Prevention and treatment of bleomycin-induced pulmonary fibrosis with the lactate dehydrogenase inhibitor gossypol.

Pulmonary fibrosis is a chronic and irreversible scarring disease in the lung with poor prognosis. Few therapies are available; therefore it is critical to identify new therapeutic targets. Our lab has previously identified the enzyme lactate dehydrogenase-A (LDHA) as a potential therapeutic target in pulmonary fibrosis. We found increases in LDHA protein and its metabolic product, lactate, in patients with idiopathic pulmonary fibrosis (IPF). Importantly, we described lactate as a novel pro-fibrotic mediator by acidifying the extracellular space, and activating latent transforming growth factor beta (TGF-ß1) in a pH-dependent manner. We propose a pro-fibrotic feed-forward loop by which LDHA produces lactate, lactate decreases pH in the extracellular space and activates TGF-ß1 which can further perpetuate fibrotic signaling. Our previous work also demonstrates that the LDHA inhibitor gossypol inhibits TGF-ß1-induced myofibroblast differentiation and collagen production in vitro. Here, we employed a mouse model of bleomycin-induced pulmonary fibrosis to test whether gossypol inhibits pulmonary fibrosis in vivo. We found that gossypol dose-dependently inhibits bleomycin-induced collagen accumulation and TGF-ß1 activation in mouse lungs when treatment is started on the same day as bleomycin administration. Importantly, gossypol was also effective at treating collagen accumulation when delayed 7 days following bleomycin. Our results demonstrate that inhibition of LDHA with the inhibitor gossypol is effective at both preventing and treating bleomycin-induced pulmonary fibrosis, and suggests that LDHA may be a potential therapeutic target for pulmonary fibrosis.

Normal Human Lung Epithelial Cells Inhibit Transforming Growth Factor-ß Induced Myofibroblast Differentiation via Prostaglandin E2.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with very few effective treatments. The key effector cells in fibrosis are believed to be fibroblasts, which differentiate to a contractile myofibroblast phenotype with enhanced capacity to proliferate and produce extracellular matrix. The role of the lung epithelium in fibrosis is unclear. While there is evidence that the epithelium is disrupted in IPF, it is not known whether this is a cause or a result of the fibroblast pathology. We hypothesized that healthy epithelial cells are required to maintain normal lung homeostasis and can inhibit the activation and differentiation of lung fibroblasts to the myofibroblast phenotype. To investigate this hypothesis, we employed a novel co-culture model with primary human lung epithelial cells and fibroblasts to investigate whether epithelial cells inhibit myofibroblast differentiation. MEASUREMENTS AND MAIN RESULTS: In the presence of transforming growth factor (TGF)-ß, fibroblasts co-cultured with epithelial cells expressed significantly less α-smooth muscle actin and collagen and showed marked reduction in cell migration, collagen gel contraction, and cell proliferation compared to fibroblasts grown without epithelial cells. Epithelial cells from non-matching tissue origins were capable of inhibiting TGF-ß induced myofibroblast differentiation in lung, keloid and Graves' orbital fibroblasts. TGF-ß promoted production of prostaglandin (PG) E2 in lung epithelial cells, and a PGE2 neutralizing antibody blocked the protective effect of epithelial cell co-culture. CONCLUSIONS: We provide the first direct experimental evidence that lung epithelial cells inhibit TGF-ß induced myofibroblast differentiation and pro-fibrotic phenotypes in fibroblasts. This effect is not restricted by tissue origin, and is mediated, at least in part, by PGE2. Our data support the hypothesis that the epithelium plays a crucial role in maintaining lung homeostasis, and that damaged and/ or dysfunctional epithelium contributes to the development of fibrosis.

Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase.

Pulmonary fibrosis is a common and dose-limiting side-effect of ionizing radiation used to treat cancers of the thoracic region. Few effective therapies are available for this disease. Pulmonary fibrosis is characterized by an accumulation of myofibroblasts and excess deposition of extracellular matrix proteins. Although prior studies have reported that ionizing radiation induces fibroblast to myofibroblast differentiation and collagen production, the mechanism remains unclear. Transforming growth factor-ß (TGF-ß) is a key profibrotic cytokine that drives myofibroblast differentiation and extracellular matrix production. However, its activation and precise role in radiation-induced fibrosis are poorly understood. Recently, we reported that lactate activates latent TGF-ß through a pH-dependent mechanism. Here, we wanted to test the hypothesis that ionizing radiation leads to excessive lactate production via expression of the enzyme lactate dehydrogenase-A (LDHA) to promote myofibroblast differentiation. We found that LDHA expression is increased in human and animal lung tissue exposed to ionizing radiation. We demonstrate that ionizing radiation induces LDHA, lactate production, and extracellular acidification in primary human lung fibroblasts in a dose-dependent manner. We also demonstrate that genetic and pharmacologic inhibition of LDHA protects against radiation-induced myofibroblast differentiation. Furthermore, LDHA inhibition protects from radiation-induced activation of TGF-ß. We propose a profibrotic feed forward loop, in which radiation induces LDHA expression and lactate production, which can lead to further activation of TGF-ß to drive the fibrotic process. These studies support the concept of LDHA as an important therapeutic target in radiation-induced pulmonary fibrosis.

Transglutaminase 2 and its role in pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a deadly progressive disease with few treatment options. Transglutaminase 2 (TG2) is a multifunctional protein, but its function in pulmonary fibrosis is unknown. OBJECTIVES: To determine the role of TG2 in pulmonary fibrosis. METHODS: The fibrotic response to bleomycin was compared between wild-type and TG2 knockout mice. Transglutaminase and transglutaminase-catalyzed isopeptide bond expression was examined in formalin-fixed human lung biopsy sections by immunohistochemistry from patients with IPF. In addition, primary human lung fibroblasts were used to study TG2 function in vitro. MEASUREMENTS AND MAIN RESULTS: TG2 knockout mice developed significantly reduced fibrosis compared with wild-type mice as determined by hydroxyproline content and histologic fibrosis score (P < 0.05). TG2 expression and activity are increased in lung biopsy sections in humans with IPF compared with normal control subjects. In vitro overexpression of TG2 led to increased fibronectin deposition, whereas transglutaminase knockdown led to defects in contraction and adhesion. The profibrotic cytokine transforming growth factor-ß causes an increase in membrane-localized TG2, increasing its enzymatic activity. CONCLUSIONS: TG2 is involved in pulmonary fibrosis in a mouse model and in human disease and is important in normal fibroblast function. With continued research on TG2, it may offer a new therapeutic target.

Peroxisome proliferator-activated receptor-gamma ligands induce heme oxygenase-1 in lung fibroblasts by a PPARgamma-independent, glutathione-dependent mechanism.

Oxidative stress plays an important role in the pathogenesis of pulmonary fibrosis. Heme oxygenase-1 (HO-1) is a key antioxidant enzyme, and overexpression of HO-1 significantly decreases lung inflammation and fibrosis in animal models. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that the PPARgamma ligands 15d-PGJ2 and 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), which have potent antifibrotic effects in vitro, also strongly induce HO-1 expression in primary human lung fibroblasts. Pharmacological and genetic approaches are used to demonstrate that induction of HO-1 is PPARgamma independent. Upregulation of HO-1 coincides with decreased intracellular glutathione (GSH) levels and can be inhibited by N-acetyl cysteine (NAC), a thiol antioxidant and GSH precursor. Upregulation of HO-1 is not inhibited by Trolox, a non-thiol antioxidant, and does not involve the transcription factors AP-1 or Nrf2. CDDO and 15d-PGJ2 contain an alpha/beta unsaturated ketone that acts as an electrophilic center that can form covalent bonds with free reduced thiols. Rosiglitazone, a PPARgamma ligand that lacks an electrophilic center, does not induce HO-1. These data suggest that in human lung fibroblasts, 15d-PGJ2 and CDDO induce HO-1 via a GSH-dependent mechanism involving the formation of covalent bonds between 15d-PGJ2 or CDDO and GSH. Inhibiting HO-1 upregulation with NAC has only a small effect on the antifibrotic properties of 15d-PGJ2 and CDDO in vitro. These results suggest that CDDO and similar electrophilic PPARgamma ligands may have great clinical potential as antifibrotic agents, not only through direct effects on fibroblast differentiation and function, but indirectly by bolstering antioxidant defenses.