Background Pulmonary hypertension (PH) is a life-threatening disease characterized by vascular remodeling and increased pulmonary vascular resistance. Chronic alveolar hypoxia in animals is often used to decipher pathways being regulated in PH. Here, we aimed to investigate whether chronic hypoxia-induced PH in mice can be reversed by reoxygenation and whether possible regression can be used to identify pathways activated during the reversal and development of PH by genome-wide screening.Methods and Results Mice exposed to chronic hypoxia (21 days, 10% O-2) were reoxygenated for up to 42 days. Full reversal of PH during reoxygenation was evident by normalized right ventricular pressure, right heart hypertrophy, and muscularization of small pulmonary vessels. Microarray analysis from these mice revealed s-adenosylmethionine decarboxylase 1 (AMD-1) as one of the most downregulated genes. In situ hybridization localized AMD-1 in pulmonary vessels. AMD-1 silencing decreased the proliferation of pulmonary arterial smooth muscle cells and diminished phospholipase C1 phosphorylation. Compared with the respective controls, AMD-1 depletion by heterozygous in vivo knockout or pharmacological inhibition attenuated PH during chronic hypoxia. A detailed molecular approach including promoter analysis showed that AMD-1 could be regulated by early growth response 1, transcription factor, as a consequence of epidermal growth factor stimulation. Key findings from the animal model were confirmed in human idiopathic pulmonary arterial hypertension.Conclusions Our study indicates that genome-wide screening in mice from a PH model in which full reversal of PH occurs can be useful to identify potential key candidates for the reversal and development of PH. Targeting AMD-1 may represent a promising strategy for PH therapy.

Die pulmonale Hypertonie (PH) ist eine lebensbedrohliche Erkrankung, die unbehandelt zum Tod führt. Sie ist durch einen erhöhten pulmonal-arteriellen Druck, erhöhten pulmonal-vaskulären Widerstand sowie die mit PH einhergehenden Gefäßumbauprozesse charakterisiert, die letztendlich zum Rechtsherzversagen führen können. Die PH kann experimentell durch chronische hypoxische alveoläre Hypoxie induziert werden. Das Ziel dieser Arbeit ist es zu überprüfen, ob eine normoxische Reexposition chronisch hypoxischer Mäuse eine vollständige Gesundung der Versuchstiere induzieren kann. Des Weiteren sollen Gene, die in diesem Heilungsprozess involviert sind, anhand genomweiter Analysen identifiziert und charakterisiert werden.

AIMS:Chronic hypoxia induces pulmonary hypertension (PH) that is concomitant with pulmonary vascular remodeling. Reactive oxygen species (ROS) are thought to play a major role in this. Recent findings suggest that ROS production by NADPH oxidase 4 (Nox4) is important in this remodeling. We investigated whether ROS production by Nox is also important in an inflammatory model of monocrotaline (MCT)-induced PH. We examined ROS production, their possible sources, and their impact on the function of pulmonary arterial smooth muscle cells (PASMC) isolated from MCT-treated and healthy rats.RESULTS:MCT-PASMC showed increased intracellular superoxide production, migration, and proliferation compared with healthy controls due to increased Nox1 expression. A comparison of PASMC from MCT- and nontreated rats revealed an up-regulation of Sod2, Nrf2, cyclin D1, and matrix metalloproteinase-9 (MMP-9) as well as an increased phosphorylation of cofilin and extracellular signal-regulated kinases (Erk). Expression of Sod2, Nrf2, and cyclin D1 and phosphorylation of cofilin and Erk were Nox1 dependent.INNOVATION:The role of ROS in PH is not fully understood. Mitochondria and Nox have been suggested as sources of altered ROS generation in PH, yet it remains unclear whether increased or decreased ROS contributes to the development of PH. Our studies provide evidence that for different triggers of PH, different Nox isoforms regulate proliferation and migration of PASMC.CONCLUSION:In contrast to hypoxia-induced PH, Nox1 but not Nox4 is responsible for pathophysiological proliferation and migration of PASMC in an inflammatory model of MCT-induced PH via increased superoxide production. Thus, different Nox isoforms may be targeted in different forms of PH.

Rationale: Chronic alveolar hypoxia induces pulmonary hypertension (PH), concomitant with pulmonary vascular remodeling. Reactive oxygen species (ROS) are thought to play a major role in this process. Our recent findings suggest that ROS production by NAD(P)H-oxidase 4 (NOX4) is important in this remodeling process.

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.

Pulmonary hypertension (PH) is a progressive and life-threatening disease, characterized by increased pulmonary vascular resistance. PH can be induced by chronic alveolar hypoxia, which results in a vascular remodeling process, including a thickening of the vessel wall and a reduced vascular lumen.

Rationale: Chronic alveolar hypoxia induces pulmonary hypertension (PH), concomitant with pulmonary vascular remodeling. Reactive oxygen species (ROS) are thought to play a major role in this process. Our recent findings suggest that ROS production by the NADPH-oxidase (NOX) subunits play an important role in this remodeling process. Objectives: We investigated whether similar mechanisms are active for non-hypoxia induced PH, namely monocrotaline (MCT) induced PH in rats. The aim was to examine ROS production and their possible source. Further we adressed the expression of NOX subunits, antioxidative enzymes SOD1, SOD2, and catalase in pulmonary arterial smooth muscle cells (PASMC), isolated from MCT-treated rats. Methods/Results: Comparing PASMC from MCT-treated and non-treated rats by real-time PCR analysis revealed an upregulation of NOX1 and p22phox (n=6). Upregulation of NOX1 was confirmed by western blot. RT-PCR and immunofluorescence showed an increased SOD2 expression after MCT treatment. ROS measurements using ESR spectroscopy showed an increased intracellular production of ROS in total, but also especially superoxide in PASMC after MCT treatment in comparison to healthy controls. Stimulation with the NOX activator phorbol myristate acetate (PMA) induced an additional ROS increase. Results were validated by dihydroethidium staining. Conclusion: Our results show an increased ROS production in PASMC of MCT-treated rats which may contribute to vascular remodeling. Data from 1) enhanced ROS production upon PMA stimulation, 2) increased expression of NOX1 and 3) the increased SOD2 expression suggests a contribution of both, mitochondria and NOX1 as possible sources of ROS release in MCT treated rats.

Einleitung: Pulmonale arterielle Hypertonie (PAH) ist eine lebensbedrohliche Krankheit, die durch einen erhöhten vaskulären Widerstand charakterisiert ist. PAH kann durch chronische alveoläre Hypoxie induziert werden, welche einen Gefäßumbauprozess (remodeling) bewirkt. Das Ziel dieser Studie war herauszufinden, ob das ausgeprägte vaskuläre remodeling im Mausmodell der Hypoxie-induzierten PAH durch anschließende Re-exposition zu Normoxie rückgängig gemacht werden kann (reverse remodeling) und welche Gene in diesem Prozess involviert sind. Methoden: Mäuse wurden chronischer Hypoxie ausgesetzt (21 Tage, 10% O2) und anschließend (bis zu 42 Tage) unter normoxischen Bedingungen gehalten. Das vaskuläre reverse remodeling wurde anhand der Veränderung der Rechtsherzhypertrophie, des rechtsventrikulären Druckes sowie des Muskularisierungsgrades der kleinen arteriellen Lungengefäße beurteilt. Von Mauslungenhomogenat wurden Microarrays und quantitative PCR durchgeführt. Ergebnisse: Nach Re-exposition zu Normoxie zeigte sich ein Rückgang der Rechtsherzhypertrophie sowie des rechtsventrikulären Druckes. Darüber hinaus konnte ein verringerter Muskularisierungsgrad der kleinen pulmonalarteriellen Gefäße beobachtet werden. Bei den Untersuchungen zur Genexpression erwies sich S-Adenosylmethionindecarboxylase (Amd-1) als einer der am stärksten herunterregulierten Kandidaten nach erhöhter Expression unter chronischer Hypoxie. Weitere Untersuchungen zeigten je eine Bindestelle des Hypoxie-induzierbaren Transkriptionsfaktors (HIF) in der Promotorregion von Amd-1 in Maus und Human. Diskussion: Zusammengefasst zeigen die Untersuchungen, dass in dem Tiermodell der Hypoxie-induzierten PAH das ausgeprägte vaskuläre remodeling durch die Re-exposition zu Normoxie rückgängig gemacht werden kann. Darüberhinaus weisen unsere Daten darauf hin, dass Amd-1 dabei sowohl am vaskulären remodeling als auch am reverse remodeling nach Re-eposition zu Normoxie beteiligt sein könnte. Somit könnte die Inhibierung von Amd-1 eine vielversprechende Zielstruktur für die Behandlung von PAH sein.

Einleitung: Superoxidradikale (O2 -.) gelten als Mediatoren der hypoxischen pulmonalen Vasokonstriktion (HPV), einem essentiellen Mechanismus der Lunge, der die Blutperfusion an die alveoläre Ventilation anpasst. Es ist unklar, ob die O2 --Bildung während einer Hypoxie ansteigt oder abfällt und woher die O2 -.-Radikale stammen. Diese gegensätzlichen Hypothesen hängen nicht zuletzt auch mit technischen Schwierigkeiten bei der Detektion von reaktiven Sauerstoffspezies (ROS) zusammen.

RATIONALE:Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism by which pulmonary gas exchange is optimized by the adaptation of blood flow to alveolar ventilation. In chronic hypoxia, in addition to HPV a vascular remodeling process leads to pulmonary hypertension. A complex of heme oxygenase-2 (HO-2) and the BK channel has been suggested as a universal oxygen sensor system.OBJECTIVES:We investigated whether this complex serves as an oxygen sensor for the vascular effects of alveolar hypoxia in the lung.METHODS:The investigations were performed in chronically hypoxic mice, in isolated perfused and ventilated lungs, and on the cellular level, including HO-2- and BK-channel deficient mice.MEASUREMENTS AND MAIN RESULTS:Immunohistochemical analysis of mouse lungs identified HO-2 mainly in pulmonary arteries, the bronchial epithelium, and alveolar epithelial cells. BK channel alpha-subunit (BKalpha) immunoreactivity was found primarily in the bronchial and vascular smooth muscle layer. Immunofluorescence staining and coimmunoprecipitation suggested only a weak complexation of HO-2 and BKalpha in pulmonary arterial smooth muscle cells. The strength of acute and sustained HPV, determined in isolated perfused and ventilated lungs, was not different among wild-type, HO-2-deficient, and BKalpha-deficient mice. Exposure of mice to 3 weeks of chronic hypoxia resulted in a slight down-regulation of HO-2 and no alteration in BKalpha expression. The degree of pulmonary hypertension that developed, quantified on the basis of right ventricular pressure, right-heart hypertrophy, and the degree of muscularization of precapillary pulmonary arteries, was not different among wild-type, HO-2-deficient, and BKalpha-deficient mice.CONCLUSIONS:It is demonstrated that neither deletion of HO-2 nor BK channels affect acute, sustained, and chronic vascular responses to alveolar hypoxia in the lung.

Einleitung: Während akute Hypoxie in der Lunge zu einer Vasokonstriktion führt, findet unter chronischer Hypoxie ein Gefäßremodeling mit konsekutiver pulmonaler Hypertonie (PH) statt. Als mögliche Sauerstoffsensoren kommen verschiedene Kandidaten in Betracht. Jedoch ist bis heute die Frage nach dem Sauerstoffsensor in der Lunge nicht geklärt. Hämoxygenase 2 (HO2) und der kalziumabhängige Kaliumkanal MaxiKα sind Sauerstoffsensor- bzw. Effektorkandidaten in der Lunge. Der Abbau von Häm durch die HO2 zu Biliverdin führt zur Generation von CO, das MaxiKα direkt beeinflusst. Daraus ergibt sich vor dem Hintergrund aktueller Literaturdaten die Frage, ob die beiden Proteine auch am Prozess des Gefäßremodelings in der chronisch hypoxischen Lunge beteiligt sind.

Rationale: Hypoxia sensitive voltage gated K++ channels have been widely hypothesised for their role in regulating the contractility and growth of pulmonary artery smooth muscle cells (PASMC). Acute hypoxia leads to the inhibition of the hypoxia sensitive Kv channels whereas chronic hypoxia down-regulates their expression at both, mRNA and protein level in isolated rat PASMCs. Regarding the regulation of various Kv channels in hypoxia the role of reactive oxygen species (ROS) has been discussed but the mechanism of its action is still unclear.

Introduction: COPD is a progressive disease having non-fully reversible airflow limitation associated with chronic obstructive bronchitis and emphysema. Genetic factors, environmental pollution and tobacco smoke inhalation are the major factors for induction of this disease. To identify pathogenetic mechanisms of lung emphysema development, the aim of this study was to establish a mouse model of tobacco-smoke induced COPD.