Ralph Theo Schermuly

Publications of Ralph Theo Schermuly

• 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.

• Phosphodiesterase 5 (PDE5) inhibition, ANP and NO rapidly reduce epididymal duct contractions, but long-term PDE5 inhibition in vivo does not.

  Authors: Andrea Mietens, Sabine Tasch, Caroline Feuerstacke, Gerrit Eichner, Johanna Volkmann, Ralph Theo Schermuly, Friedrich Grimminger, Dieter Müller, Ralf Middendorff

  Molecular and cellular endocrinology. 02/2012; 349(2):145-53.

  Contractility of the peritubular smooth muscle layer ensures the transit of immotile spermatozoa through the epididymal duct to acquire their fertilizing capacity. Atrial natriuretic peptide (ANP)Contractility of the peritubular smooth muscle layer ensures the transit of immotile spermatozoa through the epididymal duct to acquire their fertilizing capacity. Atrial natriuretic peptide (ANP) and nitric oxide (NO) affect contractility via cGMP signals that are controlled by phosphodiesterases (PDEs). Sildenafil inhibits the cGMP-hydrolyzing PDE5 and thereby promotes relaxation of smooth muscle cells. While sildenafil is increasingly used in young patients for the treatment of pulmonary hypertension, virtually no knowledge exists about PDEs in the epididymis. Western blotting, immunohistochemistry and RT-PCR analyses after laser capture microdissection localized PDE5 to smooth muscle cells, but not to epithelial cells, of the epididymal duct in man and rat. Sildenafil, ANP and NO significantly slowed spontaneous contractions of rat epididymal duct segments in organ bath studies. Sildenafil effects were additive to ANP and NO. Long-term exposure to sildenafil in vivo did not change the PDE5 expression or the observed contractility pattern with the rapid relaxing response toward ANP, NO and sildenafil. Data demonstrate that PDE5 is an important member of cGMP signaling pathways regulating the finely orchestrated process of epididymal duct contractility and suggest, however, that in the epididymis side effects of therapeutically used sildenafil are unlikely.

• 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.

• 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.

• 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.

• 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.

• Increased expression of 5-hydroxytryptamine2A/B receptors in idiopathic pulmonary fibrosis: a rationale for therapeutic intervention.

  Authors: Melanie Königshoff, Rio Dumitrascu, Sergey Udalov, Oana Veronica Amarie, Rudolf Reiter, Friedrich Grimminger, Werner Seeger, Ralph Theo Schermuly, Oliver Eickelberg

  Thorax. 11/2010; 65(11):949-55.

  Idiopathic pulmonary fibrosis (IPF) has a poor prognosis and limited responsiveness to available treatments. It is characterised by epithelial cell injury, fibroblast activation and proliferation andIdiopathic pulmonary fibrosis (IPF) has a poor prognosis and limited responsiveness to available treatments. It is characterised by epithelial cell injury, fibroblast activation and proliferation and extracellular matrix deposition. Serotonin (5-hydroxytryptamine; 5-HT) induces fibroblast proliferation via the 5-HTR(2A) and 5-HTR(2B) receptors, but its pathophysiological role in IPF remains unclear. A study was undertaken to determine the expression of 5-HT receptors in IPF and experimental lung fibrosis and to investigate the effects of therapeutic inhibition of 5-HTR(2A/B) signalling on lung fibrosis in vivo and in vitro. Quantitative RT-PCR showed that the expression of 5-HTR(1A/B) and 5-HTR(2B) was significantly increased in the lungs of patients with IPF (n=12) and in those with non-specific interstitial pneumonia (NSIP, n=6) compared with transplant donors (n=12). The expression of 5-HTR(2A) was increased specifically in IPF lungs but not in NSIP lungs. While 5-HTR(2A) protein largely localised to fibroblasts, 5-HTR(2B) localised to the epithelium. To assess the effects of 5HTR(2A/B) inhibition on fibrogenesis in vivo, mice were subjected to bleomycin-induced lung fibrosis and treated with the 5-HTR(2A/B) antagonist terguride (or vehicle) in a therapeutic approach (days 14-28 after bleomycin). Terguride-treated mice had significantly improved lung function and histology and decreased collagen content compared with vehicle-treated mice. Functional in vitro studies showed that terguride is a potent inhibitor of transforming growth factor β(1)- or WNT3a-induced collagen production. The studies revealed an increased expression of 5-HTR(2A) specifically in IPF. Blockade of 5-HTR(2A/B) signalling by terguride reversed lung fibrosis and is thus a promising therapeutic approach for IPF.

• Activation of the WNT/β-catenin pathway attenuates experimental emphysema.

  Authors: Nikolaus Kneidinger, Ali Önder Yildirim, Jens Callegari, Shinji Takenaka, Maria Magdalena Stein, Rio Dumitrascu, Alexander Bohla, Ken R Bracke, Rory E Morty, Guy G Brusselle, Ralph Theo Schermuly, Oliver Eickelberg, Melanie Königshoff

  American journal of respiratory and critical care medicine. 10/2010; 183(6):723-33.

  Chronic obstructive pulmonary disease (COPD) is a devastating disease, for which no causal therapy is available. To characterize WNT/β-catenin signaling in COPD in humans and elucidate its potentialChronic obstructive pulmonary disease (COPD) is a devastating disease, for which no causal therapy is available. To characterize WNT/β-catenin signaling in COPD in humans and elucidate its potential role as a preventive and therapeutic target in experimental emphysema in mice. The expression, localization, and activity of WNT/β-catenin signaling was assessed in 12 COPD and 12 transplant donor samples using quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, and Western blotting. The role of WNT/β-catenin signaling was assessed in elastase- and cigarette smoke-induced emphysema and therapeutic modulation thereof in elastase-induced emphysema in TOPGAL reporter and wild-type mice in vivo. No differences in the mRNA expression profile of the main WNT/β-catenin signaling components were observed comparing COPD and donor lung homogenates. Immunohistochemical analysis revealed reduced numbers of nuclear β-catenin-positive alveolar epithelial cells in COPD. Similarly, WNT/β-catenin signaling was down-regulated in both experimental emphysema models. Preventive and therapeutic, WNT/β-catenin activation by lithium chloride attenuated experimental emphysema, as assessed by decreased airspace enlargement, improved lung function, reduced collagen content, and elevated expression of alveolar epithelial cell markers. Decreased WNT/β-catenin signaling is involved in parenchymal tissue destruction and impaired repair capacity in emphysema. These data indicate a crucial role of WNT/β-catenin signaling in lung repair mechanisms in vivo, and highlight WNT/β-catenin activation as a future therapeutic approach for emphysema.

• PDGF receptor and its antagonists: role in treatment of PAH.

  Authors: Friedrich Grimminger, Ralph Theo Schermuly

  Advances in experimental medicine and biology. 01/2010; 661:435-46.

  Pulmonary Hypertension is a severe lung disease, which is characterized by vasoconstriction and remodelling of the vessel wall. Mostly addressing the increased vascular tone, prostacyclin and itsPulmonary Hypertension is a severe lung disease, which is characterized by vasoconstriction and remodelling of the vessel wall. Mostly addressing the increased vascular tone, prostacyclin and its analogues, endothelin-receptor antagonists and phosphodiesterase type 5 inhibitors have been approved for treatment of PAH and represent the current therapeutic options. Mechanistically, these vasodilators decrease pulmonary vascular resistance and reduce thereby shear stress, which is a strong proliferative stimulus per se. Beside the development of new vasodilators, current research focuses on the development of causal treatment regimens aiming a normalization of the vessel structure. Mechanistically, increased proliferation, migration and a resistance to apoptosis of vascular cells represent key events in disease progression. In this context, tyrosine kinase inhibitors like imatinib have been shown to possess reverse remodelling potential in preclinical models of pulmonary hypertension by inducing apoptosis and blocking proliferation. This book chapter describes the role of the platelet derived growth factor (PDGF) receptor and its antagonists for treatment of pulmonary hypertension.

• Targeting cancer with phosphodiesterase inhibitors.

  Authors: Rajkumar Savai, Soni Savai Pullamsetti, Gamal-Andre Banat, Norbert Weissmann, Hossein Ardeschir Ghofrani, Friedrich Grimminger, Ralph Theo Schermuly

  Expert opinion on investigational drugs. 01/2010; 19(1):117-31.

  Importance of the field: For many cancers, there has been a shift from management with traditional, nonspecific cytotoxic chemotherapies to treatment with molecule-specific targeted therapies thatImportance of the field: For many cancers, there has been a shift from management with traditional, nonspecific cytotoxic chemotherapies to treatment with molecule-specific targeted therapies that are used either alone or in combination with traditional chemotherapy and radiation therapy. Accumulating data suggest that multi-targeted agents may produce greater benefits than those observed with single-targeted therapies, may have acceptable tolerability profiles, and may be active against a broader range of tumour types. Thus, regulation of cyclic nucleotide signalling is properly regarded as a composite of multiple component pathways involved in diverse aspects of tumour cell function. The impairment of cAMP and/or cGMP generation by overexpression of PDE isoforms that has been described in various cancer pathologies, and the effects of PDE inhibitors in tumour models in vitro and in vivo, may offer promising insight into future cancer treatments because of the numerous advantages of PDE inhibitors. Areas covered in this review: In this review, we focus on the expression and regulation of cyclic nucleotide phosphodiesterases (PDEs) in tumour progression and provide evidence that PDE inhibitors may be effective agents for treating cancer; the review covers literature from the past several years. What the reader will gain: PDEs have been studied in a variety of tumours; data have suggested that the levels of PDE activity are elevated and, therefore, the ratio of cGMP to cAMP is affected. In addition, PDE inhibitors may be potential targets for tumour cell growth inhibition and induction of apoptosis. This review explores the prospects of targeting PDEs with therapeutic agents for cancer, as well as the shortcomings of this approach such as dose-limiting side effects, toxicity/efficacy ratio and selectivity towards tumour tissue. In addition, it includes opinions and suggestion for developing PDE inhibition for cancer treatment from initial concept to potential therapeutic application and final relevance in clinical use. Take home message: Impaired cAMP and/or cGMP generation upon overexpression of PDE isoforms has been described in various cancer pathologies. Inhibition of selective PDE isoforms, which raises the levels of intracellular cAMP and/or cGMP, induces apoptosis and cell cycle arrest in a broad spectrum of tumour cells and regulates the tumour microenvironment. Therefore, the development and clinical application of inhibitors specific for individual PDE isoenzymes may selectively restore normal intracellular signalling, providing antitumour therapy with reduced adverse effects.

• Role of Epidermal Growth Factor Inhibition in Experimental Pulmonary Hypertension.

  Authors: Bhola Kumar Dahal, Teodora Cornitescu, Aleksandra Tretyn, Soni Savai Pullamsetti, Djuro Kosanovic, Rio Dumitrascu, Hossein Ardeschir Ghofrani, Norbert Weissmann, Robert Voswinckel, Gamal-Andre Banat, Werner Seeger, Friedrich Grimminger, Ralph Theo Schermuly

  American journal of respiratory and critical care medicine. 10/2009;

  RATIONALE: Epidermal growth factor (EGF) and its receptors play a role in cell proliferation and survival and are implicated in the pathobiology of pulmonary arterial hypertension (PAH). ObjectiveRATIONALE: Epidermal growth factor (EGF) and its receptors play a role in cell proliferation and survival and are implicated in the pathobiology of pulmonary arterial hypertension (PAH). Objective and methods: We investigated a) the effects of three clinically approved EGF receptor (EGFR) antagonists in vitro on rat pulmonary arterial smooth muscle cell (PASMC) proliferation and in vivo on experimental pulmonary hypertension (PH) induced by monocrotaline injection in rats and by chronic hypoxia in mice, and b) the expression of EGFR in the lung tissues from experimental and clinical PH. MEASUREMENTS AND MAIN RESULTS: The EGFR inhibitors, gefitinib, erlotinib and lapatinib inhibited the EGF-induced proliferation of PASMCs. In rats with established PH, gefitinib and erlotinib significantly reduced right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH). In addition, the medial wall thickness and muscularization of pulmonary arteries were improved. In contrast, lapatinib did not provide therapeutic benefit. These EGFR antagonists at their highest tolerable dose did not yield significant improvement in RVSP, RVH and pulmonary vascular remodeling in mice with chronic hypoxic PH. Moreover, no significant alteration in the EGFR expression was detected in the lung tissues from patients with idiopathic PAH (IPAH). CONCLUSION: The partial therapeutic efficacy of the EGFR antagonists in animal models of pulmonary hypertension and the absence of significant alteration in EGFR expression in the lungs from IPAH patients suggest that the EGFR do not represent a promising target for the treatment of pulmonary hypertension.

• Expression and Activity of Phosphodiesterase Isoforms during Epithelial Mesenchymal Transition - The Role of PDE4.

  Authors: Ewa Kolosionek, Rajkumar Savai, Hossein Ardeschir Ghofrani, Norbert Weissmann, Andreas Guenther, Friedrich Grimminger, Werner Seeger, Gamal-Andre Banat, Ralph Theo Schermuly, Soni Savai Pullamsetti

  Molecular biology of the cell. 09/2009;

  Monitoring Editor: Keith E. Mostov Epithelial-to-mesenchymal transition (EMT) has emerged as a critical event in the pathogenesis of organ fibrosis and cancer, and is typically induced by theMonitoring Editor: Keith E. Mostov Epithelial-to-mesenchymal transition (EMT) has emerged as a critical event in the pathogenesis of organ fibrosis and cancer, and is typically induced by the multifunctional cytokine transforming growth factor-beta1 (TGF-beta1). The present study was undertaken to evaluate the potential role of phosphodiesterases (PDEs) in TGF-beta1 induced EMT in the human alveolar epithelial type-II cell line-A549. Stimulation of A549 with TGF-beta1 induced EMT by morphological alterations and by expression changes of the epithelial phenotype markers, E-cadherin, cytokeratin-18, zona occludens-1, and the mesenchymal phenotype markers, collagen I, fibronectin, alpha-smooth muscle actin. Interestingly, TGF-beta1 stimulation caused twofold increase in total cAMP-PDE activity, contributed mostly by PDE4. Further, mRNA and protein expression demonstrated upregulation of PDE4A and PDE4D isoforms in TGF-beta1 stimulated cells. Most importantly, treatment of TGF-beta1 stimulated epithelial cells with the PDE4 selective inhibitor Rolipram or PDE4 siRNA potently inhibited EMT changes in a Smad-independent manner by decreasing reactive oxygen species (ROS), p38 and ERK phosphorylation. In contrast, the ectopic overexpression of PDE4A and/or PDE4D resulted in a significant loss of epithelial marker E-cadherin, but did not result in changes of mesenchymal markers. Additionally, Rho kinase signaling activated by TGF-beta1 during EMT demonstrated to be a positive regulator of PDE4. Collectively, the findings presented herein suggest that TGF-beta1 mediated up-regulation of PDE4 promotes EMT in alveolar epithelial cells. Thus, targeting PDE4 isoforms may be a novel approach to attenuating EMT-associated lung diseases such as pulmonary fibrosis and lung cancer.

• The soluble guanylate cyclase activator HMR1766 reverses hypoxia-induced experimental pulmonary hypertension in mice.

  Authors: Norbert Weissmann, Sascha Hackemack, Bhola Kumar Dahal, Soni Savai Pullamsetti, Rajkumar Savai, Manish Mittal, Beate Fuchs, Thomas Medebach, Rio Dumitrascu, Martin van Eickels, Hossein Ardeschir Ghofrani, Werner Seeger, Friedrich Grimminger, Ralph Theo Schermuly

  American journal of physiology. Lung cellular and molecular physiology. 08/2009;

  Severe pulmonary hypertension (PH) is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. In mice with PH induced by chronic hypoxia,Severe pulmonary hypertension (PH) is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. In mice with PH induced by chronic hypoxia, we examined the acute and chronic effects of the soluble guanylate cyclase (sGC) activator HMR1766 on hemodynamics and pulmonary vascular remodeling. In isolated perfused mouse lungs from control animals, HMR1766 dose-dependently inhibited the pressor response of acute hypoxia. This dose response curve was shifted leftward when investigating the effects of HMR1766 in isolated lungs from chronic hypoxic animals for 21 days at 10% oxygen. Mice exposed for 21 or 35 days to chronic hypoxia developed pulmonary hypertension, right heart hypertrophy and pulmonary vascular remodeling. Treatment with HMR1766 (10mg/kg/day), after full establishment of pulmonary hypertension from day 21 to day 35, significantly reduced pulmonary hypertension, as measured continuously by telemetry. In addition, right ventricular (RV) hypertrophy and structural remodeling of the lung vasculature were reduced. Pharmacological activation of oxidized sGC partially reverses hemodynamic and structural changes in chronic hypoxia-induced experimental PH. Key words: Pulmonary hypertension, soluble guanylate cyclase (sGC), Hypoxia, Vascular remodeling.

• Heme Oxygenase-2 and Large-conductance Ca2+ activated K+ Channels: Lung Vascular Effects of Hypoxia.

  Authors: Markus Roth, Markus Rupp, Simone Hofmann, Manish Mittal, Beate Fuchs, Natascha Sommer, Nirmal Parajuli, Karin Quanz, Dominic Schubert, Eva Dony, Ralph Theo Schermuly, Hossein Ardeschir Ghofrani, Ulrike Sausbier, Katrin Rutschmann, Sarah Wilhelm, Werner Seeger, Peter Ruth, Friedrich Grimminger, Matthias Sausbier, Norbert Weissmann

  American journal of respiratory and critical care medicine. 07/2009;

  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,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. Recently, a complex of heme oxygenase-2 (HO-2) and the BK channel has been suggested as a universal oxygen sensor system. OBJECTIVE: We investigated, whether this complex serves as an oxygen sensor for the vascular effects of alveolar hypoxia in the lung. METHODS and 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 co-immunoprecipitation 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 between the wild-type, HO-2- and BKalpha-deficient mice. Exposure of mice to three weeks of chronic hypoxia resulted in a slight downregulation of HO-2 and no alteration in BKalpha expression. The degree of pulmonary hypertension which developed, quantified from right ventricular pressure, right heart hypertrophy, and the degree of muscularization of precapillary pulmonary arteries, was not different between the wild-type, HO-2- or BKalpha-deficient mice. CONCLUSION: It is demonstrated that neither deletion of HO-2 nor BK channels affects the acute, sustained and chronic vascular responses to alveolar hypoxia in the lung.

• Novel soluble guanylyl cyclase stimulator BAY 41-2272 attenuates ischemia/reperfusion induced lung injury.

  Authors: Bakytbek Egemnazarov, Akylbek Sydykov, Ralph Theo Schermuly, Norbert Weissmann, Johannes-Peter Stasch, Akpai S Sarybaev, Werner Seeger, Friedrich Grimminger, Hossein Ardeschir Ghofrani

  American journal of physiology. Lung cellular and molecular physiology. 01/2009;

  The protective effects of nitric oxide (NO), a physiological activator of soluble guanylyl cyclase (sGC), have been reported in ischemia/reperfusion (I/R) syndrome of the lung. Therefore, we studiedThe protective effects of nitric oxide (NO), a physiological activator of soluble guanylyl cyclase (sGC), have been reported in ischemia/reperfusion (I/R) syndrome of the lung. Therefore, we studied the effects of BAY 41-2272, a novel sGC stimulator, on I/R injury of the lung in an isolated intact organ model. Lung injury was assessed by measuring weight gain and microvascular permeability (capillary filtration coefficient, Kfc). Release of reactive oxygen species (ROS) into the perfusate was measured during early reperfusion by ESR spectroscopy. Rabbit lungs were treated with BAY 41-2272, L-NMMA 400, or NO to evaluate the effects on I/R induced lung injury. In untreated lungs a dramatic rise in Kfc values and weight gain during reperfusion were observed, these results were associated with increased ROS production. Both, BAY 41-2272 and L-NMMA significantly attenuated vascular leakage and suppressed ROS release. Additional experiments showed that BAY 41-2272 diminished phorbol-12-myristrate-13-acetate induced ROS production by NADPH-oxidase. A pharmacological inhibition of the enzyme with consequent reduction in ROS levels decreased I/R injury. Nitric oxide had only marginal effect on I/R injury. Thus, BAY 41-2272 protects against I/R induced lung injury by interfering with the activation of NADPH-oxidases. Key words: acute lung injury, nitric oxide, oxidative stress, ROS.

• Evaluation of angiogenesis using micro-computed tomography in a xenograft mouse model of lung cancer.

  Authors: Rajkumar Savai, Alexander Claus Langheinrich, Ralph Theo Schermuly, Soni Savai Pullamsetti, Rio Dumitrascu, Horst Traupe, Wigbert Stephan Rau, Werner Seeger, Friedrich Grimminger, Gamal-Andre Banat

  Neoplasia (New York, N.Y.). 01/2009; 11(1):48-56.

  Quantitative evaluation of lung tumor angiogenesis using immunohistochemical techniques has been limited by difficulties in generating reproducible data. To analyze intrapulmonary tumor angiogenesis,Quantitative evaluation of lung tumor angiogenesis using immunohistochemical techniques has been limited by difficulties in generating reproducible data. To analyze intrapulmonary tumor angiogenesis, we used high-resolution micro-computed tomography (micro-CT) of lung tumors of mice inoculated with mouse Lewis lung carcinoma (LLC1) or human adenocarcinoma (A549) cell lines. The lung vasculature was filled with the radiopaque silicone rubber, Microfil, through the jugular vein (in vivo application) or pulmonary artery (ex vivo application). In addition, human adenocarcinoma lung tumor-bearing mice treated site-specifically with humanized monoclonal antibody (bevacizumab) against vascular endothelial growth factor. Quantitative analysis of lung tumor microvessels imaged with micro-CT showed that more vessels (mainly small, <0.02 mm(2)) were filled using the in vivo (5.4%) compared with the ex vivo (2.1%) method. Furthermore, bevacizumab-treated lung tumor-bearing mice showed significantly reduced lung tumor volume and lung tumor angiogenesis compared with untreated mice as assessed by micro-CT. Interestingly, microvascularization of mainly the smaller vessels (<0.02 mm(2)) was reduced after bevacizumab treatment. This observation with micro-CT was nicely correlated with immunohistochemical measurement of microvessels. Therefore, micro-CT is a novel method for investigating lung tumor angiogenesis, and this might be considered as an additional complementary tool for precise quantification of angiogenesis.

• The Non-canonical WNT-pathway Is Operative in Idiopathic Pulmonary Arterial Hypertension.

  Authors: Isabel Pia Laumanns, Ludger Fink, Jochen Wilhelm, Jens C Wolff, Rita Mitnacht-Kraus, Sabine Graef-Hoechst, Maria M Stein, Rainer M Bohle, Walter Klepetko, Mir Ali Reza Hoda, Ralph Theo Schermuly, Friedrich Grimminger, Werner Seeger, Robert Voswinckel

  American journal of respiratory cell and molecular biology. 12/2008;

  Idiopathic pulmonary arterial hypertension (IPAH) is a fatal disease, which is comprised of sustained vasoconstriction, enhanced proliferation of pulmonary vascular cells and in situ thrombosis. TheIdiopathic pulmonary arterial hypertension (IPAH) is a fatal disease, which is comprised of sustained vasoconstriction, enhanced proliferation of pulmonary vascular cells and in situ thrombosis. The discovery of several contributing signaling pathways in recent years has resulted in an expanding array of novel therapies; however IPAH remains a progressive disease with poor outcome in most instances. To identify new regulatory pathways of vascular remodeling in IPAH, we performed transcriptome wide expression profiling in laser-microdissected pulmonary arterial resistance vessels derived from explanted IPAH and healthy human lungs. Statistical analysis of the data derived from six individuals in each group showed significant regulation of several mediators of the canonical and non-canonical wnt-pathway. As to the non-canonical wnt-pathway, the planar cell polarity (PCP) pathway, the ras homolog gene family member A (RHOA) and ras-related C3 botulinum toxin substrate-1 (RAC1) were strongly upregulated. Real-time PCR of laser-microdissected pulmonary arteries confirmed these array results and showed in addition significant upregulation of further PCP mediators wingless member 11 (WNT11), disheveled associated activator of morphogenesis-1 (DAAM1), disheveled (DSV), and rho-kinase (ROCK). Immunohistochemical staining and semiquantitative expression analysis confirmed the markedly enhanced expression of the PCP mediators in the pulmonary resistance vessels, in particular in the endothelial layer in IPAH. Therefore we propose the PCP pathway to be critically involved in the regulation of vascular remodeling in IPAH.

• Acute effects of the combination of sildenafil and inhaled treprostinil on haemodynamics and gas exchange in pulmonary hypertension.

  Authors: Robert Voswinckel, Frank Reichenberger, Beate Enke, Andre Kreckel, Stefanie Krick, Henning Gall, Ralph Theo Schermuly, Friedrich Grimminger, Lewis J Rubin, Horst Olschewski, Werner Seeger, Hossein A Ghofrani

  Pulmonary pharmacology & therapeutics. 10/2008; 21(5):824-32.

  Inhaled treprostinil was recently developed for the treatment of pulmonary arterial hypertension (PAH). We investigated the safety and acute haemodynamic effects of the combination oral sildenafilInhaled treprostinil was recently developed for the treatment of pulmonary arterial hypertension (PAH). We investigated the safety and acute haemodynamic effects of the combination oral sildenafil and inhaled treprostinil in an open label study in patients with precapillary pulmonary hypertension. Inhaled nitric oxide (20ppm; n=50), sildenafil (50mg; n=50) and inhaled treprostinil (15microg; n=25 or 30microg; n=25) were applied in subsequent order during right heart catheter investigation to consecutive patients with pulmonary arterial hypertension (PAH; n=28), non-operable chronic thromboembolic pulmonary hypertension (CTEPH; n=17) and pulmonary fibrosis associated pulmonary hypertension (n=5). Inhaled nitric oxide reduced pulmonary vascular resistance (PVR) to 87.3+/-5.1% of baseline values, reduced mean pulmonary arterial pressure (PAP) to 89.7+/-3.5% and increased cardiac output (CO) to 102.4+/-2.9%. Sildenafil reduced PVR to 80.1+/-5.0%, mPAP to 86.5+/-2.9% and increased CO to 103.8+/-3.2%. Treprostinil, inhaled 1h after sildenafil, reduced PVR to 66.3+/-3.8%, mPAP to 77.8+/-3.3%, and increased CO to 107.1+/-3.3% (mean+/-95% confidence interval). Subgroup analysis showed similar acute haemodynamic effects in PAH and CTEPH patients. Ventilation/perfusion distribution measurement in six patients with pre-existing gas exchange limitations was not changed by sildenafil and treprostinil. Relevant side effects were not observed. The combination of sildenafil and inhaled treprostinil was well tolerated and induced additive, pulmonary selective vasodilatation in pulmonary hypertension patients. This could be of relevance also for long-term treatment of PAH and CTEPH patients.

• Role of the prostanoid EP4 receptor in iloprost-mediated vasodilatation in pulmonary hypertension.

  Authors: Ying-Ju Lai, Soni Savai Pullamsetti, Eva Dony, Norbert Weissmann, Ghazwan Butrous, Gamal-Andre Banat, Hossein Ardeschir Ghofrani, Werner Seeger, Friedrich Grimminger, Ralph Theo Schermuly

  American journal of respiratory and critical care medicine. 08/2008; 178(2):188-96.

  RATIONALE: Iloprost is effective for the treatment of pulmonary hypertension. It acts through elevation of cAMP by binding to the prostacyclin receptor (IP receptor). However, there is evidence thatRATIONALE: Iloprost is effective for the treatment of pulmonary hypertension. It acts through elevation of cAMP by binding to the prostacyclin receptor (IP receptor). However, there is evidence that patients with severe pulmonary hypertension have decreased expression of the IP receptor in the remodeled pulmonary arterial smooth muscle. OBJECTIVES: We hypothesized that prostanoid receptors other than the IP receptor are involved in signal transduction by iloprost. METHODS: Immunoblotting was used to detect the IP and prostanoid EP4 receptor in lung tissue from patients with idiopathic pulmonary arterial hypertension, and immunohistochemistry was used to detect these receptors in lung sections from rats treated with monocrotaline (MCT28d). Protein and mRNA were isolated from pulmonary arterial smooth muscle cells (PASMCs) from control and MCT28d rats treated with AH6809 (an EP2 receptor antagonist) and AH23848 (an EP4 receptor antagonist) in combination with iloprost. Intracellular cAMP was also assessed in these tissues. Measurements and Main Results: IP receptor expression was reduced in idiopathic pulmonary arterial hypertension patient lung samples and MCT28d rat lungs compared with the controls. Reverse transcriptase-polymerase chain reaction and immunoblotting of MCT28d rat PASMC extracts revealed scant expression of the IP receptor but stable expression of EP4 receptor, compared with controls. Iloprost-induced elevation in intracellular cAMP in PASMCs was dose-dependently reduced by AH23848, but not by AH6809. CONCLUSIONS: Iloprost mediates vasodilatory functions via the EP4 receptor in the case of low IP receptor expression associated with pulmonary arterial hypertension. This is a previously unrecognized mechanism for iloprost, and illustrates that the EP4 receptor may be a novel therapeutic approach for the treatment of pulmonary arterial hypertension.