Evaluation of an ex vivo fibrogenesis model using human lung slices prepared from small tissues.

BACKGROUND: In recent years, there have been breakthroughs in the preclinical research of respiratory diseases, such as organoids and organ tissue chip models, but they still cannot provide insight into human respiratory diseases well. Human lung slices model provides a promising in vitro model for the study of respiratory diseases because of its preservation of lung structure and major cell types. METHODS: Human lung slices were manually prepared from small pieces of lung tissues obtained from lung cancer patients subjected to lung surgery. To evaluate the suitability of this model for lung fibrosis research, lung slices were treated with CdCl2 (30 µM), TGF-ß1 (1 ng/ml) or CdCl2 plus TGF-ß1 for 3 days followed by toxicity assessment, gene expression analysis and histopathological observations. RESULTS: CdCl2 treatment resulted in a concentration-dependent toxicity profile evidenced by MTT assay as well as histopathological observations. In comparison with the untreated group, CdCl2 and TGF-ß1 significantly induces MMP2 and MMP9 gene expression but not MMP1. Interestingly, CdCl2 plus TGF-ß1 significantly induces the expression of MMP1 but not MMP2, MMP7 or MMP9. Microscopic observations reveal the pathogenesis of interstitial lung fibrosis in the lung slices of all groups; however, CdCl2 plus TGF-ß1 treatment leads to a greater alveolar septa thickness and the formation of fibroblast foci-like pathological features. The lung slice model is in short of blood supply and the inflammatory/immune-responses are considered minimal. CONCLUSIONS: The results are in favor of the hypothesis that idiopathic pulmonary fibrosis (IPF) is mediated by tissue damage and abnormal repair. Induction of MMP1 gene expression and fibroblast foci-like pathogenesis suggest that this model might represent an early stage of IPF.

An ex vivo approach to the differential parenchymal responses induced by cigarette whole smoke and its vapor phase.

Using a rat lung slice model, this study compared the stress responses induced by cigarette whole smoke (WS) to that induced by the vapor phase (VP) of the smoke. Following a 3-day exposure, lung slices exposed to 4, 10 and 20% WS retained 85, 42 and 16% relative survival respectively in comparison to the air-exposed ones. Consistently, histological observations revealed concentration-related alveolar damages in the lung slices. Expression of 5 stress-response genes was examined following a single 30 min exposure to 4% WS or VP. WS exposure resulted in 4, 11 and 50-fold induction of IL-1ß, kinin type I receptor (B1R) and CYP1A1 genes, respectively, while CYP1B1 and TNF-α genes expression was found only two times higher in comparison to VP group. Since cigarette WS consists of particulate and vapor phases, these results highlight the preferential or synergistic role of the particulate phase in the induction of IL-1ß, B1R and CYP1A1 genes and that VP did not have comparable effects on expression of these genes. However, both phases fairly contributed to the induction of CYP1B1 and TNF-α genes. VP was the fraction responsible for the toxic effect since WS did not produce further toxicity. The 4% whole smoke deposited about 7.1 µg/cm² of total particulate matter (TPM) to the exposure chamber which may account for observed differential stress responses in the lung slices.

Cigarette smoke-induced kinin B1 receptor promotes NADPH oxidase activity in cultured human alveolar epithelial cells.

Pulmonary inflammation is an important pathological feature of tobacco smoke-related lung diseases. Kinin B1 receptor (B1R) is up-regulated in the rat trachea chronically exposed to cigarette-smoke. This study aimed at determining (1) whether exposure to total particulate matter of the cigarette smoke (TPM) can induce B1R in human alveolar epithelial A549 cells, (2) the mechanism of B1R induction, (3) the functionality of de novo synthesized B1R, and (4) the role of B1R in TPM-induced increase of superoxide anion (O2(●⁻)) level. Results show that A549 cells exposed to 10 µg/ml TPM increased O2(●⁻) level along with B1R (protein and mRNA) and IL-1ß mRNA. In contrast, B2R and TNF-α mRNA were not affected by TPM. The increasing effect of TPM on O2(●⁻) level was not significantly affected by the B1R antagonist SSR240612. TPM-increased B1R mRNA was prevented by co-treatments with N-acetyl-l-cysteine (potent antioxidant), diphenyleneiodonium (NADPH oxidase inhibitor), IL-1Ra (interleukin-1R antagonist) and SN-50 (specific inhibitor of NF-kB activation) but not by pentoxifylline (TNF-α release inhibitor), indomethacin and niflumic acid (COX-1 and -2 inhibitors). Stimulation of B1R with a selective agonist (des-Arg9-BK, 10 µM; 30 min) increased O2(●⁻)production which was prevented by apocynin and diphenyleneiodonium (NADPH oxidase inhibitors). Data suggest that the increased expression of B1R by TPM in A549 cells is mediated by oxidative stress, IL-1ß and NF-kB but not by cyclooxygenases or TNF-α. The amplification of O2(●⁻) levels via the activation of B1R-NADPH oxidase may exacerbate pulmonary inflammation and contribute to the chronicity of tobacco smoke-related lung diseases.

Mechanism of cigarette smoke-induced kinin B(1) receptor expression in rat airways.

Pulmonary inflammation is an important pathological feature of tobacco smoke related lung diseases such as chronic obstructive pulmonary disease (COPD). Kinin type 1 and type 2 receptors (B(1)R, B(2)R) are known to be associated with inflammatory responses of the lungs and other organs. In this study, we investigated whether cigarette smoke-induced airway inflammation could up-regulate B(1)R and B(2)R in correlation with IL-1ß and TNF-α. Rat lung slices treated with 5 µg/ml total particulate matter (TPM) of cigarette smoke for 24 h showed an enhanced expression of B(1)R and IL-1ß by 5-fold and 30-fold, respectively, in comparison to vehicle treatment (dimethyl sulfoxide). However, higher concentrations of TPM failed to induce B(1)R. No significant increase of B(2)R or TNF-α gene induction was observed. IL-1 receptor antagonist (IL-1Ra, 2 ng/ml) significantly blocked B(1)R gene induction by TPM, while 500 µM pentoxifylline, TNF-α inhibitor, reduced it partially. Western blot analysis showed a 2-fold enhanced expression of B(1)R in rat lung slices treated with 5 µg/ml TPM for 24 h and such protein expression was totally blocked by a co-treatment with IL-1Ra but not with pentoxifylline. In addition to the lower airways, rat trachea subchronically exposed to cigarette whole smoke exhibited 11-fold B(1)R gene induction in comparison with those exposed only to air. Our results demonstrate the involvement of B(1)R in cigarette smoke-induced airway inflammation through a mechanism which is mediated by the pro-inflammatory cytokine IL-1ß.