Study of lung fibrogenesis induced by several noxae in vivo and in vitro models

Pulmonary diseases, like chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), are chronic and progressive disorders that severely affect the airways and other structures of the respiratory system. COPD and IPF, characterized by quite distinct clinical and pathological features, are both related to long-term inhalation of tobacco smoking. Fibrogenesis is a common pathological feature of many chronic respiratory diseases but the specific mechanisms are still unclear. Repeated insults and/or aberrant repair events are the main processes that lead to fibrosis, and ultimately result in either non-reversible airways obstruction, typical of COPD, and impaired gas exchange and parenchymal consolidation, commonly observed in IPF patients. A variety of pathogenic mechanisms has been proposed for these diseases, including oxidative stress and accelerated cellular senescence. In order to achieve new advances in the knowledge of the pathogenetic mechanisms underlying lung fibrogenesis, the study of the fibrogenic process was carried out in vivo and in vitro models, in which the fibrotic alterations were induced by several etiological agents. The first study completed our previous works on airways remodelling by using two mice strains, C57 BL/6J and DBA/2. We previously demonstrated that these mice strains present different phenotypical responses after the exposure to cigarette smoke. In particular, the characteristics of the two strains influenced the different changes in parenchymal and airway areas observed at different time points of cigarette smoke exposure. We also demonstrated a correspondence between oxidants-induced DNA damage, cellular senescence and the presence of senescence secreted factors involved in the onset of fibrous remodelling. Along with these markers in the current study, we investigated whether alterations in the expression of histone deacetylases (SIRT1 and HDAC-2) and factors involved in the inflammatory process and progression of pulmonary lesions (p-p38 and NF-kB) contribute to the pathophysiology of airway remodelling. An earlier upregulation of p-p38 and NF-kB, together with a lower expression of SIRT1 and HDAC-2 is in line with a higher sensitivity to oxidative stress observed in the mice strains that may be one of the key factors in the development of airways fibrotic remodelling. In another in vivo study, the effect of environmental tobacco smoke (ETS) in a model of bleomycin-induced pulmonary fibrosis in C57 BL/6J mice was evaluated, in order to understand the contribution of ETS in fibrogenesis. The combined effect of ETS and BLM was able to seriously compromise the lung regenerative potential, when compared with the effects promoted by the treatment with only bleomycin. In this study, lung fibrosis was accompanied by an increase of extracellular matrix (ECM) proteins (collagens, fibronectin and elastin) and enzymes in charge of ECM reorganization (MMPs and LOXs). Interestingly, overexpression of the Complement system was also observed, together with other senescence-associated markers (cyclin-dependent kinases inhibitors, SERPINE1, and Sirtuins) suggesting an earlier induction of cellular senescence. Finally, in vitro studies were performed by using normal human lung fibroblast (NHLF) cells. A first model evaluated the involvement of the Complement system in the fibrotic remodelling. NHLF cells presented a significant increase in gene expression of COL1A1, ACTA2 and FN1 after the treatment with C3 Complement component. These results highlighted an initial stage of fibroblast differentiation into myofibroblast, a crucial event for the progression of the fibrotic process. A foremost role of the Complement system was further confirmed by exposing fibroblasts to a Complement 3a receptor (C3aR) antagonist, SB290157, which induced a decline in the gene expression of COL1A1, ACTA2 and FN1 in a dose-response manner. In a different model we exposed NHLF cells to cigarette smoke extract (CSE) followed by transforming growth factor-β (TGF-β) treatment. The results obtained from this study suggest the existence of an interference of several stimuli in the fibrogenesis. Notwithstanding, further studies are still needed to ascertain which pathways promote fibrogenesis and to understand if they can be used as therapeutic targets for both COPD and IPF.