Abstract-Nonphagocytic NADPH oxidases have recently been suggested to play a major role in the regulation of physiological and pathophysiological processes, in particular, hypertrophy, remodeling, and angiogenesis in the systemic circulation. Moreover, NADPH oxidases have been suggested to serve as oxygen sensors in the lung. Chronic hypoxia induces vascular remodeling with medial hypertrophy leading to the development of pulmonary hypertension. We screened lung tissue for the expression of NADPH oxidase subunits. NOX1, NOXA1, NOXO1, p22phox, p47phox, p40phox, p67phox, NOX2, and NOX4 were present in mouse lung tissue. Comparing mice maintained for 21 days under hypoxic (10% O 2 ) or normoxic (21% O 2 ) conditions, an upregulation exclusively of NOX4 mRNA was observed under hypoxia in homogenized lung tissue, concomitant with increased levels in microdissected pulmonary arterial vessels. In situ hybridization and immunohistological staining for NOX4 in mouse lungs revealed a localization of NOX4 mRNA and protein predominantly in the media of small pulmonary arteries, with increased labeling intensities after chronic exposure to hypoxia. In isolated pulmonary arterial smooth muscle cells (PASMCs), NOX4 was localized primarily to the perinuclear space and its expression levels were increased after exposure to hypoxia. Treatment of PASMCs with siRNA directed against NOX4 decreased NOX4 mRNA levels and reduced PASMC proliferation as well as generation of reactive oxygen species. In lungs from patients with idiopathic pulmonary arterial hypertension (IPAH), expression levels of NOX4, which was localized in the vessel media, were 2.5-fold upregulated. These results support an important role for NOX4 in the vascular remodeling associated with development of pulmonary hypertension. (Circ Res. 2007;101:258-267.)Key Words: hypoxia Ⅲ hypoxic pulmonary vasoconstriction Ⅲ NADPH oxidase Ⅲ pulmonary hypertension Ⅲ vascular smooth muscle cell proliferation T he NADPH oxidases are superoxide-generating enzymes that release superoxide by electron transfer from NADPH to oxygen. The classical leukocyte NADPH oxidase plays an important role in host defense against bacterial and fungal pathogens. 1,2 This phagocytic type of NADPH oxidase consists of 2 membrane-bound subunits, gp91 phox and p22 phox which form the flavocytochrome b 558 complex, together with the cyctosolic subunits p40 phox , p47 phox , and p67 phox . Superoxide production by this complex is induced by assembly of the cytosolic and membrane-bound subunits. Such an assembly can be induced by the phosphorylation of p47 phox . 3 Rac GTPases are also involved in this activation process. Recently, several additional isoforms of the membrane-bound subunit gp91phox have been described. The first described homolog of gp91 phox , called mox1 (later NOX1), is primarily expressed in the colon and is suggested to be involved in mitogenic activity. 4 Additional homologs, including NOX3, NOX4 (Renox), NOX5, Duox1, and Duox2, were subsequently described. [5][6][7][8] According to ...

Chronic obstructive pulmonary disease (COPD) is one of the most common causes of death worldwide. We report in an emphysema model of mice chronically exposed to tobacco smoke that pulmonary vascular dysfunction, vascular remodeling, and pulmonary hypertension (PH) precede development of alveolar destruction. We provide evidence for a causative role of inducible nitric oxide synthase (iNOS) and peroxynitrite in this context. Mice lacking iNOS were protected against emphysema and PH. Treatment of wild-type mice with the iNOS inhibitor N(6)-(1-iminoethyl)-L-lysine (L-NIL) prevented structural and functional alterations of both the lung vasculature and alveoli and also reversed established disease. In chimeric mice lacking iNOS in bone marrow (BM)-derived cells, PH was dependent on iNOS from BM-derived cells, whereas emphysema development was dependent on iNOS from non-BM-derived cells. Similar regulatory and structural alterations as seen in mouse lungs were found in lung tissue from humans with end-stage COPD.

Abstract-The excitability of pulmonary artery smooth muscle cells (PASMC) is regulated by potassium (K ϩ ) conductances. Although studies suggest that background K ϩ currents carried by 2-pore domain K ϩ channels are important regulators of resting membrane potential in PASMC, their role in human PASMC is unknown. Our study tested the hypothesis that TASK-1 leak K ϩ channels contribute to the K ϩ current and resting membrane potential in human PASMC. We used the whole-cell patch-clamp technique and TASK-1 small interfering RNA (siRNA). Noninactivating K ϩ current performed by TASK-1 K ϩ channels were identified by current characteristics and inhibition by anandamide and acidosis (pH 6.3), each resulting in significant membrane depolarization. Moreover, we showed that TASK-1 is blocked by moderate hypoxia and activated by treprostinil at clinically relevant concentrations. This is mediated via protein kinase A (PKA)-dependent phosphorylation of TASK-1. To further confirm the role of TASK-1 channels in regulation of resting membrane potential, we knocked down TASK-1 expression using TASK-1 siRNA. The knockdown of TASK-1 was reflected by a significant depolarization of resting membrane potential. Treatment of human PASMC with TASK-1 siRNA resulted in loss of sensitivity to anandamide, acidosis, alkalosis, hypoxia, and treprostinil. These results suggest that (1) TASK-1 is expressed in human PASMC; (2) TASK-1 is hypoxia-sensitive and controls the resting membrane potential, thus implicating an important role for TASK-1 K ϩ channels in the regulation of pulmonary vascular tone; and (3) treprostinil activates TASK-1 at clinically relevant concentrations via PKA, which might represent an important mechanism underlying the vasorelaxing properties of prostanoids and their beneficial effect in vivo. Key Words: pulmonary circulation Ⅲ potassium channels Ⅲ TASK-1 Ⅲ treprostinil Ⅲ hypoxic pulmonary vasoconstriction T he membrane potential of pulmonary artery smooth muscle cells (PASMC) is an important regulator of arterial tone. These cells have a resting membrane potential of approximately Ϫ65 to Ϫ50 mV in vitro, close to the predicted equilibrium potential for potassium (K ϩ ) ions. The opening of K ϩ channels in the PASMC membrane increases K ϩ efflux, which causes membrane hyperpolarization. This closes voltage-dependent Ca 2ϩ channels, decreasing Ca 2ϩ entry and leading to vasodilatation. Conversely, inhibition of K ϩ channels causes membrane depolarization, Ca 2ϩ entry, cell contraction, and vasoconstriction.Background or leak K ϩ -selective channels, as defined by a lack of time and voltage dependency, play an essential role in setting the resting membrane potential and input resistance in excitable cells. Two-pore domain K ϩ (2-PK) channels have been shown to conduct several leak K ϩ currents. The activity of 2-PK channels is strongly regulated by protons, protein kinases, and hypoxia. Alteration of K ϩ conductance can influence cellular activity via membrane potential changes.Both RT-PCR and Northern blot analyses p...

Summary Idiopathic pulmonary fibrosis (IPF) is a form of progressive interstitial lung disease with unknown etiology. Due to a lack of effective treatment, IPF is associated with a high mortality rate. The hallmark feature of this disease is the accumulation of activated myofibroblasts that excessively deposit extracellular matrix proteins, thus compromising lung architecture and function and hindering gas exchange. Here we investigated the origin of activated myofibroblasts and the molecular mechanisms governing fibrosis formation and resolution. Genetic engineering in mice enables the time-controlled labeling and monitoring of lipogenic or myogenic populations of lung fibroblasts during fibrosis formation and resolution. Our data demonstrate a lipogenic-to-myogenic switch in fibroblastic phenotype during fibrosis formation. Conversely, we observed a myogenic-to-lipogenic switch during fibrosis resolution. Analysis of human lung tissues and primary human lung fibroblasts indicates that this fate switching is involved in IPF pathogenesis, opening potential therapeutic avenues to treat patients.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease in which the intricate alveolar network of the lung is progressively replaced by fibrotic scars. Myofibroblasts are the effector cells that excessively deposit extracellular matrix proteins thus compromising lung structure and function. Emerging literature suggests a correlation between fibrosis and metabolic alterations in IPF. In this study, we show that the first-line antidiabetic drug metformin exerts potent antifibrotic effects in the lung by modulating metabolic pathways, inhibiting TGFβ1 action, suppressing collagen formation, activating PPARγ signaling and inducing lipogenic differentiation in lung fibroblasts derived from IPF patients. Using genetic lineage tracing in a murine model of lung fibrosis, we show that metformin alters the fate of myofibroblasts and accelerates fibrosis resolution by inducing myofibroblast-to-lipofibroblast transdifferentiation. Detailed pathway analysis revealed a two-arm mechanism by which metformin accelerates fibrosis resolution. Our data report an antifibrotic role for metformin in the lung, thus warranting further therapeutic evaluation.

-Prematurely born infants who require oxygen therapy often develop bronchopulmonary dysplasia (BPD), a debilitating disorder characterized by pronounced alveolar hypoplasia. Hyperoxic injury is believed to disrupt critical signaling pathways that direct lung development, causing BPD. We investigated the effects of normobaric hyperoxia on transforming growth factor (TGF)-␤ and bone morphogenetic protein (BMP) signaling in neonatal C57BL/6J mice exposed to 21% or 85% O2 between postnatal days P1 and P28. Growth and respiratory compliance were significantly impaired in pups exposed to 85% O2, and these pups also exhibited a pronounced arrest of alveolarization, accompanied by dysregulated expression and localization of both receptor (ALK-1, ALK-3, ALK-6, and the TGF-␤ type II receptor) and Smad (Smads 1, 3, and 4) proteins. TGF-␤ signaling was potentiated, whereas BMP signaling was impaired both in the lungs of pups exposed to 85% O2 as well as in MLE-12 mouse lung epithelial cells and NIH/3T3 and primary lung fibroblasts cultured in 85% O2. After exposure to 85% O2, primary alveolar type II cells were more susceptible to TGF-␤-induced apoptosis, whereas primary pulmonary artery smooth muscle cells were unaffected. Exposure of primary lung fibroblasts to 85% O2 significantly enhanced the TGF-␤-stimulated production of the ␣1 subunit of type I collagen (I␣1), tissue inhibitor of metalloproteinase-1, tropoelastin, and tenascin-C. These data demonstrated that hyperoxia significantly affects TGF-␤/BMP signaling in the lung, including processes central to septation and, hence, alveolarization. The amenability of these pathways to genetic and pharmacological manipulation may provide alternative avenues for the management of BPD.

Background-Mutations in the bmpr2 gene, encoding the type II bone morphogenetic protein (BMP) receptor, have been identified in patients with pulmonary arterial hypertension (PAH), implicating BMP signaling in PAH. The aim of this study was to assess BMP signaling and its physiological effects in a monocrotaline (MCT) model of PAH. Methods and Results-Expression of BMP receptors Ib and II, and Smads 4, 5, 6, and 8, was downregulated in lungs but not kidneys of MCT-treated rats. Smad1 phosphorylation and expression of BMP/Smad target genes id1 and id3 was also reduced, although ERK1/2 and p38 MAPK phosphorylation remained unaffected. BMP receptor and Smad expression, Smad1 phosphorylation, and induction of the BMP/Smad-responsive element of the id1 promoter were reduced in pulmonary artery smooth muscle cells (PASMCs) from MCT-treated rats. As a consequence of impaired BMP/Smad signaling, PASMCs from MCT-treated rats were resistant to apoptosis induced by BMP-4 and BMP-7, and were also resistant to BMP-4 antagonism of proliferation induced by platelet-derived growth factor. Conclusion-BMP signaling and BMP-regulated physiological phenomena are perturbed in MCT-treated rats, lending solid support to the proposed roles for BMP signaling in the pathogenesis of human PAH.

Increasing evidence points towards an inflammatory component underlying pulmonary hypertension. However, the conclusive characterisation of multiple inflammatory cell populations in the lung is challenging due to the complexity of marker specificity and tissue inaccessibility. We used an unbiased computational flow cytometry approach to delineate the inflammatory landscape of idiopathic pulmonary arterial hypertension (IPAH) and healthy donor lungs.Donor and IPAH samples were discriminated clearly using principal component analysis to reduce the multidimensional data obtained from single-cell flow cytometry analysis. In IPAH lungs, the predominant CD45 cell type switched from neutrophils to CD3 T-cells, with increases in CD4, CD8 and γδT-cell subsets. Additionally, diversely activated classical myeloid-derived dendritic cells (CD14HLA-DRCD11cCD1a) and nonclassical plasmacytoid dendritic cells (pDCs; CD14CD11cCD123HLA-DR), together with mast cells and basophils, were more abundant in IPAH samples. We describe, for the first time, the presence and regulation of two cell types in IPAH, γδT-cells and pDCs, which link innate and adaptive immunity.With our high-throughput flow cytometry with multidimensional dataset analysis, we have revealed the interactive interplay between multiple inflammatory cells is a crucial part of their integrative network. The identification of γδT-cells and pDCs in this disease potentially provides a missing link between IPAH, autoimmunity and inflammation.