Norbert Weissmann

Publications of Norbert Weissmann

• Role of Src Tyrosine Kinases in Experimental Pulmonary Hypertension.

  Authors: Soni Savai Pullamsetti, Eva Maria Berghausen, Swati Dabral, Alexandra Tretyn, Elsa Butrous, Rajkumar Savai, Ghazwan Butrous, Bhola Kumar Dahal, Ralf P Brandes, Hossein Ardeschir Ghofrani, Norbert Weissmann, Friedrich Grimminger, Werner Seeger, Stephan Rosenkranz, Ralph Theo Schermuly

  Arteriosclerosis, thrombosis, and vascular biology. 04/2012;

  OBJECTIVE: Pulmonary arterial hypertension is a progressive pulmonary vascular disorder with high morbidity and mortality. Compelling evidence suggests that receptor tyrosine kinases, such asOBJECTIVE: Pulmonary arterial hypertension is a progressive pulmonary vascular disorder with high morbidity and mortality. Compelling evidence suggests that receptor tyrosine kinases, such as platelet-derived growth factor (PDGF) are closely involved in the pathogenesis of pulmonary arterial hypertension. We investigated the effects of 2 novel PDGF inhibitors, nilotinib/AMN107 (Abl kinases/PDGF receptor inhibitor) and dasatinib/BMS-354825 (Abl kinases/PDGF receptor/Src inhibitor), on the proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) and on the hemodynamics and pulmonary vascular remodeling in experimental pulmonary hypertension, and determined the expression and regulation of Src family kinases. METHODS AND RESULTS: Human PASMCs were stimulated by PDGF alone or multiple growth factors to induce proliferation and migration in vitro. Dasatinib (0.03 μmol/L), nilotinib (0.3 μmol/L), and imatinib (1 μmol/L) potently inhibited PDGF-induced Human PASMCs were stimulated by PDGF alone or multiple growth factors to induce proliferation and migration in vitro. Dasatinib (0.03 μmol/L), nilotinib (0.3 μmol/L), and imatinib (1 μmol/L) potently inhibited PDGF-induced signal transducer and activator of transcription 3 and Akt phosphorylation. All 3 inhibitors decreased PDGF-induced proliferation, cell cycle gene regulation, and migration. In contrast, only dasatinib inhibited multiple growth factor-induced PASMC proliferation, and this was associated with the inhibition of Src phosphorylation. Combination of specific Src inhibitors (phosphoprotein phosphatase 1, phosphoprotein phosphatase 2) with either imatinib or nilotinib reduced multiple growth factor-induced proliferation to a similar extent as dasatinib. Importantly, Src phosphorylation increased in pulmonary arterial hypertension PASMCs compared with control PASMCs. Finally, in vivo dasatinib (15 mg/kg per bw) treatment caused a complete reversal of pulmonary vascular remodeling and achieved similar effectiveness as imatinib (100 mg/kg per bw) in both monocrotaline- and hypoxia-induced pulmonary hypertension models. phosphatase 1, phosphoprotein phosphatase 2) with either imatinib or nilotinib reduced multiple growth factor-induced proliferation to a similar extent as dasatinib. Importantly, Src phosphorylation increased in pulmonary arterial hypertension PASMCs compared with control PASMCs. Finally, in vivo dasatinib (15 mg/kg per bw) treatment caused a complete reversal of pulmonary vascular remodeling and achieved similar effectiveness as imatinib (100 mg/kg per bw) in both monocrotaline- and hypoxia-induced pulmonary hypertension models. CONCLUSIONS: We suggest that dual inhibition of PDGF receptor and Src kinases potently inhibits mitogenic and motogenic responses to growth factors in PASMCs and pulmonary vascular remodeling in vivo so that dual inhibition may represent an alternative therapeutic approach for pulmonary arterial hypertension.

• Nox4 Is a Protective Reactive Oxygen Species Generating Vascular NADPH Oxidase.

  Authors: Katrin Schröder, Min Zhang, Sebastian Benkhoff, Anja Mieth, Rainer Pliquett, Judith Kosowski, Christoph Kruse, Peter Lüdike, U Ruth Michaelis, Norbert Weissmann, Stefanie Dimmeler, Ajay M Shah, Ralf P Brandes

  Circulation research. 03/2012;

  Rationale:The function of Nox4, a source of vascular H(2)O(2), is unknown. Other Nox proteins were identified as mediators of endothelial dysfunction.Objective:We determined the function of Nox4 inRationale:The function of Nox4, a source of vascular H(2)O(2), is unknown. Other Nox proteins were identified as mediators of endothelial dysfunction.Objective:We determined the function of Nox4 in situations of increased stress induced by ischemia or angiotensin II with global and tamoxifen-inducible Nox4(-/-) mice.Methods and Results:Nox4 was highly expressed in the endothelium and contributed to H(2)O(2) formation. Nox4(-/-) mice exhibited attenuated angiogenesis (femoral artery ligation) and PEG-catalase treatment in control mice had a similar effect. Tube formation in cultured Nox4(-/-) lung endothelial cells (LECs) was attenuated and restored by low concentrations of H(2)O(2,) whereas PEG-catalase attenuated tube formation in control LECs. Angiotensin II infusion was used as a model of oxidative stress. Compared to wild-type, aortas from inducible Nox4-deficient animals had development of increased inflammation, media hypertrophy, and endothelial dysfunction. Mechanistically, loss of Nox4 resulted in reduction of endothelial nitric oxide synthase expression, nitric oxide production, and heme oxygenase-1 (HO-1) expression, which was associated with apoptosis and inflammatory activation. HO-1 expression is controlled by Nrf-2. Accordingly, Nox4-deficient LECs exhibited reduced Nrf-2 protein level and deletion of Nox4 reduced Nrf-2 reporter gene activity. In vivo treatment with hemin, an inducer of HO-1, blocked the vascular hypertrophy induced by Nox4 deletion in the angiotensin II infusion model and carbon monoxide, the product of HO-1, blocked the Nox4-deletion-induced apoptosis in LECs.Conclusion:Endogenous Nox4 protects the vasculature during ischemic or inflammatory stress. Different from Nox1 and Nox2, this particular NADPH oxidase therefore may have a protective vascular function.

• Hypoxia induces K(v) channel current inhibition by increased NADPH oxidase-derived reactive oxygen species.

  Authors: Manish Mittal, Xiang Q Gu, Oleg Pak, Matthew E Pamenter, Daniela Haag, D Beate Fuchs, Ralph T Schermuly, H A Ghofrani, Ralf P Brandes, Werner Seeger, Friedrich Grimminger, Gabriel G Haddad, Norbert Weissmann

  Free radical biology & medicine. 03/2012; 52(6):1033-42.

  There is current discussion whether reactive oxygen species are up- or downregulated in the pulmonary circulation during hypoxia, from which sources (i.e., mitochondria or NADPH oxidases) they areThere is current discussion whether reactive oxygen species are up- or downregulated in the pulmonary circulation during hypoxia, from which sources (i.e., mitochondria or NADPH oxidases) they are derived, and what the downstream targets of ROS are. We recently showed that the NADPH oxidase homolog NOX4 is upregulated in hypoxia-induced pulmonary hypertension in mice and contributes to the vascular remodeling in pulmonary hypertension. We here tested the hypothesis that NOX4 regulates K(v) channels via an increased ROS formation after prolonged hypoxia. We showed that (1) NOX4 is upregulated in hypoxia-induced pulmonary hypertension in rats and isolated rat pulmonary arterial smooth muscle cells (PASMC) after 3days of hypoxia, and (2) that NOX4 is a major contributor to increased reactive oxygen species (ROS) after hypoxia. Our data indicate colocalization of K(v)1.5 and NOX4 in isolated PASMC. The NADPH oxidase inhibitor and ROS scavenger apocynin as well as NOX4 siRNA reversed the hypoxia-induced decrease in K(v) current density whereas the protein levels of the channels remain unaffected by siNOX4 treatment. Determination of cysteine oxidation revealed increased NOX4-mediated K(v)1.5 channel oxidation. We conclude that sustained hypoxia decreases K(v) channel currents by a direct effect of a NOX4-derived increase in ROS.

• Effects of hypercapnia and NO synthase inhibition in sustained hypoxic pulmonary vasoconstriction.

  Authors: Farzaneh Ketabchi, Hossein A Ghofrani, Ralph T Schermuly, Werner Seeger, Friedrich Grimminger, Bakytbek Egemnazarov, S Mostafa Shid-Moosavi, Gholam A Dehghani, Norbert Weissmann, Natascha Sommer

  Respiratory research. 01/2012; 13(1):7.

  ABSTRACT: BACKGROUND: Acute respiratory disorders may lead to sustained alveolar hypoxia with hypercapnia resulting in impaired pulmonary gas exchange. Hypoxic pulmonary vasoconstriction (HPV)ABSTRACT: 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 1400W, 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 1400W. 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 is iNOS dependent.

• Mitochondrial complex II is essential for hypoxia-induced pulmonary vasoconstriction of intra- but not of pre-acinar arteries.

  Authors: Renate Paddenberg, Martina Tiefenbach, Petra Faulhammer, Anna Goldenberg, Barbara Gries, Uwe Pfeil, Katrin S Lips, José I Piruat, José López-Barneo, Ralph T Schermuly, Norbert Weissmann, Wolfgang Kummer

  Cardiovascular research. 01/2012; 93(4):702-10.

  Alveolar hypoxia acutely elicits contraction of pulmonary arteries, leading to a rise in pulmonary arterial pressure (PAP) and shifting blood to better ventilated areas of the lung. The molecularAlveolar hypoxia acutely elicits contraction of pulmonary arteries, leading to a rise in pulmonary arterial pressure (PAP) and shifting blood to better ventilated areas of the lung. The molecular mechanisms underlying this hypoxic pulmonary vasoconstriction (HPV) are still incompletely understood. Here, we investigated the role of succinate dehydrogenase (SDH; synonymous to mitochondrial complex II) in HPV, with particular emphasis on regional differences along the vascular bed and consequences for PAP and perfusion-to-ventilation matching, using mutant mice heterozygous for the SDHD subunit of complex II (SDHD(+/-)). Western blots revealed reduced protein content of complex II subunits SDHA, SDHB, and SDHC in lungs of SDHD(+/-) mice, despite unaffected mRNA content as determined by real-time PCR. Hypoxic pulmonary vasoconstriction of small (20-50 µm) intra-acinar and larger (51-100 µm) pre-acinar arteries was evaluated by videomorphometric analysis of precision-cut lung slices. The hypoxic response was detectable in pre-acinar arteries but absent from intra-acinar arteries of SDHD(+/-) mice. In isolated perfused lungs, basal PAP and its hypoxia-induced increase were indistinguishable between both mouse strains. Arterial oxygenation was measured after provocation of regional ventilatory failure by tracheal fluid instillation in anaesthetized mice, and it declined more in SDHD(+/-) than in wild-type mice. SDHD is required for the formation of a stable mitochondrial complex II and it is selectively important for HPV of intra-acinar vessels. This specialized vascular segment participates in perfusion-to-ventilation matching but does not significantly contribute to the acute hypoxic rise in PAP that results from more proximal vasoconstriction.

• Activation of TRPC6 channels is essential for lung ischaemia-reperfusion induced oedema in mice.

  Authors: Norbert Weissmann, Akylbek Sydykov, Hermann Kalwa, Ursula Storch, Beate Fuchs, Michael Mederos Y Schnitzler, Ralf P Brandes, Friedrich Grimminger, Marcel Meissner, Marc Freichel [......] Oleg Pak, Karl-Heinz Krause, Ralph T Schermuly, Alison C Brewer, Harald H H W Schmidt, Werner Seeger, Ajay M Shah, Thomas Gudermann, Hossein A Ghofrani, Alexander Dietrich

  Nature communications. 01/2012; 3:649.

  Lung ischaemia-reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lackingLung ischaemia-reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2(y/-)) or the classical transient receptor potential channel 6 (TRPC6(-/-)) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca(2+) influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2(y/-) cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE.

• Inhibition of MicroRNA-17 Improves Lung and Heart Function in Experimental Pulmonary Hypertension.

  Authors: Soni S Pullamsetti, Carmen Doebele, Ariane Fischer, Rajkumar Savai, Baktybek Kojonazarov, Bhola K Dahal, Hossein A Ghofrani, Norbert Weissmann, Friedrich Grimminger, Angelika Bonauer, Werner Seeger, Andreas M Zeiher, Stefanie Dimmeler, Ralph T Schermuly

  American journal of respiratory and critical care medicine. 12/2011; 185(4):409-19.

  Rationale: MicroRNAs (miRs) control various cellular processes in tissue homeostasis and disease by regulating gene expression on the posttranscriptional level. Recently, it was demonstrated that theRationale: MicroRNAs (miRs) control various cellular processes in tissue homeostasis and disease by regulating gene expression on the posttranscriptional level. Recently, it was demonstrated that the expression of miR-21 and members of the miR-17-92 cluster was significantly altered in experimental pulmonary hypertension (PH). Objectives: To evaluate the therapeutic efficacy and antiremodeling potential of miR inhibitors in the pathogenesis of PH. Methods: We first tested the effects of miR inhibitors (antagomirs), which were specifically designed to block miR-17 (A-17), miR-21 (A-21), and miR-92a (A-92a) in chronic hypoxia-induced PH in mice and A-17 in monocrotaline-induced PH in rats. Moreover, biological function of miR-17 was analyzed in cultured pulmonary artery smooth muscle cells. Measurements and Main Results: In the PH mouse model, A-17 and A-21 reduced right ventricular systolic pressure, and all antagomirs decreased pulmonary arterial muscularization. However, only A-17 reduced hypoxia-induced right ventricular hypertrophy and improved pulmonary artery acceleration time. In the monocrotaline-induced PH rat model, A-17 treatment significantly decreased right ventricular systolic pressure and total pulmonary vascular resistance index, increased pulmonary artery acceleration time, normalized cardiac output, and decreased pulmonary vascular remodeling. Among the tested miR-17 targets, the cyclin-dependent kinase inhibitor 1A (p21) was up-regulated in lungs undergoing A-17 treatment. Likewise, in human pulmonary artery smooth muscle cells, A-17 increased p21. Overexpression of miR-17 significantly reduced p21 expression and increased proliferation of smooth muscle cells. Conclusions: Our data demonstrate that A-17 improves heart and lung function in experimental PH by interfering with lung vascular and right ventricular remodeling. The beneficial effects may be related to the up-regulation of p21. Thus, inhibition of miR-17 may represent a novel therapeutic concept to ameliorate disease state in PH.

• Inducible NOS inhibition reverses tobacco-smoke-induced emphysema and pulmonary hypertension in mice.

  Authors: Michael Seimetz, Nirmal Parajuli, Alexandra Pichl, Florian Veit, Grazyna Kwapiszewska, Friederike C Weisel, Katrin Milger, Bakytbek Egemnazarov, Agnieszka Turowska, Beate Fuchs [......] Peter Jaksch, Rio Dumitrascu, Holger Garn, Robert Voswinckel, Sawa Kostin, Werner Seeger, Ralph T Schermuly, Friedrich Grimminger, Hossein A Ghofrani, Norbert Weissmann

  Cell. 10/2011; 147(2):293-305.

  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 vascularChronic 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.

• Therapeutic efficacy of TBC3711 in monocrotaline-induced pulmonary hypertension.

  Authors: Djuro Kosanovic, Baktybek Kojonazarov, Himal Luitel, Bhola K Dahal, Akylbek Sydykov, Teodora Cornitescu, Wiebke Janssen, Ralf P Brandes, Neil Davie, Hossein A Ghofrani, Norbert Weissmann, Friedrich Grimminger, Werner Seeger, Ralph T Schermuly

  Respiratory research. 06/2011; 12:87.

  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 theEndothelin-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. 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. 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. 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.

• The role of dimethylarginine dimethylaminohydrolase in idiopathic pulmonary fibrosis.

  Authors: Soni Savai Pullamsetti, Rajkumar Savai, Rio Dumitrascu, Bhola Kumar Dahal, Jochen Wilhelm, Melanie Konigshoff, Dariusz Zakrzewicz, Hossein Ardeschir Ghofrani, Norbert Weissmann, Oliver Eickelberg, Andreas Guenther, James Leiper, Werner Seeger, Friedrich Grimminger, Ralph Theo Schermuly

  Science translational medicine. 06/2011; 3(87):87ra53.

  Idiopathic pulmonary fibrosis (IPF) is a progressive, dysregulated response to alveolar injury that culminates in compromised lung function from excess extracellular matrix production. AssociatedIdiopathic pulmonary fibrosis (IPF) is a progressive, dysregulated response to alveolar injury that culminates in compromised lung function from excess extracellular matrix production. Associated with high morbidity and mortality, IPF is generally refractory to current pharmacological therapies. We examined fibrotic lungs from mice and from patients with IPF and detected increased expression of dimethylarginine dimethylaminohydrolases (DDAHs)--key enzymes that metabolize asymmetric dimethylarginine (ADMA), which is an endogenous inhibitor of nitric oxide synthase, to form l-citrulline and dimethylamine. DDAHs are up-regulated in primary alveolar epithelial type II cells from these mice and patients where they are colocalized with inducible nitric oxide synthase. In cultured alveolar epithelial type II cells from bleomycin-induced fibrotic mouse lungs, inhibition of DDAH suppressed proliferation and induced apoptosis in an ADMA-dependent manner. In addition, DDAH inhibition reduced collagen production by fibroblasts in an ADMA-independent but transforming growth factor/SMAD-dependent manner. In mice with bleomycin-induced pulmonary fibrosis, the DDAH inhibitor L-291 reduced collagen deposition and normalized lung function. In bleomycin-induced fibrosis, inducible nitric oxide synthase inhibition decreased fibrosis, but an even stronger reduction was observed after inhibition of DDAH. Thus, DDAH inhibition reduces fibroblast-induced collagen deposition in an ADMA-independent manner and reduces abnormal epithelial proliferation in an ADMA-dependent manner, offering a possible therapeutic avenue for attenuation of pulmonary fibrosis.

• Hypoxic pulmonary hypertension in mice with constitutively active platelet-derived growth factor receptor-β.

  Authors: Bhola K Dahal, Rainer Heuchel, Soni Savai Pullamsetti, Jochen Wilhelm, Hossein A Ghofrani, Norbert Weissmann, Werner Seeger, Friedrich Grimminger, Ralph T Schermuly

  Pulmonary circulation. 04/2011; 1(2):259-68.

  Platelet-derived growth factor (PDGF) has been implicated in the pathobiology of vascular remodeling. The multikinase inhibitor imatinib that targets PDGF receptor (PDGFR), c-kit and Abl kinases,Platelet-derived growth factor (PDGF) has been implicated in the pathobiology of vascular remodeling. The multikinase inhibitor imatinib that targets PDGF receptor (PDGFR), c-kit and Abl kinases, shows therapeutic efficacy against experimental pulmonary hypertension (PH); however, the role of PDGFR-b in experimental PH has not been examined by genetic approach. We investigated the chronic hypoxia-induced PH in mice carrying an activating point mutation of PDGFR-β (D849N) and evaluated the therapeutic efficacy of imatinib. In addition, we studied pulmonary global gene expression and confirmed the expression of identified genes by immunohistochemistry. Chronically hypoxic D849N mice developed PH and strong pulmonary vascular remodeling that was improved by imatinib (100 mg/kg/day) as evident from the significantly reduced right ventricular systolic pressure, right ventricular hypertrophy and muscularization of peripheral pulmonary arteries. Global gene expression analysis revealed that stromal cell derived factor SDF)-1α was significantly upregulated, which was confirmed by immunohistochemistry. Moreover, an enhanced immunoreactivity for SDF-1α, PDGFR-β and CXCR4, the receptor for SDF-1α was localized to the α-smooth muscle cell (SMC) actin positive pulmonary vascular cells in hypoxic mice and patients with idiopathic pulmonary arterial hypertension (IPAH). In conclusion, our findings substantiate the major role of PDGFR activation in pulmonary vascular remodeling by a genetic approach. Immunohistochemistry findings suggest a role for SDF-1α/CXCR4 axis in pulmonary vascular remodeling and point to a potential interaction between the chemokine SDF-1 and the growth factor PDGF signaling. Future studies designed to elucidate an interaction between the chemokine SDF-1 and the PDGF system may uncover novel therapeutic targets.

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• cAMP phosphodiesterase inhibitors increases nitric oxide production by modulating dimethylarginine dimethylaminohydrolases.

  Authors: Soni Savai Pullamsetti, Rajkumar Savai, Martina Barbara Schaefer, Jochen Wilhelm, Hossein Ardeschir Ghofrani, Norbert Weissmann, Christian Schudt, Ingrid Fleming, Konstantin Mayer, James Leiper, Werner Seeger, Friedrich Grimminger, Ralph Theo Schermuly

  Circulation. 03/2011; 123(11):1194-204.

  Pulmonary arterial hypertension is characterized by a progressive increase in pulmonary vascular resistance caused by endothelial dysfunction, inward vascular remodeling, and severe loss ofPulmonary arterial hypertension is characterized by a progressive increase in pulmonary vascular resistance caused by endothelial dysfunction, inward vascular remodeling, and severe loss of precapillary pulmonary vessel cross-sectional area. Asymmetrical dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, and its metabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) play important roles in endothelial dysfunction. We investigated whether combined phosphodiesterase (PDE) 3 and 4 inhibition ameliorates endothelial function by regulating the ADMA-DDAH axis. We investigated the effects of the PDE3/4 inhibitor tolafentrine in vitro on endothelial cell survival, proliferation, and apoptosis. Effects of tolafentrine on the endothelial nitric oxide synthase/nitric oxide pathway, DDAH expression, DDAH promoter activity, and cytokine release from endothelial cells and their subsequent influence on DDAH expression were investigated. In monocrotaline-induced pulmonary arterial hypertension in rats, the effects of inhaled tolafentrine on DDAH expression and activity were investigated. Real-time-polymerase chain reaction, immunocytochemistry, and PDE activity assays suggested high expression of PDE3 and PDE4 isoforms in endothelial cells. Treatment of endothelial cells with PDE3/4 inhibitor significantly decreased ADMA-induced apoptosis via a cAMP/PKA-dependent pathway by induction of DDAH2. Chronic nebulization of PDE3/4 inhibitor significantly attenuated monocrotaline-induced hemodynamic, gas exchange abnormalities, vascular remodeling, and right heart hypertrophy. Interestingly, PDE3/4 inhibitor treatment reduced ADMA and elevated nitric oxide/cGMP levels. Mechanistically, this could be attributed to direct modulatory effects of cAMP on the promoter region of DDAH2, which was consequently found to be increased in expression and activity. Furthermore, PDE3/4 inhibitor suppressed apoptosis in endothelial cells and increased vascularization in the lung. Combined inhibition of PDE3 and 4 regresses development of pulmonary hypertension and promotes endothelial regeneration by modulating the ADMA-DDAH axis.

• Riociguat for the treatment of pulmonary hypertension.

  Authors: Ralph T Schermuly, Wiebke Janssen, Norbert Weissmann, Johannes-Peter Stasch, Friedrich Grimminger, Hossein Ardeschir Ghofrani

  Expert opinion on investigational drugs. 03/2011; 20(4):567-76.

  INTRODUCTION: Pulmonary hypertension (PH) is a severe condition with a poor prognosis despite recent treatment advances. Therapies with new mechanisms of action are needed. AREAS COVERED: This reviewINTRODUCTION: Pulmonary hypertension (PH) is a severe condition with a poor prognosis despite recent treatment advances. Therapies with new mechanisms of action are needed. AREAS COVERED: This review will help readers understand the mechanism of action of the soluble guanylate cyclase (sGC) stimulator riociguat (BAY 63-2521) and will provide a comprehensive summary regarding efficacy and safety of this drug in the management of PH. The most relevant publications up to December 2010 were used as sources for this review. EXPERT OPINION: Cyclic guanosine monophosphate (cGMP) is an important mediator of the preferential perfusion of well-ventilated regions throughout the lung. Drugs that increase cGMP levels could promote pulmonary vasorelaxation while maintaining optimal gas exchange. cGMP is generated by sGC, which can be stimulated by nitric oxide (NO). Riociguat stimulates sGC independently of NO and increases the sensitivity of sGC to NO, resulting in increased cGMP levels. Results to date suggest rapid, potent and prolonged efficacy and good tolerability in different types of PH. Phase III clinical trials are evaluating the long-term safety and clinical effectiveness of riociguat in pulmonary arterial hypertension (PAH) and chronic thromboembolic PH. Riociguat has the potential to become an important drug for the treatment of patients with PH.

• Neurotrophic tyrosine kinase receptor B/neurotrophin 4 signaling axis is perturbed in clinical and experimental pulmonary fibrosis.

  Authors: Sibel Avcuoglu, Małgorzata Wygrecka, Leigh M Marsh, Andreas Günther, Werner Seeger, Norbert Weissmann, Ludger Fink, Rory E Morty, Grażyna Kwapiszewska

  American journal of respiratory cell and molecular biology. 02/2011; 45(4):768-80.

  The neurotrophins (NTs) are emerging as exciting new participants in normal lung physiology, as well as in several pathological processes in diseased lungs. In this study, the increased expression ofThe neurotrophins (NTs) are emerging as exciting new participants in normal lung physiology, as well as in several pathological processes in diseased lungs. In this study, the increased expression of NT4/5 and of its cognate receptor, the neurotrophic tyrosine kinase receptor Type 2 (TrkB), was observed in human lungs explanted from patients with idiopathic pulmonary fibrosis (IPF), and in lungs from mice with bleomycin-induced pulmonary fibrosis. The expression of NT4/5 and TrkB localized to hyperplastic alveolar Type II cells (ATII) and fibroblastic foci in affected lungs. Increased concentrations of NT4/5 and TrkB were evident in ATII isolated from the lungs of bleomycin-treated mice. Primary ATII were shown to secrete NT4/5 into the cell culture medium. The profibrotic cytokine transforming growth factor-β1, stimulated TrkB, but not NT4/5 gene expression, suggesting that perturbed profibrotic growth factor signaling in affected lungs may drive the expression of TrkB. NT4/5 enhanced the proliferation of ATII through a TrkB/extracellular-regulated kinase/protein kinase B pathway, and could also drive the proliferation of primary human and murine lung fibroblasts, through TrkB-dependent and protein kinase B-dependent pathways. Taken together, these data suggest that a dysregulated TrkB/NT4/5 axis may contribute to several of the pathological lesions associated with pulmonary fibrosis, including ATII hyperplasia and the proliferation of fibroblasts, and we would add IPF to the list of disorders, such as pain and cancer, for which therapeutic targeting of the TrkB/neurotrophin axis has been proposed for further investigation.

• Glycogen synthase kinase 3beta contributes to proliferation of arterial smooth muscle cells in pulmonary hypertension.

  Authors: Piotr Sklepkiewicz, Ralph Theo Schermuly, Xia Tian, Hossein Ardeschir Ghofrani, Norbert Weissmann, Daniel Sedding, Tarek Kashour, Werner Seeger, Friedrich Grimminger, Soni Savai Pullamsetti

  PloS one. 01/2011; 6(4):e18883.

  Pulmonary arterial hypertension (PAH) is a rare progressive pulmonary vascular disorder associated with vascular remodeling and right heart failure. Vascular remodeling involves numerous signalingPulmonary arterial hypertension (PAH) is a rare progressive pulmonary vascular disorder associated with vascular remodeling and right heart failure. Vascular remodeling involves numerous signaling cascades governing pulmonary arterial smooth muscle cell (PASMC) proliferation, migration and differentiation. Glycogen synthase kinase 3beta (GSK3ß) is a serine/threonine kinase and can act as a downstream regulatory switch for numerous signaling pathways. Hence, we hypothesized that GSK3ß plays a crucial role in pulmonary vascular remodeling. All experiments were done with lung tissue or isolated PASMCs in a well-established monocrotaline (MCT)-induced PAH rat model. The mRNA expression of Wnt ligands (Wnt1, Wnt3a, Wnt5a), upstream Wnt signaling regulator genes (Frizzled Receptors 1, 2 and secreted Frizzled related protein sFRP-1) and canonical Wnt intracellular effectors (GSK3ß, Axin1) were assessed by real-time polymerase chain reaction and protein levels of GSK3ß, phospho-GSK3ß (ser 9) by western blotting and localization by immunohistochemistry. The role of GSK3ß in PASMCs proliferation was assessed by overexpression of wild-type GSK3ß (WT) and constitutively active GSK3ß S9A by [(3)H]-thymidine incorporation assay. Increased levels of total and phosphorylated GSK3ß (inhibitory phosphorylation) were observed in lungs and PASMCs isolated from MCT-induced PAH rats compared to controls. Further, stimulation of MCT-PASMCs with growth factors induced GSK3ß inactivation. Most importantly, treatment with the PDGFR inhibitor, Imatinib, attenuated PDGF-BB and FCS induced GSK3ß phosphorylation. Increased expression of GSK3ß observed in lungs and PASMC isolated from MCT-induced PAH rats was confirmed to be clinically relevant as the same observation was identified in human iPAH lung explants. Overexpression of GSK3ß significantly increased MCT-PASMCs proliferation by regulating ERK phosphorylation. Constitutive activation of GSK3ß (GSK3ß S9A, 9th serine replaced to alanine) inhibited MCT-PASMCs proliferation by decreasing ERK phosphorylation. This study supports a central role for GSK3ß in vascular remodeling processes and suggests a novel therapeutic opportunity for the treatment of PAH.

• Involvement of mast cells in monocrotaline-induced pulmonary hypertension in rats.

  Authors: Bhola K Dahal, Djuro Kosanovic, Christina Kaulen, Teodora Cornitescu, Rajkumar Savai, Julia Hoffmann, Irwin Reiss, Hossein A Ghofrani, Norbert Weissmann, Wolfgang M Kuebler, Werner Seeger, Friedrich Grimminger, Ralph T Schermuly

  Respiratory research. 01/2011; 12:60.

  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 tyrosineMast 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. 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. 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. 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.

• Diacylglycerol regulates acute hypoxic pulmonary vasoconstriction via TRPC6.

  Authors: Beate Fuchs, Markus Rupp, Hossein A Ghofrani, Ralph T Schermuly, Werner Seeger, Friedrich Grimminger, Thomas Gudermann, Alexander Dietrich, Norbert Weissmann

  Respiratory research. 01/2011; 12:20.

  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 acuteHypoxic 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. 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. 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. These findings support the conclusion that the TRPC6-dependency of acute HPV is induced via DAG.

• Phosphodiesterase 10A upregulation contributes to pulmonary vascular remodeling.

  Authors: Xia Tian, Christina Vroom, Hossein Ardeschir Ghofrani, Norbert Weissmann, Ewa Bieniek, Friedrich Grimminger, Werner Seeger, Ralph Theo Schermuly, Soni Savai Pullamsetti

  PloS one. 01/2011; 6(4):e18136.

  Phosphodiesterases (PDEs) modulate the cellular proliferation involved in the pathophysiology of pulmonary hypertension (PH) by hydrolyzing cAMP and cGMP. The present study was designed to determinePhosphodiesterases (PDEs) modulate the cellular proliferation involved in the pathophysiology of pulmonary hypertension (PH) by hydrolyzing cAMP and cGMP. The present study was designed to determine whether any of the recently identified PDEs (PDE7-PDE11) contribute to progressive pulmonary vascular remodeling in PH. All in vitro experiments were performed with lung tissue or pulmonary arterial smooth muscle cells (PASMCs) obtained from control rats or monocrotaline (MCT)-induced pulmonary hypertensive (MCT-PH) rats, and we examined the effects of the PDE10 inhibitor papaverine (Pap) and specific small interfering RNA (siRNA). In addition, papaverine was administrated to MCT-induced PH rats from day 21 to day 35 by continuous intravenous infusion to examine the in vivo effects of PDE10A inhibition. We found that PDE10A was predominantly present in the lung vasculature, and the mRNA, protein, and activity levels of PDE10A were all significantly increased in MCT PASMCs compared with control PASMCs. Papaverine and PDE10A siRNA induced an accumulation of intracellular cAMP, activated cAMP response element binding protein and attenuated PASMC proliferation. Intravenous infusion of papaverine in MCT-PH rats resulted in a 40%-50% attenuation of the effects on pulmonary hypertensive hemodynamic parameters and pulmonary vascular remodeling. The present study is the first to demonstrate a central role of PDE10A in progressive pulmonary vascular remodeling, and the results suggest a novel therapeutic approach for the treatment of PH.

• Redox signaling and reactive oxygen species in hypoxic pulmonary vasoconstriction.

  Authors: Beate Fuchs, Natascha Sommer, Alexander Dietrich, Ralph Theo Schermuly, Hossein Ardeschir Ghofrani, Friedrich Grimminger, Werner Seeger, Thomas Gudermann, Norbert Weissmann

  Respiratory physiology & neurobiology. 12/2010; 174(3):282-91.

  Hypoxic pulmonary vasoconstriction (HPV) is an essential physiological mechanism of the lung that matches blood perfusion with alveolar ventilation to optimize gas exchange. Perturbations of HPV, asHypoxic pulmonary vasoconstriction (HPV) is an essential physiological mechanism of the lung that matches blood perfusion with alveolar ventilation to optimize gas exchange. Perturbations of HPV, as may occur in pneumonia or adult respiratory distress syndrome, can cause life-threatening hypoxemia. Despite intensive research for decades, the molecular mechanisms of HPV have not been fully elucidated. Reactive oxygen species (ROS) and changes in the cellular redox state are proposed to link O2 sensing and pulmonary arterial smooth muscle cell contraction underlying HPV. In this regard, mitochondria and NAD(P)H oxidases are discussed as sources of ROS. However, there is controversy whether ROS levels decrease or increase during hypoxia. With this background we summarize the current knowledge on the role of ROS and redox state in HPV.

• Hypoxia enhances platelet-derived growth factor signaling in the pulmonary vasculature by down-regulation of protein tyrosine phosphatases.

  Authors: Henrik ten Freyhaus, Markus Dagnell, Maike Leuchs, Marius Vantler, Eva M Berghausen, Evren Caglayan, Norbert Weissmann, Bhola K Dahal, Ralph T Schermuly, Arne Ostman, Kai Kappert, Stephan Rosenkranz

  American journal of respiratory and critical care medicine. 12/2010; 183(8):1092-102.

  Platelet-derived growth factor (PDGF) plays a pivotal role in the pathobiology of pulmonary hypertension (PH) because it promotes pulmonary vascular remodeling. PH is frequently associated withPlatelet-derived growth factor (PDGF) plays a pivotal role in the pathobiology of pulmonary hypertension (PH) because it promotes pulmonary vascular remodeling. PH is frequently associated with pulmonary hypoxia. To investigate whether hypoxia alters PDGF β receptor (βPDGFR) signaling in the pulmonary vasculature. The impact of chronic hypoxia on signal transduction by the βPDGFR was measured in human pulmonary arterial smooth muscle cells (hPASMC) in vitro, and in mice with hypoxia-induced PH in vivo. Chronic hypoxia significantly enhanced PDGF-BB-dependent proliferation and chemotaxis of hPASMC. Pharmacologic inhibition of PI3 kinase (PI3K) and PLCγ abrogated these events under both normoxia and hypoxia. Although hypoxia did not affect βPDGFR expression, it increased the ligand-induced tyrosine phosphorylation of the receptor, particularly at binding sites for PI3K (Y751) and PLCγ (Y1021). The activated βPDGFR is dephosphorylated by protein tyrosine phosphatases (PTPs). Interestingly, hypoxia decreased expression of numerous PTPs (T cell PTP, density-enhanced phosphatase-1, PTP1B, and SH2 domain-containing phosphatase-2), resulting in reduced PTP activity. Hypoxia-inducible factor (HIF)-1α is involved in this regulation of gene expression, because hypoxia-induced βPDGFR hyperphosphorylation and PTP down-regulation were abolished by HIF-1α siRNA and by the HIF-1α inhibitor 2-methoxyestradiol. βPDGFR hyperphosphorylation and PTP down-regulation were also present in vivo in mice with chronic hypoxia-induced PH. Hypoxia reduces expression and activity of βPDGFR-antagonizing PTPs in a HIF-1α-dependent manner, thereby enhancing receptor activation and proliferation and chemotaxis of hPASMC. Because hyperphosphorylation of the βPDGFR and down-regulation of PTPs occur in vivo, this mechanism likely has significant impact on the development and progression of PH and other hypoxia-associated diseases.