Werner Seeger

Publications of Werner Seeger

• Matrix metalloproteinases and their inhibitors in pulmonary hypertension.

  Authors: Prakash Chelladurai, Werner Seeger, Soni Savai Pullamsetti

  The European respiratory journal : official journal of the European Society for Clinical Respiratory Physiology. 04/2012;

  Pulmonary hypertension (PH) is a severe and progressive disease, characterized by high pulmonary artery pressure, usually culminating in right heart failure. Current therapeutic approaches in PHPulmonary hypertension (PH) is a severe and progressive disease, characterized by high pulmonary artery pressure, usually culminating in right heart failure. Current therapeutic approaches in PH largely provide symptomatic relief while the prognosis rate is lower due to the lack of specific molecular targets and involvement of multitude of factors in the development of PH. Numerous studies have suggested crucial role of matrix metalloproteinase (MMP) axis during development and disease states specifically with regard to extracellular matrix (ECM) remodeling and vascular homeostasis. Increased MMP activity has been demonstrated in experimental animal models of PH, and MMP inhibition has been shown to either attenuate or enhance vascular remodeling. Moreover, several studies emphasize that restoration of deregulated MMPs to physiological MMP/TIMP (Tissue Inhibitor of MMPs) ratios would potentiate reverse remodeling process in PH. This review will highlight the pathophysiological role of MMPs in vascular remodeling and establishment of PH. In particular, this review will focus on the MMP expression and regulation in pulmonary vasculature and pulmonary vascular remodeling. Later, this review provides an overview of recent clinical and experimental findings and their impact on achieving maximal reversal of PH, current issues and future perspectives.

• 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.

• 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.

• Nebulization performance of biodegradable sildenafil-loaded nanoparticles using the Aeroneb Pro: formulation aspects and nanoparticle stability to nebulization.

  Authors: Moritz Beck-Broichsitter, Pia Kleimann, Tobias Gessler, Werner Seeger, Thomas Kissel, Thomas Schmehl

  International journal of pharmaceutics. 01/2012; 422(1-2):398-408.

  Polymeric nanoparticles meet the increasing interest for drug delivery applications and hold great promise to improve controlled drug delivery to the lung. Here, we present a series of investigationsPolymeric nanoparticles meet the increasing interest for drug delivery applications and hold great promise to improve controlled drug delivery to the lung. Here, we present a series of investigations that were carried out to understand the impact of formulation variables on the nebulization performance of novel biodegradable sildenafil-loaded nanoparticles designed for targeted aerosol therapy of life-threatening pulmonary arterial hypertension. Narrowly distributed poly(D,L-lactide-co-glycolide) nanoparticles (size: ∼200 nm) were prepared by a solvent evaporation technique using poly(vinyl alcohol) (PVA) as stabilizer. The aerodynamic and output characteristics using the Aeroneb Pro nebulizer correlated well with the dynamic viscosity of the employed fluids for nebulization. The nebulization performance was mainly affected by the amount of employed stabilizer, rather than by the applied nanoparticle concentration. Nanoparticles revealed physical stability against forces generated during aerosolization, what is attributed to the adsorbed PVA layer around the nanoparticles. Sildenafil was successfully encapsulated into nanoparticles (encapsulation efficiency: ∼80%). Size, size distribution and sildenafil content of nanoparticles were not affected by nebulization and the in vitro drug release profile demonstrated a sustained sildenafil release over ∼120 min. The current study suggests that the prepared sildenafil-loaded nanoparticles are a promising pharmaceutical for the therapy of pulmonary arterial hypertension.

• 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.

• Long-term effects of inhaled treprostinil in patients with pulmonary arterial hypertension: the Treprostinil Sodium Inhalation Used in the Management of Pulmonary Arterial Hypertension (TRIUMPH) study open-label extension.

  Authors: Raymond L Benza, Werner Seeger, Vallerie V McLaughlin, Richard N Channick, Robert Voswinckel, Victor F Tapson, Ivan M Robbins, Horst Olschewski, Lewis J Rubin

  The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation. 12/2011; 30(12):1327-33.

  Inhaled treprostinil improved functional capacity as add-on therapy in the short-term management of patients with pulmonary arterial hypertension (PAH). This study investigated the long-term effectsInhaled treprostinil improved functional capacity as add-on therapy in the short-term management of patients with pulmonary arterial hypertension (PAH). This study investigated the long-term effects of inhaled treprostinil in patients concurrently receiving oral background therapy. A total of 206 patients (81% women) completing the 12-week double-blind phase of the Treprostinil Sodium Inhalation Used in the Management of Pulmonary Arterial Hypertension (TRIUMPH) study transitioned into an open-label extension. Patients were assessed every 3 months for changes in 6-minute walk distance (6MWD), Borg dyspnea score, New York Heart Association (NYHA) functional class, quality of life (QOL) scores, and signs and symptoms of PAH. Patients were primarily NYHA class III (86%), with a mean baseline 6MWD of 349 ± 81 meters. A median change in 6MWD of 28, 31, 32, and 18 meters in patients continuing therapy was observed at 6, 12, 18, and 24 months, respectively. This effect was more prominent in those patients originally allocated to active therapy in the double-blind phase. Survival rates for patients remaining on therapy were 97%, 94%, and 91% at 12, 18, and 24 months, respectively. In addition, 82%, 74%, and 69% of patients maintained treatment benefit as evidenced by lack of clinical worsening at 12, 18, and 24 months. The most common adverse events were known effects of prostanoid therapy (headache [34%], nausea [21%], and vomiting [10%]) or were due to the route of administration (cough [53%], pharyngolaryngeal pain [13%], and chest pain [13%]). Long-term therapy with inhaled treprostinil demonstrated persistent benefit for PAH patients who remained on therapy for up to 24 months.

• Dynamic hyperinflation during exercise in patients with precapillary pulmonary hypertension.

  Authors: Manuel J Richter, Robert Voswinckel, Henning Tiede, Richard Schulz, Christian Tanislav, Andreas Feustel, Rory E Morty, Hossein A Ghofrani, Werner Seeger, Frank Reichenberger

  Respiratory medicine. 11/2011; 106(2):308-13.

  Patients with pulmonary arterial hypertension often present with a mild obstructive lung pattern, however, the functional consequences are not known. We analysed flow volume loops during exercise inPatients with pulmonary arterial hypertension often present with a mild obstructive lung pattern, however, the functional consequences are not known. We analysed flow volume loops during exercise in 61 patients with precapillary pulmonary hypertension (PH) (age 55 ± 14 years) in comparison with 21 patients with COPD (60 ± 12 years), 39 patients with pulmonary fibrosis (58 ± 11 years) and 38 healthy controls (HC) (39 ± 15 years). Inspiratory capacity (IC) was measured at rest, and during maximum exercise (max). HC exhibited a stable IC of 3.0 ± 0.9 l at rest, and at max. A reduction in IC of 2.6 ± 0.8 l at rest to 2.0 ± 0.7 l at max was observed in patients with COPD. Patients with PH exhibited a significant reduction in IC from 2.3 ± 0.6 l at rest to 2.1 ± 0.6 l at max, while patients with pulmonary fibrosis exhibited a stable IC of 1.8 ± 0.6 at rest and 1.7 ± 0.6 l at max. In patients with PH, a weak negative correlation was drawn between the change in IC (%) and peak VO2 (r = -0.29, p = 0.01), as well as with PVR (r = -0.27, p = 0.02). Patients with PH demonstrate a characteristic change in IC during exercise, which might contribute to impaired exercise tolerance.

• Characterization of novel spray-dried polymeric particles for controlled pulmonary drug delivery.

  Authors: Moritz Beck-Broichsitter, Christoph Schweiger, Thomas Schmehl, Tobias Gessler, Werner Seeger, Thomas Kissel

  Journal of controlled release : official journal of the Controlled Release Society. 10/2011; 158(2):329-35.

  Numerous studies have addressed the controlled pulmonary drug delivery properties of colloidal particles. However, only scant information on the potential of spray-drying for submicron particleNumerous studies have addressed the controlled pulmonary drug delivery properties of colloidal particles. However, only scant information on the potential of spray-drying for submicron particle preparation is available. By exploiting the advantages of spray-drying, the characteristics of submicron particles can be optimized to meet the requirements necessary for lung application. Submicron particles were prepared from organic poly(d,l-lactide-co-glycolide) (PLGA) solutions, and composite particles were spray-dried from aqueous PLGA nanosuspensions. The feed concentration, as well as the spray-mesh diameter influenced the resulting particle sizes. Nanoparticles were virtually unaffected after spray-drying. The aerodynamic characteristics of both particle species revealed aerosol particle sizes suitable for deposition in the deep lungs (≤4μm). While the entrapped drug was released within ~90min from the composite particles, extensive drug retardation (~480min) was observed for PLGA particles spray-dried from organic solution. These results suggest that nanospray-drying is a convenient method to prepare submicron, controlled drug delivery vehicles useful for pulmonary application potentially allowing access to alveolar tissue.

• 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.

• Development of a biodegradable nanoparticle platform for sildenafil: Formulation optimization by factorial design analysis combined with application of charge-modified branched polyesters.

  Authors: Moritz Beck-Broichsitter, Thomas Schmehl, Tobias Gessler, Werner Seeger, Thomas Kissel

  Journal of controlled release : official journal of the Controlled Release Society. 09/2011; 157(3):469-77.

  Biodegradable nanoparticles have gained tremendous attraction as carriers for controlled drug delivery to the lung. Despite numerous advances in the field, e.g. development of suitable methods forBiodegradable nanoparticles have gained tremendous attraction as carriers for controlled drug delivery to the lung. Despite numerous advances in the field, e.g. development of suitable methods for pulmonary administration of polymeric nanoparticles, a sufficient association of the therapeutic agent with the carrier system as well as drug release in a controlled fashion remain considerable challenges. Hence, this study examines the optimization of biodegradable sildenafil-loaded nanoparticle formulations intended for aerosol treatment of pulmonary hypertension. A factorial design analysis was employed to identify the important experimental factors involved in the preparation of nanoparticles by the solvent evaporation technique. The effect of tailored charge-modified branched polyesters on drug loading and in vitro drug release from nanoparticles was also evaluated. Moreover, colloidal stability of obtained nanoparticles was assessed, and stabilization of nanoparticles by lyophilization was accomplished without additional excipients. Essential experimental factors were identified and optimized to allow the preparation of nanoparticles composed of linear polyesters with a sildenafil content of ~5wt.%. The in vitro drug release profile from these nanoparticles demonstrated a sustained release of sildenafil over ~90min. Application of charge-modified branched polyesters enhanced the drug content in nanoparticles and drug release profile, according to the charge-density present in the employed polymer. Accordingly an increase in drug loading by a factor of ~1.4, a prolonged drug release profile from nanoparticles over ~240min was achieved. Sildenafil release from nanoparticles made of linear and charge-modified branched polyesters was governed by a diffusion process. The obtained drug diffusion coefficients were decreased as the charge-density present in the applied polymer was increased, which promotes the strategy to improve drug loading and release rates by electrostatic interactions between polymer and drug. In addition, nanoparticles showed high colloidal stability in different media of importance for pulmonary application and were successfully stabilized by lyophilization. In conclusion, optimization of the nanoparticle preparation process together with the application of tailored polymeric materials facilitated the synthesis of promising drug carriers for sildenafil that permit a novel treatment modality for severe pulmonary hypertension.

• Hemodynamic and clinical onset in patients with hereditary pulmonary arterial hypertension and BMPR2 mutations.

  Authors: Nicole Pfarr, Justyna Szamalek-Hoegel, Christine Fischer, Katrin Hinderhofer, Christian Nagel, Nicola Ehlken, Henning Tiede, Horst Olschewski, Frank Reichenberger, Ardeschir H A Ghofrani, Werner Seeger, Ekkehard Grünig

  Respiratory research. 07/2011; 12:99.

  Mutations in the bone morphogenetic protein receptor 2 (BMPR2) gene can lead to idiopathic pulmonary arterial hypertension (IPAH). This study prospectively screened for BMPR2 mutations in a largeMutations in the bone morphogenetic protein receptor 2 (BMPR2) gene can lead to idiopathic pulmonary arterial hypertension (IPAH). This study prospectively screened for BMPR2 mutations in a large cohort of PAH-patients and compared clinical features between BMPR2 mutation carriers and non-carriers. Patients have been assessed by right heart catheterization and genetic testing. In all patients a detailed family history and pedigree analysis have been obtained. We compared age at diagnosis and hemodynamic parameters between carriers and non-carriers of BMPR2 mutations. In non-carriers with familial aggregation of PAH further genes/gene regions as the BMPR2 promoter region, the ACVRL1, Endoglin, and SMAD8 genes have been analysed. Of the 231 index patients 22 revealed a confirmed familial aggregation of the disease (HPAH), 209 patients had sporadic IPAH. In 49 patients (86.3% of patients with familial aggregation and 14.3% of sporadic IPAH) mutations of the BMPR2 gene have been identified. Twelve BMPR2 mutations and 3 unclassified sequence variants have not yet been described before. Mutation carriers were significantly younger at diagnosis than non-carriers (38.53 ± 12.38 vs. 45.78 ± 11.32 years, p < 0.001) and had a more severe hemodynamic compromise. No gene defects have been detected in 3 patients with HPAH. This study identified in a large prospectively assessed cohort of PAH- patients new BMPR2 mutations, which have not been described before and confirmed previous findings that mutation carriers are younger at diagnosis with a more severe hemodynamic compromise. Thus, screening for BMPR2 mutations may be clinically useful.

• 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.

• Comparison of the effects of carbon ion and photon irradiation on the angiogenic response in human lung adenocarcinoma cells.

  Authors: Florentine Kamlah, Jörg Hänze, Andrea Arenz, Ulrike Seay, Diya Hasan, Janko Juricko, Birgit Bischoff, Oana R Gottschald, Claudia Fournier, Gisela Taucher-Scholz, Michael Scholz, Werner Seeger, Rita Engenhart-Cabillic, Frank Rose

  International journal of radiation oncology, biology, physics. 05/2011; 80(5):1541-9.

  Radiotherapy resistance is a commonly encountered problem in cancer treatment. In this regard, stabilization of endothelial cells and release of angiogenic factors by cancer cells contribute to thisRadiotherapy resistance is a commonly encountered problem in cancer treatment. In this regard, stabilization of endothelial cells and release of angiogenic factors by cancer cells contribute to this problem. In this study, we used human lung adenocarcinoma (A549) cells to compare the effects of carbon ion and X-ray irradiation on the cells' angiogenic response. A549 cells were irradiated with biologically equivalent doses for cell survival of either carbon ions (linear energy transfer, 170 keV/μm; energy of 9.8 MeV/u on target) or X-rays and injected with basement membrane matrix into BALB/c nu/nu mice to generate a plug, allowing quantification of angiogenesis by blood vessel enumeration. The expression of angiogenic factors (VEGF, PlGF, SDF-1, and SCF) was assessed at the mRNA and secreted protein levels by using real-time reverse transcription-PCR and enzyme-linked immunosorbent assay. Signal transduction mediated by stem cell factor (SCF) was assessed by phosphorylation of its receptor c-Kit. For inhibition of SCF/c-Kit signaling, a specific SCF/c-Kit inhibitor (ISCK03) was used. Irradiation of A549 cells with X-rays (6 Gy) but not carbon ions (2 Gy) resulted in a significant increase in blood vessel density (control, 20.71 ± 1.55; X-ray, 36.44 ± 3.44; carbon ion, 16.33 ± 1.03; number per microscopic field). Concordantly, irradiation with X-rays but not with carbon ions increased the expression of SCF and subsequently caused phosphorylation of c-Kit in endothelial cells. ISCK03 treatment of A549 cells irradiated with X-rays (6 Gy) resulted in a significant decrease in blood vessel density (X-ray, 36.44 ± 3.44; X-ray and ISCK03, 4.33 ± 0.71; number of microscopic field). These data indicate that irradiation of A549 cells with X-rays but not with carbon ions promotes angiogenesis. The present study provides evidence that SCF is an X-ray-induced mediator of angiogenesis in A549 cells, a phenomenon that could not be observed with carbon ion irradiation. Thus, in this model system evaluating angiogenesis, carbon ion irradiation may have a therapeutic advantage. This observation should be confirmed in orthotopic lung tumor models.

• Air travel can be safe and well tolerated in patients with clinically stable pulmonary hypertension.

  Authors: Melanie Thamm, Robert Voswinckel, Henning Tiede, Friederike Lendeckel, Friedrich Grimminger, Werner Seeger, Hossein A Ghofrani

  Pulmonary circulation. 04/2011; 1(2):239-43.

  Our aim was to determine what proportion of patients with pulmonary hypertension (PH) has undertaken air travel contrary to the general medical advice and to characterize these patients according toOur aim was to determine what proportion of patients with pulmonary hypertension (PH) has undertaken air travel contrary to the general medical advice and to characterize these patients according to disease severity and medical treatment. In cooperation with Pulmonale Hypertonie e.V., the German patient organization, a questionnaire was distributed. In total, 430 of 720 questionnaires were returned completed. Of the 179 patients who travelled at least once by air, the distribution of New York Heart Association functional classes I/ II/ III/ IV was 2/ 77/ 74/ 8, respectively; 83 patients were receiving monotherapy; 58 patients were receiving a combination of two or more therapies; 57 patients were on long-term ambulatory oxygen treatment; and 29 patients used supplemental oxygen while travelling. Overall, 20 adverse events were reported, mostly of mild to moderate severity (i.e., peripheral edema, dyspnea), with need of medical intervention in only 7 cases. The 251 patients who did not travel by air were, on average, in more advanced stages of disease and/or clinically unstable. In conclusion, a majority of patients (159 out of 179) did not experience any complications during or directly after the flight even though no special precautions were taken. Thus we conclude that for patients with PH in a stable clinical condition, air travel can be safe and well tolerated.

• 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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• Shedding of low-density lipoprotein receptor–related protein-1 in acute respiratory distress syndrome.

  Authors: Malgorzata Wygrecka, Jochen Wilhelm, Ewa Jablonska, Dariusz Zakrzewicz, Klaus T Preissner, Werner Seeger, Andreas Guenther, Philipp Markart

  American journal of respiratory and critical care medicine. 03/2011; 184(4):438-48.

  Low-density lipoprotein receptor–related protein-1 (LRP-1) mediates the endocytic clearance of various proteinases, including matrix metalloproteinases (MMPs). The ectodomain of LRP-1 can be shedLow-density lipoprotein receptor–related protein-1 (LRP-1) mediates the endocytic clearance of various proteinases, including matrix metalloproteinases (MMPs). The ectodomain of LRP-1 can be shed from the cell surface, releasing a soluble form of this receptor (sLRP-1), which antagonizes ligand endocytosis by cellular LRP-1. To assess if increased LRP-1 shedding occurs in the lungs of patients with acute respiratory distress syndrome (ARDS) and may lead to the accumulation of MMPs and subsequent tissue injury. We determined sLRP-1 levels in bronchoalveolar lavage fluids (BALF) from 46 patients with ARDS and their correlation with MMP concentration and disease severity. In complementary in vitro studies, we investigated the mechanisms underlying the LRP-1 release from the cell surface and its impact on MMP cellular uptake. sLRP-1 levels were significantly elevated in BALF but not in plasma from patients with ARDS compared with control subjects and further increased in the later course of the disease. Baseline BALF sLRP-1 concentration was positively correlated with disease severity and significantly higher in nonsurvivors compared with survivors. The presence of ARDS BALF enhanced LRP-1 shedding from cultured lung fibroblasts but not from alveolar type II cells or macrophages. This process was blocked when ARDS BALF was supplemented with metalloproteinase inhibitor resulting in enhanced cellular uptake and degradation of MMP-2 and -9. Accordingly, sLRP-1 BALF concentration in patients with ARDS was positively correlated with MMP levels and laminin, a marker of basement membrane disruption. Increased LRP-1 shedding prevents the cellular clearance of MMPs and might thereby contribute to tissue destruction in ARDS lungs.

• 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.