Expression of inducible nitric oxide synthase in healthy pleura and in malignant mesothelioma

In this study we investigated the immunohistochemical expression of inducible nitric oxide synthase (iNOS) in a set of normal pleural mesothelial tissues, malignant mesotheliomas, mesothelioma cell lines and metastatic pleural adenocarcinomas. Furthermore, the expression of mRNA was assessed in four malignant mesothelioma cell lines in culture. Apoptosis and vascular density in malignant mesotheliomas was assessed by the TUNEL method and by immunohistochemistry with an antibody against FVIII-related antigen. Immunohistochemically mesothelial cells in non-neoplastic healthy pleural tissues were mostly negative for iNOS. Positivity for iNOS was observed in 28/38 (74%) and 24/25 (96%) of malignant mesotheliomas and metastatic pleural adenocarcinomas, respectively. Epithelial and mixed mesotheliomas expressed more often strong iNOS immunoreactivity compared to the sarcomatoid subtype (P = 0.023). Moreover, metastatic adenocarcinomas expressed more often iNOS positivity than mesotheliomas (P = 0.021). Experiments with the cell lines confirmed that malignant mesothelioma cells are capable of synthesizing iNOS. No significant association was found between iNOS expression and apoptosis or vascular density in malignant mesotheliomas. The higher expression of iNOS in the epithelial subtype of mesothelioma and pleural metastatic adenocarcinoma might be due to an increased sensitivity of these cell types to cytokine-mediated iNOS upregulation. The strong expression of iNOS suggests a putative role for NO in the growth and progression of these tumours. © 2000 Cancer Research Campaig

Modulation of glutaredoxin in the lung and sputum of cigarette smokers and chronic obstructive pulmonary disease

BACKGROUND: One typical feature in chronic obstructive pulmonary disease (COPD) is the disturbance of the oxidant/antioxidant balance. Glutaredoxins (Grx) are thiol disulfide oxido-reductases with antioxidant capacity and catalytic functions closely associated with glutathione, the major small molecular weight antioxidant of human lung. However, the role of Grxs in smoking related diseases is unclear. METHODS: Immunohistochemical and Western blot analyses were conducted with lung specimens (n = 45 and n = 32, respectively) and induced sputum (n = 50) of healthy non-smokers and smokers without COPD and at different stages of COPD. RESULTS: Grx1 was expressed mainly in alveolar macrophages. The percentage of Grx1 positive macrophages was significantly lower in GOLD stage IV COPD than in healthy smokers (p = 0.021) and the level of Grx1 in total lung homogenate decreased both in stage I–II (p = 0.045) and stage IV COPD (p = 0.022). The percentage of Grx1 positive macrophages correlated with the lung function parameters (FEV1, r = 0.45, p = 0.008; FEV1%, r = 0.46, p = 0.007, FEV/FVC%, r = 0.55, p = 0.001). Grx1 could also be detected in sputum supernatants, the levels being increased in the supernatants from acute exacerbations of COPD compared to non-smokers (p = 0.013) and smokers (p = 0.051). CONCLUSION: The present cross-sectional study showed that Grx1 was expressed mainly in alveolar macrophages, the levels being decreased in COPD patients. In addition, the results also demonstrated the presence of Grx1 in extracellular fluids including sputum supernatants. Overall, the present study suggests that Grx1 is a potential redox modulatory protein regulating the intracellular as well as extracellular homeostasis of glutathionylated proteins and GSH in human lung

Heme oxygenase-1 and carbon monoxide in pulmonary medicine

Heme oxygenase-1 (HO-1), an inducible stress protein, confers cytoprotection against oxidative stress in vitro and in vivo. In addition to its physiological role in heme degradation, HO-1 may influence a number of cellular processes, including growth, inflammation, and apoptosis. By virtue of anti-inflammatory effects, HO-1 limits tissue damage in response to proinflammatory stimuli and prevents allograft rejection after transplantation. The transcriptional upregulation of HO-1 responds to many agents, such as hypoxia, bacterial lipopolysaccharide, and reactive oxygen/nitrogen species. HO-1 and its constitutively expressed isozyme, heme oxygenase-2, catalyze the rate-limiting step in the conversion of heme to its metabolites, bilirubin IXα, ferrous iron, and carbon monoxide (CO). The mechanisms by which HO-1 provides protection most likely involve its enzymatic reaction products. Remarkably, administration of CO at low concentrations can substitute for HO-1 with respect to anti-inflammatory and anti-apoptotic effects, suggesting a role for CO as a key mediator of HO-1 function. Chronic, low-level, exogenous exposure to CO from cigarette smoking contributes to the importance of CO in pulmonary medicine. The implications of the HO-1/CO system in pulmonary diseases will be discussed in this review, with an emphasis on inflammatory states

Pleiotropic effect of the proton pump inhibitor esomeprazole leading to suppression of lung inflammation and fibrosis

Background: The beneficial outcome associated with the use of proton pump inhibitors (PPIs) in idiopathic pulmonary fibrosis (IPF) has been reported in retrospective studies. To date, no prospective study has been conducted to confirm these outcomes. In addition, the potential mechanism by which PPIs improve measures of lung function and/or transplant-free survival in IPF has not been elucidated. Methods: Here, we used biochemical, cell biological and preclinical studies to evaluate regulation of markers associated with inflammation and fibrosis. In our in vitro studies, we exposed primary lung fibroblasts, epithelial and endothelial cells to ionizing radiation or bleomycin; stimuli typically used to induce inflammation and fibrosis. In addition, we cultured lung fibroblasts from IPF patients and studied the effect of esomeprazole on collagen release. Our preclinical study tested efficacy of esomeprazole in a rat model of bleomycin-induced lung injury. Furthermore, we performed retrospective analysis of interstitial lung disease (ILD) databases to examine the effect of PPIs on transplant-free survival. Results: The cell culture studies revealed that esomeprazole controls inflammation by suppressing the expression of pro-inflammatory molecules including vascular cell adhesion molecule-1, inducible nitric oxide synthase, tumor necrosis factor-alpha (TNF-alpha) and interleukins (IL-1 beta and IL-6). The antioxidant effect is associated with strong induction of the stress-inducible cytoprotective protein heme oxygenase-1 (HO1) and the antifibrotic effect is associated with potent inhibition of fibroblast proliferation as well as downregulation of profibrotic proteins including receptors for transforming growth factor beta (TGF beta), fibronectin and matrix metalloproteinases (MMPs). Furthermore, esomeprazole showed robust effect in mitigating the inflammatory and fibrotic responses in a murine model of acute lung injury. Finally, retrospective analysis of two ILD databases was performed to assess the effect of PPIs on transplant-free survival in IPF patients. Intriguingly, this data demonstrated that IPF patients on PPIs had prolonged survival over controls (median survival of 3.4 vs 2 years). Conclusions: Overall, these data indicate the possibility that PPIs may have protective function in IPF by directly modulating the disease process and suggest that they may have other clinical utility in the treatment of extra-intestinal diseases characterized by inflammatory and/or fibrotic phases.Stanford School of Medicine [1049528-149- KAVFB]; Tobacco-Related Disease Research Program of the University of California [20FT-0090]; National Institutes of Health National Heart, Lung, and Blood Institute [5K01HL118683, P01HL114470]; Houston Methodist Research Institute [25150001]; Stanford SPARK Translational Research ProgramSCI(E)[email protected]

Expression of oxidant and antioxidant enzymes in human lung and interstitial lung diseases

Abstract Antioxidants function as blockers of radical processes and eliminate harmful reactive oxygen species (ROS) produced during normal cellular metabolism. A complex antioxidant defence system has evolved to protect the cellular homeostasis. This system includes antioxidant enzymes (AOEs), such as superoxide dismutases (SODs), which are intracellular MnSOD and CuZnSOD and extracellular ECSOD, H2O2 scavenging enzymes catalase and glutathione peroxidase, and hemeoxygenase-1 (HO-1), an important enzyme in heme metabolism, which has also been suggested to have antioxidant capacities. ROS play an important role in the pathogenesis of interstitial lung diseases. These diseases represent a group of disorders with different etiology, histopathology, treatment and prognosis. Sarcoidosis, extrinsic allergic alveolitis and two different forms of idiopathic pulmonary fibrosis, usual interstitial pneumonia (UIP) and desquamative interstitial pneumonia (DIP) were included in this study. The purpose of this research was to evaluate the expressions of inducible nitric oxide synthase (i-NOS), endothelial nitric oxide synthase (e-NOS) and xanthine oxidase (XAO), oxidant generating enzymes commonly associated with tissue injury, and, on the other hand, the expressions of AOEs suggested to be involved in the defence of lung tissue against oxidant stress. The methods included immunohistochemistry on lung biopsies (n=48) and Western blotting, Northern blotting or reverse polymerase chain reaction (RT-PCR) on human inflammatory cells and cells obtained from bronchoalveolar lavage. I-NOS was intensively expressed in inflammatory, but not in fibrotic lesions, similar e-NOS expression was found in control lung and in all interstitial lung diseases, while XAO was mainly negative. MnSOD and HO-1 were highly expressed in the granulomas of sarcoidosis. In contrast the expressions of MnSOD and HO-1 in late fibrotic lesions of UIP were low or undetectable by immunohistochemistry. CuZnSOD and catalase showed similar immunoreactivity in healthy and diseased lung. A cell specific expression and regulation of various enzymes may play an important role during acute inflammatory diseases and also in the progression of lung fibrogenesis

Cell specific expression of peroxiredoxins in human lung and pulmonary sarcoidosis

Background: Six proteins of the peroxiredoxin (Prx) family have recently been characterised which have the capacity to decompose hydrogen peroxide in vivo and in vitro. These proteins may have an important role in the protection of human lung against endogenous and exogenous oxidant stress. However, the expression and distribution of these proteins in healthy human lung and diseased lung tissue is unknown. Methods: The cell specific expression of Prxs in healthy lung tissue from four non-smokers and in parenchymal tissue from 10 subjects with pulmonary sarcoidosis was investigated by immunohistochemistry, and expression of these proteins in various cultured lung cells and cells of bronchoalveolar lavage (BAL) fluid of controls and patients with sarcoidosis was assessed by Western blot analysis. Results: All six Prxs could be synthesised in cultured human lung cells. The bronchial epithelium showed moderate to high expression of Prxs I, III, V and VI, the alveolar epithelium expressed mainly Prxs V and VI, and alveolar macrophages expressed mainly Prxs I and III. Granulomas of subjects with sarcoidosis expressed mainly Prxs I and III. Samples of BAL fluid from controls and from subjects with sarcoidosis had very similar findings, except that Prxs II and III had a tendency for increased immunoreactivity in sarcoidosis tissue. Conclusions: Prxs I, III, V, and VI, in particular, have prominent and cell specific expression in human lung tissue. High expression of Prxs I and III in granulomas and alveolar macrophages of sarcoidosis parenchyma may have a significant effect on the oxidant burden and the progression of lung injury in this disease