Journal Article

S91 Defining the antifibrotic mechanisms of treprostinil in pulmonary fibrosis

Jenkins, R G, May, J, Yao, H, John, A E, Rahman, L, Mitchell, JA, Liu, B

2025

Overview

IntroductionIdiopathic Pulmonary Fibrosis (IPF) is a progressive fibrosing interstitial lung disease with limited treatment options and a grave prognosis. Treprostinil, a synthetic prostacyclin analogue used in pulmonary hypertension, is under investigation for its antifibrotic potential. The antifibrotic mechanisms of Treprostinil are yet to be fully defined. Treprostinil activates the IP receptor to generate cAMP but may also target intracellular PPARβδ receptors. PPARβδ activation can inhibit the RhoA pathway which is amplified in IPF. These studies aim to explore the antifibrotic mechanisms of Treprostinil.MethodsExpression of IP and PPARβδ receptors in primary human lung fibroblasts (pHLFs) was confirmed via RT-qPCR and Western blotting. RhoA activation was measured using G-LISAä and TGFβ activity via TMLC coculture assay and Western blotting for phospho-SMAD2. Profibrotic gene expression was measured by RT-qPCR. Fibroblast proliferation was assessed using BrdU incorporation by ELISA and time-lapse live cell imaging. In IPF patient-derived precision-cut lung slices (PCLS), nuclear phospho-SMAD2 accumulation was assessed via immunofluorescence and hydroxyproline levels quantified using high-performance liquid chromatography in PCLS conditioned media.ResultsBoth IP and PPARβδ receptors were present in pHLFs, with PPARβδ more abundant. Treprostinil inhibited LPA-induced RhoA activation and this effect was not reversed by the presence of an IP antagonist. Treprostinil did not inhibit LPA-induced TGFβ activation in the TMLC assay or SMAD2 phosphorylation. However, Treprostinil depletes total SMAD2/3 likely via cAMP and this correlates with a rise in phospho-YAP. Fibroblast proliferation was also inhibited in a concentration-dependent manner by Treprostinil and there were additive effects when combined with nintedanib, pirfenidone, or nerandomilast. Treprostinil downregulated TGFβ-response genes in pHLFs including FN and SERPINE1, as well as nuclear phospho-SMAD2 and hydroxyproline content levels in the PCLS model.ConclusionsTreprostinil inhibits RhoA activation and this may be via PPARβδ activation. Treprostinil did not affect SMAD2 phosphorylation but did deplete total SMAD2/3 in a cAMP dependent manner in pHLFs. Antifibrotic effects of Treprostinil were detected in IPF derived PCLS. Further studies are ongoing to establish the underlying mechanism as there were additive effects of Treprostinil with established antifibrotic agents which may have important clinical implications.

• Publisher: BMJ Publishing Group LTD
• Subject: Fibroblasts / Phosphorylation / Pulmonary fibrosis