Background— Increased production of reactive oxygen species (ROSs) by angiotensin II (Ang II) is involved in the initiation and progression of cardiovascular diseases. NADPH oxidase is a major source of superoxide generated in vascular tissues. Although Nox1 has been identified in vascular smooth muscle cells as a new homolog of gp91phox (Nox2), a catalytic subunit of NADPH oxidase, the pathophysiological function of Nox1-derived ROSs has not been fully elucidated. To clarify the role of Nox1 in Ang II–mediated hypertension, we generated Nox1-deficient ( −/Y ) mice. Methods and Results— No difference in the baseline blood pressure was observed between Nox1 +/Y and Nox1 −/Y . Infusion of Ang II induced a significant increase in mean blood pressure, accompanied by augmented expression of Nox1 mRNA and superoxide production in the aorta of Nox1 +/Y , whereas the elevation in blood pressure and production of superoxide were significantly blunted in Nox1 −/Y . Conversely, the infusion of pressor as well as subpressor doses of Ang II did elicit marked hypertrophy in the thoracic aorta of Nox1 −/Y similar to Nox1 +/Y . Administration of a nitric oxide synthase inhibitor (L-NAME) to Nox1 +/Y did not affect the Ang II–mediated increase in blood pressure, but it abolished the suppressed pressor response to Ang II in Nox1 −/Y . Finally, endothelium-dependent relaxation and the level of cGMP in the isolated aorta were preserved in Nox1 −/Y infused with Ang II. Conclusions— A pivotal role for ROSs derived from Nox1/NADPH oxidase was suggested in the pressor response to Ang II by reducing the bioavailability of nitric oxide.

Abstract-We investigated whether the mobilization of endothelial progenitor cells (EPCs) by exogenous erythropoietin (Epo) promotes the repair of injured endothelium. Recombinant human Epo was injected (1000 IU/kg for the initial 3 days) after wire injury of the femoral artery of mice. Neointimal formation was inhibited by Epo to 48% of the control (PϽ0.05) in an NO-dependent manner. Epo induced a 1.4-fold increase in reendothelialized area of day 14 denuded vessels, 55% of which was derived from bone marrow (BM) cells. Epo increased the circulating Sca-1 ϩ /Flk-1 ϩ EPCs (2.0-fold, PϽ0.05) with endothelial properties NO dependently. BM replacement by GFP-or ␤-galactosidaseoverexpressing cells showed that Epo stimulated both differentiation of BM-derived EPCs and proliferation of resident ECs. BM-derived ECs increased 2.2-to 2.7-fold (PϽ0.05) in the Epo-induced neoendothelium, where the expression of Epo receptor was upregulated. Epo induced Akt/eNOS phosphorylation and NO synthesis on EPCs and exerted an antiapoptotic action on wire-injured arteries. In conclusion, Epo treatment inhibits the neointimal hyperplasia after arterial injury in an NO-dependent manner by acting on the injured vessels and mobilizing EPCs to the neo-endothelium.

The involvement of reactive oxygen species (ROS) in an augmented sensitivity to painful stimuli (hyperalgesia) during inflammation has been suggested, yet how and where ROS affect the pain signaling remain unknown. Here we report a novel role for the superoxidegenerating NADPH oxidase in the development of hyperalgesia. In mice lacking Nox1 (Nox1 Ϫ/Y ), a catalytic subunit of NADPH oxidase, thermal and mechanical hyperalgesia was significantly attenuated, whereas no change in nociceptive responses to heat or mechanical stimuli was observed.

Canonical Wnt signaling critically regulates cell fate and proliferation in developmental stages and adult tissues. Redox regulation through nucleoredoxin (NRX) has recently been identified in canonical Wnt signaling. However, the source of reactive oxygen species (ROS) affecting the redox state of NRX remains elusive. Our principal aim in this study was to investigate whether superoxide-generating NADPH oxidase1 (Nox1) is involved in NRX-regulated Wnt signaling in intestinal and colon epithelial cells. Here, we demonstrate that Wnt treatment of mouse intestinal cells induces production of ROS through Nox1. This Nox1 action is regulated by Rac1 GTPase through Wnt-induced activation of the Rac1 guanine nucleotide exchange factor Vav2 by Src-mediated tyrosine phosphorylation. Nox1-generated ROS oxidize and inactivate NRX, thereby releasing the NRX-dependent suppression of Wnt-β-catenin signaling through dissociation of NRX from Dvl. Nox1 small-interference RNA inhibits cell response to Wnt, including stabilization of β-catenin, expression of cyclin D1 and c-Myc via the TCF transcription factor, and accelerated cell proliferation. Nox1 mediates Wnt-induced cell growth in colon cancer cells with the normal Wnt pathway, but not in APC-deficient colon cancer cells, which are constitutively active in Wnt signaling. Together, these results suggest the mediating role of Nox1 in redox-dependent regulation of canonical Wnt-β-catenin signaling and provide further insight into the regulatory mechanism of the Wnt pathway.

Among multiple isoforms of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase expressed in the liver, the phagocytic NOX2 isoform in hepatic stellate cells (HSCs) has been demonstrated to play a key role in liver fibrogenesis. The aim of this study was to clarify the role of NOX1, a nonphagocytic form of NADPH oxidase, in the development of fibrosis using Nox1-deficient mice (Nox1KO). Liver injury and fibrosis were induced by bile duct ligation (BDL) and carbon tetrachloride in Nox1KO and wildtype littermate mice (WT). Primary HSCs were isolated to characterize the NOX1-induced signaling cascade involved in liver fibrogenesis. Following BDL, a time-dependent increase in NOX1 messenger RNA (mRNA) was demonstrated in WT liver. Compared with those in WT, levels of collagen-1a mRNA and hydroxyproline were significantly suppressed in Nox1KO with a reduced number of activated HSCs and less severe fibrotic lesions. The expression levels of a-smooth muscle actin, a marker of HSCs activation, were similar in cultured HSCs isolated from both genotypes. However, cell proliferation was significantly attenuated in HSCs isolated from Nox1KO. In these cells, the expression of p27 kip1 , a cell cycle suppressor, was significantly up-regulated. Concomitantly, a significant reduction in phosphorylated forms of Akt and forkhead box O (FOXO) 4, a downstream effector of Akt that regulates the transcription of p27 kip1 gene, was demonstrated in Nox1KO. Finally, the level of the oxidized inactivated form of phosphatase and tensin homolog (PTEN), a negative regulator of PI3K/Akt pathway, was significantly attenuated in HSCs of Nox1KO. Conclusion: These findings indicate that reactive oxygen species derived from NOX1/NADPH oxidase oxidize and inactivate PTEN to positively regulate the Akt/FOXO4/ p27 kip1 signaling pathway. NOX1 may thus promote proliferation of HSCs and accelerate the development of fibrosis following BDL-induced liver injury. (HEPATOLOGY 2011;54:949-958)

Matrilysin expression could be an important marker of activity and could be used for the prediction of subsequent transformation in patients with ulcerative colitis.

Activated oncogenes induce premature cellular senescence, a permanent state of proliferative arrest in primary rodent and human fibroblasts. Recent studies suggest that generation of reactive oxygen species (ROS) is involved in oncogenic Ras-induced premature senescence. However, the signaling mechanism controlling this oxidant-mediated irreversible growth arrest is not fully understood. Here, we show that through the Ras/MEK pathway, Ras oncogene upregulated the expression of superoxide-generating oxidases, Nox1 in rat REF52 cells and Nox4 in primary human lung TIG-3 cells, leading to an increase in intracellular level of ROS. Ablation of Nox1 and Nox4 by small interfering RNAs (siRNAs) blocked the RasV12 senescent phenotype including b-galactosidase activity, growth arrest and accumulation of tumor suppressors such as p53 and p16Ink4a . This suggests that Nox-generated ROS transduce senescence signals by activating the p53 and p16Ink4a pathway. Furthermore, Nox1 and Nox4 siRNAs inhibited both Ras-induced DNA damage response and p38MAPK activation, whereas overexpression of Nox1 and Nox4 alone was able to induce senescence. The involvement of Nox1 in Ras-induced senescence was also confirmed with embryonic fibroblasts derived from Nox1 knockout mice. Together, these findings suggest that Nox1-and Nox4-generated ROS play an important role in Ras-induced premature senescence, which may involve DNA damage response and p38MAPK signaling pathways.

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