Patricia J. Sime

Transforming growth factor (TGF)-beta1 has been implicated in the pathogenesis of fibrosis based upon its matrix-inducing effects on stromal cells in vitro, and studies demonstrating increased expression of total TGF-beta1 in fibrotic tissues from a variety of organs. The precise role in vivo of this cytokine in both its latent and active forms, however, remains unclear. Using replication-deficient adenovirus vectors to transfer the cDNA of porcine TGF-beta1 to rat lung, we have been able to study the effect of TGF-beta1 protein in the respiratory tract directly. We have demonstrated that transient overexpression of active, but not latent, TGF-beta1 resulted in prolonged and severe interstitial and pleural fibrosis characterized by extensive deposition of the extracellular matrix (ECM) proteins collagen, fibronectin, and elastin, and by emergence of cells with the myofibroblast phenotype. These results illustrate the role of TGF-beta1 and the importance of its activation in the pulmonary fibrotic process, and suggest that targeting active TGF-beta1 and steps involved in TGF-beta1 activation are likely to be valuable antifibrogenic therapeutic strategies. This new and versatile model of pulmonary fibrosis can be used to study such therapies.

Numerous compounds have shown efficacy in limiting development of pulmonary fibrosis using animal models, yet few of these compounds have replicated these beneficial effects in clinical trials. Given the challenges associated with performing clinical trials in patients with idiopathic pulmonary fibrosis (IPF), it is imperative that preclinical data packages be robust in their analyses and interpretations to have the best chance of selecting promising drug candidates to advance to clinical trials. The American Thoracic Society has convened a group of experts in lung fibrosis to discuss and formalize recommendations for preclinical assessment of antifibrotic compounds. The panel considered three major themes (choice of animal, practical considerations of fibrosis modeling, and fibrotic endpoints for evaluation). Recognizing the need for practical considerations, we have taken a pragmatic approach. The consensus view is that use of the murine intratracheal bleomycin model in animals of both genders, using hydroxyproline measurements for collagen accumulation along with histologic assessments, is the best-characterized animal model available for preclinical testing. Testing of antifibrotic compounds in this model is recommended to occur after the acute inflammatory phase has subsided (generally after Day 7). Robust analyses may also include confirmatory studies in human IPF specimens and validation of results in a second system using in vivo or in vitro approaches. The panel also strongly encourages the publication of negative results to inform the lung fibrosis community. These recommendations are for preclinical therapeutic evaluation only and are not intended to dissuade development of emerging technologies to better understand IPF pathogenesis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a complex disease for which the pathogenesis is poorly understood. In this study, we identified lactic acid as a metabolite that is elevated in the lung tissue of patients with IPF. OBJECTIVES: This study examines the effect of lactic acid on myofibroblast differentiation and pulmonary fibrosis. METHODS: We used metabolomic analysis to examine cellular metabolism in lung tissue from patients with IPF and determined the effects of lactic acid and lactate dehydrogenase-5 (LDH5) overexpression on myofibroblast differentiation and transforming growth factor (TGF)-β activation in vitro. MEASUREMENTS AND MAIN RESULTS: Lactic acid concentrations from healthy and IPF lung tissue were determined by nuclear magnetic resonance spectroscopy; α-smooth muscle actin, calponin, and LDH5 expression were assessed by Western blot of cell culture lysates. Lactic acid and LDH5 were significantly elevated in IPF lung tissue compared with controls. Physiologic concentrations of lactic acid induced myofibroblast differentiation via activation of TGF-β. TGF-β induced expression of LDH5 via hypoxia-inducible factor 1α (HIF1α). Importantly, overexpression of both HIF1α and LDH5 in human lung fibroblasts induced myofibroblast differentiation and synergized with low-dose TGF-β to induce differentiation. Furthermore, inhibition of both HIF1α and LDH5 inhibited TGF-β-induced myofibroblast differentiation. CONCLUSIONS: We have identified the metabolite lactic acid as an important mediator of myofibroblast differentiation via a pH-dependent activation of TGF-β. We propose that the metabolic milieu of the lung, and potentially other tissues, is an important driving force behind myofibroblast differentiation and potentially the initiation and progression of fibrotic disorders.

Pulmonary fibrosis is a progressive life-threatening disease for which no effective therapy exists. Myofibroblasts are one of the key effector cells in pulmonary fibrosis and are the primary source of extracellular matrix production. Drugs that inhibit the differentiation of fibroblasts to myofibroblasts have potential as antifibrotic therapies. Peroxisome proliferator-activated receptor (PPAR)-gamma is a transcription factor that upon ligation with PPARgamma agonists activates target genes containing PPAR response elements. PPARgamma agonists have anti-inflammatory activities and may have potential as antifibrotic agents. In this study, we examined the abilities of PPARgamma agonists to block two of the most important profibrotic activities of TGF-beta on pulmonary fibroblasts: myofibroblast differentiation and production of excess collagen. Both natural (15d-PGJ2) and synthetic (ciglitazone and rosiglitazone) PPARgamma agonists inhibited TGF-beta-driven myofibroblast differentiation, as determined by alpha-smooth muscle actin-specific immunocytochemistry and Western blot analysis. PPARgamma agonists also potently attenuated TGF-beta-driven type I collagen protein production. A dominant-negative PPARgamma partially reversed the inhibition of myofibroblast differentiation by 15d-PGJ2 and rosiglitazone, but the irreversible PPARgamma antagonist GW-9662 did not, suggesting that the antifibrotic effects of the PPARgamma agonists are mediated through both PPARgamma-dependent and independent mechanisms. Thus PPARgamma agonists have novel and potent antifibrotic effects in human lung fibroblasts and may have potential for therapy of fibrotic diseases in the lung and other tissues.