common cause of heart failure in the developed world. Following MI, reactive oxygen species (ROS) play a key role in the pathogenesis of cardiac remodeling leading to impaired ventricular function. Oxidative stress at high levels lead to many of the injury associated changes: proinflammatory cytokine release, myocyte apoptosis, cardiac fibrosis and hypertrophy. Nicotinamide adenine denucleotide phosphate (NADPH) oxidase with Nox2 as the catalytic subunit, is a major source for cardiac ROS production. After MI, Nox2 expression is significantly increased in the infarcted myocardium. Moreover, mice lacking the Nox2 gene are protected from ischemic injury. We used polyketals, a new class of acid-degradable polymers, as delivery vehicles for Nox2-siRNA to the post-MI environment. When engaged by macrophages, present in high quantities during MI, these particles have been shown to be taken up by macrophages and contents released within cells in active form. Nox2-siRNA was ion-paired to the cationic lipid DOTAP, and spherical particles averaging 500nm diameter were made by a single emulsion procedure with polyketal PK3 using PVA as surfactant. While a commercially available transfection reagent yielded 7% uptake as measured by flow cytometery, 81% of macrophages were positive for fluorescently-labeled siRNA in PK3-siNOX2 treated cells. These data were confirmed with confocal microscopy. Macrophages treated with PK3-siNOX2 demonstrated a significant 43% knockdown in Nox2 gene expression at 24 hours, while no reduction was seen with scrambled siRNA or empty particles. Functional activity was assessed by a fluorescent dihyroethidium dye based HPLC quantification after phorbol 12-myristate 13-acetate stimulation following 72 hours of particle treatment to measure superoxide production. PK3-siNox2 treated cells exhibited a 41% reduction in activity, with no significant changes seen with scrambled siRNA or empty particle treatment. Currently, the therapeutic potential of the Nox2-siRNA particles is being evaluated in a mouse model of ischemia-reperfusion. Successful completion of these studies could lead to a novel treatment for post-infarction injury.

Pulmonary Oxidative Stress Is Increased in Cyclooxygenase-2 Knockdown Mice with Mild Pulmonary Hypertension Induced by Monocrotaline

The aim of this study was to examine the role of cyclooxygenase-2 (COX-2) and downstream signaling of prostanoids in the pathogenesis of pulmonary hypertension (PH) using mice with genetically manipulated COX-2 expression. COX-2 knockdown (KD) mice, characterized by 80–90% suppression of COX-2, and wild-type (WT) control mice were treated weekly with monocrotaline (MCT) over 10 weeks. Mice were examined for cardiac hypertrophy/function and right ventricular pressure. Lung histopathological analysis was performed and various assays were carried out to examine oxidative stress, as well as gene, protein, cytokine and prostanoid expression. We found that MCT increased right ventricular systolic and pulmonary arterial pressures in comparison to saline-treated mice, with no evidence of cardiac remodeling. Gene expression of endothelin receptor A and thromboxane synthesis, regulators of vasoconstriction, were increased in MCT-treated lungs. Bronchoalveolar lavage fluid and lung sections demonstrated mild inflammation and perivascular edema but activation of inflammatory cells was not predominant under the experimental conditions. Heme oxygenase-1 (HO-1) expression and indicators of oxidative stress in lungs were significantly increased, especially in COX-2 KD MCT-treated mice. Gene expression of NOX-4, but not NOX-2, two NADPH oxidase subunits crucial for superoxide generation, was induced by ∼4-fold in both groups of mice by MCT. Vasodilatory and anti-aggregatory prostacyclin was reduced by ∼85% only in MCT-treated COX-2 KD mice. This study suggests that increased oxidative stress-derived endothelial dysfunction, vasoconstriction and mild inflammation, exacerbated by the lack of COX-2, contribute to the pathogenesis of early stages of PH when mild hemodynamic changes are evident and not yet accompanied by vascular and cardiac remodeling.

Piper sarmentosum inhibits ICAM-1 and Nox4 gene expression in oxidative stress-induced human umbilical vein endothelial cells

BackgroundAqueous extract of Piper sarmentosum (AEPS) is known to possess antioxidant and anti-atherosclerotic activities but the mechanism responsible for it remains unclear. In early part of atherosclerosis, nuclear factor-kappa B (NF-κB) induces the expression of cellular adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1) and E-selectin. NADPH oxidase 4 (Nox4) is the predominant source of superoxide in the endothelial cells whereas superoxide dismutase 1 (SOD1), catalase (CAT) and glutathione peroxidase (GPx) are the antioxidant enzymes responsible for inactivating reactive oxygen species. The present study aimed to investigate the effects of AEPS on the gene expression of NF-κB, VCAM-1, ICAM-1, E-selectin, Nox4, SOD1, CAT and GPx in cultured human umbilical vein endothelial cells (HUVECs).MethodsHUVECs were divided into four groups:- control; treatment with 180 μM hydrogen peroxide (H2O2); treatment with 150 μg/mL AEPS and concomitant treatment with AEPS and H2O2 for 24 hours. Total RNA was extracted from all the groups of HUVEC using TRI reagent. Subsequently, qPCR was carried out to determine the mRNA expression of NF-κB, VCAM-1, ICAM-1, E-selectin, Nox4, SOD1, CAT and GPx. The specificity of the reactions was verified using melting curve analysis and agarose gel electrophoresis.ResultsWhen stimulated with H2O2, HUVECs expressed higher level of ICAM-1 (1.3-fold) and Nox4 (1.2-fold) mRNA expression. However, AEPS treatment led to a reduction in the mRNA expression of ICAM-1 (p < 0.01) and Nox4 (p < 0.05) in the H2O2-induced HUVECs. AEPS also upregulated the mRNA expression of SOD1 (p < 0.05), CAT (p < 0.01) and GPx (p < 0.05) in oxidative stress-induced HUVECs. There was no significant change in the mRNA expression of VCAM-1 and E-selectin.ConclusionThe expressional suppression of ICAM-1 and Nox4 and induction of antioxidant enzymes might be an important component of the vascular protective effect of AEPS.

Mineralocorticoid Accelerates Transition to Heart Failure With Preserved Ejection Fraction Via “Nongenomic Effects”

Mechanisms promoting the transition from hypertensive heart disease to heart failure with preserved ejection fraction are poorly understood. When inappropriate for salt status, mineralocorticoid (deoxycorticosterone acetate) excess causes hypertrophy, fibrosis, and diastolic dysfunction. Because cardiac mineralocorticoid receptors are protected from mineralocorticoid binding by the absence of 11-beta hydroxysteroid dehydrogenase, salt-mineralocorticoid-induced inflammation is postulated to cause oxidative stress and to mediate cardiac effects. Although previous studies have focused on salt/nephrectomy in accelerating mineralocorticoid-induced cardiac effects, we hypothesized that hypertensive heart disease is associated with oxidative stress and sensitizes the heart to mineralocorticoid, accelerating hypertrophy, fibrosis, and diastolic dysfunction. Cardiac structure and function, oxidative stress, and mineralocorticoid receptor-dependent gene transcription were measured in sham-operated and transverse aortic constriction (studied 2 weeks later) mice without and with deoxycorticosterone acetate administration, all in the setting of normal-salt diet. Compared with sham mice, sham plus deoxycorticosterone acetate mice had mild hypertrophy without fibrosis or diastolic dysfunction. Transverse aortic constriction mice displayed compensated hypertensive heart disease with hypertrophy, increased oxidative stress (osteopontin and NOX4 gene expression), and normal systolic function, filling pressures, and diastolic stiffness. Compared with transverse aortic constriction mice, transverse aortic constriction plus deoxycorticosterone acetate mice had similar left ventricular systolic pressure and fractional shortening but more hypertrophy, fibrosis, and diastolic dysfunction with increased lung weights, consistent with heart failure with preserved ejection fraction. There was progressive activation of markers of oxidative stress across the groups but no evidence of classic mineralocorticoid receptor-dependent gene transcription. Pressure-overload hypertrophy sensitizes the heart to mineralocorticoid excess, which promotes the transition to heart failure with preserved ejection fraction independently of classic mineralocorticoid receptor-dependent gene transcription.

PPARγ regulates hypoxia-induced Nox4 expression in human pulmonary artery smooth muscle cells through NF-κB

NADPH oxidases are a major source of superoxide production in the vasculature. The constitutively active Nox4 subunit, which is selectively upregulated in the lungs of human subjects and experimental animals with pulmonary hypertension, is highly expressed in vascular wall cells. We demonstrated that rosiglitazone, a synthetic agonist of the peroxisome proliferator-activated receptor-γ (PPARγ), attenuated hypoxia-induced pulmonary hypertension, vascular remodeling, Nox4 induction, and reactive oxygen species generation in the mouse lung. The current study examined the molecular mechanisms involved in PPARγ-regulated, hypoxia-induced Nox4 expression in human pulmonary artery smooth muscle cells (HPASMC). Exposing HPASMC to 1% oxygen for 72 h increased Nox4 gene expression and H(2)O(2) production, both of which were reduced by treatment with rosiglitazone during the last 24 h of hypoxia exposure or by treatment with small interfering RNA (siRNA) to Nox4. Hypoxia also increased HPASMC proliferation as well as the activity of a Nox4 promoter luciferase reporter, and these increases were attenuated by rosiglitazone. Chromatin immunoprecipitation assays demonstrated that hypoxia increased binding of the NF-κB subunit, p65, to the Nox4 promoter and that binding was attenuated by rosiglitazone treatment. The role of NF-κB in Nox4 regulation was further supported by demonstrating that overexpression of p65 stimulated Nox4 promoter activity, whereas siRNA to p50 or p65 attenuated hypoxic stimulation of Nox4 promoter activity. These results provide novel evidence for NF-κB-mediated stimulation of Nox4 expression in HPASMC that can be negatively regulated by PPARγ. These data provide new insights into potential mechanisms by which PPARγ activation inhibits Nox4 upregulation and the proliferation of cells in the pulmonary vascular wall to ameliorate pulmonary hypertension and vascular remodeling in response to hypoxia.

PPARγ regulates hypoxia‐induced Nox4 expression in human pulmonary artery smooth muscle cells through NF‐κB

The Nox4 NADPH oxidase, which is highly expressed in vascular wall cells, is selectively upregulated in the lungs of human subjects and experimental animals with pulmonary hypertension. We demonstrated that rosiglitazone, a synthetic PPARγ agonist, attenuated hypoxia‐induced pulmonary hypertension, vascular remodeling, Nox4 induction and reactive oxygen species generation in the mouse lung. The current study examined PPARγ‐regulated, hypoxia‐induced Nox4 expression in human pulmonary artery smooth muscle cells (HPASMC). Exposure to 1% oxygen for 72 hours increased Nox4 gene expression and HPASMC H2O2 production, both of which were reduced by treatment with rosiglitazone or with siRNA to Nox4. Rosiglitazone also attenuated hypoxia‐induced HPASMC proliferation and the activity of a Nox4 promoter luciferase reporter. Chromatin immunoprecipitation assays demonstrated that hypoxic‐induced binding of the NF‐κB subunits, p65 and C‐rel, to the Nox4 promoter was attenuated by rosiglitazone. The role of NF‐κB in Nox4 regulation was further supported by demonstrating that overexpression of p65 stimulated Nox4 promoter activity whereas siRNA to p50 or p65 attenuated hypoxic‐stimulated Nox4 promoter activity. These results provide novel evidence for NF‐κB‐mediated stimulation of Nox4 expression in HPASMC that can be negatively regulated by PPARγ.

Role of caveolin and heat shock protein 70 interaction in the antioxidative effects of an angiotensin II type 1 receptor blocker in spontaneously hypertensive rats proximal tubules

Role of caveolin and heat shock protein 70 interaction in the antioxidative effects of an angiotensin II type 1 receptor blocker in spontaneously hypertensive rats proximal tubules

Irreversibly glycated LDL induce oxidative and inflammatory state in human endothelial cells; added effect of high glucose

In diabetes, hyperglycemia and the associated formation of advanced glycation end-products (AGE) and AGE-modified low density lipoproteins (AGE-LDL) can directly affect the cells of the vascular wall. We hypothesize that AGE-LDL may act directly and induce oxidant and inflammatory alterations in human endothelial cells (HEC), this effect being amplified by high glucose. To test this assumption, the activity of NADPH oxidase (NADPHox) was evaluated and the expression of its subunits (p22 phox, NOX4, and p67 phox), of the AGE receptor (RAGE), and of the monocyte chemoattractant protein-1 (MCP-1) were assessed by real-time PCR and Western blot in confluent EA.hy926 cells incubated with AGE-LDL for 24 and 48 h, in normal and high glucose conditions. Exposure of HEC for 48 h to AGE-LDL in 5 mM glucose induced an increase of RAGE expression (50%), NADPHox activity (107%), p22 phox and NOX4 mRNA (50% and 188%, respectively) and MCP-1 expression (80%). AGE-LDL-stimulated p22 phox expression by activating p38 MAP kinase and NF-kB, and MCP-1 expression by activating NF-kB, as demonstrated by the use of specific inhibitors (SB203580 and Bay11-7085). The addition of 25 mM glucose in the culture medium enhanced the effect of AGE-LDL, but also of nLDL, on RAGE, p22 phox, NOX4, p67 phox, and MCP-1 gene expression. In conclusion, AGE-LDL induce an oxidative stress and a pro-inflammatory state in human endothelial cells. Both AGE-LDL and nLDL in the presence of high glucose amplify their effect, revealing a link between hyperlipidemia, diabetes, and endothelial cell dysfunction.

Hepatitis C Virus (HCV) Proteins Induce NADPH Oxidase 4 Expression in a Transforming Growth Factor β-Dependent Manner: a New Contributor to HCV-Induced Oxidative Stress

Viral hepatitis-induced oxidative stress accompanied by increased levels of transforming growth factor beta (TGF-beta) and hepatic fibrosis are hallmarks of hepatitis C virus (HCV) infection. The mechanisms of redox regulation in the pathogenesis of HCV-induced liver disease are not clearly understood. The results of our current studies suggest that reactive oxygen species (ROS) derived from Nox4, a member of the NADPH oxidase (Nox) family, could play a role in HCV-induced liver disease. We found that the expression of HCV (genotype 1a) cDNA constructs (full-length and subgenomic), core protein alone, viral RNA, or replicating HCV (JFH-AM2) induced Nox4 mRNA expression and ROS generation in human hepatocyte cell lines (Huh-7, Huh-7.5, HepG2, and CHL). Conversely, hepatocytes expressing Nox4 short hairpin RNA (shRNA) or an inactive dominant negative form of Nox4 showed decreased ROS production when cells were transfected with HCV. The promoters of both human and murine Nox4 were used to demonstrate transcriptional regulation of Nox4 mRNA by HCV, and a luciferase reporter tied to an approximately 2-kb promoter region of Nox4 identified HCV-responsive regulatory regions modulating the expression of Nox4. Furthermore, the human Nox4 promoter was responsive to TGF-beta1, and the HCV core-dependent induction of Nox4 was blocked by antibody against TGF-beta or the expression of dominant negative TGF-beta receptor type II. These findings identified HCV as a regulator of Nox4 gene expression and subsequent ROS production through an autocrine TGF-beta-dependent mechanism. Collectively, these data provide evidence that HCV-induced Nox4 contributes to ROS production and may be related to HCV-induced liver disease.

Getting Radical About Obesity

Visceral obesity has become the most frequent preventable condition accounting for diseases such as hypertension and diabetes, two of the major risk factors for coronary artery disease, stroke, and chronic renal failure.1–3 Despite the awareness aiming to reduce the prevalence of obesity, the number of obese patients continues to rise and now includes even developing countries, and increasingly children are affected.4,5 In fact, if this trend continues, 26 million children in Europe will be overweight or obese by the year 2010, rising by approximately 1.3 million per year.5,6 The annual health costs related to obesity have been estimated close to 100 billion USD in the United States,7,8 illustrating the enormous economic burden the disease obesity carries. See accompanying article in ATVB 2009;29:239–245. What are the mechanisms contributing to the high cardiovascular risk brought about by obesity? Obesity is a low-grade inflammatory condition9 and typically characterized by increase in visceral adipose tissue. Visceral fat is both a source and target of inflammatory cytokines and growth factors, which directly may affect preadipocyte differentiation via signaling cascades implicated in cell growth such as Ras-Raf-ERK-1/2.10 Adipose tissue also contains inflammatory cells, including cytokine-producing macrophages.9 In addition, adipocytes express a fully functional NADPH oxidase …

Angiotensin II AT1 blockade reduces the lipopolysaccharide-induced innate immune response in rat spleen

ANG II AT(1) receptor blockade reduces inflammation in hypertension. To determine whether ANG II AT(1) receptor blockers (ARBs) influence the innate immune inflammatory response in normotensive rats, we studied rat plasma and spleen after a 3-day subcutaneous pretreatment with the ARB candesartan followed by a single dose of the bacterial endotoxin LPS (50 microg/kg ip). Peripheral administration of LPS to rodents produced a generalized inflammatory response with increased release of TNF-alpha, IL-1beta, and IL-6 into the circulation. Candesartan pretreatment reduced the LPS-induced release of TNF-alpha, IL-1beta, and IL-6 into the circulation. The red pulp of rat spleen expressed large numbers of AT(1) receptors and the LPS receptors Toll-like receptor 4 and CD14. Candesartan administration significantly blocked AT(1) receptors. The ARB reduced the LPS-induced upregulation of CD14 gene expression; expression of TNF-alpha and IL-6 mRNA and protein; expression of IL-1beta and IkappaB-alpha mRNA; COX-2 mRNA and protein expression and PGE(2) concentration; inducible nitric oxide synthase (iNOS) gene and protein expression and iNOS activity; and Nox2 gene expression and 8-isoprostane levels. In addition, candesartan reduced the CD14 protein expression in saline- and LPS-treated rats. Our results suggest that AT(1) receptors are essential for the development of the full innate immune response to bacterial endotoxin. The ARB decreased the general peripheral inflammatory reaction to LPS and partially decreased the inflammatory response in the spleen. An unrestricted innate immune response to the bacterial endotoxin may have deleterious effects for the organism and may lead to development of chronic inflammatory disease. We postulate that ARBs may have therapeutic effects on inflammatory conditions.

NOX4 mediates hypoxia-induced proliferation of human pulmonary artery smooth muscle cells: the role of autocrine production of transforming growth factor-β1 and insulin-like growth factor binding protein-3

Persistent hypoxia can cause pulmonary arterial hypertension that may be associated with significant remodeling of the pulmonary arteries, including smooth muscle cell proliferation and hypertrophy. We previously demonstrated that the NADPH oxidase homolog NOX4 mediates human pulmonary artery smooth muscle cell (HPASMC) proliferation by transforming growth factor-beta1 (TGF-beta1). We now show that hypoxia increases HPASMC proliferation in vitro, accompanied by increased reactive oxygen species generation and NOX4 gene expression, and is inhibited by antioxidants, the flavoenzyme inhibitor diphenyleneiodonium (DPI), and NOX4 gene silencing. HPASMC proliferation and NOX4 expression are also observed when media from hypoxic HPASMC are added to HPASMC grown in normoxic conditions, suggesting autocrine stimulation. TGF-beta1 and insulin-like growth factor binding protein-3 (IGFBP-3) are both increased in the media of hypoxic HPASMC, and increased IGFBP-3 gene expression is noted in hypoxic HPASMC. Treatment with anti-TGF-beta1 antibody attenuates NOX4 and IGFBP-3 gene expression, accumulation of IGFBP-3 protein in media, and proliferation. Inhibition of IGFBP-3 expression with small interfering RNA (siRNA) decreases NOX4 gene expression and hypoxic proliferation. Conversely, NOX4 silencing does not decrease hypoxic IGFBP-3 gene expression or secreted protein. Smad inhibition does not but the phosphatidylinositol 3-kinase (PI3K) signaling pathway inhibitor LY-294002 does inhibit NOX4 and IGFBP-3 gene expression, IGFBP-3 secretion, and cellular proliferation resulting from hypoxia. Immunoblots from hypoxic HPASMC reveal increased TGF-beta1-mediated phosphorylation of the serine/threonine kinase (Akt), consistent with hypoxia-induced activation of PI3K/Akt signaling pathways to promote proliferation. We conclude that hypoxic HPASMC produce TGF-beta1 that acts in an autocrine fashion to induce IGFBP-3 through PI3K/Akt. IGFBP-3 increases NOX4 gene expression, resulting in HPASMC proliferation. These observations add to our understanding hypoxic pulmonary vascular remodeling.

Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2

NADPH oxidase (Nox4) produces reactive oxygen species (ROS) that are important for vascular smooth muscle cell (SMC) behavior, but the potential impact of Nox4 in stem cell differentiation is unknown. When mouse embryonic stem (ES) cells were plated on collagen IV-coated dishes/flasks, a panel of SMC-specific genes was significantly and consistently upregulated. Nox4 expression was markedly correlated with such a gene induction as confirmed by real-time PCR, immunofluorescence, and Western blot analysis. Overexpression of Nox4 specifically resulted in increased SMC marker production, whereas knockdown of Nox4 induced a decrease. Furthermore, SMC-specific transcription factors, including serum response factor (SRF) and myocardin were activated by Nox4 gene expression. Moreover, Nox4 was demonstrated to drive SMC differentiation through generation of H(2)O(2). Confocal microscopy analysis indicates that SRF was translocated into the nucleus during SMC differentiation in which SRF was phosphorylated. Additionally, autosecreted transforming growth factor (TGF)-beta(1) activated Nox4 and promoted SMC differentiation. Interestingly, cell lines generated from stem cells by Nox4 transfection and G418 selection displayed a characteristic of mature SMCs, including expression of SMC markers and cells with contractile function. Thus we demonstrate for the first time that Nox4 is crucial for SMC differentiation from ES cells, and enforced Nox4 expression can maintain differentiation status and functional features of stem cell-derived SMCs, highlighting its impact on vessel formation in vivo and vascular tissue engineering in the future.

Differential roles of PKCα and PKCɛ in controlling the gene expression of Nox4 in human endothelial cells

NADPH oxidases are major sources of superoxide in the vascular wall. This study investigates the role of protein kinase C (PKC) in regulating gene expression of NADPH oxidases. Treatment of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 endothelial cells with phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate led to a PKC-dependent biphasic expression of the gp91phox homolog Nox4. A downregulation of Nox4 was observed at 6 h and an upregulation at 48 h after phorbol ester treatment. The early Nox4 downregulation was associated with a reduced superoxide production, whereas the late Nox4 upregulation was accompanied by a clear enhancement of superoxide. PMA activated the PKC isoforms α and ɛ in HUVEC and EA.hy 926 cells. Knockdown of PKCɛ by siRNA prevented the early downregulation of Nox4, whereas knockdown of PKCα selectively abolished the late Nox4 upregulation. Vascular endothelial growth factor (VEGF), which activates PKCα but not PKCɛ in HUVEC, increased Nox4 expression without the initial downregulation. VEGF-induced Nox4 upregulation was associated with an enhanced proliferation and angiogenesis of HUVEC. Both effects could be reduced by inhibition of NADPH oxidase. Thus, a selective inhibition/knockdown of PKCα may represent a novel therapeutic strategy for vascular disease.