Reactive Oxygen Species Are Required for Maintenance and Differentiation of Primary Lung Fibroblasts in Idiopathic Pulmonary Fibrosis

Abstract

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive and fatal illness whose pathogenesis remains poorly understood. Recent evidence suggests oxidative stress as a key player in the establishment/progression of lung fibrosis in animal models and possibly in human IPF. The aim of the present study was to characterize the cellular phenotype of fibroblasts derived from IPF patients and identify underlying molecular mechanisms.Methodology/Principal FindingsWe first analyzed the baseline differentiation features and growth ability of primary lung fibroblasts derived from 7 histology proven IPF patients and 4 control subjects at different culture passages. Then, we focused on the redox state and related molecular pathways of IPF fibroblasts and investigated the impact of oxidative stress in the establishment of the IPF phenotype. IPF fibroblasts were differentiated into alpha-smooth muscle actin (SMA)-positive myofibroblasts, displayed a pro-fibrotic phenotype as expressing type-I collagen, and proliferated lower than controls cells. The IPF phenotype was inducible upon oxidative stress in control cells and was sensitive to ROS scavenging. IPF fibroblasts also contained large excess of reactive oxygen species (ROS) due to the activation of an NADPH oxidase-like system, displayed higher levels of tyrosine phosphorylated proteins and were more resistant to oxidative-stress induced cell death. Interestingly, the IPF traits disappeared with time in culture, indicating a transient effect of the initial trigger.Conclusions/SignificanceRobust expression of α-SMA and type-I collagen, high and uniformly-distributed ROS levels, resistance to oxidative-stress induced cell death and constitutive activation of tyrosine kinase(s) signalling are distinctive features of the IPF phenotype. We suggest that this phenotype can be used as a model to identify the initial trigger of IPF.

Trichostatin A Inhibits Transforming Growth Factor-β–Induced Reactive Oxygen Species Accumulation and Myofibroblast Differentiation via Enhanced NF-E2-Related Factor 2-Antioxidant Response Element Signaling

“…Moreover, both the intracellular GSH level and total antioxidant capacity were decreased after treatment with TGF-b, which suggests that TGF-b may disturb the redox balance of corneal fibroblasts and promote the shifting of the cells into a state of oxidative stress. The results are consistent with previous studies reporting that TGF-b increased ROS production in numerous types of nonphagocytic cells, including aortic smooth muscle cells; prostatic stromal cells; hepatic stellate cells; and cardiac, lung, and skin fibroblasts (Hecker et al, 2009;Bocchino et al, 2010;Bondi et al, 2010;Michaeloudes et al, 2011;Sampson et al, 2011;Baek et al, 2012). It has been shown that TGF-b increases the activity of NAD(P)H oxidase and the expression of Nox2 and Nox4, and that both of these two homologs of the Nox family are the ones that mainly mediate the TGFb-induced ROS accumulation (Hecker et al, 2009;Jiang et al, 2010;Crestani et al, 2011;Sampson et al, 2011).…”

“…TGF-b stimulates reactive oxygen species generation, which in turn mediates myofibroblast differentiation (Sharma et al, 2009;Bocchino et al, 2010). To examine whether the myofibroblast differentiation of corneal fibroblasts by TGF-b treatment was mediated through enhanced ROS generation and accumulation, the cells were exposed to the NADPH oxidase (Nox) inhibitor DPI, the antioxidant NAC, or the Nrf2-ARE signaling activator sulforaphane, before the addition of TGF-b.…”

“…Accumulating evidence indicates that increased oxidative stress is critical for the development of fibrotic diseases in humans, such as various pulmonary fibrotic disorders, liver fibrosis/cirrhosis, renal fibrosis, and fibrotic cardiac repair/remodeling after infarction (Liu and Gaston Pravia, 2010). The essential cytokine in fibrosis development, TGF-b, increases reactive oxygen species (ROS) production in fibroblasts, whereas the generated ROS in turn stimulates fibroblast activation and myofibroblast differentiation (Amara et al, 2010;Bocchino et al, 2010;Barnes and Gorin, 2011;Crestani et al, 2011;Sampson et al, 2011).…”

The matricellular protein CCN1 enhances TGF‐β1/SMAD3‐dependent profibrotic signaling in fibroblasts and contributes to fibrogenic responses to lung injury

“…IPF fibroblasts are known to maintain a profibrotic phenotype in ex vivo culture (53), and express constitutively higher nuclear SMAD3 (54,55). There have been very few studies that have investigated the regulatory mechanisms of constitutively active IPF fibroblasts (53,(55)(56)(57).…”

Matrices of Physiologic Stiffness Potently Inactivate Idiopathic Pulmonary Fibrosis Fibroblasts

“…These efforts have demonstrated, among a host of changes, that IPF fibroblasts are more contractile (6), express higher concentrations of a-smooth muscle actin (a-SMA) (7,8), Type I collagen (8)(9)(10), and tissue inhibitors of metalloproteinase (11) than do normal lung tissue-derived fibroblasts, and are characterized by pathologic integrin signaling (12) and an invasive phenotype (13). Relative to normal cells, IPF-derived fibroblasts have also been shown to express lower concentrations of cyclooxygenase-2 (COX-2), produce less of the antifibrotic lipid mediator prostaglandin E 2 (PGE 2 ) (14,15), and exhibit resistance to this antifibrotic lipid mediator applied exogenously (16).…”