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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.

The stress-responsive serum- and glucocorticoid-inducible kinase Sgk-1 is involved in osmoregulation and cell survival and may contribute to fibrosis and hypertension. However, the function of Sgk-1 in vascular remodeling and thrombosis, 2 major determinants of pulmonary hypertension (PH), has not been elucidated. We investigated the role of Sgk-1 in thrombin signaling and tissue factor (TF) expression and activity in pulmonary artery smooth muscle cells (PASMC). Thrombin increased Sgk-1 activity and mRNA and protein expression. H2O2 similarly induced Sgk-1 expression. Antioxidants, dominant-negative Rac, and depletion of the NADPH oxidase subunit p22phox diminished thrombin-induced Sgk-1 expression. Inhibition of p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and phosphoinositide-dependent kinase-1 prevented thrombin-induced Sgk-1 expression. Thrombin or Sgk-1 overexpression enhanced TF expression and procoagulant activity, whereas TF upregulation by thrombin was diminished by kinase-deficient Sgk-1 and was not detectable in fibroblasts from mice deficient in sgk-1 (sgk1(-/-)). Similarly, dexamethasone treatment failed to induce TF expression and activity in lung tissue from sgk1(-/-) mice. Transcriptional induction of TF by Sgk-1 was mediated through nuclear factor kappaB. Finally, Sgk-1 and TF proteins were detected in the media of remodeled pulmonary vessels associated with PH. These data show that thrombin potently induces Sgk-1 involving NADPH oxidases, phosphatidylinositol 3-kinase, p38 mitogen-activated protein kinase, and phosphoinositide-dependent kinase-1, and that activation of nuclear factor kappaB by Sgk-1 mediates TF expression and activity by thrombin. Because enhanced procoagulant activity can promote pulmonary vascular remodeling, and Sgk-1 and TF were present in the media of remodeled pulmonary vessels, this pathway may play a critical role in vascular remodeling in PH.

The p21-activated serine/threonine kinases (PAK) play an important role in a variety of cellular functions. However, their role in the smooth muscle response to thrombin, which is activated upon vascular injury and promotes vascular remodelling processes, is not resolved. Here we investigated the role of PAK in thrombin signalling and regulation of tissue factor (TF), the activator of the extrinsic coagulation cascade, in pulmonary artery smooth muscle cells (PASMC), the main cell type responsible for vascular remodelling in pulmonary hypertension. PAK was rapidly phosphorylated in response to thrombin. Thrombin and active PAKT423E phosphorylated p38 MAP kinase (p38MAPK), ERK1/2, phosphatidylinositol-dependent kinase-1 (PDK1) and protein kinase B/Akt (PKB) whereas kinase-deficient PAK1 prevented activation of these kinases by thrombin. In addition, kinase- deficient MKK3 inhibited activation of PDK1 and PKB by thrombin. Further, thrombin and active PAK1 induced TF expression and promoter activity while kinase-deficient PAK1 diminished thrombin-induced TF upregulation. Moreover, kinase-deficient MKK3, PDK1 and PKB inhibited thrombin- and PAK-dependent TF expression and promoter activity. Together these findings show that PAK is a critical element of thrombin signalling in PASMC which is involved in the regulation of TF expression by sequentially activating MKK3/p38MAPK, PDK1 and PKB. Thus, PAK may play an important role in promoting vascular remodelling processes in pulmonary hypertension.

Endothelial dysfunction is characterized by increased levels of reactive oxygen species (ROS) and a prothrombotic state. The mechanisms linking thrombosis to ROS production in the endothelium are not well understood. We investigated the role of thrombin in regulating NADPH oxidase-dependent ROS production and expression of its subunit p22phox in the endothelial cell line EaHy926. Thrombin elicited a biphasic increase in ROS generation peaking within 15 min, but also at 3 h. The delayed response was accompanied by increased p22phox mRNA and protein expression. Two-photon confocal laser microscopy showed colocalization between p22phox and ROS production. Antioxidant treatment with vitamin C or diphenyleneiodonium abrogated thrombin-induced ROS production and p22phox expression, whereas H2O2 elevated ROS production and p22phox levels. Both responses were dependent on p38 MAP kinase and phosphatidylinositol-3-kinase (PI3 kinase)/Akt. Finally, p22phox was required for thrombin- or H2O2-stimulated proliferation. These data show that thrombin rapidly increases ROS production in endothelial cells, resulting, via activation of p38 MAP kinase and PI3 kinase/Akt, in upregulation of p22phox accompanied by a delayed increase in ROS generation and enhanced proliferation. These findings suggest a positive feedback mechanism whereby ROS, possibly generated by the NADPH oxidase, lead to elevated levels of p22phox and, thus, sustained ROS generation as is observed in endothelial dysfunction.

Pulmonary hypertension is associated with enhanced thrombogenicity of the vessel wall contributing to vascular remodeling. However, the signaling mechanisms promoting this prothrombotic state are not resolved. Here we investigated the role of the GTPase Rac in the regulation of tissue factor (TF) expression and activity in response to thrombin in pulmonary artery smooth muscle cells (PASMC). TF mRNA and protein expression and surface procoagulant activity were increased by thrombin in PASMC. These responses were enhanced in the presence of the constitutively active Rac mutant RacG12V, but were abrogated in cells expressing dominant-negative RacT17N. Thrombin and RacG12V also increased human TF promoter activity primarily involving a sequence between -636 and -111 bp containing a distal, nuclear factor-kappaB (NFkappaB)-dependent enhancer element. Indeed, thrombin and RacG12V stimulated NFkappaB-dependent transcriptional activity, and overexpression of p50/p65 significantly increased human TF promoter activity. Moreover, in RacG12V-overexpressing cells, TF promoter activity was significantly decreased by coexpression of dominant-negative mutants of IkappaBalpha and IkappaBKalpha, which prevent NFkappaB activation. As enhanced NFkappaB activity has been observed in patients with pulmonary hypertension, Rac-dependent activation of the NFkappaB pathway may be a critical element promoting thrombin-induced TF expression and activity, and thus a prothrombotic state in pulmonary hypertension.

Background: Tissue factor (TF) initiates the extrinsic coagulation cascade leading to thrombin formation. Thrombin induces TF mRNA in vascular smooth muscle cells (VSMCs), thereby contributing to the prolonged procoagulant activity and enhanced thrombogenicity at sites of vascular injury. However, the signaling mechanisms mediating this thrombogenic cycle are unclear. Characteristically, vascular injury promotes the generation of reactive oxygen species (ROS). Because ROS exert signaling functions, we investigated whether the NADPH oxidase, an important source of ROS in VSMCs, contributes to upregulation of TF by thrombin., Methods and Results: Thrombin not only stimulated TF mRNA expression, but also TF-dependent surface procoagulant activity in cultured human VSMCs. This response was attenuated by antioxidants; the flavin inhibitor diphenylene-iodonium, Clostridium difficile toxin B, which inhibits Rho GTPases, p22phox antisense oligonucleotides, or the dominant-negative RacT17N mutant. Inhibitors of p38 MAP kinase and phosphatidylinositol (PI) 3-kinase also prevented thrombin-stimulated TF mRNA expression. Furthermore, thrombin stimulated the phosphorylation of the PI 3-kinase target protein kinase B/Akt in a redox-sensitive and NADPH oxidase-dependent manner., Conclusion: These findings indicate that the NADPH oxidase is essentially involved in the redox-sensitive induction of TF mRNA expression and surface procoagulant activity by thrombin. This response is mediated by NADPH oxidase-dependent activation of p38 MAP kinase and the PI 3-kinase/protein kinase B/Akt pathway. Given that active TF promotes thrombin formation, the NADPH oxidase may play a crucial role in perpetuating the thrombogenic cycle in the injured vessel wall.

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-α 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. [ABSTRACT FROM AUTHOR]

Abstract: NADPH oxidases have been identified as sources of reactive oxygen species (ROS) in vascular cells. In addition to the initially described enzyme containing gp91phox (NOX2), several homologues to NOX2 have been identified. Whereas NOX1, NOX2, and NOX4 are expressed in endothelial cells, a functional role of NOX5 containing additional N-terminal calcium-binding domains of varying sequences has not been reported in these cells. NOX5 protein was found in the endoplasmic reticulum of human microvascular endothelial cells (HMEC-1) and in the vascular wall. HMEC-1 cells expressed NOX5β and NOX5δ as well as a variant lacking calcium-binding domains (NOX5S). NOX5β and NOX5S increased basal ROS levels. Ionomycin exclusively enhanced NOX5β-mediated ROS production. Although p22phox, when overexpressed, interacted with both NOX5 proteins, it was not essential for NOX5-mediated ROS production. NOX5 proteins stimulated endothelial cell proliferation and the formation of capillary-like structures whereas depletion of NOX5 by siRNA prevented these responses to thrombin. These data show that endothelial cells express different NOX5 variants including NOX5S lacking calcium-binding domains. NOX5 proteins are functional, promoting endothelial ROS production, proliferation, and the formation of capillary-like structures and contribute to the endothelial response to thrombin. These findings suggest that NOX5 variants play a novel role in controlling ROS-dependent processes in the vasculature. [Copyright &y& Elsevier]

An NADPH oxidase is thought to be a main source of vascular superoxide (O2−) production. The functional role of this oxidase, however, and the contribution of the different subunits of the enzyme to cellular signaling are still incompletely understood. We determined the role of the p47phox subunit of the oxidase in O2− generation and signaling in aortic rings and cultured smooth muscle cells (SMC) from wild-type (WT) and p47phox-deficient (p47phox -/-) mice. Basal O2− levels in aortae of p47phox -/- mice were lower than those in WT aortae. Infusion of [val5]-angiotensin II increased O2− levels in aortae from WT more than in aortae from p47phox -/- mice. O2− generation was similar in quiescent SMC from WT and p47phox -/- mice. However, exposure to thrombin selectively increased O2− generation in VSMC from WT, but not from p47phox -/- mice. Thrombin-activated redox-mediated signal transduction and gene expression was attenuated in VSMC from p47phox -/- compared to cells from WT mice as determined by p38 MAP kinase activation and VEGF gene expression. We conclude that p47phox is important for vascular ROS production and redox-modulated signaling and gene expression in VSMC. [Copyright &y& Elsevier]