Although prostaglandins and their receptors have been studied extensively in pulmonary fibrosis, there is a paucity of data regarding thromboxane A2 (TXA2) and the thromboxane-prostanoid receptor (TPr) in the lungs. We found that TPr is expressed in lung fibroblasts and that expression of this receptor is upregulated in fibroblasts from patients with idiopathic pulmonary fibrosis (IPF), as well as lung fibroblasts from mice treated with bleomycin. Genetic deletion of TPr in mice or treatment with a TPr antagonist (Ifetroban) markedly attenuated bleomycin-induced lung fibrosis. In addition, TPr deficiency or Ifetroben treatment reduced Smad2/3 phosphorylation, ?-smooth muscle actin (?-SMA) expression, and collagen 1 production in lung tissue and isolated lung fibroblasts following bleomycin treatment, without effects on inflammation or epithelial apoptosis. In contrast, treatment with a thromboxane synthesis inhibitor (Ozagrel) was minimally effective at inhibiting lung fibrosis. These findings, along with data showing that thromboxane expression was only transiently upregulated following bleomycin treatment, suggested that TPr activation in fibrosis is mediated through an alternative ligand. F2-isoprostanes (F2-isoPs) are a non-enzymatic product of reactive oxygen species (ROS)-induced peroxidation of arachidonic acid that have structural similarities to TXA2 and can activate TPr signaling. Following treatment with bleomycin, F2-isoPs in mouse lungs were persistently upregulated, suggesting that these ROS products could mediate lung fibrosis via TPr activation. To further investigate mechanisms by which TPr regulates fibrosis, we exposed mouse lung fibroblasts to F2-isoPs (or the specific TPr agonist U-46619) and observed myofibroblast differentiation, increased proliferation, and Smad2/3 phosphorylation, and collagen production, all of which were blocked by deletion of TPr or Ifetroban treatment. Further, in primary lung fibroblasts from IPF patients, we found that TPr antagonism reduced cell proliferation and expression of ?-smooth muscle actin and collagen 1. Together, these data support the hypothesis that reactive oxygen species produced in the lungs of IPF patients generate F2-isoprostanes which activate TPr signaling in lung fibroblasts, leading to myofibroblast differentiation and persistent collagen and matrix production through downstream activation of the Smad/TGF-? pathway. Interventions that block TPr signaling could provide novel therapeutic options to limit progressive pulmonary fibrosis.
Specific Aims will: 1) determine the role of TPr signaling in lung fibroblasts in relevant pre-clinical models of lung fibrosis, 2) identify mechanisms by which TPr signaling regulates myofibroblast differentiation and activation, and 3) examine the anti-fibrotic effects of TPr inhibition in human lung fibroblasts and 3-D pulmosphere cultures. Since TPr antagonists, including Ifetroban, are currently available for human use, these studies are likely to set the stage for future clinical studies targeting this pathway (alone or in combination with current drugs) to improve outcomes in IPF and related diseases characterized by progressive pulmonary fibrosis.
Despite the hard work of many investigators, the underlying mechanisms of progressive fibrosis in idiopathic pulmonary fibrosis (IPF) remain incompletely understood. In this proposal, we have outlined a comprehensive approach to evaluate thromboxane receptor signaling as a novel pathway that contributes to persistent fibroblast activation in the lungs. If successful, these studies could pave the way for rapid translation into clinical studies with the goal of improving outcomes in IPF and related fibrotic diseases.
