Clemens Ruppert

Vitos Gießen-Marburg, Giessen, Hesse, Germany

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Publications (103)365.81 Total impact

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    ABSTRACT: Background: Activation and differentiation of fibroblasts into contractile protein-expressing myofibroblasts and their acquired apoptosis-resistant phenotype are critical factors towards the development of idiopathic pulmonary fibrosis (IPF), a fatal disease characterised by distorted pulmonary structure and excessive extracellular matrix (ECM) deposition. The molecular mechanisms underlying these processes in IPF remain incompletely understood. We investigated the possible implication of aberrant overexpression and activity of histone deacetylases (HDACs) in IPF. Methods: We analysed lung tissues from patients with sporadic IPF (n=26) and non-diseased control lungs (n=16) for expression of class I and II HDACs. Primary IPF fibroblasts were treated with HDAC inhibitors (HDACi) LBH589 or valproic acid (VPA). Results: Compared to control lungs, protein levels of class I (HDAC1, HDAC2, HDAC3, HDAC8) and class II HDACs (HDAC4, HDAC 5, HDAC 7, HDAC 9) were significantly elevated in IPF lungs. Using immunohistochemistry, strong induction of nearly all HDAC enzymes was observed in myofibroblasts of fibroblast foci and in abnormal bronchiolar basal cells at sites of aberrant re-epithelialisation in IPF lungs, but not in controls. Treatment of primary IPF fibroblasts with the pan-HDACi LBH589 resulted in significantly reduced expression of genes associated with ECM synthesis, proliferation and cell survival, as well as in suppression of HDAC7, and was paralleled by induction of endoplasmic reticulum stress and apoptosis. The profibrotic and apoptosis-resistant phenotype of IPF fibroblasts was also partly attenuated by the class I HDACi VPA. Conclusions: Aberrant overexpression of HDACs in basal cells of IPF lungs may contribute to the bronchiolisation process in this disease. Similarly, generation and apoptosis resistance of IPF fibroblasts are mediated by enhanced activity of HDAC enzymes. Therefore, pan-HDAC inhibition by LBH589 may present a novel therapeutic option for patients with IPF.
    Thorax 09/2015; 70(11). DOI:10.1136/thoraxjnl-2014-206411 · 8.29 Impact Factor

  • European Respiratory Journal 09/2015; 46(suppl 59):PA3831. DOI:10.1183/13993003.congress-2015.PA3831 · 7.64 Impact Factor

  • Pneumologie 07/2015; 69(07). DOI:10.1055/s-0035-1556650
  • S Backhaus · S Wilker · A Zakrzewicz · M Küllmar · W Padberg · C Ruppert · V Grau ·

    Pneumologie 07/2015; 69(07). DOI:10.1055/s-0035-1556631
  • I Shalashova · M Hühn · C Ruppert · O Klymenko · I Henneke · A Günther ·

    Pneumologie 07/2015; 69(07). DOI:10.1055/s-0035-1556651
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    ABSTRACT: Impaired immune function contributes to the development of chronic obstructive pulmonary disease (COPD). Disease progression is further exacerbated by pathogen infections due to impaired immune responses. Elimination of infected cells is achieved by cytotoxic CD8(+) T cells that are activated by MHC I-mediated presentation of pathogen-derived antigenic peptides. The immunoproteasome, a specialized form of the proteasome, improves generation of antigenic peptides for MHC I presentation thereby facilitating anti-viral immune responses. However, immunoproteasome function in the lung has not been investigated in detail yet. In this study, we comprehensively characterized the function of immunoproteasomes in the human and murine lung. Parenchymal cells of the lung express low constitutive levels of immunoproteasomes, while they are highly and specifically expressed in alveolar macrophages. Immunoproteasome expression is not altered in whole lung tissue of COPD patients. Novel activity-based probes and native gel analysis revealed that immunoproteasome activities are specifically and rapidly induced by IFNγ treatment in respiratory cells in vitro and by virus infection of the lung in mice. Our results suggest that the lung is potentially capable of mounting an immunoproteasome-mediated efficient adaptive immune response to intracellular infections.
    Scientific Reports 05/2015; 5:10230. DOI:10.1038/srep10230 · 5.58 Impact Factor
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    ABSTRACT: Chronic injury of alveolar epithelial type II cells (AE2 cells) represents a key event in the development of lung fibrosis in animal models and in humans, such as Idiopathic Pulmonary Fibrosis (IPF). Intratracheal delivery of amiodarone to mice results in a profound injury and macroautophagy dependent apoptosis of AE2 cells. Increased autophagy manifested in AE2 cells by disturbances of the intracellular surfactant . Hence, we hypothesized that ultrastructural alterations of the intracellular surfactant pool are signs of epithelial stress correlating with the severity of fibrotic remodeling. Using design based stereology the amiodarone model of pulmonary fibrosis in mice was characterized at the light and ultrastructural level during progression. Mean volume of AE2 cells, volume of lamellar bodies per AE2 cell and mean size of lamellar bodies were correlated to structural parameters reflecting severity of fibrosis like collagen content. Within 2 weeks amiodarone leads to an increase in septal wall thickness and a decrease in alveolar numbers due to irreversible alveolar collapse associated with alveolar surfactant dysfunction. Progressive hypertrophy of AE2 cells, increase in mean individual size and total volume of lamellar bodies per AE2 cell were observed. A high positive correlation of these AE2 cell-related ultrastructural changes and the deposition of collagen fibrils within septal walls were established. Qualitatively, similar alterations could be found in IPF samples with mild to moderate fibrosis. We conclude that ultrastructural alterations of AE2 cells including the surfactant system are tightly correlated with the progression of fibrotic remodeling. Copyright © 2014, American Journal of Physiology - Lung Cellular and Molecular Physiology.
    AJP Lung Cellular and Molecular Physiology 05/2015; 309(1):ajplung.00279.2014. DOI:10.1152/ajplung.00279.2014 · 4.08 Impact Factor

  • Medical Informatics Europe 2015, Madrid; 05/2015
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    ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a devastating disease, and its pathogenic mechanisms remain incompletely understood. Peroxisomes are known to be important in ROS and proinflammatory lipid degradation, and their deficiency induces liver fibrosis. However, altered peroxisome functions in IPF pathogenesis have never been investigated. By comparing peroxisome-related protein and gene expression in lung tissue and isolated lung fibroblasts between human control and IPF patients, we found that IPF lungs exhibited a significant down-regulation of peroxisomal biogenesis and metabolism (e.g., PEX13p and acyl-CoA oxidase 1). Moreover, in vivo the bleomycin-induced down-regulation of peroxisomes was abrogated in transforming growth factor beta (TGF-β) receptor II knockout mice indicating a role for TGF-β signaling in the regulation of peroxisomes. Furthermore, in vitro treatment of IPF fibroblasts with the profibrotic factors TGF-β1 or tumor necrosis factor alpha (TNF-α) was found to down-regulate peroxisomes via the AP-1 signaling pathway. Therefore, the molecular mechanisms by which reduced peroxisomal functions contribute to enhanced fibrosis were further studied. Direct down-regulation of PEX13 by RNAi induced the activation of Smad-dependent TGF-β signaling accompanied by increased ROS production and resulted in the release of cytokines (e.g., IL-6, TGF-β) and excessive production of collagen I and III. In contrast, treatment of fibroblasts with ciprofibrate or WY14643, PPAR-α activators, led to peroxisome proliferation and reduced the TGF-β-induced myofibroblast differentiation and collagen protein in IPF cells. Taken together, our findings suggest that compromised peroxisome activity might play an important role in the molecular pathogenesis of IPF and fibrosis progression, possibly by exacerbating pulmonary inflammation and intensifying the fibrotic response in the patients.
    Proceedings of the National Academy of Sciences 04/2015; 112(16). DOI:10.1073/pnas.1415111112 · 9.67 Impact Factor
  • Majeed RW · Kuhn S · Ruppert C · Günther A · Röhrig R ·

    Jahrestagung der Deutschen Gesellschaft für Medizinische Informatik, Biometrie und Epidemiologie e.V. (GMDS), Göttingen; 09/2014
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    ABSTRACT: Amiodarone (AD) is a highly efficient antiarrhythmic drug with potentially serious side effects. Severe pulmonary toxicity is reported in patients receiving AD even at low doses, and may cause interstitial pneumonia as well as lung fibrosis. Apoptosis of alveolar epithelial type II cells (AECII) has been suggested to play an important role in this disease. In the current study, we aimed to establish a murine model of AD induced lung fibrosis and analyze surfactant homeostasis, lysosomal and endoplasmic reticulum stress in this model. AD/ vehicle was instilled intratracheally into C57BL/6 mice, which were sacrificed on days 7, 14, 21 and 28. Extent of lung fibrosis development was assessed by trichrome staining and hydroxyproline measurement. Cytotoxicity was assessed by lactate dehydrogenase assay. Phospholipids (PL) were analyzed by mass spectrometry. Surfactant proteins (SP) and markers for apoptosis, lysosomal & ER stress were studied by western blotting and immunohistochemistry. AECII morphology was evaluated by electron microscopy. Extensive lung fibrosis and AECII hyperplasia was observed in AD treated mice already at day7. Surfactant PL and SP accumulated in AECII over time. In parallel, induction of apoptosis, lysosomal and ER stress was encountered in AECII of mice lungs and in MLE12 cells treated with AD. In vitro, si RNA mediated knock down of cathepsin D did not alter the AD induced apoptotic response. Our data suggest that mice exposed to intratracheal AD develop severe pulmonary fibrosis, exhibit extensive surfactant alterations and cellular stress, but AD induced AECII apoptosis is not mediated primarily via cathepsin D.
    Toxicological Sciences 08/2014; 142(1). DOI:10.1093/toxsci/kfu177 · 3.85 Impact Factor
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    ABSTRACT: The current study investigated the mechanisms involved in the process of biophysical inhibition of pulmonary surfactant by polymeric nanoparticles0020(NP). The minimal surface tension of diverse synthetic surfactants was monitored in the presence of bare and surface-decorated (i.e. poloxamer 407) sub-100 nm poly(lactide) NP. Moreover, the influence of NP on surfactant composition (i.e. surfactant protein (SP) content) was studied. Dose-elevations of SP advanced the biophysical activity of the tested surfactant preparation. SP-C supplemented phospholipid mixtures (PLM-C) were shown to be more susceptible to biophysical inactivation by bare NP than PLM-B and PLM-B/C. Surfactant function was hindered due to a drastic depletion of the SP content upon contact with bare NP. By contrast, surface-modified NP were capable to circumvent unwanted surfactant inhibition. Surfactant constitution influences the extent of biophysical inhibition by polymeric NP. Steric shielding of the NP surface minimizes unwanted NP-surfactant interactions, which represents an option for the development of surfactant-compatible nanomedicines.
    Acta Biomaterialia 07/2014; 10(11). DOI:10.1016/j.actbio.2014.07.026 · 6.03 Impact Factor
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    ABSTRACT: Rationale: Idiopathic pulmonary fibrosis (IPF) and bleomycin-induced pulmonary fibrosis are associated with surfactant-system dysfunction, alveolar collapse (derecruitment), and collapse induration (irreversible collapse). These events play undefined roles in the loss of lung function. Objective: To quantify how surfactant inactivation, alveolar collapse and collapse induration lead to degradation of lung function. Methods and measurements: Design-based stereology and invasive pulmonary function tests were performed 1, 3, 7, and 14 days (D) following intratracheal bleomycin-instillation in rats. The number and size of open alveoli was correlated to mechanical properties. Main Results: Active surfactant subtypes declined by D1, associated with a progressive alveolar derecruitment and a decrease in compliance. Alveolar epithelial damage was more pronounced in closed alveoli compared to ventilated alveoli. Collapse induration occurred on D7 and D14 as indicated by collapsed alveoli overgrown by a hyperplastic alveolar epithelium. This pathophysiology was also observed for the first time in human IPF lung explants. Prior to the onset of collapse induration, distal airspaces were easily recruited, and lung elastance could be kept low after recruitment by positive end-expiratory pressure (PEEP). At later time points the recruitable fraction of the lung was reduced by collapse induration, causing elastance to be elevated at high levels of PEEP. Conclusion: Surfactant inactivation leading to alveolar collapse and subsequent collapse induration might be the primary pathway for the loss of alveoli in this animal model. Loss of alveoli is highly correlated with the degradation of lung function. Our ultrastructural observations suggest that collapse induration is important in human IPF.
    American Journal of Respiratory Cell and Molecular Biology 07/2014; 52(2). DOI:10.1165/rcmb.2014-0078OC · 3.99 Impact Factor

  • Pneumologie 06/2014; 68(06). DOI:10.1055/s-0034-1376807
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    ABSTRACT: Amiodarone (AD) is an iodinated benzofuran derivative, especially known for its antiarrhythmic properties. It exerts serious side-effects even in patients receiving low doses. AD is well-known to induce apoptosis of type II alveolar epithelial cells (AECII), a mechanism that has been suggested to play an important role in AD-induced lung fibrosis. The precise molecular mechanisms underlying this disease are however still unclear. Because of its amphiphilic nature, AD becomes enriched in the lysosomal compartments, affecting the general functions of these organelles. Hence, in this study, we aimed to assess the role of autophagy, a lysosome dependent homeostasis mechanism, in driving AECII apoptosis in response to AD. In vitro, AD-treated MLE12 and primary AECII cells showed increased proSP-C and LC3B positive vacuolar structures and underwent LC3B-dependent apoptosis. In addition, AD-induced autophagosome-lysosome fusion and increased autophagy flux were observed. In vivo, in C57BL/6 mice, LC3B was localized at the limiting membrane of lamellar bodies, which were closely connected to the autophagosomal structures in AECIIs. Our data suggest that AD causes activation of macroautophagy in AECIIs and extensive autophagy-dependent apoptosis of alveolar epithelial cells. Targeting the autophagy pathway may therefore represent an attractive treatment modality in AD-induced lung fibrosis. This article is protected by copyright. All rights reserved.
    Pneumologie 06/2014; 68(06). DOI:10.1055/s-0034-1376801
  • Conference Paper: DZL-Platform Biobanking
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    ABSTRACT: Broad, coordinated access to biomaterials is essential for the translation of research findings into patient therapies. A centrally-organized DZL Biobanking Platform will guarantee that member of the DZL as well as external partners will have easy and direct access to biomaterials from patients with pulmonary disease. The DZL Biobanking Platform will capitalize on existing biobanking structures within DZL sites and will be connected to the Technology and Methods Platform for Network Research in Medicine (TMF e.V.) and Biobanking and Biomolecular Resources Research Infrastructure (BBMRI) catalogues. The biomaterial banks of DZL sites are not homogeneous. They are varying regarding structure and organizational standards methods of biomaterial collection, sample, data and quality management. In addition to implementing the DZL Biobanking portal in order to provide an overview of existing collections and biomaterials, the DZL Biobanking initiative aims to harmonize operating procedures and policies across DZL sites. These harmonization efforts include standardization of informed consent procedures standardization of sample procurement, processing, and handling, as well as the development of harmonized of phenotyping tools and sample management. Member of the platform compiled a forward-looking broad informed consent form allowing for collection, unlimited storage, and unrestricted use of biomaterials and phenotyping data. For a prospective collection of biomaterials and phenotyping data an overarching data management structure was considered including a centralized patient registration and pseudonymization service and a data warehouse for integrating phenotyping, imaging an experimental data.
    Pneumologie; 06/2014
  • O Klymenko · I Henneke · C Ruppert · W Seeger · A Guenther · M Korfei ·

    Pneumologie 06/2014; 68(06). DOI:10.1055/s-0034-1376806
  • S Skwarna · I Henneke · W Seeger · T Geiser · A Günther · C Ruppert ·

    Pneumologie 06/2014; 68(06). DOI:10.1055/s-0034-1376808

  • Pneumologie 02/2014; 68(S 01). DOI:10.1055/s-0034-1367944
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    ABSTRACT: Reasonable suspicion has accumulated that inhaled nano-scale particulate matter influences the biophysical function of the pulmonary surfactant system. Hence, it is evident to provide novel insights into the extent and mechanisms of nanoparticle-surfactant interactions in order to facilitate the fabrication of safe nanomedicines suitable for pulmonary applications. Negatively- and positively-charged poly(styrene) nanoparticles (diameters of ~100nm) served as model carriers. Nanoparticles were incubated with several synthetic and naturally-derived pulmonary surfactants to characterize the sensitivity of each preparation to biophysical inactivation. Changes in surface properties (i.e. adsorption and dynamic surface tension behavior) were monitored in a pulsating bubble surfactometer. Both nanoparticle formulations revealed a dose-dependent influence on the biophysical behavior of all investigated pulmonary surfactants. However, the surfactant sensitivity towards inhibition depended on both the carrier type, where negatively-charged nanoparticles showed increased inactivation potency compared to their positively-charged counterparts, and surfactant composition. Among the surfactants tested, synthetic mixtures (i.e. phospholipids, phospholipids supplemented with surfactant protein B, and Venticute®) were more susceptible to surface-activity inhibition as the more complex naturally-derived preparations (i.e. Alveofact® and large surfactant aggregates isolated from rabbit bronchoalveolar lavage fluid). Overall, nanoparticle characteristics and surfactant constitution both influence the extent of biophysical inhibition of pulmonary surfactants.
    Biochimica et Biophysica Acta 10/2013; 1838(1). DOI:10.1016/j.bbamem.2013.10.016 · 4.66 Impact Factor