Background Persistence of myofibroblasts is believed to contribute to the development of fibrosis in idiopathic pulmonary fibrosis (IPF). Transforming growth factor b1 (TGFb1) irreversibly converts fibroblasts into pathological myofibroblasts, which express smooth muscle a-actin (a-SMA) and produce extracellular matrix proteins, such as procollagen I (a1). Reactive oxygen species produced by NADPH oxidases (NOXs) have been shown to regulate cell differentiation. It was hypothesised that NOX could be expressed in parenchymal pulmonary fibroblasts and could mediate TGFb1-stimulated conversion of fibroblasts into myofibroblasts. Methods Fibroblasts were cultured from the lung of nine controls and eight patients with IPF. NOX4, a-SMA and procollagen I (a1) mRNA and protein expression, reactive oxygen species production and Smad2/3 phosphorylation were quantified, in the absence and in the presence of incubation with TGFb1. Migration of platelet-derived growth factor (PDGF)-induced fibroblasts was also assessed. Results It was found that (1) NOX4 mRNA and protein expression was upregulated in pulmonary fibroblasts from patients with IPF and correlated with mRNA expression of a-SMA and procollagen I (a1) mRNA; (2) TGFb1 upregulated NOX4, a-SMA and procollagen I (a1) expression in control and IPF fibroblasts; (3) the change in a-SMA and procollagen I (a1) expression in response to TGFb1 was inhibited by antioxidants and by a NOX4 small interfering RNA (siRNA); (4) NOX4 modulated a-SMA and procollagen I (a1) expression by controlling activation of Smad2/3; and (5) NOX4 modulated PDGF-induced fibroblast migration. Conclusion NOX4 is critical for modulation of the pulmonary myofibroblast phenotype in IPF, probably by modulating the response to TGFb1 and PDGF.

We investigated a possible beneficial role for bilirubin, one of the products of heme degradation by the cytoprotective enzyme heme oxygenase-1 in counteracting Escherichia coli endotoxin-mediated toxicity. Homozygous jaundice Gunn rats, which display high plasma bilirubin levels due to deficiency of glucuronyl transferase activity, and Sprague-Dawley rats subjected to sustained exogenous bilirubin administration were more resistant to endotoxin (LPS)-induced hypotension and death compared with nonhyperbilirubinemic rats. LPS-stimulated production of nitric oxide (NO) was significantly decreased in hyperbilirubinemic rats compared with normal animals; this effect was associated with reduction of inducible NO synthase (NOS2) expression in renal, myocardial, and aortic tissues. Furthermore, NOS2 protein expression and activity were reduced in murine macrophages stimulated with LPS and preincubated with bilirubin at concentrations similar to that found in the serum of hyperbilirubinemic animals. This effect was secondary to inhibition of NAD(P)H oxidase since 1) inhibition of NAD(P)H oxidase attenuated NOS2 induction by LPS, 2) bilirubin decreased NAD(P)H oxidase activity in vivo and in vitro, and 3) down-regulation of NOS2 by bilirubin was reversed by addition of NAD(P)H. These findings indicate that bilirubin can act as an effective agent to reduce mortality and counteract hypotension elicited by endotoxin through mechanisms involving a decreased NOS2 induction secondary to inhibition of NAD(P)H oxidase.