Davide Scozzi

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.

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.

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.

Modified expression of peripheral blood lymphocyte muscarinic cholinergic receptors in asthmatic children.

Lymphocytes possess an independent cholinergic system. We assessed the expression of muscarinic cholinergic receptors in lymphocytes from 49 asthmatic children and 10 age matched controls using Western blot. We demonstrated that CD4+ and CD8+ T cells expressed M2 and M4 muscarinic receptors which density were significantly increased in asthmatic children in comparison with controls. M2 and M4 receptor increase was strictly related with IgE and fraction of exhaled nitric oxide (FeNO) measurements and with impairment in objective measurements of airway obstruction. Increased lymphocyte muscarinic cholinergic receptor expression may concur with lung cholinergic dysfunction and with inflammatory molecular framework in asthma.

Spleen-derived classical monocytes mediate lung ischemia-reperfusion injury through IL-1β

&NA; Ischemia‐reperfusion injury, a form of sterile inflammation, is the leading risk factor for both short‐term mortality following pulmonary transplantation and chronic lung allograft dysfunction. While it is well recognized that neutrophils are critical mediators of acute lung injury, processes that guide their entry into pulmonary tissue are not well understood. Here, we found that CCR2+ classical monocytes are necessary and sufficient for mediating extravasation of neutrophils into pulmonary tissue during ischemia‐reperfusion injury following hilar clamping or lung transplantation. The classical monocytes were mobilized from the host spleen, and splenectomy attenuated the recruitment of classical monocytes as well as the entry of neutrophils into injured lung tissue, which was associated with improved graft function. Neutrophil extravasation was mediated by MyD88‐dependent IL‐1&bgr; production by graft‐infiltrating classical monocytes, which downregulated the expression of the tight junction‐associated protein ZO‐2 in pulmonary vascular endothelial cells. Thus, we have uncovered a crucial role for classical monocytes, mobilized from the spleen, in mediating neutrophil extravasation, with potential implications for targeting of recipient classical monocytes to ameliorate pulmonary ischemia‐reperfusion injury in the clinic.