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.

Abstract-Cyclic nucleotide phosphodiesterases (PDEs) control the levels of the second messengers cAMP and cGMP in many cell types including endothelial cells. Although PDE2 has the unique property to be activated by cGMP but to hydrolyze cAMP, its role in endothelial function is only poorly understood. Reactive oxygen species (ROS) have been recognized as signaling molecules controlling many endothelial functions. We thus investigated whether PDE2 would link to ROS generation and proliferative responses in human umbilical vein endothelial cells in response to thrombin. Thrombin stimulated the GTPase Rac1, known to activate NADPH oxidases, and enhanced ROS formation, whereas PDE2 inhibition or depletion by short hairpin (sh)RNA prevented these responses. Similar observations were made with 8-Br-cGMP or atrial natriuretic peptide. In agreement, thrombin elevated cGMP but decreased cAMP levels, whereas db-cAMP or forskolin diminished Rac1 activity and ROS production. Subsequently, PDE2 overexpression activated Rac1, increased ROS generation, and enhanced proliferation and in vitro capillary formation. These responses were not observed in the presence of inactive Rac1 or shRNA against the NADPH oxidase subunit NOX2. Inhibition or depletion of PDE2 also prevented thrombin-induced proliferation and capillary formation. Importantly, downregulation of PDE2 by lentiviral shRNA or PDE2 inhibition prevented vessel sprouting from mouse aortic explants and in vivo angiogenesis in a mouse model, respectively. In summary, PDE2 promotes activation of NADPH oxidase-dependent ROS production and subsequent endothelial proliferation and angiogenesis. Targeting PDE2 may provide a new therapeutic approach in diseases associated with endothelial dysfunction, oxidative stress, vascular proliferation, and angiogenesis.

Pulmonary hypertension and vascular remodeling processes are associated with oxidative stress, hypoxia and enhanced levels of thrombin and vascular endothelial growth factor (VEGF). The hypoxia-inducible transcription factor HIF regulates the expression of VEGF under hypoxia. The HIF pathway is also activated by thrombin or CoCl2, likely via reactive oxygen species (ROS). In this study we investigated whether the redox-modifying enzymes superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase affect HIF levels and the expression of VEGF mRNA in pulmonary artery smooth muscle cells (PASMC). Stimulation of PASMC with thrombin or CoCl2 increased ROS production and enhanced HIF-alpha protein and VEGF mRNA levels as well as HIF-dependent reporter gene activity. These responses were inhibited by vitamin C and by overexpression of GPX and catalase, whereas the opposite effects were observed in SOD-expressing cells. These findings suggest that an 'antioxidant' state with reduced levels of H2O2 limits the activation of the HIF pathway, whereas a 'prooxidant' state allowing elevated H2O2 levels promotes it. Thus, shifting the redox balance to a more reduced environment, thereby limiting VEGF expression, may be beneficial for treating remodeling processes during pulmonary hypertension.

Various cardiovascular diseases including thrombosis, atherosclerosis, (pulmonary) hypertension and diabetes, are associated with disturbed coagulation. Alterations in the vessel wall common to many cardiovascular disorders have been shown to initiate the activity of the coagulation system, but also to be the result of an abnormal coagulation system. The primary link between the coagulation and the vascular system appears to be tissue factor (TF), which is induced on the surface of vascular cells and initiates the extrinsic pathway of the blood coagulation cascade, leading to the formation of thrombin. Thrombin can also interact with the vascular wall via specific receptors and can increase vascular TF expression. Such a "thrombogenic cycle" may be essentially involved in the pathogenesis of cardiovascular disorders associated with an abnormal coagulation. Therefore, the identification of the signaling pathways regulating this cycle and each of its relevant connecting links is of fundamental importance for the understanding of these disorders and their putative therapeutic potential. Reactive oxygen species (ROS) and the ROS-generating NADPH oxidases have been shown to play important roles as signaling molecules in the vasculature. In this review, we summarize the data supporting a substantial role of ROS in promoting a thrombogenic cycle in the vascular system.

SummaryUrotensin-II (U-II) has been considered as one of the most potent vasoactive peptides, although its physiological and pathophysiological role is still not finally resolved. Recent evidence suggests that it promotes angiogenic responses in endothelial cells, although the underlying signalling mechanisms are unclear. Reactive oxygen species derived from NADPH oxidases are major signalling molecules in the vasculature. Because NOX2 is functional in endothelial cells, we investigated the role of the NOX2-containing NADPH oxidase in U-II-induced angiogenesis and elucidated a possible contribution of hypoxia-inducible factor-1 (HIF-1), the master regulator of hypoxic angiogenesis, in the response to U-II. We found that U-II increases angiogenesis in vitro and in vivo, and these responses were prevented by antioxidants, NOX2 knockdown and in Nox2 -/-mice. In addition, U-II-induced angiogenesis was dependent on HIF-1. Interestingly, U-II increased NOX2 transcription involving HIF-1, and chromatin immunoprecipitation confirmed NOX2 as a target gene of HIF-1. In support, NOX2 levels were greatly diminished in U-II-stimulated isolated vessels derived from mice deficient in endothelial HIF-1. Conversely, reactive oxygen species derived from NOX2 were required for U-II activation of HIF and upregulation of HIF-1. In line with this, U-II-induced upregulation of HIF-1 was absent in Nox2 -/-vessels. Collectively, these findings identified HIF-1 and NOX2 as partners acting in concert to promote angiogenesis in response to U-II. Because U-II has been found to be elevated in cardiovascular disorders and in tumour tissues, this feed-forward mechanism could be an interesting anti-angiogenic therapeutic option in these disorders.