Junya Kuroda

NADPH oxidase 4 (Nox4) is a major source of oxidative stress in the failing heart

NAD(P)H oxidases (Noxs) produce O2− and play an important role in cardiovascular pathophysiology. The Nox4 isoform is expressed primarily in the mitochondria in cardiac myocytes. To elucidate the function of endogenous Nox4 in the heart, we generated cardiac-specific Nox4−/− (c-Nox4−/−) mice. Nox4 expression was inhibited in c-Nox4−/− mice in a heart-specific manner, and there was no compensatory up-regulation in other Nox enzymes. These mice exhibited reduced levels of O2− in the heart, indicating that Nox4 is a significant source of O2− in cardiac myocytes. The baseline cardiac phenotype was normal in young c-Nox4−/− mice. In response to pressure overload (PO), however, increases in Nox4 expression and O2− production in mitochondria were abolished in c-Nox4−/− mice, and c-Nox4−/− mice exhibited significantly attenuated cardiac hypertrophy, interstitial fibrosis and apoptosis, and better cardiac function compared with WT mice. Mitochondrial swelling, cytochrome c release, and decreases in both mitochondrial DNA and aconitase activity in response to PO were attenuated in c-Nox4−/− mice. On the other hand, overexpression of Nox4 in mouse hearts exacerbated cardiac dysfunction, fibrosis, and apoptosis in response to PO. These results suggest that Nox4 in cardiac myocytes is a major source of mitochondrial oxidative stress, thereby mediating mitochondrial and cardiac dysfunction during PO.

Upregulation of Nox4 by Hypertrophic Stimuli Promotes Apoptosis and Mitochondrial Dysfunction in Cardiac Myocytes

Rationale: NADPH oxidases are a major source of superoxide (O2−) in the cardiovascular system. The function of Nox4, a member of the Nox family of NADPH oxidases, in the heart is poorly understood. Objective: The goal of this study was to elucidate the role of Nox4 in mediating oxidative stress and growth/death in the heart. Methods and Results: Expression of Nox4 in the heart was increased in response to hypertrophic stimuli and aging. Neither transgenic mice with cardiac specific overexpression of Nox4 (Tg-Nox4) nor those with catalytically inactive Nox4 (Tg-Nox4-P437H) showed an obvious baseline cardiac phenotype at young ages. Tg-Nox4 gradually displayed decreased left ventricular (LV) function with enhanced O2− production in the heart, which was accompanied by increased apoptosis and fibrosis at 13 to 14 months of age. On the other hand, the level of oxidative stress was attenuated in Tg-Nox4-P437H. Although the size of cardiac myocytes was significantly greater in Tg-Nox4 than in nontransgenic, the LV weight/tibial length was not significantly altered in Tg-Nox4 mice. Overexpression of Nox4 in cultured cardiac myocytes induced apoptotic cell death but not hypertrophy. Nox4 is primarily localized in mitochondria and upregulation of Nox4 enhanced both rotenone- and diphenyleneiodonium-sensitive O2− production in mitochondria. Cysteine residues in mitochondrial proteins, including aconitase and NADH dehydrogenases, were oxidized and their activities decreased in Tg-Nox4. Conclusions: Upregulation of Nox4 by hypertrophic stimuli and aging induces oxidative stress, apoptosis and LV dysfunction, in part because of mitochondrial insufficiency caused by increased O2− production and consequent cysteine oxidation in mitochondrial proteins.

The superoxide‐producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells

The superoxide‐producing NAD(P)H oxidase Nox4 was initially identified as an enzyme that is highly expressed in the kidney and is possibly involved in oxygen sensing and cellular senescence. Although the oxidase is also abundant in vascular endothelial cells, its role remains to be elucidated. Here we show that Nox4 preferentially localizes to the nucleus of human umbilical vein endothelial cells (HUVECs), by immunocytochemistry and immunoelectron microscopy using three kinds of affinity‐purified antibodies raised against distinct immunogens from human Nox4. Silencing of Nox4 by RNA interference (RNAi) abrogates nuclear signals given with the antibodies, confirming the nuclear localization of Nox4. The nuclear fraction of HUVECs exhibits an NAD(P)H‐dependent superoxide‐producing activity in a manner dependent on Nox4, which activity can be enhanced upon cell stimulation with phorbol 12‐myristate 13‐acetate. This stimulant also facilitates gene expression as estimated in the present transfection assay of HUVECs using a reporter regulated by the Maf‐recognition element MARE, a DNA sequence that constitutes a part of oxidative stress response. Both basal and stimulated transcriptional activities are impaired by RNAi‐mediated Nox4 silencing. Thus Nox4 appears to produce superoxide in the nucleus of HUVECs, thereby regulating gene expression via a mechanism for oxidative stress response.

Regulation of myocardial growth and death by NADPH oxidase

The NADPH oxidases (Nox) are transmembrane proteins dedicated to producing reactive oxygen species (ROS), including superoxide and hydrogen peroxide, by transferring electrons from NAD(P)H to molecular oxygen. Nox2 and Nox4 are expressed in the heart and play an important role in mediating oxidative stress at baseline and under stress. Nox2 is primarily localized on the plasma membrane, whereas Nox4 is found primarily on intracellular membranes, on mitochondria, the endoplasmic reticulum or the nucleus. Although Nox2 plays an important role in mediating angiotensin II-induced cardiac hypertrophy, Nox4 mediates cardiac hypertrophy and heart failure in response to pressure overload. Expression of Nox4 is upregulated by hypertrophic stimuli, and Nox4 in mitochondria plays an essential role in mediating oxidative stress during pressure overload-induced cardiac hypertrophy. Upregulation of Nox4 induces oxidation of mitochondrial proteins, including aconitase, thereby causing mitochondrial dysfunction and myocardial cell death. On the other hand, Noxs also appear to mediate physiological functions, such as erythropoiesis and angiogenesis. In this review, we discuss the role of Noxs in mediating oxidative stress and both pathological and physiological functions of Noxs in the heart.

NAD(P)H Oxidases in Rat Basilar Arterial Endothelial Cells

Background and Purpose— Reactive oxygen species (ROS) may play a critical role in the regulation of vascular tone and development of vascular diseases, such as stroke. NAD(P)H oxidase is a major source of ROS in vascular cells, including endothelial cells. It has been considered that Nox2 and Nox4 are exclusively expressed among Nox homologues in the endothelial cells of noncerebral blood vessels. However, the precise molecular identity of the NAD(P)H oxidase in the endothelial cells of the cerebral arteries is not fully understood. We examined the expression of Nox homologues and their activation mechanism in the endothelial cells of the cerebral arteries. Methods— We isolated and cultured basilar artery endothelial cells (BAECs) of Sprague-Dawley rats. Expression of NAD(P)H oxidase was examined by reverse-transcription-polymerase chain reaction (RT-PCR) and immunohistological staining. Results— RT-PCR disclosed abundant expression of Nox4 with marginal Nox2 in BAEC. In addition, Nox1 was expressed highly both at mRNA and protein levels in BAECs. Immunohistological staining also showed the prominent expression of Nox1 in the endothelial cells of the basilar artery. With respect to the cytosolic components of NAD(P)H oxidases, BAECs expressed p67phox and, to a lesser extent, p47phox, Noxo1, and Noxa1. Both NADH and NADPH induced superoxide production of the BAEC membranes. The phagocyte-type cytosolic components, p47phox and p67phox, significantly enhanced the NADH-induced superoxide production of the BAEC membranes, whereas the components failed to increase the NADPH-induced superoxide production. Conclusions— Nox1 is highly expressed in the endothelial cells of the cerebral arteries along with Nox2 and Nox4, and the endothelial NAD(P)H oxidase of the cerebral arteries may have a unique activation mechanism by the phagocyte-type cytosolic components.

Increased Oxidative Stress in the Nucleus Caused by Nox4 Mediates Oxidation of HDAC4 and Cardiac Hypertrophy

Rationale: Oxidation of cysteine residues in class II histone deacetylases (HDACs), including HDAC4, causes nuclear exit, thereby inducing cardiac hypertrophy. The cellular source of reactive oxygen species responsible for oxidation of HDAC4 remains unknown. Objective: We investigated whether nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4), a major nicotinamide adenine dinucleotide phosphate oxidase, mediates cysteine oxidation of HDAC4. Methods and Results: Phenylephrine (100 &mgr;mol/L), an &agr;1 adrenergic agonist, induced upregulation of Nox4 (1.5-fold; P<0.05) within 5 minutes, accompanied by increases in O2− (3.5-fold; P<0.01) from the nuclear membrane and nuclear exit of HDAC4 in cardiomyocytes. Knockdown of Nox4, but not Nox2, attenuated O2− production in the nucleus and prevented phenylephrine-induced oxidation and nuclear exit of HDAC4. After continuous infusion of phenylephrine (20 mg/kg per day) for 14 days, wild-type and cardiac-specific Nox4 knockout mice exhibited similar aortic pressures. Left ventricular weight/tibial length (5.7±0.2 versus 6.4±0.2 mg/mm; P<0.05) and cardiomyocytes cross-sectional area (223±13 versus 258±12 &mgr;m2; P<0.05) were significantly smaller in cardiac-specific Nox4 knockout than in wild-type mice. Nuclear O2−production in the heart was significantly lower in cardiac-specific Nox4 knockout than in wild-type mice (4116±314 versus 7057±1710 relative light unit; P<0.05), and cysteine oxidation of HDAC4 was decreased. HDAC4 oxidation and cardiac hypertrophy were also attenuated in cardiac-specific Nox4 knockout mice 2 weeks after transverse aortic constriction. Conclusions: Nox4 plays an essential role in mediating cysteine oxidation and nuclear exit of HDAC4, thereby mediating cardiac hypertrophy in response to phenylephrine and pressure overload.

Increased expression of gp91phox homologues of NAD(P)H oxidase in the aortic media during chronic hypertension: involvement of the renin-angiotensin system.

Although vascular cells express multiple members of the Nox family of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase, including gp91phox, Nox1, and Nox4, the reasons for the different expressions and specific roles of these members in vascular injury in chronic hypertension have remained unclear. Thus, we quantified the mRNA expressions of these NAD(P)H oxidase components by real-time polymerase chain reaction and evaluated superoxide production and morphological changes in the aortas of 32-week-old stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wistar Kyoto rats (WKY). The aortic media of SHRSP had an approximately 2.5-fold greater level of Nox4 mRNA and an approximately 10-fold greater level of Nox1 mRNA than WKY. The mRNA expressions of gp91phox and p22phox in SHRSP and WKY were comparable. SHRSP were treated from 24 weeks of age for 8 weeks with either high or low doses of candesartan (4 mg/kg/day or 0.2 mg/kg/day), or a combination of hydralazine (30 mg/kg/day) and hydrochlorothiazide (4.5 mg/kg/day). The high-dose candesartan or the hydralazine plus hydrochlorothiazide decreased the blood pressure of SHRSP to that of WKY, whereas the low-dose candesartan exerted no significant antihypertensive action. Media thickening and fibrosis, as well as the increased production of superoxide in SHRSP, were nearly normalized with high-dose candesartan and partially corrected with low-dose candesartan or hydralazine plus hydrochlorothiazide. These changes by antihypertensive treatment paralleled the decrease in mRNA expression of Nox4 and Nox1. These results suggest that blood pressure and angiotensin II type 1 receptor activation are involved in the up-regulation of Nox1 and Nox4 expression, which could contribute to vascular injury during chronic hypertension.

Enhanced expression of NADPH oxidase Nox4 in human gliomas and its roles in cell proliferation and survival

Reactive oxygen species (ROS) have been attracting attention as mediators of various cell‐signaling pathways. Nox‐family NADPH oxidases have proven to be a major source of ROS production in various cell types and have crucial roles in various physiological and pathological processes. In this study, we show that Nox4, a member of Nox family, is prominently expressed in various neuroepithelial tumors by reverse transcription‐polymerase chain reaction (RT‐PCR) and immunohistochemical studies. We quantified Nox4 mRNA expression by real‐time PCR in tumor specimens from 58 patients with astrocytomas and found that the expression levels of Nox4 mRNA in glioblastomas (WHO grade IV) were significantly higher than those in other astrocytomas (WHO grade II and III). In addition, we show that specific knockdown of Nox4 expression by RNA interference results in cell‐growth inhibition and enhances induction of apoptosis by chemotherapeutic agents, such as cisplatin, in cultured glioma cell lines. Based on these observations, enhanced expression of Nox4 appears to be involved in cell proliferation and survival in glioma cells.

Broad Suppression of NADPH Oxidase Activity Exacerbates Ischemia/Reperfusion Injury Through Inadvertent Downregulation of Hypoxia-inducible Factor-1α and Upregulation of Peroxisome Proliferator–activated Receptor-α

Rationale: Nox2 and Nox4 are major components of the NADPH oxidase (Nox) family, which purposefully produce reactive oxidative species (ROS), namely O 2- and H 2 O 2 , in the heart. The isoform-specific contribution of Nox2 and Nox4 to ischemia/reperfusion (I/R) injury is poorly understood. Objective: We investigated the role of Nox2 and Nox4 in mediating oxidative stress and myocardial injury during I/R using loss of function mouse models. Methods and Results: Systemic (s) Nox2 KO, sNox4 KO, and cardiac-specific (c) Nox4 KO mice were subjected to I (30 min)/R (24 h). Both myocardial infarct size/area at risk (MI/AAR) and O 2- production were lower in sNox2 KO, sNox4 KO, and cNox4 KO than in wild-type (WT) mice. Unexpectedly, however, the MI/AAR was greater, despite less O2- production, in sNox2 KO+cNox4 KO (DKO) mice and transgenic mice with cardiac-specific expression of dominant-negative Nox (Tg-DN-Nox), which suppresses both Nox2 and Nox4, than in WT or single KO mice. Hypoxia-inducible factor-1α (HIF-1α) was downregulated while peroxisome proliferator-activated receptor-alpha (PPARα was upregulated in Tg-DN-Nox mice. A cross with mice deficient in prolyl hydroxylase 2, which hydroxylates HIF-1α, rescued the I/R injury and prevented upregulation of PPARα in Tg-DN-Nox mice. A cross with PPARα KO mice also attenuated the injury in Tg-DN-Nox mice. Conclusions: nBoth Nox2 and Nox4 contribute to the increase in ROS and injury by I/R. However, low levels of ROS produced by either Nox2 or Nox4 regulate HIF-1α and PPARα thereby protecting the heart against I/R, suggesting that Noxs also act as a physiological sensor for myocardial adaptation.Rationale: NADPH oxidase (Nox) 2 and Nox4 are major components of the Nox family which purposefully produce reactive oxidative species, namely O2− and H2O2, in the heart. The isoform-specific contribution of Nox2 and Nox4 to ischemia/reperfusion (I/R) injury is poorly understood. Objective: We investigated the role of Nox2 and Nox4 in mediating oxidative stress and myocardial injury during I/R using loss-of-function mouse models. Methods and Results: Systemic (s) Nox2 knockout (KO), sNox4 KO, and cardiac-specific (c) Nox4 KO mice were subjected to I/R (30 minutes/24 hours, respectively). Both myocardial infarct size/area at risk and O2− production were lower in sNox2 KO, sNox4 KO, and cNox4 KO than in wild-type mice. Unexpectedly, however, the myocardial infarct size/area at risk was greater, despite less O2− production, in sNox2 KO+cNox4 KO (double-KO) mice and transgenic mice (Tg) with cardiac-specific expression of dominant-negative Nox, which suppresses both Nox2 and Nox4, than in wild-type or single KO mice. Hypoxia-inducible factor-1&agr; was downregulated whereas peroxisome proliferator–activated receptor-&agr; was upregulated in Tg-dominant-negative Nox mice. A cross with mice deficient in prolyl hydroxylase 2, which hydroxylates hypoxia-inducible factor-1&agr;, rescued the I/R injury and prevented upregulation of peroxisome proliferator–activated receptor-&agr; in Tg-dominant–negative Nox mice. A cross with peroxisome proliferator–activated receptor-&agr; KO mice also attenuated the injury in Tg- dominant–negative Nox mice. Conclusions: Both Nox2 and Nox4 contribute to the increase in reactive oxidative species and injury by I/R. However, low levels of reactive oxidative species produced by either Nox2 or Nox4 regulate hypoxia-inducible factor-1&agr; and peroxisome proliferator–activated receptor-&agr;, thereby protecting the heart against I/R, suggesting that Noxs also act as a physiological sensor for myocardial adaptation.

PDGF Receptor β Signaling in Pericytes Following Ischemic Brain Injury

Platelet derived growth factor (PDGF)-B plays a neuroprotective role in brain damages, including ischemic stroke. It has been suggested recently that PDGF receptor β (PDGFRβ) expressed in brain pericytes as well as in neurons and astrocytes may mediate the neuroprotective role of PDGF-B. The aims of this study were to elucidate the roles of PDGFRβ signaling in brain pericytes after ischemic stroke. In a rat middle cerebral artery occlusion (MCAO) model, PDGFRβ expression was induced specifically in the pericytes in peri-infarct areas and its level was gradually increased. PDGF-B induced marked phosphorylation of Akt in cultured brain pericytes. Consistently, PDGF-B was upregulated in endothelial cells in per-infarct areas and Akt was strongly phosphorylated in the PDGFRβ-expressing pericytes in periinfarct areas after MCAO. In the cultured pericytes, PDGF-B induced cell growth and anti-apoptotic responses through Akt. Furthermore, PDGF-B significantly increased the expression of nerve growth factor (NGF) and neurotrophin-3 (NT-3) through Akt in the pericytes. Thus, the PDGFRβ-Akt signaling in brain pericytes may play various important roles leading to neuroprotection after ischemic stroke.