Dysregulated Proinflammatory and Fibrogenic Phenotype of Fibroblasts in Cystic Fibrosis

Morbi-mortality in cystic fibrosis (CF) is mainly related to chronic lung infection and inflammation, uncontrolled tissue rearrangements and fibrosis, and yet the underlying mechanisms remain largely unknown. We evaluated inflammatory and fibrosis responses to bleomycin in F508del homozygous and wild-type mice, and phenotype of fibroblasts explanted from mouse lungs and skin. The effect of vardenafil, a cGMP-specific phosphodiesterase type 5 inhibitor, was tested in vivo and in culture. Responses of proinflammatory and fibrotic markers to bleomycin were enhanced in lungs and skin of CF mice and were prevented by treatment with vardenafil. Purified lung and skin fibroblasts from CF mice proliferated and differentiated into myofibroblasts more prominently and displayed higher sensitivity to growth factors than those recovered from wild-type littermates. Under inflammatory stimulation, mRNA and protein expression of proinflammatory mediators were higher in CF than in wild-type fibroblasts, in which CFTR expression reached similar levels to those observed in other non-epithelial cells, such as macrophages. Increased proinflammatory responses in CF fibroblasts were reduced by half with submicromolar concentrations of vardenafil. Proinflammatory and fibrogenic functions of fibroblasts are upregulated in CF and are reduced by vardenafil. This study provides compelling new support for targeting cGMP signaling pathway in CF pharmacotherapy

Quantitative determination of 5-hydroxy-N-methylpyrrolidone in urine for biological monitoring of N-methylpyrrolidone exposure.

The aim of this work was to validate a sensitive method for quantitative analysis of 5-hydroxy-N-methylpyrrolidone (5-HNMP) in urine. This compound has been recommended as a marker for biological monitoring of N-methylpyrrolidone (NMP) exposure. Different solvents and alternative methods of extraction including liquid-liquid extraction (LLE) on Chem Elut and solid-phase extraction (SPE) on Oasis HLB columns were tested. The most efficient extraction of 5-HNMP in urine was LLE with Chem Elut columns and dichloromethane as a solvent (consistently 22% of recovery). The urinary extracts were derivatized by bis(trimethylsilyl)trifluoroacetamide and analysed by gas chromatography-mass spectrometry (GC-MS) with tetradeutered 5-HNMP as an internal standard. The detection limit of this method is 0.017 mg/l urine with an intraassay precision of 1.6-2.6%. The proposed method of extraction is simple and reproducible. Four different m/z signal ratios of TMS-5-HNMP and tetralabelled TMS-5-HNMP have been validated and could be indifferently used in case of unexpected impurities from urine matrix

The role of vitamin E in the susceptibility of rat lung and liver microsomes to iron-stimulated peroxidation.

The production of thiobarbituric acid-reactive substances (TBA-RS) and ethane, two markers of the lipid peroxidation process, was evaluated in rat lung and liver microsomal membranes incubated in the presence of either ferrous ions or a mixture of ferric ions and ascorbate. Microsomal fractions isolated from lung tissue were more resistant than those isolated from the liver. Compared to Fe2+, the association of Fe3+/ascorbate seemed to be totally ineffective in stimulating peroxidation of lung microsomes. The fatty acid profile of lung and liver microsomal membranes could not be responsible for their different susceptibility to free radical degradation. The microsomal fraction isolated from lung showed a higher vitamin E concentration than the liver. The importance of vitamin E in protecting lung membranes was assessed by using lung and liver isolated from vitamin E-deficient and vitamin E-supplemented rats. For both lung and liver microsomal fractions an inverse relationship between vitamin E concentrations and the extent of lipid peroxidation was observed. However, although the vitamin E concentrations in lung and liver microsomes isolated from rats submitted to a vitamin E-deficient diet were not different, lung microsomes still exhibited a lower production of TBA-RS and ethane than liver. In addition to vitamin E, other factors must be involved to explain the resistance of lung microsomes to lipid peroxidation

Assessment of lipid peroxidation associated with lung damage induced by oxidative stress. In vivo and in vitro studies

The lung thiobarbituric acid-reactive substances (TBA-RS) content and the amount of ethane exhaled, two potential markers of the lipid peroxidation process, were measured in rats following intratracheal administration of chemicals stimulating the production of free radicals, i.e. paraquat, phorbol myristate acetate and ferrous ions. Five hours after treatment, autopsy revealed gross pulmonary damage but the lung TBA-RS and the ethane exhalation were not different from control animals. On the contrary, a large increase in ethane production was observed 2 hr after intraperitoneal administration of the hepatotoxic carbon tetrachloride. In vitro, incubation of lung and liver homogenates from control rats with ferrous iron led to the development of a lipid peroxidation process in both tissues but the accumulation of TBA-RS and ethane was much lower with homogenates from lung as compared to liver tissue. Those results suggest that the lung may be more resistant than the liver to the initiation and/or propagation of a lipid peroxidation process. The possibility that others markers than ethane and TBA-RS are more appropriate to detect this process in the lung must also be considered

Effect of oxygen concentration on production of ethane and thiobarbituric acid-reactive substances by peroxidizing lung and liver homogenates and formation of ethanol by peroxidizing docosahexaenoic acid preparations under hyperoxic conditions.

The oxygen dependence of ethane formation was investigated in rat lung and liver homogenates, incubated in sealed flasks, in which the peroxidation was stimulated by the addition of ferrous ions. For both tissues, the production of ethane was maximal under a 20% oxygenated gas phase, while hyperoxic conditions led to a decreased ethane in the gas phase. The formation of thiobarbituric acid-reactive substances (TBA-RS), another marker of the lipid peroxidation process, in the homogenates of lung and liver was strongly stimulated at 100% compared to 20% oxygen. Experiments were also carried out on iron-stimulated peroxidation of pure docosahexaenoic acid preparations, which under air led to a large production of ethane. As for tissue homogenates, the TBA-RS content was increased in the presence of 100% oxygen. Those conditions, however, did not induce an increase in ethane production but led to the formation of ethanol. Therefore, the quenching of ethyl radical by molecular oxygen seems to be a very attractive hypothesis to explain the lack of increased ethane production in favor of ethanol when iron-induced lipid peroxidation was stimulated by oxygen

Evaluation of three methods for the detection of DNA single-strand breaks in human lymphocytes: alkaline elution, nick translation, and single-cell gel electrophoresis.

The aim of this study is to assess the ability of three methods, alkaline elution (AE), nick translation (NT), and single-cell gel electrophoresis (SCGE), to detect DNA single-strand breaks (ssb) in human peripheral blood lymphocytes (HPBL) exposed in vitro to three genotoxic agents; gamma-rays, ethyl methanesulfonate (EMS) and benzo[a]pyrene diol epoxide (BPDE). The ultimate objective is to select the most feasible, sensitive, and reproducible method for the monitoring of populations exposed to genotoxic agents. AE and NT do not seem suitable assays. AE is able to detect DNA lesions induced by the three compounds, but only at relatively high doses (2 Gy, 5 mM EMS and 20 microM BPDE). With NT, DNA alterations induced by gamma-rays are not detected and ssb are only evidenced after exposure to EMS (80 mM), which already alters the viability of the lymphocytes. Nick translation is able to detect ssb induced by 10 microM BPDE. Compared to the other assays, the sensitivity of the SCGE assay is significantly higher since statistically significant changes were detected after incubation with 0.5 mM EMS and 1.25 microM BDPE. SCGE is a relatively simple method, not time-consuming and applicable to a large number of samples per working day. In conclusion, on the basis of the results of this in vitro comparison, SCGE seems a promising method for the monitoring of populations exposed to genotoxic chemicals