Respiratory motoneuron response to hypoxia is reflex in nature and carotid body sensory receptor constitutes the afferent limb of this reflex. Recent studies showed that repetitive exposures to hypoxia evokes long term facilitation of sensory nerve discharge (sLTF) of the carotid body in rodents exposed to chronic intermittent hypoxia (CIH). Although studies with anti-oxidants suggested the involvement of reactive oxygen species (ROS)-mediated signaling in eliciting sLTF, the source of and the mechanisms associated with ROS generation have not yet been investigated. We tested the hypothesis that ROS generated by NADPH oxidase (NOX) mediate CIH-evoked sLTF. Experiments were performed on ex vivo carotid bodies from rats and mice exposed either to 10 d of CIH or normoxia. Acute repetitive hypoxia evoked a ϳ12-fold increase in NOX activity in CIH but not in control carotid bodies, and this effect was associated with upregulation of NOX2 mRNA and protein, which was primarily localized to glomus cells of the carotid body. sLTF was prevented by NOX inhibitors and was absent in mice deficient in NOX2. NOX activation by CIH required 5-HT release and activation of 5-HT 2 receptors coupled to PKC signaling. Studies with ROS scavengers revealed that H 2 O 2 generated from O 2 ⅐ Ϫ contributes to sLTF. Priming with H 2 O 2 elicited sLTF of carotid bodies from normoxic control rats and mice, similar to that seen in CIH-treated animals. These observations reveal a novel role for NOX-induced ROS signaling in mediating sensory plasticity of the carotid body.

In vascular endothelium, the major research focus has been on reactive oxygen species (ROS) derived from Nox2. The role of Nox4 in endothelial signal transduction, ROS production, and cytoskeletal reorganization is not well defined. In this study, we show that human pulmonary artery endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) express higher levels of Nox4 and p22 phox compared to Nox1, Nox2, Nox3, or Nox5. Immunofluorescence microscopy and Western blot analysis revealed that Nox4 and p22 phox , but not Nox2 or p47 phox , are localized in nuclei of HPAECs. Further, knockdown of Nox4 with siRNA decreased Nox4 nuclear expression significantly. Exposure of HPAECs to hyperoxia (3-24 h) enhanced mRNA and protein expression of Nox4, and Nox4 siRNA decreased hyperoxia-induced ROS production. Interestingly, Nox4 or Nox2 knockdown with siRNA upregulated the mRNA and protein expression of the other, suggesting activation of compensatory mechanisms. A similar upregulation of Nox4 mRNA was observed in Nox2 Ϫ/Ϫ ko mice. Downregulation of Nox4, or pretreatment with N-acetylcysteine, attenuated hyperoxia-induced cell migration and capillary tube formation, suggesting that ROS generated by Nox4 regulate endothelial cell motility. These results indicate that Nox4 and Nox2 play a physiological role in hyperoxia-induced ROS production and migration of ECs. Antioxid. Redox Signal. 11,[747][748][749][750][751][752][753][754][755][756][757][758][759][760][761][762][763][764]

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease, wherein transforming growth factor β (TGF-β) and sphingosine-1-phosphate (S1P) contribute to the pathogenesis of fibrosis. However, the in vivo contribution of sphingosine kinase (SphK) in fibrotic processes has not been documented. Microarray analysis of blood mononuclear cells from patients with IPF and SphK1- or SphK2-knockdown mice and SphK inhibitor were used to assess the role of SphKs in fibrogenesis. The expression of SphK1/2 negatively correlated with lung function and survival in patients with IPF. Also, the expression of SphK1 was increased in lung tissues from patients with IPF and bleomycin-challenged mice. Knockdown of SphK1, but not SphK2, increased survival and resistance to pulmonary fibrosis in bleomycin-challenged mice. Administration of SphK inhibitor reduced bleomycin-induced mortality and pulmonary fibrosis in mice. Knockdown of SphK1 or treatment with SphK inhibitor attenuated S1P generation and TGF-β secretion in a bleomycin-induced lung fibrosis mouse model that was accompanied by reduced phosphorylation of Smad2 and MAPKs in lung tissue. In vitro, bleomycin-induced expression of SphK1 in lung fibroblast was found to be TGF-β dependent. Taken together, these data indicate that SphK1 plays a critical role in the pathology of lung fibrosis and is a novel therapeutic target.

Abstract-In mammalian organs under normoxic conditions, O 2 concentration ranges from 12% to Ͻ0.5%, with O 2 Ϸ14%in arterial blood and Ͻ10% in the myocardium. During mild hypoxia, myocardial O 2 drops to Ϸ1% to 3% or lower. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of PO 2 results in perceived hyperoxia. These effects were independent of NADPH oxidase function. CFs exposed to high O 2 exhibited higher levels of reactive oxygen species production. The molecular signature response to perceived hyperoxia included (1) induction of p21, cyclin D1, cyclin D2, cyclin G1, Fos-related antigen-2, and transforming growth factor-␤1, (2) lowered telomerase activity, and (3) Key Words: redox Ⅲ free radicals Ⅲ heart Ⅲ cell culture C ellular O 2 concentrations are maintained within a narrow range (normoxia) because of the risk of oxidative damage from excess O 2 (hyperoxia) and of metabolic demise from insufficient O 2 (hypoxia). 1 PO 2 ranges from 90 to Ͻ3 mm Hg in mammalian organs under normoxic conditions, with arterial PO 2 of Ϸ100 mm Hg or Ϸ14% O 2 . 2 Thus, "normoxia" for cells is a variable that is dependent on the specific localization of the cell in organs and functional status of the specific tissue. O 2 sensing is required to adjust to physiological or pathophysiological variations in PO 2 . Current work in this field is almost exclusively focused on the study of hypoxia. Reoxygenation, on the other hand, has been mostly investigated in the context of oxidative injury. Over 25 years ago, it was observed that PO 2 beyond the comfort of the "perceived normoxic range" is a significant stressor, leading to growth arrest. 3 The molecular bases of such observations remain to be characterized in light of current knowledge of signal transduction.During chronic hypoxia in the heart, cells adjust their normoxic set point such that the return to normoxic PO 2 after chronic hypoxia is perceived as relative hyperoxia. 4,5 We hypothesized that such challenge triggers changes in signal transduction processes. Although acute insult caused during reperfusion may be lethal to cells localized at the focus of insult, elevation of O 2 tension in the surrounding ischemic tissue triggers phenotypic changes in the surviving cells that may be associated with tissue remodeling.Ischemia in the heart results in a hypoxic area containing a central focus of near-zero O 2 pressure bordered by tissue with diminished but nonzero O 2 pressures. These border zones extend for several millimeters from the hypoxic core, with the O 2 pressures progressively increasing from the focus to the normoxic region. 6 Moderate hypoxia is associated with a 30% to 60% decrease (Ϸ1% to 3% O 2 ) in PO 2 . 7 Cardiac fibroblasts (CFs) are mainly responsible for the synthesis of major extracellular matrix (ECM) in the myocardium, including fibrillar collagen types I and III and fibronectin. More than 90% of the interstitial cells of the myocardium are fibroblasts, 8 which actively e...