Treprostinil reduces clinically relevant fibrosis biomarkers in a Scar-in-a-Jar model using a fibrotic cocktail

<bold>Aim:</bold> Fibroblast activation and collagen deposition is pivotal in development of fibrotic interstitial lung disease (ILD). Therefore, inhibiting these processes is crucial for anti-fibrotic therapies. This study aims to improve the Scar-in-a-Jar model by introducing a fibrotic cocktail (FC) of nine cytokines to better mimic the fibrogenic microenvironment. We examined how the FC affects collagen synthesis and explored Treprostinil's potential as an anti-fibrotic agent. Treprostinil, approved by the FDA for pulmonary arterial hypertension (PAH) and pulmonary hypertension in ILD (PH-ILD), is currently in phase 3 trials for the treatment of idiopathic pulmonary fibrosis, underlining its therapeutic prospect. <bold>Methods:</bold> Primary lung fibroblasts were exposed to the FC and Treprostinil (10-3000 nM) or vehicle for 12 days. Supernatants were collected every fourth day for quantification of type III and VI collagen synthesis (PRO-C3, PRO-C6) using the clinically relevant biomarker immunoassays NordicPRO-3^TM and NordicPRO-C6^TM. <bold>Results:</bold> Stimulating fibroblasts with the FC significantly increased PRO-3 and PRO-C6 levels (11.0 and 2.8-fold, p<0.0001) compared to non-stimulated cells at day 12. Treprostinil (10-3000 nM) significantly reduced PRO-C3 and PRO-C6 (2.95-8.4-fold and 1.7-3.3-fold, p<0.0001) in a concentration dependent manner compared to vehicle at day 12. <bold>Conclusions:</bold> The FC stimulates type III and type VI collagen synthesis in the Scar-in-a-Jar model which could be inhibited by Treprostinil. This model gives insight into collagen synthesis and therapeutic efficacy using clinically relevant biomarkers, contributing to development of novel anti-fibrotic therapies.