Objective-Reactive oxygen species have been implicated as signaling molecules modulating the activity of redoxsensitive transcription factors such as nuclear factor kappa B (NF-B). Recently, the transcription factor hypoxiainducible factor-1 (HIF-1), known to mediate gene expression by hypoxia, has been found to be also activated by nonhypoxic factors in a redox-sensitive manner. We therefore aimed to elucidate the link between these 2 important redox-sensitive transcription factors. Methods and Results-In pulmonary artery smooth muscle cells, reactive oxygen species generated either by exogenous H 2 O 2 or by a NOX4-containing NADPH oxidase stimulated by thrombin activated or induced NF-B and HIF-1␣. The reactive oxygen species-mediated HIF-1␣ induction occurred on the transcriptional level and was dependent on NF-B.Transfection experiments with wild-type or mutant HIF-1␣ promoter constructs revealed the presence of a yet unidentified NF-B binding element. Gel shift analyses and chromatin immunoprecipitation verified binding of NF-B to this site. Furthermore, reactive oxygen species enhanced expression of plasminogen activator inhibitor-1, which was prevented by dominant-negative IB or mutation of the HIF-1 binding site within the plasminogen activator inhibitor-1 promoter. Conclusion-These findings show for the first time to our knowledge that reactive oxygen species directly link HIF-1␣and NF-B, implicating an important pathophysiological role of this novel pathway in disorders associated with elevated levels of reactive oxygen species. Key Words: hypoxia-inducible factor Ⅲ NADPH oxidase Ⅲ nuclear factor kappa B Ⅲ reactive oxygen species Ⅲ thrombin O xidative stress has been implicated to play an important role in the pathophysiology of many cardiovascular diseases including systemic and pulmonary hypertension and atherosclerosis as well as in tumor progression and vascularization. [1][2][3] Moderate levels of reactive oxygen species (ROS), especially superoxide anions and hydrogen peroxide, have been shown to activate signaling cascades mediating the responses to vasoactive peptides, growth factors, cytokines, hormones, and coagulation factors, as well as to physical and chemical stress. ROS participate in the regulation of vascular proliferation, migration, apoptosis, modification of the extracellular matrix, and procoagulant activity. 4 -9 Moreover, ROS can activate angiogenesis, 10 a process known to be primarily mediated by vascular endothelial growth factor under hypoxia. Under hypoxia, vascular endothelial growth factor expression is induced by the transcription factor hypoxia-inducible factor-1 (HIF-1). 11 Aside from vascular endothelial growth factor, HIF-1 regulates Ͼ100 genes encoding for metabolic enzymes, growth factors, and factors contributing to modulation of extracellular matrix and thrombosis such as plasminogen activator inhibitor-1 (PAI-1). [12][13][14][15] HIF-1 is composed of an inducible ␣-subunit (HIF-1␣) and a constitutive ␤-subunit (also termed ARNT). 12 HIF-1␣ contains an oxygen-de...

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Increased levels of reactive oxygen species (ROS) contribute to many cardiovascular diseases. In neutrophils, ROS are generated by a NADPH oxidase containing p22phox and NOX2. NADPH oxidases are also major sources of vascular ROS. Whereas an active NOX2-containing enzyme has been described in endothelial cells, the contribution of recently identified NOX homologues to endothelial ROS production and proliferation has been controversial. The authors, therefore, compared the role of NOX2 with NOX4 and NOX1 in endothelial EaHy926 and human microvascular endothelial cells. NOX2 and NOX4 were abundantly expressed, whereas NOX1 expression was less prominent. NOX2, NOX4, and NOX1 were simultaneously present in a single cell in a perinuclear compartment. NOX2 and NOX4 co-localized with the endoplasmic reticulum (ER) marker calreticulin. Additionally, NOX2 co-localized with F-actin at the plasma membrane. NOX2 and NOX4, which interacted with p22phox, as was shown by bimolecular fluorescent complementation, contributed equally to endothelial ROS production and proliferation, whereas NOX1 depletion did not alter ROS levels under basal conditions. These data show that endothelial cells simultaneously express NOX2, NOX4, and NOX1. NOX2 and NOX4, but not NOX1, equally contributed to ROS generation and proliferation under basal conditions, indicating that a complex relation between NOX homologues controls endothelial function.

Background-Human urotensin II (hU-II) is a potent vasoactive peptide possibly involved in pulmonary hypertension.Because the signaling mechanisms activated by this peptide in the pulmonary vasculature are largely unknown, we investigated the role of hU-II in the activation of NADPH oxidase and the control of redox-sensitive kinase pathways, expression of plasminogen activator inhibitor-1 (PAI-1), and proliferation in pulmonary artery smooth muscle cells (PASMCs). Methods and Results-hU-II upregulated expression of the NADPH oxidase subunits p22phox and NOX4 and increased the levels of reactive oxygen species (ROS), which were abrogated by transfecting p22phox or NOX4 antisense vectors. p22phox and NOX4 also contributed to hU-II-induced activation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and protein kinase B (Akt). Furthermore, hU-II increased the expression of PAI-1 and enhanced PASMC proliferation in an NADPH oxidase-and kinase-dependent manner. Conclusions-hU-II is a potent activator of ROS generation by NADPH oxidase in PASMCs, leading to redox-sensitive activation of mitogen-activated protein kinases and Akt and subsequently to enhanced PAI-1 expression and increased proliferation. These findings suggest that hU-II may play a novel role in pulmonary hypertension by promoting remodeling processes via activation of NADPH oxidases.