Oxidant stress has been implicated in the pathogenesis of chronic lung disorders like idiopathic pulmonary fibrosis. However, mechanisms that link oxidant stress to fibrogenesis remain partially elucidated. Emerging data suggest an important role for the extracellular thiol/disulfide redox environment. The cysteine (Cys)/cystine (CySS) redox couple represents the predominant low-molecular-weight thiol/disulfide pool found in plasma and is sensitive to aging, smoking, and other host factors. We hypothesized that an oxidized extracellular Cys/CySS redox potential (E(h) Cys/CySS) affects lung fibroblasts by inducing intracellular signals that stimulate proliferation and matrix expression. We tested this hypothesis in primary murine lung fibroblasts and found that an oxidized E(h) Cys/CySS (-46 mV) stimulated lung fibroblast proliferation. Furthermore, it stimulated their expression of fibronectin, a matrix glycoprotein highly expressed in fibrotic lung diseases and implicated in lung injury. This stimulatory effect was dependent on protein kinase C activation. Oxidant stress also increased the phosphorylation of cAMP response element binding protein, a transcription factor known for its ability to stimulate fibronectin expression, and increased the expression of mRNAs and proteins coding for the transcription factors nuclear factor (NF)-kappaB and mothers against decapentaplegic homolog 3. Fibroblasts cultured in normal (-80 mV) or reduced (-131 mV) E(h) Cys/CySS showed less induction. Furthermore, fibronectin expression in response to an oxidized E(h) Cys/CySS was associated with expression of transforming growth factor-beta1 (TGF-beta1) and was inhibited by an anti-TGF-beta1 antibody and SB-431542, a TGF-beta1 receptor inhibitor. These studies suggest that extracellular oxidant stress activates redox-sensitive pathways that stimulate lung fibroblast proliferation and matrix expression through upregulation of TGF-beta1.

Background The objective of this study was to evaluate the factors predictive of 28-day mortality and admission to Intensive Care Unit (ICU) in patients with ANCA-related pulmonary vasculitis. Methods We reviewed the medical records and imaging studies of 65 patients diagnosed with ANCA-related vasculitis hospitalized with pulmonary complications between February 1985 and November 2002. All patients underwent open or video-assisted thoracoscopic lung biopsy, had a positive ANCA serology, and were negative for glomerular basement membrane antibodies. Results At presentation, 72% had dyspnea, 68% fever, 47% cough, 45% elevated blood pressure, 32.3% hemoptysis, 26.1% sinus involvement, 15% renal failure, and 4.6% scleritis. Pathological findings included alveolar hemorrhage (60%), granulomatous inflammation (46%), and capillaritis (38%). A significant number required mechanical ventilation (27.7%), hemodialysis (24.6%), continuous renal replacement therapy (3.1%), and plasmapheresis (3.1%). The 28-day mortality was 16.9% (11/65). Mechanical ventilation (OR 68, P < 0.005), admission to ICU (OR 18.5, P < 0.01), and blood transfusion (OR 22.4, P < 0.004) were strong predictors of increased mortality within 28 days after admission. Respiratory failure (OR 31, P < 0.0007), hemoptysis (OR 2.9, P < 0.06), smoking (OR 5.9, P < 0.02), and acute renal failure (OR 7.8, P < 0.01) were also predictors for admission to the ICU. Conclusion In patients with ANCA-related pulmonary vasculitis several clinical factors, but not pathologic findings or ANCA titers, are associated with ICU admission and/or 28-day mortality.

Insulin receptor-substrate-1 (IRS-1) is a docking protein for several tyrosine kinase receptors. Upon tyrosine phosphorylation, IRS-1 binds to signaling molecules that express Src homology 2 (SH-2) binding domains, including phosphatidylinositol 3-kinase (PI 3-kinase), phosphotyrosine phosphatase SHP-2 (Syp), Nck, Crk and Grb-2. Hydrogen peroxide (H(2)O(2)) induces tyrosine phosphorylation of key signaling mediators presumably by inhibition of tyrosine phosphatases. In many cell types, the activation of extracellular signal-related kinases (e.g. MAPK) and other protein kinases by H(2)O(2) leads to transcriptional activation. In the current study, we examined the effect of H(2)O(2) on IRS-1 tyrosine phosphorylation in primary cultured rat cerebellar granule neurons. H(2)O(2) stimulated the rapid tyrosine phosphorylation of IRS-1 and p42/p44 MAP kinase, and induced its association with PI 3-kinase. H(2)O(2)-induced IRS-1 phosphorylation was rapidly reversible (5 min) whereas MAPK phosphorylation persisted for up to 1 h. NMDA reversed H(2)O(2)-mediated tyrosine phosphorylation of IRS-1 and its association with PI 3-kinase. The dephosphorylation of IRS-1 by NMDA was calcium-dependent and was inhibited by the calcineurin inhibitor cyclosporine. Calmodulin-dependent tyrosine phosphatase activity of calcineurin was observed in vitro using both immunoprecipitated and recombinant tyrosine-phosphorylated IRS-1 as substrates. These data highlight the role of multiple phosphatases in the regulation of IRS-1 tyrosine phosphorylation and identify a novel functional property of calcineurin.

Insulin receptor‐substrate‐1 (IRS‐1) is a docking protein for several tyrosine kinase receptors. Upon tyrosine phosphorylation, IRS‐1 binds to signaling molecules that express Src homology 2 (SH‐2) binding domains, including phosphatidylinositol 3‐kinase (PI 3‐kinase), phosphotyrosine phosphatase SHP‐2 (Syp), Nck, Crk and Grb‐2. Hydrogen peroxide (H2O2) induces tyrosine phosphorylation of key signaling mediators presumably by inhibition of tyrosine phosphatases. In many cell types, the activation of extracellular signal‐related kinases (e.g. MAPK) and other protein kinases by H2O2 leads to transcriptional activation. In the current study, we examined the effect of H2O2 on IRS‐1 tyrosine phosphorylation in primary cultured rat cerebellar granule neurons. H2O2 stimulated the rapid tyrosine phosphorylation of IRS‐1 and p42/p44 MAP kinase, and induced its association with PI 3‐kinase. H2O2‐induced IRS‐1 phosphorylation was rapidly reversible (5 min) whereas MAPK phosphorylation persisted for up to 1 h. NMDA reversed H2O2‐mediated tyrosine phosphorylation of IRS‐1 and its association with PI 3‐kinase. The dephosphorylation of IRS‐1 by NMDA was calcium‐dependent and was inhibited by the calcineurin inhibitor cyclosporine. Calmodulin‐dependent tyrosine phosphatase activity of calcineurin was observed in vitro using both immunoprecipitated and recombinant tyrosine‐phosphorylated IRS‐1 as substrates. These data highlight the role of multiple phosphatases in the regulation of IRS‐1 tyrosine phosphorylation and identify a novel functional property of calcineurin.

Oxidant stress has been implicated in the pathogenesis of chronic fibrosing lung disorders like idiopathic pulmonary fibrosis, and environmental and host factors capable of causing chronic oxidant stress have been identified. However, the mechanisms that link oxidant stress to fibrogenesis remain only partially elucidated. We hypothesize that extracellular oxidant stress affects lung fibroblasts directly by inducing intracellular pathways (ie, redox signaling) that stimulate their proliferation and matrix expression. In experiments designed to test this hypothesis, we showed that extracellular oxidant stress caused by oxidation of the thiol disulfide cysteine (Cys)/cystine (CySS) redox couple stimulates lung fibroblast proliferation. This is intriguing because most of the available research on oxidant stress has focused on intracellular oxidant stress and glutathione, the most abundant low-molecular-weight thiol in cells. However, evidence suggests that the extracellular thiol/disulfide redox environment may also be important. The Cys/CySS redox couple represents the predominant low-molecular-weight thiol/disulfide pool found in plasma, and it is sensitive to many host factors (eg, aging and smoking). Further work revealed that lung fibroblasts cultured in the setting of oxidized extracellular Cys/CySS redox (246 mV) showed increased expression of fibronectin, a matrix glycoprotein highly expressed in fibrotic lung disease and implicated in lung injury and repair. This stimulatory effect was related to increased fibronectin gene transcription, was enhanced by nicotine, and was blocked by inhibitors of protein kinase C activation. Fibroblasts cultured on normal medium (280 mV) or reduced medium (2130) medium showed no induction. Lung fibroblasts cultured in oxidized extracellular Cys/CySS redox also showed phosphorylation of CREB, a transcription factor known for its ability to stimulate fibronectin expression. Other transcription factors stimulated were NFkB and Smad3. Together, these studies suggest that extracellular oxidant stress, through oxidation of the thiol/disulfide couple Cys/CySS, activates redox-sensitive pathways that stimulate the differential expression of genes that enhance fibroblast proliferation and matrix deposition.