Immunohistochemical studies of pulmonary remodeling in mice exposed to chronic cigarette smoke

Chronic Obstructive Pulmonary Disease (COPD) is a progressive and debilitating disease, associated primarily with cigarette smoke exposure, and it is characterized by chronic inflammation of the airways and lung parenchyma and changes in the pulmonary vasculature. Four anatomic lesions are recognizable in COPD: emphysema, small airway remodeling (SAR), vascular remodeling, which may be associated with pulmonary hypertension, and chronic bronchitis, characterized by excessive mucus secretion. Several mechanisms are presumed to cause these changes and support related symptoms. In particular, chronic inflammation and its related consequences, which include epithelial necrosis and apoptosis, changes in cell phenotype and function, proliferation and compartmentalization of specific cells in some pulmonary areas, and deposition of excessive extracellular matrix, have been implicated as the cause of the different clinical presentations of COPD. Although a lot of studies have been carried out in the last twenty years, many facets of the pathogenesis of COPD are not fully understood. Some murine strains mirror some human phenotypes after smoke exposure. Therefore, it was of interest to investigate in these strains whether changes in different endogenous factors, whose expression can influence alveolar destruction, repair and anatomical remodeling, are associated with changes characterizing different phenotypes of the disease. This study has been approached by using immunohistochemistry in order to have information on the expression and distribution of these factors in pulmonary structures at selected time points after the start of smoke exposure. By using this methodological approach, the expression of important fibrogenic cytokines (i.e. TGF-β, PDGF-B and CTGF) at various time points after cigarette smoke exposure have been investigated in C57 Bl/6J mice, which develop significant emphysema, and DBA/2 mice that develop changes similar to those of the "pulmonary fibrosis /emphysema syndrome”. Some other factors that are indicative of apoptosis (cleaved caspase-3), senescence (p16ink4A and p21), regeneration (PCNA and Ki-67) or are implicated in fibrosis resolution and in apoptosis resistance (MyoD) were studied. In order to evaluate the involvement of these factors in parenchymal (i.e., vascular and peri-bronchiolar fibrosis) and airways remodeling (such as goblet cell metaplasia, fibrous and muscular remodeling), we used specific staining techniques (i.e., Masson’s trichrome and PAS staining) or an immunohistochemical analysis for -SMA, at the various experimental time points. A further investigation has been carried out to investigate the expression of three purinergic receptors (i.e. P2X4, P2X7 and P2Y2), which have been recently involved in COPD pathogenesis by acting as danger signals and important mediators of inflammation. The data obtained suggest that apoptosis, senescence and proliferation, that are induced at different rate and time points by inflammatory and fibrotic cytokines, play a role in early or late appearance of the remodeling processes that we observe in these strains of mice. Additionally, the necrosis of alveolar epithelial cells caused by enzyme release and oxidative damage (as revealed by MMP-9 and 8-OHdG positivity) characterize, at different extent, the lung responses of C57 Bl/6J and DBA/2 mice. The activation of purinergic receptors may be due to the release of alarmins, such as ATP and UTP, (which are the main ligands for P2X4, P2X7 and P2Y2) following the necrosis of alveolar epithelial cells.