Comparative proteome analysis of lung tissue from patients with idiopathic pulmonary fibrosis (IPF), non-specific interstitial pneumonia (NSIP) and organ donors

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Loss of SMAD3 Promotes Vascular Remodeling in Pulmonary Arterial Hypertension via MRTF Disinhibition.

RATIONALE: Vascular remodeling in pulmonary arterial hypertension (PAH) results from smooth muscle cell hypertrophy and proliferation of vascular cells. Loss of BMPR-II (bone morphogenetic protein receptor 2) signaling and increased signaling via TGF-β (transforming growth factor β) and its downstream mediators SMAD (small body size [a C. elegans protein] mothers against decapentaplegic [a Drosophila protein family])-2/3 has been proposed to drive lung vascular remodeling; yet, proteomic analyses indicate a loss of SMAD3 in PAH. OBJECTIVES: We proposed that SMAD3 may be dysregulated in PAH and that loss of SMAD3 may present a pathophysiological master switch by disinhibiting its interaction partner, MRTF (myocardin-related transcription factor), which drives muscle protein expression. METHODS: SMAD3 levels were measured in lungs from PAH patients, rats treated either with Sugen/hypoxia or monocrotaline (MCT), and in mice carrying a BMPR2 mutation. In vitro, effects of SMAD3 or BMPR2 silencing or SMAD3 overexpression on cell proliferation or smooth muscle hypertrophy were assessed. In vivo, the therapeutic and prophylactic potential of CCG1423, an inhibitor of MRTF, was investigated in Sugen/hypoxia rats. MEASUREMENTS AND MAIN RESULTS: SMAD3 was downregulated in lungs of patients with PAH and in pulmonary arteries of three independent PAH animal models. TGF-β treatment replicated the loss of SMAD3 in human pulmonary artery smooth muscle cells (huPASMCs) and human pulmonary artery endothelial cells. SMAD3 silencing increased proliferation and migration in huPASMCs and human pulmonary artery endothelial cells. Coimmunoprecipitation revealed reduced interaction of MRTF with SMAD3 in TGF-β-treated huPASMCs and pulmonary arteries of PAH animal models. In huPASMCs, loss of SMAD3 or BMPR-II increased smooth muscle actin expression, which was attenuated by MRTF inhibition. Conversely, SMAD3 overexpression prevented TGF-β-induced activation of an MRTF reporter and reduced actin stress fibers in BMPR2-silenced huPASMCs. MRTF inhibition attenuated PAH and lung vascular remodeling in Sugen/hypoxia rats. CONCLUSIONS: Loss of SMAD3 presents a novel pathomechanism in PAH that promotes vascular cell proliferation and-via MRTF disinhibition-hypertrophy of huPASMCs, thereby reconciling the parallel induction of a synthetic and contractile huPASMC phenotype

Targeting TMEM16A to reverse vasoconstriction and remodelling in idiopathic PAH

Our systematic analysis of anion channels and transporters in idiopathic pulmonary arterial hypertension (IPAH) showed marked upregulation of the Cl- channel TMEM16A gene.We hypothesised that TMEM16A overexpression might represent a novel vicious circle in the molecular pathways causing PAH.We investigated healthy donor lungs (n=40) and recipient lungs with IPAH (n=38) for the expression of anion channel and transporter genes in small pulmonary arteries and pulmonary arterial smooth muscle cells (PASMC). In IPAH, TMEM16A was strongly upregulated and patch-clamp recordings confirmed an increased Cl- current in PASMC (n=9-10). These cells were depolarised and could be repolarized by TMEM16A inhibitors or knock-down experiments (n=6-10). Inhibition/knock-down of TMEM16A reduced proliferation of IPAH-PASMC (n=6). Conversely, overexpression of TMEM16A in healthy donor PASMC produced an IPAH-like phenotype. Chronic application of benzbromarone in two independent animal models significantly decreased right ventricular pressure and reversed remodelling of established PH.Our findings suggest that increased TMEM16A expression and activity comprise an important pathologic mechanism underlying vasoconstriction and remodelling of pulmonary arteries in PAH. Inhibition of TMEM16A represents a novel therapeutic approach to achieve reverse remodelling in PAH

Subtype-specific KRAS mutations in advanced lung adenocarcinoma: A retrospective study of patients treated with platinum-based chemotherapy

Background: Platinum-based chemotherapy is the most common treatment in advanced-stage lung adenocarcinoma. Because the clinical significance of KRAS mutational status in this setting has not yet been clearly determined, a mutation subtype-specific analysis was performed in the so far largest cohort of Caucasian patients with KRAS mutant advanced-stage lung adenocarcinoma treated with platinum-based chemotherapy. Methods: 505 Caucasian stage III-IV lung adenocarcinoma patients with known amino acid substitution-specific KRAS mutational status and treated with platinum-based chemotherapy were included. The correlations of subtype-specific KRAS mutations with smoking status, progression-free and overall survival (PFS and OS, respectively) and therapeutic response were analysed. Results: Among 338 KRAS wild-type, 147 codon 12 mutant and 20 codon 13 mutant patients, there were no mutation-related significant differences in PFS or OS (P values were 0.534 and 0.917, respectively). Eastern Cooperative Oncology Group (ECOG) status and clinical stage were significant independent prognostic factors. KRAS mutation showed a significant correlation with smoking status (P = 0.018). Importantly, however, G12V KRAS mutant patients were significantly more frequent among never-smokers than all other codon 12 KRAS mutant (G12x) subtypes (P = 0.016). Furthermore, this subgroup tended to have a higher response rate (66% versus 47%; P = 0.077). A modestly longer median PFS was also found in the G12V mutant cohort (233 days; versus 175 days in the G12x group; P = 0.145). Conclusions: While KRAS mutation status per se is neither prognostic nor predictive in stage III-IV lung adenocarcinoma, subtype-specific analysis may indeed identify clinically relevant subgroups of patients that may ultimately influence treatment decisions. © 2014 The Authors

Identification and evaluation of prognostic and predictive biomarkers in malignant pleural mesothelioma

Hintergrund: Das maligne Pleuramesotheliom (MPM) ist eine seltene aber therapieresistente Erkrankung. Etablierte prognostische und prädiktive Biomarker fehlen derzeit in der klinischen Routine. Ziel dieser Dissertation war daher die Erforschung von klinisch nützlichen Biomarkern im MPM. Methoden: Es wurden drei internationale retrospektive multizentrische Studien im Rahmen dieser Dissertation durchgeführt. In den ersten beiden Arbeiten wurden die Routine-Laborparameter C-reaktives Protein (CRP) und Fibrinogen untersucht. In der dritten und letzten Arbeit wurde die Ki67 Expression mittels Immunhistochemie in Paraffin-Blöcken aus vier verschiedenen Ländern analysiert. Resultate: In der ersten Studie konnte gezeigt werden, dass CRP ein unabhängiger prognostischer Marker im MPM ist (Hazard Ratio [HR] 2.07, 95% Konfidenz Intervall [CI 1.23-3.46; p=0.01) und auch den Erfolg multimodaler Therapie vorhersagte. So hatten Patienten mit normalen CRP einen Überlebensvorteil nach multimodaler Therapie mit radikaler Tumorchirurgie (Medianes Gesamtüberleben [OS] 35.93 Monate) wohingegen Patienten mit erhöhtem CRP nach multimodaler Therapie ein ähnliches OS zeigten wie nach alleiniger Chemo- und/oder Strahlentherapie (OS 7.86 Monate; p<0.001). Vergleichbare Resultate wurden in der zweiten Studie für Fibrinogen gezeigt. Fibrinogen war ein unabhängiger prognostischer Marker (HR 1.81, CI 1.23-2.65, Grenzwert: Median der Studienkohorte) und prädiktiv, wenn die 75th Perzentile der Studienkohorte als Grenzwert verwendet wurde (Interaktionswert: p=0.034). Schlussendlich konnten wir zeigen, dass Ki67 ein unabhängiger prognostischer Biomarker (HR: 2.1, 95% CI: 1.4-3.2, p<0.001) war. Diese Ergebnisse waren in einer weiteren unabhängigen Kohorte reproduzierbar (p=0.048). Schlussfolgerung: In der vorliegenden Dissertation konnten wir zeigen, dass Routine-Entzündungsparametern in MPM Patienten nicht nur prognostisch sondern auch prädiktiv für multimodale Therapieansätze waren. Des Weiteren konnte Ki67 als unabhängiger und reproduzierbar prognostischer Biomarker gezeigt werden.Background: Malignant pleural mesothelioma (MPM) is a seldom and treatment resistant disease. Biomarkers with established prognostic and/or predictive value are missing in clinical routine so far but would be of urgent need. Therefore, the major aim of the present thesis is the identification of clinical useful biomarkers in MPM. Methods: Three international retrospective multicenter studies have been performed. In the first two studies, the routine inflammatory markers C-reactive protein (CRP) and fibrinogen have been investigated and in the third study, paraffin embedded tissue samples from four different nations have been stained for Ki67 to evaluate this routine proliferation marker for its prognostic power in MPM. Results: In the first study, our research group was able to show, that serum CRP was not only a biomarker, showing independent prognostic value in MPM (hazard ratio [HR] 2.07, 95% confidence interval [CI] 1.23-3.46; p=0.01), but was furthermore able to predict survival benefit, achieved by surgery within multimodality therapy. Only patients with normal CRP values benefit from multimodality treatment including surgery (median overall survival [OS] 35.93 months) whereas patients with elevated CRP receiving multimodality therapy had a comparable poor outcome as patients receiving chemo- and/or radiotherapy alone (OS 7.86 months; p<0.001). Similar results have been shown in my second study where we proved, that fibrinogen, another proinflammatory maker, was also found to be independent prognostic (HR 1.81, CI 1.23-2.65, cut off: median of the whole cohort) and predictive, when the 75th percentile of our study cohort was used as cut point (interaction term: p=0.034). Finally, in my last study our group demonstrated Ki67 as an independent (HR: 2.1, 95% CI: 1.4-3.2, p<0.001) and reproducible (p=0.048) prognostic factor in MPM. Conclusion: In the present thesis, we were able to show, that a proinflammatory status of MPM patients is not only prognostic, but predictive for treatment benefit achieved by surgery within multimodality therapy. Furthermore, Ki67 proved to be an independent and reproducible prognostic marker in MPM.submitted by Bahil GhanimAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2015OeBB(VLID)171484

Biomarkers for Malignant Pleural Mesothelioma—A Novel View on Inflammation

Malignant pleural mesothelioma (MPM) is an aggressive disease with limited treatment response and devastating prognosis. Exposure to asbestos and chronic inflammation are acknowledged as main risk factors. Since immune therapy evolved as a promising novel treatment modality, we want to reevaluate and summarize the role of the inflammatory system in MPM. This review focuses on local tumor associated inflammation on the one hand and systemic inflammatory markers, and their impact on MPM outcome, on the other hand. Identification of new biomarkers helps to select optimal patient tailored therapy, avoid ineffective treatment with its related side effects and consequently improves patient’s outcome in this rare disease. Additionally, a better understanding of the tumor promoting and tumor suppressing inflammatory processes, influencing MPM pathogenesis and progression, might also reveal possible new targets for MPM treatment. After reviewing the currently available literature and according to our own research, it is concluded that the suppression of the specific immune system and the activation of its innate counterpart are crucial drivers of MPM aggressiveness translating to poor patient outcome

Transbronchial lung cryobiopsy: prospective safety evaluation and 90-day mortality after a standardized examination protocol

Background: Transbronchial lung cryobiopsy (TBLC) is a new method of bronchoscopic tissue sampling in patients with unclear diffuse parenchymal lung disease (DPLD). While not the gold standard, TBLC has a good diagnostic correlation with surgical lung biopsy, and retrospective analyses of peri-interventional complications and mortality are promising. However, prospective reports on 90-day mortality are lacking. Objectives: This study addresses morbidity and 30- and 90-day mortality in TBLC after a standardized protocol. Methods: In this prospective study, 75 patients with DPLD requiring tissue sampling were included. A standardized protocol (including prophylactic use of an endobronchial balloon, postinterventional observation, and minimum sampling requirements) was used in all patients. Adverse events (pneumothorax, bronchial bleeding, premature discontinuation, prolonged monitoring at ICU, and fatal outcome) and 30- and 90-day mortality rates were recorded. Results: A total of 308 cryobiopsies were performed in 75 patients. Peri- and postinterventional pneumothorax were observed in 20% (9.3% mild and 10.7% moderate with the necessity of chest drainage), and bronchial bleeding was found in 29.3% (22.7% moderate and 6.7% severe). Total lung capacity below normal value was associated with the risk of pneumothorax ( p  = 0.009), and diffusion limitation for carbon monoxide below normal value was associated with the risk of bronchial bleeding ( p  = 0.044). No fatal events were observed within 30 days, and the 90-day mortality rate was 1.3%, but not related to the procedure itself. Conclusion: As it gradually becomes the invasive procedure of choice in unclear DPLD, TBLC is a safe procedure with a low 30- and 90-day mortality. Trial registration ID: DRKS00026746 (German Clinical Trial Register

Biomarkers for Malignant Pleural Mesothelioma—A Novel View on Inflammation

Malignant pleural mesothelioma (MPM) is an aggressive disease with limited treatment response and devastating prognosis. Exposure to asbestos and chronic inflammation are acknowledged as main risk factors. Since immune therapy evolved as a promising novel treatment modality, we want to reevaluate and summarize the role of the inflammatory system in MPM. This review focuses on local tumor associated inflammation on the one hand and systemic inflammatory markers, and their impact on MPM outcome, on the other hand. Identification of new biomarkers helps to select optimal patient tailored therapy, avoid ineffective treatment with its related side effects and consequently improves patient’s outcome in this rare disease. Additionally, a better understanding of the tumor promoting and tumor suppressing inflammatory processes, influencing MPM pathogenesis and progression, might also reveal possible new targets for MPM treatment. After reviewing the currently available literature and according to our own research, it is concluded that the suppression of the specific immune system and the activation of its innate counterpart are crucial drivers of MPM aggressiveness translating to poor patient outcome

Treprostinil inhibits proliferation and extracellular matrix deposition by fibroblasts through cAMP activation

Abstract Idiopathic pulmonary fibrosis (IPF) is characterized by peripheral lung fibrosis and increased interstitial extracellular matrix (ECM) deposition. In IPF, tumor growth factor (TGF)-β1 which is the major stimulus of ECM deposition, and platelet derived growth factor (PDGF)-BB is a potent stimulus of fibrosis. Thus, the effect of Treprostinil on TGF-ß1 and PDGF-induced fibroblast proliferation and ECM deposition was investigated. Human peripheral lung fibroblasts of seven IPF patients and five lung donors were stimulated by PDGF, or TGF-β1, or the combination. Cells were pre-incubated (30 min) with either Treprostinil, forskolin, di-deoxyadenosine (DDA), or vehicle. Treprostinil time dependently activated cAMP thereby preventing PDGF-BB induced proliferation and TGF-β1 secretion. Cell counts indicated proliferation; α-smooth muscle actin (α-SMA) indicted differentiation, and collagen type-1 or fibronectin deposition remodeling. Myo-fibroblast indicating α-SMA expression was significantly reduced and its formation was altered by Treprostinil. Collagen type-I and fibronectin deposition were also reduced by Treprostinil. The effect of Treprostinil on collagen type-I deposition was cAMP sensitive as it was counteracted by DDA, while the effect on fibronectin was not cAMP mediated. Treprostinil antagonized the pro-fibrotic effects of both PDGF-BB and TGF-β1 in primary human lung fibroblasts. The data presented propose a therapeutic relevant anti-fibrotic effect of Treprostinil in IPF

Trabectedin Is Active against Two Novel, Patient-Derived Solitary Fibrous Pleural Tumor Cell Lines and Synergizes with Ponatinib

Solitary fibrous tumor of the pleura (SFT) is a rare disease. Besides surgery combined with radiotherapy in nondisseminated stages, curative options are currently absent. Out of fourteen primo-cell cultures, established from surgical SFT specimens, two showed stable in vitro growth. Both cell models harbored the characteristic NAB2-STAT6 fusion and were further investigated by different preclinical methods assessing cell viability, clone formation, and protein regulation upon single-drug treatment or in response to selected treatment combinations. Both fusion-positive cell models showed&mdash;in line with the clinical experience and the literature&mdash;a low to moderate response to most of the tested cytotoxic and targeted agents. However, the multi-tyrosine kinase inhibitors ponatinib and dasatinib, as well as the anti-sarcoma compound trabectedin, revealed promising activity against SFT growth. Furthermore, both cell models spontaneously presented strong FGFR downstream signaling targetable by ponatinib. Most interestingly, the combination of either ponatinib or dasatinib with trabectedin showed synergistic effects. In conclusion, this study identified novel trabectedin-based treatment combinations with clinically approved tyrosine kinase inhibitors, using two newly established NAB2-STAT6 fusion-positive cell models. These findings can be the basis for anti-SFT drug repurposing approaches in this rare and therapy-refractory disease