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Background: Chronic obstructive lung disease (COPD) is a common cause of death in industrialized countries often induced by exposure to tobacco smoke. A substantial number of patients with COPD also suffer from pulmonary hypertension that may be caused by hypoxia or other hypoxia-independent stimuli - inducing pulmonary vascular remodeling. The Ca(2+) binding protein, S100A4 is known to play a role in non-COPD-driven vascular remodeling of intrapulmonary arteries. Therefore, we have investigated the potential involvement of S100A4 in COPD induced vascular remodeling., Methods: Lung tissue was obtained from explanted lungs of five COPD patients and five non-transplanted donor lungs. Additionally, mice lungs of a tobacco-smoke-induced lung emphysema model (exposure for 3 and 8 month) and controls were investigated. Real-time RT-PCR analysis of S100A4 and RAGE mRNA was performed from laser-microdissected intrapulmonary arteries. S100A4 immunohistochemistry was semi-quantitatively evaluated. Mobility shift assay and siRNA knock-down were used to prove hypoxia responsive elements (HRE) and HIF binding within the S100A4 promoter., Results: Laser-microdissection in combination with real-time PCR analysis revealed higher expression of S100A4 mRNA in intrapulmonary arteries of COPD patients compared to donors. These findings were mirrored by semi-quantitative analysis of S100A4 immunostaining. Analogous to human lungs, in mice with tobacco-smoke-induced emphysema an up-regulation of S100A4 mRNA and protein was observed in intrapulmonary arteries. Putative HREs could be identified in the promoter region of the human S100A4 gene and their functionality was confirmed by mobility shift assay. Knock-down of HIF1/2 by siRNA attenuated hypoxia-dependent increase in S100A4 mRNA levels in human primary pulmonary artery smooth muscle cells. Interestingly, RAGE mRNA expression was enhanced in pulmonary arteries of tobacco-smoke exposed mice but not in pulmonary arteries of COPD patients., Conclusions: As enhanced S100A4 expression was observed in remodeled intrapulmonary arteries of COPD patients, targeting S100A4 could serve as potential therapeutic option for prevention of vascular remodeling in COPD patients.

Background: Acute respiratory disorders may lead to sustained alveolar hypoxia with hypercapnia resulting in impaired pulmonary gas exchange. Hypoxic pulmonary vasoconstriction (HPV) optimizes gas exchange during local acute (0-30 min), as well as sustained (> 30 min) hypoxia by matching blood perfusion to alveolar ventilation. Hypercapnia with acidosis improves pulmonary gas exchange in repetitive conditions of acute hypoxia by potentiating HPV and preventing pulmonary endothelial dysfunction. This study investigated, if the beneficial effects of hypercapnia with acidosis are preserved during sustained hypoxia as it occurs, e.g in permissive hypercapnic ventilation in intensive care units. Furthermore, the effects of NO synthase inhibitors under such conditions were examined., Method: We employed isolated perfused and ventilated rabbit lungs to determine the influence of hypercapnia with or without acidosis (pH corrected with sodium bicarbonate), and inhibitors of endothelial as well as inducible NO synthase on acute or sustained HPV (180 min) and endothelial permeability., Results: In hypercapnic acidosis, HPV was intensified in sustained hypoxia, in contrast to hypercapnia without acidosis when HPV was amplified during both phases. L-NG-Nitroarginine (L-NNA), a non-selective NO synthase inhibitor, enhanced acute as well as sustained HPV under all conditions, however, the amplification of sustained HPV induced by hypercapnia with or without acidosis compared to normocapnia disappeared. In contrast 1400 W, a selective inhibitor of inducible NO synthase (iNOS), decreased HPV in normocapnia and hypercapnia without acidosis at late time points of sustained HPV and selectively reversed the amplification of sustained HPV during hypercapnia without acidosis. Hypoxic hypercapnia without acidosis increased capillary filtration coefficient (Kfc). This increase disappeared after administration of 1400 W., Conclusion: Hypercapnia with and without acidosis increased HPV during conditions of sustained hypoxia. The increase of sustained HPV and endothelial permeability in hypoxic hypercapnia without acidosis was iNOS dependent.

Background: Endothelin-1 signalling plays an important role in pathogenesis of pulmonary hypertension. Although different endothelin-A receptor antagonists are developed, a novel therapeutic option to cure the disease is still needed. This study aims to investigate the therapeutic efficacy of the selective endothelin-A receptor antagonist TBC3711 in monocrotaline-induced pulmonary hypertension in rats., Methods: Monocrotaline-injected male Sprague-Dawley rats were randomized and treated orally from day 21 to 35 either with TBC3711 (Dose: 30 mg/kg body weight/day) or placebo. Echocardiographic measurements of different hemodynamic and right-heart hypertrophy parameters were performed. After day 35, rats were sacrificed for invasive hemodynamic and right-heart hypertrophy measurements. Additionally, histologic assessment of pulmonary vascular and right-heart remodelling was performed., Results: The novel endothelin-A receptor antagonist TBC3711 significantly attenuated monocrotaline-induced pulmonary hypertension, as evident from improved hemodynamics and right-heart hypertrophy in comparison with placebo group. In addition, muscularization and medial wall thickness of distal pulmonary vessels were ameliorated. The histologic evaluation of the right ventricle showed a significant reduction in fibrosis and cardiomyocyte size, suggesting an improvement in right-heart remodelling., Conclusion: The results of this study suggest that the selective endothelin-A receptor antagonist TBC3711 demonstrates therapeutic benefit in rats with established pulmonary hypertension, thus representing a useful therapeutic approach for treatment of pulmonary hypertension.

Background: Mast cells (MCs) are implicated in inflammation and tissue remodeling. Accumulation of lung MCs is described in pulmonary hypertension (PH); however, whether MC degranulation and c-kit, a tyrosine kinase receptor critically involved in MC biology, contribute to the pathogenesis and progression of PH has not been fully explored., Methods: Pulmonary MCs of idiopathic pulmonary arterial hypertension (IPAH) patients and monocrotaline-injected rats (MCT-rats) were examined by histochemistry and morphometry. Effects of the specific c-kit inhibitor PLX and MC stabilizer cromolyn sodium salt (CSS) were investigated in MCT-rats both by the preventive and therapeutic approaches. Hemodynamic and right ventricular hypertrophy measurements, pulmonary vascular morphometry and analysis of pulmonary MC localization/counts/activation were performed in animal model studies., Results: There was a prevalence of pulmonary MCs in IPAH patients and MCT-rats as compared to the donors and healthy rats, respectively. Notably, the perivascular MCs were increased and a majority of them were degranulated in lungs of IPAH patients and MCT-rats (p < 0.05 versus donor and control, respectively). In MCT-rats, the pharmacological inhibitions of MC degranulation and c-kit with CSS and PLX, respectively by a preventive approach (treatment from day 1 to 21 of MCT-injection) significantly attenuated right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH). Moreover, vascular remodeling, as evident from the significantly decreased muscularization and medial wall thickness of distal pulmonary vessels, was improved. However, treatments with CSS and PLX by a therapeutic approach (from day 21 to 35 of MCT-injection) neither improved hemodynamics and RVH nor vascular remodeling., Conclusions: The accumulation and activation of perivascular MCs in the lungs are the histopathological features present in clinical (IPAH patients) and experimental (MCT-rats) PH. Moreover, the accumulation and activation of MCs in the lungs contribute to the development of PH in MCT-rats. Our findings reveal an important pathophysiological insight into the role of MCs in the pathogenesis of PH in MCT-rats.

Background: Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches blood perfusion to alveolar ventilation to optimize gas exchange. Recently we have demonstrated that acute but not sustained HPV is critically dependent on the classical transient receptor potential 6 (TRPC6) channel. However, the mechanism of TRPC6 activation during acute HPV remains elusive. We hypothesize that a diacylglycerol (DAG)-dependent activation of TRPC6 regulates acute HPV., Methods: We investigated the effect of the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) on normoxic vascular tone in isolated perfused and ventilated mouse lungs from TRPC6-deficient and wild-type mice. Moreover, the effects of OAG, the DAG kinase inhibitor R59949 and the phospholipase C inhibitor U73122 on the strength of HPV were investigated compared to those on non-hypoxia-induced vasoconstriction elicited by the thromboxane mimeticum U46619., Results: OAG increased normoxic vascular tone in lungs from wild-type mice, but not in lungs from TRPC6-deficient mice. Under conditions of repetitive hypoxic ventilation, OAG as well as R59949 dose-dependently attenuated the strength of acute HPV whereas U46619-induced vasoconstrictions were not reduced. Like OAG, R59949 mimicked HPV, since it induced a dose-dependent vasoconstriction during normoxic ventilation. In contrast, U73122, a blocker of DAG synthesis, inhibited acute HPV whereas U73343, the inactive form of U73122, had no effect on HPV., Conclusion: These findings support the conclusion that the TRPC6-dependency of acute HPV is induced via DAG.

Background: Idiopathic Pulmonary Fibrosis (IPF) is an unresolved clinical issue. Phosphodiesterases (PDEs) are known therapeutic targets for various proliferative lung diseases. Lung PDE6 expression and function has received little or no attention. The present study aimed to characterize (i) PDE6 subunits expression in human lung, (ii) PDE6 subunits expression and alteration in IPF and (iii) functionality of the specific PDE6D subunit in alveolar epithelial cells (AECs)., Methodology/principal Findings: PDE6 subunits expression in transplant donor (n = 6) and IPF (n = 6) lungs was demonstrated by real-time quantitative (q)RT-PCR and immunoblotting analysis. PDE6D mRNA and protein levels and PDE6G/H protein levels were significantly down-regulated in the IPF lungs. Immunohistochemical analysis showed alveolar epithelial localization of the PDE6 subunits. This was confirmed by qRT-PCR from human primary alveolar type (AT)II cells, demonstrating the down-regulation pattern of PDE6D in IPF-derived ATII cells. In vitro, PDE6D protein depletion was provoked by transforming growth factor (TGF)-β1 in A549 AECs. PDE6D siRNA-mediated knockdown and an ectopic expression of PDE6D modified the proliferation rate of A549 AECs. These effects were mediated by increased intracellular cGMP levels and decreased ERK phosphorylation., Conclusions/significance: Collectively, we report previously unrecognized PDE6 expression in human lungs, significant alterations of the PDE6D and PDE6G/H subunits in IPF lungs and characterize the functional role of PDE6D in AEC proliferation.

Background: Generalized hypoxic pulmonary vasoconstriction (HPV) occurring during exposure to hypoxia is a detrimental process resulting in an increase in lung vascular resistance. Nebulization of sodium nitrite has been shown to inhibit HPV. The aim of this project was to investigate and compare the effects of nebulization of nitrite and different formulations of acidified sodium nitrite on acute HPV., Methods: Ex vivo isolated rabbit lungs perfused with erythrocytes in Krebs-Henseleit buffer (adjusted to 10% hematocrit) and in vivo anesthetized catheterized rabbits were challenged with periods of hypoxic ventilation alternating with periods of normoxic ventilation. After baseline hypoxic challenges, vehicle, sodium nitrite or acidified sodium nitrite was delivered via nebulization. In the ex vivo model, pulmonary arterial pressure and nitric oxide concentrations in exhaled gas were monitored. Nitrite and nitrite/nitrate were measured in samples of perfusion buffer. Pulmonary arterial pressure, systemic arterial pressure, cardiac output and blood gases were monitored in the in vivo model., Results: In the ex vivo model, nitrite nebulization attenuated HPV and increased nitric oxide concentrations in exhaled gas and nitrite concentrations in the perfusate. The acidified forms of sodium nitrite induced higher levels of nitric oxide in exhaled gas and had longer vasodilating effects compared to nitrite alone. All nitrite formulations increased concentrations of circulating nitrite to the same degree. In the in vivo model, inhaled nitrite inhibited HPV, while pulmonary arterial pressure, cardiac output and blood gases were not affected. All nitrite formulations had similar potency to inhibit HPV. The tested concentration of appeared tolerable., Conclusion: Nitrite alone and in acidified forms effectively and similarly attenuates HPV. However, acidified nitrite formulations induce a more pronounced increase in nitric oxide exhalation.

Background: The rapid desensitization of the human prostacyclin (IP) in response to agonist binding has been shown in cell culture. Phosphorylation of the IP receptor by protein kinase C (PKC) has been suggested to be involved in this process., Methods and Results: In this study we investigated the vasodilatory effects of iloprost, a stable prostacyclin analogue, in perfused rabbit lungs. Continuous infusion of the thromboxane mimetic U46619 was employed to establish stable pulmonary hypertension. A complete loss of the vasodilatory response to iloprost was observed in experiments with continuous iloprost perfusion, maintaining the intravascular concentration of this prostanoid over a 180 min period. When lungs under chronic iloprost infusion were acutely challenged with inhaled iloprost, a corresponding complete loss of vasoreactivity was observed. This desensitization was not dependent on upregulation of cAMP-specific phosphodiesterases or changes in adenylate cyclase activity, as suggested by unaltered dose-response curves to agents directly affecting these enzymes. Application of a prostaglandin E1 receptor antagonist 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809) or the PKC inhibitor bisindolylmaleimide I (BIM) enhanced the vasodilatory response to infused iloprost and partially prevented tachyphylaxis., Conclusion: A three-hour infusion of iloprost in pulmonary hypertensive rabbit lungs results in complete loss of the lung vasodilatory response to this prostanoid. This rapid desensitization is apparently not linked to changes in adenylate cyclase and phosphodiesterase activation, but may involve PKC function and co-stimulation of the EP1 receptor in addition to the IP receptor by this prostacyclin analogue.

Background: The aim of the study was to assess the chronic effects of combined phosphodiesterase 3/4 inhibitor tolafentrine, administered by inhalation, during monocrotaline-induced pulmonary arterial hypertension (PAH) in rats., Methods: CD rats were given a single subcutaneous injection of monocrotaline to induce PAH. Four weeks after, rats were subjected to inhalation of tolafentrine or sham nebulization in an unrestrained, whole body aerosol exposure system. In these animals (i) the acute pulmonary vasodilatory efficacy of inhaled tolafentrine (ii) the anti-remodeling effect of long-term inhalation of tolafentrine (iii) the effects of tolafentrine on the expression profile of 96 genes encoding cell adhesion and extracellular matrix regulation were examined. In addition, the inhibitory effect of tolafentrine on ex vivo isolated pulmonary artery SMC cell migration was also investigated., Results: Monocrotaline injection provoked severe PAH (right ventricular systolic pressure increased from 25.9 +/- 4.0 to 68.9 +/- 3.2 after 4 weeks and 74.9 +/- 5.1 mmHg after 6 weeks), cardiac output depression and right heart hypertrophy. The media thickness of the pulmonary arteries and the proportion of muscularization of small precapillary resistance vessels increased dramatically, and the migratory response of ex-vivo isolated pulmonary artery smooth muscle cells (PASMC) was increased. Micro-arrays and subsequent confirmation with real time PCR demonstrated upregulation of several extracellular matrix regulation and adhesion genes, such as matrixmetalloproteases (MMP) 2, 8, 9, 10, 11, 12, 20, Icam, Itgax, Plat and serpinb2. When chronically nebulized from day 28 to 42 (12 daily aerosol maneuvers), after full establishment of severe pulmonary hypertension, tolafentrine reversed about 60% of all hemodynamic abnormalities, right heart hypertrophy and monocrotaline-induced structural lung vascular changes, including the proportion of pulmonary artery muscularization. The upregulation of extracellular matrix regulation and adhesion genes was reduced by nearly 80% by inhalation of the tolafentrine. When assessed in vitro, tolafentrine blocked the enhanced PASMC migratory response., Conclusion: In conclusion, we demonstrate for the first time that inhalation of combined PDE3/4 inhibitor reverses pulmonary hypertension fully developed in response to monocrotaline in rats. This "reverse-remodeling" effect includes structural changes in the lung vascular wall and key molecular pathways of matrix regulation, concomitant with 60% normalization of hemodynamics.

Background: Chronic hypoxia influences gene expression in the lung resulting in pulmonary hypertension and vascular remodelling. For specific investigation of the vascular compartment, laser-microdissection of intrapulmonary arteries was combined with array profiling., Methods and Results: Analysis was performed on mice subjected to 1, 7 and 21 days of hypoxia (FiO2 = 0.1) using nylon filters (1176 spots). Changes in the expression of 29, 38, and 42 genes were observed at day 1, 7, and 21, respectively. Genes were grouped into 5 different classes based on their time course of response. Gene regulation obtained by array analysis was confirmed by real-time PCR. Additionally, the expression of the growth mediators PDGF-B, TGF-beta, TSP-1, SRF, FGF-2, TIE-2 receptor, and VEGF-R1 were determined by real-time PCR. At day 1, transcription modulators and ion-related proteins were predominantly regulated. However, at day 7 and 21 differential expression of matrix producing and degrading genes was observed, indicating ongoing structural alterations. Among the 21 genes upregulated at day 1, 15 genes were identified carrying potential hypoxia response elements (HREs) for hypoxia-induced transcription factors. Three differentially expressed genes (S100A4, CD36 and FKBP1a) were examined by immunohistochemistry confirming the regulation on protein level. While FKBP1a was restricted to the vessel adventitia, S100A4 and CD36 were localised in the vascular tunica media., Conclusion: Laser-microdissection and array profiling has revealed several new genes involved in lung vascular remodelling in response to hypoxia. Immunohistochemistry confirmed regulation of three proteins and specified their localisation in vascular smooth muscle cells and fibroblasts indicating involvement of different cells types in the remodelling process. The approach allows deeper insight into hypoxic regulatory pathways specifically in the vascular compartment of this complex organ.

Background: The sources and measurement of reactive oxygen species (ROS) in intact organs are largely unresolved. This may be related to methodological problems associated with the techniques currently employed for ROS detection. Electron spin resonance (ESR) with spin trapping is a specific method for ROS detection, and may address some these technical problems., Methods: We have established a protocol for the measurement of intravascular ROS release from isolated buffer-perfused and ventilated rabbit and mouse lungs, combining lung perfusion with the spin probe 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) and ESR spectroscopy. We then employed this technique to characterize hypoxia-dependent ROS release, with specific attention paid to NADPH oxidase-dependent superoxide formation as a possible vasoconstrictor pathway., Results: While perfusing lungs with CPH over a range of inspired oxygen concentrations (1-21 %), the rate of CP* formation exhibited an oxygen-dependence, with a minimum at 2.5 % O2. Addition of superoxide dismutase (SOD) to the buffer fluid illustrated that a minor proportion of this intravascular ROS leak was attributable to superoxide. Stimulation of the lungs by injection of phorbol-12-myristate-13-acetate (PMA) into the pulmonary artery caused a rapid increase in CP* formation, concomitant with pulmonary vasoconstriction. Both the PMA-induced CPH oxidation and the vasoconstrictor response were largely suppressed by SOD. When the PMA challenge was performed at different oxygen concentrations, maximum superoxide liberation and pulmonary vasoconstriction occurred at 5% O2. Using a NADPH oxidase inhibitor and NADPH-oxidase deficient mice, we illustrated that the PMA-induced superoxide release was attributable to the stimulation of NADPH oxidases., Conclusion: The perfusion of isolated lungs with CPH is suitable for detection of intravascular ROS release by ESR spectroscopy. We employed this technique to demonstrate that 1) PMA-induced vasoconstriction is caused "directly" by superoxide generated from NADPH oxidases and 2) this pathway is pronounced in hypoxia. NADPH oxidases thus may contribute to the hypoxia-dependent regulation of pulmonary vascular tone.

Inhaled prostanoids and phosphodiesterase (PDE) inhibitors have been suggested for treatment of severe pulmonary hypertension. In catheterized rabbits with acute pulmonary hypertension induced by continuous infusion of the stable thromboxane analogue U46619, we asked whether sildenafil (PDE1/5/6 inhibitor), motapizone (PDE3 inhibitor) or 8-Methoxymethyl-IBMX (PDE1 inhibitor) synergize with inhaled iloprost. Inhalation of iloprost caused a transient pulmonary artery pressure decline, levelling off within <20 min, without significant changes in blood gases or systemic hemodynamics. Infusion of 8-Methoxymethyl-IBMX, motapizone and sildenafil caused each a dose-dependent decrease in pulmonary artery pressure, with sildenafil possessing the highest efficacy and at the same time selectivity for the pulmonary circulation. When combining a per se ineffective dose of each PDE inhibitor (200 microg/kg x min 8-Methoxymethyl-IBMX, 1 microg/kg x min sildenafil, 5 microg/kg x min motapizone) with subsequent iloprost nebulization, marked amplification of the prostanoid induced pulmonary vasodilatory response was noted and the area under the curve of PPA reduction was nearly threefold increased with all approaches, as compared to sole iloprost administration. Further amplification was achieved with the combination of inhaled iloprost with sildenafil plus motapizone, but not with sildenafil plus 8MM-IBMX. Systemic hemodynamics and gas exchange were not altered for all combinations. We conclude that co-administration of minute systemic doses of selective PDE inhibitors with inhaled iloprost markedly enhances and prolongs the pulmonary vasodilatory response to inhaled iloprost, with maintenance of pulmonary selectivity and ventilation perfusion matching. The prominent effect of sildenafil may be operative via both PDE1 and PDE5, and is further enhanced by co-application of a PDE3 inhibitor.