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.

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.

Emerging evidence suggests that air pollution increases the risk of cardiovascular disease (CVD) and metabolic disorders, adding to the global burden of disease attributable to lifestyle and behavioral factors. Although long interspersed nucleotide elements 1 (LINE-1) methylation has been associated with these disorders, no studies have simultaneously examined the effects of diet and air pollution exposure on DNA methylation. Herein, we evaluated the association of particulate matter (PM with aerodynamic diameters of less than 10 mm) exposure and adherence to Mediterranean Diet (MD) with LINE-1 methylation. Healthy women (n = 299), aged 15 to 80 years, were enrolled in a cross-sectional study. Dietary data and adherence to MD were assessed by a Food Frequency Questionnaire (FFQ) and Mediterranean Diet Score (MDS). PM10 levels during 1-month before recruitment were recorded by monitoring stations and assigned to each woman based on their residential address and day of recruitment. LINE-1 methylation in blood samples was assessed by pyrosequencing and reported as percentage of 5-methylcytosine (5mC). The Mann–Whitney U test, Spearman’s rank correlation test and linear regression models were applied. Our results demonstrated, for the first time, an inverse association between adherence to MD and exposure to PM10 with LINE-1 methylation: while higher monthly PM10 exposure decreases LINE-1 methylation level (β = −0.121; p = 0.037), the adherence to MD increases it (β = 0.691; p < 0.001). MDS seemed to interact with PM10 levels (p = 0.002) on LINE-1 methylation, as such we confirmed that the effect of MD decreased with increasing PM10 levels (β = 0.657; p < 0.001 in the first tertile; β = 0.573; p < 0.001 in the second tertile; β = 0.551; p < 0.001 in the third tertile). Thus, we suggest that LINE-1 methylation is a possible mechanism underpinning environment-related health effects, and encourage further research to evaluate whether the adherence to the MD could counteract the negative effect of PM10 exposure.

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.