Grazyna Kwapiszewska

Publications of Grazyna Kwapiszewska

• Src tyrosine kinase is crucial for potassium channel function in human pulmonary arteries.

  Authors: Chandran Nagaraj, Bi Tang, Zoltán Bálint, Malgorzata Wygrecka, Andelko Hrzenjak, Grazyna Kwapiszewska, Elvira Stacher, Joerg Lindenmann, E Kenneth Weir, Horst Olschewski, Andrea Olschewski

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

  The potassium channel TASK-1, together with other potassium channels controls the low resting tone of pulmonary arteries. The Src family tyrosine kinase (SrcTK) may control potassium channel functionThe potassium channel TASK-1, together with other potassium channels controls the low resting tone of pulmonary arteries. The Src family tyrosine kinase (SrcTK) may control potassium channel function in human pulmonary artery smooth muscle cells (hPASMCs) in response to changes in oxygen tension and the clinical use of an SrcTK inhibitor has resulted in partly reversible pulmonary hypertension. This study aimed to determine the role of SrcTK for hypoxia-induced inhibition of potassium channels in hPASMCs.We show that SrcTK is colocalized with the TASK-1 channel. Inhibition of SrcTK decreases potassium current density and results in considerable depolarization, while activation of SrcTK increases potassium current in patch-clamp recordings. Moderate hypoxia and the SrcTK-inhibitor decrease the tyrosine-phosphorylation state of the TASK-1 channel. Hypoxia also decreases the level of phospho-SrcTK (tyr419) and reduces the co-localization of the TASK-1 channel and phospho-SrcTK. Corresponding to this, hypoxia reduces TASK-1 currents before but not after SrcTK inhibition and, in the isolated perfused mouse lung, SrcTK inhibitors increase pulmonary arterial pressure.We propose that the Src family tyrosine kinase is a crucial factor controlling potassium channels, acting as a cofactor for setting a negative resting membrane potential in hPASMCs and a low resting pulmonary vascular tone.

• Peroxisome Proliferator-Activated Receptor-β/δ, the Acute Signaling Factor in Prostacyclin-Induced Pulmonary Vasodilation.

  Authors: Yingji Li, Michelle Connolly, Chandran Nagaraj, Bi Tang, Zoltan Bálint, Helmut Popper, Freyja M Smolle-Juettner, Jörg Lindenmann, Grazyna Kwapiszewska, Philipp I Aaronson, Christoph Wohlkoenig, Katharina Leithner, Horst Olschewski, Andrea Olschewski

  American journal of respiratory cell and molecular biology. 03/2012; 46(3):372-9.

  As powerful vasodilators, prostacyclin analogues are presently the mainstay in the treatment of severe pulmonary arterial hypertension. Although the hemodynamic effects of prostacyclin analogues areAs powerful vasodilators, prostacyclin analogues are presently the mainstay in the treatment of severe pulmonary arterial hypertension. Although the hemodynamic effects of prostacyclin analogues are well known, the molecular mechanism of their acute effects on pulmonary vascular tone and systemic vascular tone remains poorly understood. Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) was previously identified as a putative receptor responsible for the modulation of target gene expression in response to prostacyclin analogues. The present study investigated the signaling pathway of prostacyclin in human pulmonary arterial smooth muscle cells (PASMCs), and sought to define the role of PPARβ/δ in the acute vasodilating effect. In human PASMCs, prostacyclin rapidly activated TWIK-related acid-sensitive K channel 1 (TASK-1) and calcium-dependent potassium channels (K(Ca)). This pathway was mediated via the prostanoid I receptor-protein kinase A pathway. The silencing of PPARβ/δ demonstrated that the downstream K(Ca) activation was exclusively dependent on PPARβ/δ signaling, whereas the activation of TASK-1 was not. In addition, the PPARβ/δ-induced activation of K(Ca) was independent of NO. The acute prostacyclin-induced K(Ca) activation is critically dependent on PPARβ/δ as a rapid signaling factor. This accounts in part for the vasodilating effect of prostacyclin in pulmonary arteries, and provides insights into a new molecular explanation for the effects of prostanoids.

• Origin of neomuscularized vessels in mice exposed to chronic hypoxia.

  Authors: Slaven Crnkovic, Andelko Hrzenjak, Leigh M Marsh, Andrea Olschewski, Grazyna Kwapiszewska

  Respiratory physiology & neurobiology. 12/2011; 179(2-3):342-5.

  Exposure of mice to chronic hypoxia is one of the most often used animal models to study pulmonary hypertension. Hypoxia exposure leads to vascular remodeling and muscularization of the smallExposure of mice to chronic hypoxia is one of the most often used animal models to study pulmonary hypertension. Hypoxia exposure leads to vascular remodeling and muscularization of the small parenchymal vessels in the lung. Due to the anatomical differences between mice and humans, it is not possible to determine whether the remodeled vessels originate from the arterial or venous side of the vasculature. By applying antibodies against specific marker molecules expressed by arterial (ephrinB2) and venous (EphB4) endothelial cells, we could show that remodeled parenchymal vessels in hypoxia-exposed mice are mostly of arterial origin with slight venous involvement. Using these tools, it is possible to further characterize remodeled vessels in other small animal models, such as transgenic or knockout mice. Particularly useful applications would include selection of parenchymal vessels for laser microdissection studies.

• 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.

• Role of protease-activated receptor-2 in idiopathic pulmonary fibrosis.

  Authors: Malgorzata Wygrecka, Grazyna Kwapiszewska, Ewa Jablonska, Susanne von Gerlach, Ingrid Henneke, Dariusz Zakrzewicz, Andreas Guenther, Klaus T Preissner, Philipp Markart

  American journal of respiratory and critical care medicine. 03/2011; 183(12):1703-14.

  Activation of the coagulation cascade has been demonstrated in pulmonary fibrosis. In addition to its procoagulant function, various coagulation proteases exhibit cellular effects that may alsoActivation of the coagulation cascade has been demonstrated in pulmonary fibrosis. In addition to its procoagulant function, various coagulation proteases exhibit cellular effects that may also contribute to fibrotic processes in the lung. To investigate the importance of protease-activated receptor (PAR)-2 and its activators, coagulation factor VIIa (FVIIa)/tissue factor (TF), in the development of idiopathic pulmonary fibrosis (IPF). Expression and localization of PAR-2 and its activators were examined in IPF lung tissue. The ability of PAR-2 to mediate various cellular processes was studied in vitro. Expression of PAR-2 was strongly elevated in IPF lungs and was attributable to alveolar type II cells and fibroblasts/myofibroblasts. Transforming growth factor-β(1), a key profibrotic cytokine, considerably enhanced PAR-2 expression in human lung fibroblasts. FVIIa stimulated proliferation of human lung fibroblasts and extracellular matrix production in a PAR-2-dependent manner, but did not initiate differentiation of fibroblasts into myofibroblasts. PAR-2/FVIIa-driven mitogenic activities were mediated via the p44/42 mitogen-activated protein kinase pathway and were independent of factor Xa and thrombin production. Proproliferative properties of FVIIa were markedly potentiated in the presence of TF and abrogated by TF antisense oligonucleotides. Hyperplastic alveolar type II cells overlying fibroblastic foci were found to be the source of FVII in IPF lungs. Moreover, TF colocalized with PAR-2 on fibroblasts/myofibroblasts in IPF lungs. The PAR-2/TF/FVIIa axis may contribute to the development of pulmonary fibrosis; thus, interference with this pathway confers novel therapeutic potential for the treatment of IPF.

• Dysregulation of the IL-13 receptor system: a novel pathomechanism in pulmonary arterial hypertension.

  Authors: Matthias Hecker, Zbigniew Zaslona, Grazyna Kwapiszewska, Gero Niess, Anna Zakrzewicz, Eduard Hergenreider, Jochen Wilhelm, Leigh M Marsh, Daniel Sedding, Walter Klepetko, Jürgen Lohmeyer, Stefanie Dimmeler, Werner Seeger, Norbert Weissmann, Ralph T Schermuly, Nikolaus Kneidinger, Oliver Eickelberg, Rory E Morty

  American journal of respiratory and critical care medicine. 09/2010; 182(6):805-18.

  Idiopathic pulmonary arterial hypertension (IPAH) is characterized by medial hypertrophy due to pulmonary artery smooth muscle cell (paSMC) hyperplasia. Inflammation is proposed to play a role inIdiopathic pulmonary arterial hypertension (IPAH) is characterized by medial hypertrophy due to pulmonary artery smooth muscle cell (paSMC) hyperplasia. Inflammation is proposed to play a role in vessel remodeling associated with IPAH. IL-13 is emerging as a regulator of tissue remodeling; however, the contribution of the IL-13 system to IPAH has not been assessed. The objective of this study was to assess the possible contribution of the IL-13 system to IPAH. Expression and localization of IL-13, and IL-13 receptors IL-4R, IL-13Rα1, and IL-13Rα2 were assessed by real-time reverse transcription-polymerase chain reaction, immunohistochemistry, and flow cytometry in lung tissue, paSMC, and microdissected vascular lesions from patients with IPAH, and in lung tissue from rodents with hypoxia- or monocrotaline-induced pulmonary hypertension. A whole-genome microarray analysis was used to study IL-13-regulated genes in paSMC. Pulmonary expression of the IL-13 decoy receptor IL-13Rα2 was up-regulated relative to that of the IL-13 signaling receptors IL-4R and IL-13Rα1 in patients with IPAH and in two animal models of IPAH. IL-13, signaling via STAT3 and STAT6, suppressed proliferation of paSMC by promoting G(0)/G(1) arrest. Whole-genome microarrays revealed that IL-13 suppressed endothelin-1 production by paSMC, suggesting that IL-13 controlled paSMC growth by regulating endothelin production. Ectopic expression of the il13ra2 gene resulted in partial loss of paSMC growth control by IL-13 and blunted IL-13 suppression of endothelin-1 production by paSMC, whereas small-interfering RNA knockdown of il13ra2 gene expression had the opposite effects. The IL-13 system is a novel regulator of paSMC growth. Dysregulation of IL-13 receptor expression in IPAH may partially underlie smooth muscle hypertrophy associated with pathological vascular remodeling in IPAH.

• Fhl-1, a new key protein in pulmonary hypertension.

  Authors: Grazyna Kwapiszewska, Malgorzata Wygrecka, Leigh M Marsh, Sigrid Schmitt, Roger Trösser, Jochen Wilhelm, Katja Helmus, Bastian Eul, Anna Zakrzewicz, Hossein A Ghofrani, Ralph T Schermuly, Rainer M Bohle, Friedrich Grimminger, Werner Seeger, Oliver Eickelberg, Ludger Fink, Norbert Weissmann

  Circulation. 10/2008; 118(11):1183-94.

  BACKGROUND: Pulmonary hypertension (PH) is a severe disease with a poor prognosis. Different forms of PH are characterized by pronounced vascular remodeling, resulting in increased vascularBACKGROUND: Pulmonary hypertension (PH) is a severe disease with a poor prognosis. Different forms of PH are characterized by pronounced vascular remodeling, resulting in increased vascular resistance and subsequent right heart failure. The molecular pathways triggering the remodeling process are poorly understood. We hypothesized that underlying key factors can be identified at the onset of the disease. Thus, we screened for alterations to protein expression in lung tissue at the onset of PH in a mouse model of hypoxia-induced PH. METHODS AND RESULTS: Using 2-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption/ionization time-of-flight analysis, we identified 36 proteins that exhibited significantly altered expression after short-term hypoxic exposure. Among these, Fhl-1, which is known to be involved in muscle development, was one of the most prominently upregulated proteins. Further analysis by immunohistochemistry, Western blot, and laser-assisted microdissection followed by quantitative polymerase chain reaction confirmed the upregulation of Fhl-1, particularly in the pulmonary vasculature. Comparable upregulation was confirmed (1) after full establishment of hypoxia-induced PH, (2) in 2 rat models of PH (monocrotaline-treated and hypoxic rats treated with the vascular endothelial growth factor receptor antagonist SU5416), and (3) in lungs from patients with idiopathic pulmonary arterial hypertension. Furthermore, we demonstrated that regulation of Fhl-1 was hypoxia-inducible transcription factor dependent. Abrogation of Fhl-1 expression in primary human pulmonary artery smooth muscle cells by small-interfering RNA suppressed, whereas Fhl-1 overexpression increased, migration and proliferation. Coimmunoprecipitation experiments identified Talin1 as a new interacting partner of Fhl-1. CONCLUSIONS: Protein screening identified Fhl-1 as a novel protein regulated in various forms of PH, including idiopathic pulmonary arterial hypertension.

• Hypoxia-dependent regulation of nonphagocytic NADPH oxidase subunit NOX4 in the pulmonary vasculature.

  Authors: Manish Mittal, Markus Roth, Peter König, Simone Hofmann, Eva Dony, Parag Goyal, Anne-Christin Selbitz, Ralph Theo Schermuly, Hossein Ardeschir Ghofrani, Grazyna Kwapiszewska, Wolfgang Kummer, Walter Klepetko, Mir Ali Reza Hoda, Ludger Fink, Jörg Hänze, Werner Seeger, Friedrich Grimminger, Harald H H W Schmidt, Norbert Weissmann

  Circulation research. 09/2007; 101(3):258-67.

  Nonphagocytic NADPH oxidases have recently been suggested to play a major role in the regulation of physiological and pathophysiological processes, in particular, hypertrophy, remodeling, andNonphagocytic NADPH oxidases have recently been suggested to play a major role in the regulation of physiological and pathophysiological processes, in particular, hypertrophy, remodeling, and angiogenesis in the systemic circulation. Moreover, NADPH oxidases have been suggested to serve as oxygen sensors in the lung. Chronic hypoxia induces vascular remodeling with medial hypertrophy leading to the development of pulmonary hypertension. We screened lung tissue for the expression of NADPH oxidase subunits. NOX1, NOXA1, NOXO1, p22phox, p47phox, p40phox, p67phox, NOX2, and NOX4 were present in mouse lung tissue. Comparing mice maintained for 21 days under hypoxic (10% O(2)) or normoxic (21% O(2)) conditions, an upregulation exclusively of NOX4 mRNA was observed under hypoxia in homogenized lung tissue, concomitant with increased levels in microdissected pulmonary arterial vessels. In situ hybridization and immunohistological staining for NOX4 in mouse lungs revealed a localization of NOX4 mRNA and protein predominantly in the media of small pulmonary arteries, with increased labeling intensities after chronic exposure to hypoxia. In isolated pulmonary arterial smooth muscle cells (PASMCs), NOX4 was localized primarily to the perinuclear space and its expression levels were increased after exposure to hypoxia. Treatment of PASMCs with siRNA directed against NOX4 decreased NOX4 mRNA levels and reduced PASMC proliferation as well as generation of reactive oxygen species. In lungs from patients with idiopathic pulmonary arterial hypertension (IPAH), expression levels of NOX4, which was localized in the vessel media, were 2.5-fold upregulated. These results support an important role for NOX4 in the vascular remodeling associated with development of pulmonary hypertension.

• Receptor for activated C-kinase 1, a novel interaction partner of type II bone morphogenetic protein receptor, regulates smooth muscle cell proliferation in pulmonary arterial hypertension.

  Authors: Anna Zakrzewicz, Matthias Hecker, Leigh M Marsh, Grazyna Kwapiszewska, Bozena Nejman, Lu Long, Werner Seeger, Ralph T Schermuly, Nicholas W Morrell, Rory E Morty, Oliver Eickelberg

  Circulation. 07/2007; 115(23):2957-68.

  BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by selective elevation of pulmonary arterial pressure. The pathological hallmark of PAH is the narrowing of pulmonary arteriolesBACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by selective elevation of pulmonary arterial pressure. The pathological hallmark of PAH is the narrowing of pulmonary arterioles secondary to endothelial cell dysfunction and smooth muscle cell proliferation. Heterozygous mutations in BMPR2, encoding the type II bone morphogenetic protein receptor (BMPRII), were identified in PAH, suggesting that alterations to BMPRII function are involved in disease onset and/or progression. METHODS AND RESULTS: We identified the receptor for activated C-kinase (RACK1) as a novel interaction partner of BMPRII by yeast 2-hybrid analyses using the kinase domain of BMPRII as a bait. Glutathione-S-transferase pull-down and coimmunoprecipitation confirmed the interaction of RACK1 with BMPRII in vitro and in vivo. RACK1-BMPRII interaction was reduced when kinase domain mutations occurring in patients with PAH were introduced to BMPRII. Immunohistochemistry of lung sections from PAH and control patients and immunofluorescence analysis of primary pulmonary arterial smooth muscle cells demonstrated colocalization of BMPRII and RACK1 in vivo. Quantitative reverse-transcription polymerase chain reaction and Western blot analysis showed significant downregulation of RACK1 expression in the rat model of monocrotaline-induced PAH but not in pulmonary arterial smooth muscle cells from PAH patients. Abrogation of RACK1 expression in pulmonary arterial smooth muscle cells led to decreased Smad1 phosphorylation and increased proliferation, whereas overexpression of RACK1 led to increased Smad1 phosphorylation and decreased proliferation. CONCLUSIONS: RACK1, a novel interaction partner of BMPRII, constitutes a new negative regulator of pulmonary arterial smooth muscle cell proliferation, suggesting a potential role for RACK1 in the pathogenesis of PAH.

• Dysregulated bone morphogenetic protein signaling in monocrotaline-induced pulmonary arterial hypertension.

  Authors: Rory E Morty, Bozena Nejman, Grazyna Kwapiszewska, Matthias Hecker, Anka Zakrzewicz, Fotini M Kouri, Dorothea M Peters, Rio Dumitrascu, Werner Seeger, Petra Knaus, Ralph T Schermuly, Oliver Eickelberg

  Arteriosclerosis, thrombosis, and vascular biology. 06/2007; 27(5):1072-8.

  BACKGROUND: Mutations in the bmpr2 gene, encoding the type II bone morphogenetic protein (BMP) receptor, have been identified in patients with pulmonary arterial hypertension (PAH), implicating BMPBACKGROUND: Mutations in the bmpr2 gene, encoding the type II bone morphogenetic protein (BMP) receptor, have been identified in patients with pulmonary arterial hypertension (PAH), implicating BMP signaling in PAH. The aim of this study was to assess BMP signaling and its physiological effects in a monocrotaline (MCT) model of PAH. METHODS AND RESULTS: Expression of BMP receptors Ib and II, and Smads 4, 5, 6, and 8, was downregulated in lungs but not kidneys of MCT-treated rats. Smad1 phosphorylation and expression of BMP/Smad target genes id1 and id3 was also reduced, although ERK1/2 and p38(MAPK) phosphorylation remained unaffected. BMP receptor and Smad expression, Smad1 phosphorylation, and induction of the BMP/Smad-responsive element of the id1 promoter were reduced in pulmonary artery smooth muscle cells (PASMCs) from MCT-treated rats. As a consequence of impaired BMP/Smad signaling, PASMCs from MCT-treated rats were resistant to apoptosis induced by BMP-4 and BMP-7, and were also resistant to BMP-4 antagonism of proliferation induced by platelet-derived growth factor. CONCLUSION: BMP signaling and BMP-regulated physiological phenomena are perturbed in MCT-treated rats, lending solid support to the proposed roles for BMP signaling in the pathogenesis of human PAH.

• Phosphodiesterase 1 upregulation in pulmonary arterial hypertension: target for reverse-remodeling therapy.

  Authors: Ralph Theo Schermuly, Soni Savai Pullamsetti, Grazyna Kwapiszewska, Rio Dumitrascu, Xia Tian, Norbert Weissmann, Hossein Ardeschir Ghofrani, Christina Kaulen, Torsten Dunkern, Christian Schudt, Robert Voswinckel, Jiang Zhou, Arun Samidurai, Walter Klepetko, Renate Paddenberg, Wolfgang Kummer, Werner Seeger, Friedrich Grimminger

  Circulation. 06/2007; 115(17):2331-9.

  BACKGROUND: Pulmonary arterial hypertension (PAH) is a life-threatening disease, characterized by vascular smooth muscle cell hyperproliferation. The calcium/calmodulin-dependent phosphodiesterase 1BACKGROUND: Pulmonary arterial hypertension (PAH) is a life-threatening disease, characterized by vascular smooth muscle cell hyperproliferation. The calcium/calmodulin-dependent phosphodiesterase 1 (PDE1) may play a major role in vascular smooth muscle cell proliferation. METHODS AND RESULTS: We investigated the expression of PDE1 in explanted lungs from idiopathic PAH patients and animal models of PAH and undertook therapeutic intervention studies in the animal models. Strong upregulation of PDE1C in pulmonary arterial vessels in the idiopathic PAH lungs compared with healthy donor lungs was noted on the mRNA level by laser-assisted vessel microdissection and on the protein level by immunohistochemistry. In chronically hypoxic mouse lungs and lungs from monocrotaline-injected rats, PDE1A upregulation was detected in the structurally remodeled arterial muscular layer. Long-term infusion of the PDE1 inhibitor 8-methoxymethyl 3-isobutyl-1-methylxanthine in hypoxic mice and monocrotaline-injected rats with fully established pulmonary hypertension reversed the pulmonary artery pressure elevation, structural remodeling of the lung vasculature (nonmuscularized versus partially muscularized versus fully muscularized small pulmonary arteries), and right heart hypertrophy. CONCLUSIONS: Strong upregulation of the PDE1 family in pulmonary artery smooth muscle cells is noted in human idiopathic PAH lungs and lungs from animal models of PAH. Inhibition of PDE1 reverses structural lung vascular remodeling and right heart hypertrophy in 2 animal models. The PDE1 family may thus offer a new target for therapeutic intervention in pulmonary hypertension.

• Hyperoxia modulates TGF-beta/BMP signaling in a mouse model of bronchopulmonary dysplasia.

  Authors: Miguel A Alejandre-Alcázar, Grazyna Kwapiszewska, Irwin Reiss, Oana V Amarie, Leigh M Marsh, Julia Sevilla-Pérez, Malgorzata Wygrecka, Bastian Eul, Silke Köbrich, Mareike Hesse, Ralph T Schermuly, Werner Seeger, Oliver Eickelberg, Rory E Morty

  American journal of physiology. Lung cellular and molecular physiology. 03/2007; 292(2):L537-49.

  Prematurely born infants who require oxygen therapy often develop bronchopulmonary dysplasia (BPD), a debilitating disorder characterized by pronounced alveolar hypoplasia. Hyperoxic injury isPrematurely born infants who require oxygen therapy often develop bronchopulmonary dysplasia (BPD), a debilitating disorder characterized by pronounced alveolar hypoplasia. Hyperoxic injury is believed to disrupt critical signaling pathways that direct lung development, causing BPD. We investigated the effects of normobaric hyperoxia on transforming growth factor (TGF)-beta and bone morphogenetic protein (BMP) signaling in neonatal C57BL/6J mice exposed to 21% or 85% O(2) between postnatal days P1 and P28. Growth and respiratory compliance were significantly impaired in pups exposed to 85% O(2), and these pups also exhibited a pronounced arrest of alveolarization, accompanied by dysregulated expression and localization of both receptor (ALK-1, ALK-3, ALK-6, and the TGF-beta type II receptor) and Smad (Smads 1, 3, and 4) proteins. TGF-beta signaling was potentiated, whereas BMP signaling was impaired both in the lungs of pups exposed to 85% O(2) as well as in MLE-12 mouse lung epithelial cells and NIH/3T3 and primary lung fibroblasts cultured in 85% O(2). After exposure to 85% O(2), primary alveolar type II cells were more susceptible to TGF-beta-induced apoptosis, whereas primary pulmonary artery smooth muscle cells were unaffected. Exposure of primary lung fibroblasts to 85% O(2) significantly enhanced the TGF-beta-stimulated production of the alpha(1) subunit of type I collagen (Ialpha(1)), tissue inhibitor of metalloproteinase-1, tropoelastin, and tenascin-C. These data demonstrated that hyperoxia significantly affects TGF-beta/BMP signaling in the lung, including processes central to septation and, hence, alveolarization. The amenability of these pathways to genetic and pharmacological manipulation may provide alternative avenues for the management of BPD.

• Laser-microdissection for cell type- and compartment-specific analyses on genomic and proteomic level.

  Authors: Ludger Fink, Grazyna Kwapiszewska, Jochen Wilhelm, Rainer M Bohle

  Experimental and toxicologic pathology : official journal of the Gesellschaft für Toxikologische Pathologie. 07/2006; 57 Suppl 2:25-9.

  Morphological study and identification of cell differentiation within tissues are the basis for assessment of physiological and pathological processes. Applying molecular techniques, the analysis ofMorphological study and identification of cell differentiation within tissues are the basis for assessment of physiological and pathological processes. Applying molecular techniques, the analysis of tissue homogenates may lead to masking of genetic deviations or expression changes of an individual cell type by the bulk of surrounding cells. To overcome the tissue heterogeneity, cells have to be isolated selectively. Therefore, microdissection techniques were developed making retrieval of target cells simple, rapid and precise. Presenting an overview of our approaches, it is demonstrated that single cell isolation is often preconditional for the investigation of splicing isoform expression. To reveal gene regulation combining microdissection of few cells and real-time RT-PCR allows to determine relative mRNA expression in a cell type-specific manner, even after immunofluorescence staining of target cells. Combination with RNA preamplification techniques and micro arrays results in cell type- or compartment-specific expression profiles that especially differ from those of tissue homogenates when minor represented cell types are investigated. For proteomic biomarker screening, the application of mass spectrometry techniques (SELDI/MALDI TOF) turned out to be feasible in combination with microdissected minute amounts of tissues. However, further identification of marker peaks still needs a remarkable effort. Strategies to deal with this problem are presented. In consequence, the isolation of cells or cell types allows a more accurate investigation of complex tissues and gives deeper insight to regulation processes and crosstalk of the respective cells.

• Systematic comparison of the T7-IVT and SMART-based RNA preamplification techniques for DNA microarray experiments.

  Authors: Jochen Wilhelm, Jai Prakash Muyal, Johannes Best, Grazyna Kwapiszewska, Maria Magdalena Stein, Werner Seeger, Rainer Maria Bohle, Ludger Fink

  Clinical chemistry. 06/2006; 52(6):1161-7.

  BACKGROUND: Small biological samples obtained from biopsies or laser microdissection often do not yield sufficient RNA for successful microarray hybridization; therefore, RNA amplification isBACKGROUND: Small biological samples obtained from biopsies or laser microdissection often do not yield sufficient RNA for successful microarray hybridization; therefore, RNA amplification is performed before microarray experiments. We compared 2 commonly used techniques for RNA amplification. METHODS: We compared 2 commercially available methods, Arcturus RiboAmp for in vitro transcription (IVT) and Clontech BD SMART for PCR, to preamplify 50 ng of total RNA isolated from mouse livers and kidneys. Amplification factors of 3 sequences were determined by real-time PCR. Differential expression profiles were compared within and between techniques as well as with unamplified samples with 10K 50mer oligomer-spotted microarrays (MWG Biotech). The microarray results were validated on the transcript and protein levels by comparison with public expression databases. RESULTS: Amplification factors for specific sequences were lower after 2 rounds of IVT than after 12 cycles of SMART. Furthermore, IVT showed a clear decrease in amplification with increasing distance of the amplified sequences from the polyA tail, indicating generation of smaller products. In the microarray experiments, reproducibility of the duplicates was highest after SMART. In addition, SMART-processed samples showed higher correlation when compared with unamplified samples as well as with expression databases. CONCLUSIONS: Whenever 1 round of T7-IVT does not yield sufficient product for microarray hybridization, which is usually the case when <200 ng of total RNA is used as starting material, we suggest the use of SMART PCR for preamplification.

• Impact of TASK-1 in human pulmonary artery smooth muscle cells.

  Authors: Andrea Olschewski, Yingji Li, Bi Tang, Jörg Hanze, Bastian Eul, Rainer M Bohle, Jochen Wilhelm, Rory E Morty, Michael E Brau, E Kenneth Weir, Grazyna Kwapiszewska, Walter Klepetko, Werner Seeger, Horst Olschewski

  Circulation research. 05/2006; 98(8):1072-80.

  The excitability of pulmonary artery smooth muscle cells (PASMC) is regulated by potassium (K+) conductances. Although studies suggest that background K+ currents carried by 2-pore domain K+ channelsThe excitability of pulmonary artery smooth muscle cells (PASMC) is regulated by potassium (K+) conductances. Although studies suggest that background K+ currents carried by 2-pore domain K+ channels are important regulators of resting membrane potential in PASMC, their role in human PASMC is unknown. Our study tested the hypothesis that TASK-1 leak K+ channels contribute to the K+ current and resting membrane potential in human PASMC. We used the whole-cell patch-clamp technique and TASK-1 small interfering RNA (siRNA). Noninactivating K+ current performed by TASK-1 K+ channels were identified by current characteristics and inhibition by anandamide and acidosis (pH 6.3), each resulting in significant membrane depolarization. Moreover, we showed that TASK-1 is blocked by moderate hypoxia and activated by treprostinil at clinically relevant concentrations. This is mediated via protein kinase A (PKA)-dependent phosphorylation of TASK-1. To further confirm the role of TASK-1 channels in regulation of resting membrane potential, we knocked down TASK-1 expression using TASK-1 siRNA. The knockdown of TASK-1 was reflected by a significant depolarization of resting membrane potential. Treatment of human PASMC with TASK-1 siRNA resulted in loss of sensitivity to anandamide, acidosis, alkalosis, hypoxia, and treprostinil. These results suggest that (1) TASK-1 is expressed in human PASMC; (2) TASK-1 is hypoxia-sensitive and controls the resting membrane potential, thus implicating an important role for TASK-1 K+ channels in the regulation of pulmonary vascular tone; and (3) treprostinil activates TASK-1 at clinically relevant concentrations via PKA, which might represent an important mechanism underlying the vasorelaxing properties of prostanoids and their beneficial effect in vivo.

• Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension.

  Authors: Grazyna Kwapiszewska, Jochen Wilhelm, Stephanie Wolff, Isabel Laumanns, Inke R Koenig, Andreas Ziegler, Werner Seeger, Rainer M Bohle, Norbert Weissmann, Ludger Fink

  Respiratory research. 02/2005; 6:109.

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

• Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension

  Authors: Grazyna Kwapiszewska, Jochen Wilhelm, Stephanie Wolff, Isabel Laumanns, Inke Koenig, Andreas Ziegler, Werner Seeger, Rainer Bohle, Norbert Weissmann, Ludger Fink

  Respiratory Research. 01/2005;

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

• Identification of proteins in laser-microdissected small cell numbers by SELDI-TOF and Tandem MS.

  Authors: Grazyna Kwapiszewska, Markus Meyer, Ralf Bogumil, Rainer M Bohle, Werner Seeger, Norbert Weissmann, Ludger Fink

  BMC biotechnology. 01/2005; 4:30.

  BACKGROUND: Laser microdissection allows precise isolation of specific cell types and compartments from complex tissues. To analyse proteins from small cell numbers, we combine laser-microdissectionBACKGROUND: Laser microdissection allows precise isolation of specific cell types and compartments from complex tissues. To analyse proteins from small cell numbers, we combine laser-microdissection and manipulation (LMM) with mass spectrometry techniques. RESULTS: Hemalaun stained mouse lung sections were used to isolate 500-2,000 cells, enough material for complex protein profiles by SELDI-TOF MS (surface enhanced laser desorption and ionization/time of flight mass spectrometry), employing different chromatographic ProteinChip Arrays. Initially, to establish the principle, we identified specific protein peaks from 20,000 laser-microdissected cells, combining column chromatography, SDS-PAGE, tryptic digestion, SELDI technology and Tandem MS/MS using a ProteinChip Tandem MS Interface. Secondly, our aim was to reduce the labour requirements of microdissecting several thousand cells. Therefore, we first defined target proteins in a few microdissected cells, then recovered in whole tissue section homogenates from the same lung and applied to these analytical techniques. Both approaches resulted in a successful identification of the selected peaks. CONCLUSION: Laser-microdissection may thus be combined with SELDI-TOF MS for generation of protein marker profiles in a cell-type- or compartment-specific manner in complex tissues, linked with mass fingerprinting and peptide sequencing by Tandem MS/MS for definite characterization.

• Characterization of platelet-specific mRNA by real-time PCR after laser-assisted microdissection.

  Authors: Ludger Fink, Hans Hölschermann, Grazyna Kwapiszewska, Jai Prakash Muyal, Björn Lengemann, Rainer Maria Bohle, Sentot Santoso

  Thrombosis and haemostasis. 10/2003; 90(4):749-56.

  Circulating anucleate platelets contain minute amounts of residual megakaryocytic-derived mRNA. To study cell type-specific gene expression in platelets, an accurate and sensitive method to detectCirculating anucleate platelets contain minute amounts of residual megakaryocytic-derived mRNA. To study cell type-specific gene expression in platelets, an accurate and sensitive method to detect and quantify platelet mRNA that excludes contamination with leukocyte RNA is mandatory. Applying laser-assisted microdissection and manipulation (LMM) we could isolate platelets from hemalaun-stained cytospins under permanent visual control and after laser-photolysis of nucleated blood cells. For mRNA quantification, the platelet-specific mRNAs were subsequently measured by real time RT-PCR. High-copy beta3 integrin and low-copy a alpha2 integrin as well as tissue factor (TF) transcripts were analyzed in LMM-harvested platelets. In 91.2% (83/91) beta3 integrin was detectable with a mean threshold cycle (CT) value of 32.5+/-3.2 (< or =50,000 cells). The low-copy a 2 integrin mRNA was positive in 84.4% (38/45) with CT mean value of 36.9+/-1.3, indicating that the relative expression of alpha2 integrin mRNA in platelets was about 130 times lower than beta3. The TF transcript was undetectable in all samples. Comparing platelet mRNA from LMM isolation to that from limiting dilution series resulted in a high accordance for beta3 integrin transcript in both, recovery (91.2% vs. 95.2%) and CT value (32.5 vs. 32.8). These results demonstrate that the combination of LMM and real-time RT-PCR is a valuable tool for precise, platelet-specific mRNA analysis without contamination of other cells.