Pierdonato Bruno

Decreased expression of autophagic beclin 1 protein in idiopathic pulmonary fibrosis fibroblasts

Autophagy is the main cellular pathway for degradation of long‐lived proteins and organelles and regulates cell fate in response to stress. Beclin 1 is a key regulator of this process. In some settings autophagy and apoptosis seem to be interconnected. Recent reports indicate that fibroblasts in idiopathic pulmonary fibrosis (IPF) acquire resistance to apoptosis. Here, we examined the expression of beclin 1, and of the anti apoptotic protein Bcl‐2 in human IPF fibroblasts using immunohistochemistry and molecular biology in bioptic sections, in primary cultures of fibroblasts taken from patients with IPF and in fibroblast cell lines. Expression of beclin 1 in fibroblasts from IPF was down‐regulated in comparison with fibroblasts from normal lungs while the anti‐apoptotic protein Bcl‐2 expression was over‐expressed. Treatment of fibroblast cell cultures with cisplatin induced a significant increase in beclin 1 and caspase 3 protein levels but a reduction in Bcl‐2 expression. These observations were confirmed by the analysis of acid compartments and transmission electron microscopy. Our results demonstrate a modified expression of the apoptotic beclin 1 Bcl‐2 proteins in human IPF fibroblasts suggesting the existence of an autophagy/apoptosis system dysfunction. J. Cell. Physiol. 228: 1516–1524, 2013.

EMT markers in lung adenocarcinoma pleural effusion spheroid cells

Epithelial‐to‐mesenchymal transition (EMT) is a process in which cells undergo a developmental switch from epithelial to mesenchymal phenotype. This process has been related to embryologic morphogenesis but also to cancer progression and metastasis. The aim of the current study was to investigate the expression of EMT‐related markers in adherent and spheroid cell cultures derived from malignant pleural effusions (MPEs) of patients affected by lung adenocarcinoma. On the basis of efficient in vitro propagation, six cases of MPEs were selected and analyzed by immunocytochemistry staining for EMT markers and by RT‐PCR for transcription factors known to orchestrate EMT. EMT markers immunostaining showed in spheroids a statistically significant correlation between the loss of E‐cadherin immunoreactivity and overexpression of N‐cadherin (P < 0.001). Likewise loss of EpCAM epithelial marker was coincident with Vimentin overexpression (P < 0.001). RT‐PCR analysis of transcription factors Snail, Slug, and Twist showed a highly variable expression, although a general trend to increase was observed. Importantly, in some selected cases it was possible to establish a precise relationship between spheroid formation, EMT switch and increased upregulation of the marker related to cancer stemness such as ALDH positivity. Therefore, MPE‐derived cell cultures, while recapitulating the heterogeneity of lung cancer, are a suitable system to study the mechanisms at the basis of EMT and to understand its relationship with the generation of cancer stem cells. J. Cell. Physiol. 228: 1720–1726, 2013.

Homeodomain-interacting protein kinase2 in human idiopathic pulmonary fibrosis.

Homeodomain‐interacting protein kinase 2 (Hipk2) is an emerging player in cell response to genotoxic agents that contributes to the cells decision between cell cycle arrest or apoptosis. HIPK2 acts as co‐regulator of an increasing number of transcription factors and modulates many different basic cellular processes such as apoptosis, proliferation, DNA damage response, differentiation. Idiopathic pulmonary fibrosis (IPF) is characterized by an anatomical disarrangement of the lung due to fibroblast proliferation, extracellular matrix deposition and lung function impairment. Although the role of inflammation is still debated, attention has been focused on lung cell functions as fibroblast phenotype and activity. Aim of the present study was to analyze the loss of heterozygosity (LOH) at HIPK2 locus 7q32.34 in human lung fibroblasts and the HIPK2 expression in 15 IPF samples and in four primary fibroblast cell cultures isolated from IPF biopsies using semi‐quantitative RT‐PCR, Western blots and immunohistochemistry. We demonstrated a frequency of LOH in IPF fibroblasts of 46% for the internal D7S6440 microsatellite and 26.6% for the external D7S2468 microsatellite. Furthermore, we demonstrated low HIPK2 protein expression in those fibroblasts from IPF patients that present the HIPK2 LOH. The restoration of HIPK2 expression in IPF derived cells induced a significant reduction of chemoresistance after treatment with cisplatin. The results obtained allow us to hypothesize that HIPK2 dysfunction may play a role in fibroblasts behavior and in IPF pathogenesis. HIPK2 may be considered as a novel potential target for anti‐fibrosis therapy. J. Cell. Physiol. 228: 235–241, 2013.

Neurotrophin system activation in pleural effusions

Neurotrophins (NTs) expression was assessed in malignant and non-malignant pleural effusions (inflammatory exudates and transudates). Enzyme-linked immunosorbent assay, in malignant exudates from small and non-small cell lung cancer (SCLC and NSCLC), detected nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), and their levels are higher as compared with inflammatory and transudative effusions. By immunoblots, in cultured cancer cells coming from malignant pleural effusions, NTs and low- and high-affinity NT receptors were detected in a percentage of SCLC and NSCLC. Proliferation assay demonstrated that BDNF significantly increased cancer cell proliferation in vitro, on the contrary, NT-3 reduced cancer cell growth rate and NGF did not modify cell growth. Moreover, NGF protects cells from death during starvation. These effects are reverted by the addition of NT receptor antagonists. Cultured cancer cells injected into the lung of immunodeficient mice generate lung tumors expressing NTs and NT receptors. These findings suggest that NTs may be able to modulate cancer cell behavior and their growth.

Reversibility Test in the Early Stages of Bronchial Asthma

In the early stages of bronchial asthma, it is frequent to find subjects with a positive history and an FEV1 or FEV1/FVC > 80% of the predicted value. This study investigated if the test of reversibility showed a reversible airway obstruction (RAO) in 291 subjects with the above clinical and functional features. Forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), and expiratory flows (PEF, MEF50, FEF25–75) were registered before and 20 minutes after salbutamol administration (200 mcg by MDI). Of 291 subjects, FEV1 increased in 73 (25%) after bronchodilator ≧12% compared to baseline; the number of subjects with a ≥ 35% increase in MEF50 or FEF25–75 were similar in terms of percentage (respectively, 29.2% and 29%), whereas those with increases in FVC ( ≥ 12%) and in PEF ( ≥ 15%) were significantly lower (respectively, 2.7% and 12.3%). The percentage of subjects with RAO (FEV1 increase after bronchodilator ≥ 12%) was lower (12%) in the subgroup (108 subjects), with an MEF50≥ 70% of the value predicted at the baseline assessment, and higher (36%) in the subjects of the subgroup (183 subjects) with an MEF50< 70%. In conclusion, it is advisable to carry out reversibility tests in all subjects with symptoms indicative of asthma even if their functional tests are “normal” because in a considerable number of cases the RAO was found to confirm the suspected diagnosis and provided a more reliable classification of the disease.

Relationship between diffuse pulmonary fibrosis, alveolar proteinosis, and granulocyte-macrophage colony stimulating factor autoantibodies

Extensive pulmonary fibrosis is a rare occurrence in pulmonary alveolar proteinosis. We report 2 cases that have interesting implications. A female patient was diagnosed with autoimmune pulmonary alveolar proteinosis that evolved over 7 years into diffuse fibrosis. In a male patient with diffuse fibrosis we incidentally detected electron microscopic features of alveolar surfactant accumulation and positive autoantibodies to granulocyte-macrophage colony stimulating factor. In the male patient we speculated that the pulmonary fibrosis might have been preceded by an asymptomatic phase of autoimmune pulmonary alveolar proteinosis, and that we should investigate the involvement of surfactant dysfunction in the pathogenesis of fibrotic lung disease.

In Vivo Evaluation of TNF-Alpha in the Lungs of Patients Affected by Sarcoidosis

Introduction. Sarcoidosis is a multisystemic granulomatous disorder characterized by multiple noncaseating granulomas involving intrathoracic lymph nodes and lung parenchyma. Recently, the use of anti-tumor necrosis factor alpha (anti-TNFα) agents has been introduced for therapy of chronic and refractory sarcoidosis with controversial results. Infliximab (Remicade) is a chimeric monoclonal antibody (mAb) that recognizes and binds TNFα, neutralizing its biological effects. In the present study,   99mTc labelled infliximab was used to study the expression of TNFα in sarcoid lesions and to evaluate its role as a predictive marker in response to therapy with Remicade. Material and Methods. A total of 10 patients with newly diagnosed sarcoidosis were enrolled together with 10 control patients affected by rheumatoid arthritis. All patients were studied by planar imaging of the chest with   99mTc-infliximab at 6 h and 24 h and total body [18F]-FDG PET/CT. Regions of interest were drawn over the lungs and the right arm and target-to-background ratios were analysed for   99mTc-infliximab. SUVmean and SUVmax were calculated over lungs for FDG. Results and Discussion. Image analysis showed low correlation between T/B ratios and BAL results in patients despite positivity at [18F]-FDG PET. Conclusion. In conclusion, patients with newly diagnosed pulmonary sarcoidosis, with FDG-PET and BAL positivity, showed a negative   99mTc-infliximab scintigraphy.

NT-proAtrial Natriuretic Peptide as a possible biomarker of cardiopulmonary involvement in sarcoidosis

BACKGROUND Lung diffusion for carbon monoxide (DLCO) has been shown to associate with the risk of pulmonary arterial hypertension development and, most likely, with right ventricular (RV) myocardial dysfunction in sarcoidosis patients. Besides its known role as a marker of left ventricular dysfunction, experimental evidence suggests a role of NT-proAtrial Natriuretic Peptide (NT-proANP) also in modulating pulmonary circulation. We therefore investigated possible relationships between NT-proANP, lung diffusion impairment and RV dysfunction. METHODS Thirty-two pulmonary sarcoidosis outpatients and eighteen volunteers underwent full clinical assessment, including full lung function tests and Doppler echocardiography integrated with tissue Doppler imaging (TDI) study. Resting circulating NT-proBNP and NT-proANP plasma levels were also determined. RESULTS NT-proANP and RV-myocardial performance index (RV-MPI) were significantly higher in those patients with the greatest DLCO impairment, whereas no differences were found for NT-proBNP values. At multivariable analysis, only DLCO (β: -0.496; standard error: 3.38; p=0.000) and RV-MPI (β: 0.373; standard error: 6.56; p=0.031) remained significantly associated with NT-proANP levels. CONCLUSIONS Our finding may support a key role of NT-proANP in the complex mechanisms underlying modulation of lung function. An early increase in pulmonary vascular resistance may stimulate NT-proANP increase, thus explaining its association with signs of early RV myocardial dysfunction. This hypothesis warrants further confirmation.

BDNF/TrkB axis activation promotes epithelial–mesenchymal transition in idiopathic pulmonary fibrosis

BackgroundNeurotrophins (NT) belongs to a family of growth factors which promotes neurons survival and differentiation. Increasing evidence show that NT and their receptor are expressed in lung tissues suggesting a possible role in lung health and disease. Here we investigated the expression and functional role of the TrkB/BDNF axis in idiopathic pulmonary fibrotic lung (myo)fibroblasts.MethodsLung fibroblast were isolated from IPF patients and characterized for the expression of mesenchymal markers in comparison to normal lung fibroblasts isolated from non-IPF controls.ResultsBDNF treatment promoted mesenchymal differentiation and this effect was counteracted by the TrkB inhibitor K252a. In this regard, we showed that K252a treatment was able to control the expression of transcription factors involved in epithelial to mesenchymal transition (EMT). Accordingly, K252a treatment reduced matrix metalloproteinase-9 enzyme activity and E-cadherin expression while increased cytoplasmic β-catenin expression.ConclusionsOur results suggest that BDNF/TrkB axis plays a role in EMT promoting the acquisition of (myo)fibroblast cell phenotype in IPF. Targeting BDNF/TrkB seems to represent a viable approach in order to prevent EMT dependent lung fibrosis.

Genetic Polymorphism of CHRM2 in COPD: Clinical Significance and Therapeutic Implications

Chronic Obstructive Pulmonary Disease (COPD) is a common preventable and treatable disease, characterized by persistent airflow limitation not fully reversible. However, a number of patients with COPD respond to bronchodilator agents. Some studies have shown polymorphisms in the b2‐adrenergic (ADRb2) and muscarinic M2 and M3 receptors (CHRM) that may participate in the modulation of the receptor responses. This study was designed to investigate the existence and the role of adrenergic and muscarinic receptor polymorphisms and their functional impact in COPD. Eighty‐two patients with COPD and 17 healthy smokers were recruited and screened for ADRb2 (T164I and R175R), for CHRM2 (rs1824024) and for CHRM3 (−513C/A and −492C/T). Among the polymorphisms studied our results was not able to demonstrate statistically significant association between the polymorphisms studied and COPD risk. Contrarily, we identified, in our COPD population, a significant association with the CHRM2 (rs1824024) polymorphism and disease severity, with lower lung function test values, frequent exacerbations, and poor response to anti‐cholinergic drugs. These results suggest the potential role of receptor polymorphism assessment to discriminate newly COPD phenotypes. J. Cell. Physiol. 231: 1745–1751, 2016.