No abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease characterized by an accumulation of fibroblasts and myofibroblasts in the alveolar wall. Even though the pathogenesis of this fatal disorder remains unclear, transforming growth factor-β (TGF-β)-induced differentiation and proliferation of myofibroblasts is recognized as a primary event. The molecular pathways involved in TGF-β signalling are generally Smad-dependent yet Smad-independent pathways, including phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), have been recently proposed. In this research we established ex-vivo cultures of human lung fibroblasts and we investigated the role of the PI3K/Akt pathway in two critical stages of the fibrotic process induced by TGF-β: fibroblast proliferation and differentiation into myofibroblasts. Here we show that the pan-inhibitor of PI3Ks LY294002 is able to abrogate the TGF-β-induced increase in cell proliferation, in α- smooth muscle actin expression and in collagen production besides inhibiting Akt phosphorylation, thus demonstrating the centrality of the PI3K/Akt pathway in lung fibroblast proliferation and differentiation. Moreover, for the first time we show that PI3K p110δ and p110γ are functionally expressed in human lung fibroblasts, in addition to the ubiquitously expressed p110α and β. Finally, results obtained with both selective inhibitors and gene knocking-down experiments demonstrate a major role of p110γ and p110α in both TGF-β-induced fibroblast proliferation and differentiation. This finding suggests that specific PI3K isoforms can be pharmacological targets in IPF.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive and fatal illness whose pathogenesis remains poorly understood. Recent evidence suggests oxidative stress as a key player in the establishment/progression of lung fibrosis in animal models and possibly in human IPF. The aim of the present study was to characterize the cellular phenotype of fibroblasts derived from IPF patients and identify underlying molecular mechanisms.Methodology/Principal FindingsWe first analyzed the baseline differentiation features and growth ability of primary lung fibroblasts derived from 7 histology proven IPF patients and 4 control subjects at different culture passages. Then, we focused on the redox state and related molecular pathways of IPF fibroblasts and investigated the impact of oxidative stress in the establishment of the IPF phenotype. IPF fibroblasts were differentiated into alpha-smooth muscle actin (SMA)-positive myofibroblasts, displayed a pro-fibrotic phenotype as expressing type-I collagen, and proliferated lower than controls cells. The IPF phenotype was inducible upon oxidative stress in control cells and was sensitive to ROS scavenging. IPF fibroblasts also contained large excess of reactive oxygen species (ROS) due to the activation of an NADPH oxidase-like system, displayed higher levels of tyrosine phosphorylated proteins and were more resistant to oxidative-stress induced cell death. Interestingly, the IPF traits disappeared with time in culture, indicating a transient effect of the initial trigger.Conclusions/SignificanceRobust expression of α-SMA and type-I collagen, high and uniformly-distributed ROS levels, resistance to oxidative-stress induced cell death and constitutive activation of tyrosine kinase(s) signalling are distinctive features of the IPF phenotype. We suggest that this phenotype can be used as a model to identify the initial trigger of IPF.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease whose molecular pathogenesis remains unclear. In a recent paper, we demonstrated a key role for the PI3K pathway in both proliferation and differentiation into myofibroblasts of normal human lung fibroblasts treated with TGF-β. In this research, we assessed the expression of class I PI3K p110 isoforms in IPF lung tissue as well as in tissue-derived fibroblast cell lines. Moreover, we investigated the in vitro effects of the selective inhibition of p110 isoforms on IPF fibroblast proliferation and fibrogenic activity. IHC was performed on normal and IPF lung tissue. Expression levels of PI3K p110 isoforms were evaluated by western blot and flow cytometry analysis. Fibroblast cell lines were established from both normal and IPF tissue and the effects of selective pharmacological inhibition as well as specific gene silencing by small interfering RNAs were studied in vitro. No significant differences between normal and IPF tissue/tissue-derived fibroblasts were observed for the expression of PI3K p110 α, β and δ isoforms whereas p110γ was more greatly expressed in both IPF lung homogenates and ex vivo fibroblast cell lines. Myofibroblasts and bronchiolar basal cells in IPF lungs exhibited strong immunoreactivity for p110γ. Positive staining for the markers of proliferation proliferating cell nuclear antigen and cyclin D1 was also shown in cells of fibrolastic foci. Furthermore, both p110γ pharmacological inhibition and gene silencing were able to significantly inhibit proliferation rate as well as α-SMA expression in IPF fibroblasts. Our data suggest that PI3K p110γ isoform may have an important role in the etio-pathology of IPF and can be a specific pharmacological target.

The authors investigated the role of resveratrol (RV), a natural poliphenolic molecule with several biological activities, in transforming growth factor-β (TGF-β)-induced proliferation and differentiation of ex vivo human pulmonary fibroblasts into myofibroblasts. The effects of RV treatment were evaluated by analyzing TGF-β-induced α-smooth muscle actin (α-SMA) expression and collagen production, as well as cell proliferation of both normal and idiopathic pulmonary fibrosis (IPF) lung fibroblasts. Results demonstrate that RV inhibits TGF-β-induced cell proliferation of both normal and pathological lung fibroblasts, attenuates α-SMA expression at both the mRNA and protein levels, and also inhibits intracellular collagen deposition. In order to understand the molecular mechanisms, the authors also investigated the effects of RV treatment on signaling pathways involved in TGF-β-induced fibrosis. The authors show that RV inhibited TGF-β-induced phosphorylation of both extracellular signal-regulated kinases (ERK1/2) and the serine/threonine kinase, Akt. Moreover, RV treatment blocked the TGF-β-induced decrease in phosphatase and tensin homolog (PTEN) expression levels.

In this study, we evaluated the protective effect and therapeutic potential of the prostaglandin E(2) (PGE(2)) synthetic analog 16,16-dimethyl-PGE(2) (dmPGE(2)) in the animal model of pulmonary fibrosis induced by bleomycin. Mice subjected to intratracheal administration of bleomycin (1 mg/kg) received a dmPGE(2) dose of 30 microg/kg/day by continuous subcutaneous infusion. Bronchoalveolar lavage (BAL); immunohistochemical analysis for IL-1, TNF-alpha, and nitrotyrosine; measurement of fluid content in lung; myeloperoxidase activity assay; and lung histology were performed 1 week later. Lung histology and Sircol assay for collagen deposition were performed 3 weeks after treatments. Changes of body weight and survival rate were also evaluated at 1 and 3 weeks. Compared with bleomycin-treated mice, dmPGE(2) co-treated mice exhibited a reduced degree of body weight loss and mortality rate as well as of lung damage and inflammation, as shown by the significant reduction of: (1) lung infiltration by leukocytes; (2) myeloperoxidase activity; (3) IL-1, TNF-alpha, and nitrotyrosine immunostaining; (4) lung edema; and (5) histologic evidence of lung injury and collagen deposition. In a separate set of experiments, dmPGE(2) treatment was started 3 days after bleomycin administration, and the evaluation of lung damage and inflammation was assessed 4 days later. Importantly, delayed administration of dmPGE(2) also was able to protect from inflammation and lung injury induced by bleomycin. These results, indicating that dmPGE(2) is able to prevent and to reduce bleomycin-induced lung injury through its regulatory and anti-inflammatory properties, encourage further research to find new options for the treatment of pulmonary fibrosis.

This is the first report that shows a Tβ4 protective role in lung toxicity associated with BLEO in a mouse model. Future studies are needed to assess its putative antifibrotic properties.

The diagnostic assessment of patients with Interstitial Lung Disease (ILD) can be challenging due to the large number of possible causes. Moreover, the diagnostic approach can be limited by the severity of the disease, which may not allow invasive exams. To overcome this issue, the referral centers for ILD organized Multidisciplinary Teams (MDTs), including physicians and experts in complementary discipline, to discuss the management of doubtful cases of ILD. MDT is currently considered the gold standard for ILD diagnosis, but it is not often simple to organize and, furthermore, rheumatologists are still not always included. In fact, even if rheumatologic conditions represent a common cause of ILD, they are sometimes difficult to recognize, considering the variegated clinical features and their association with all possible radiographic patterns of ILD. The first objective of this review is to describe the clinical, laboratory, and instrumental tests that can drive a diagnosis toward a possible rheumatic disease. The secondary objective is to propose a set of first-line tests to perform in all patients in order to recognize any possible rheumatic conditions underlying ILD.