Anti-fibrotic effects of thrombin inhibition in systemic sclerosis-associated interstitial lung disease: Proof of concept.

Abstract

Introduction: Activation of the coagulation cascade leading to generation of thrombin is well documented in various forms of lung injury including systemic sclerosis-associated interstitial lung disease (SSc-ILD). We previously demonstrated that the direct thrombin inhibitor dabigatran inhibits thrombin-induced profibrotic signaling in lung fibroblasts isolated from scleroderma patients. The objective of this study was to characterize and compare lung fibroblasts from an SSc-ILD patient at baseline and after dabigatran treatment to ascertain this therapy's differential effects on fibrogenic gene expression.

Materials and methods: Lung fibroblasts isolated by bronchoalveolar lavage (BAL) from a SSc-ILD patient before and after receiving dabigatran (Pradaxa^®) 75 mg twice daily for 6 months (ClinicalTrials.gov Identifier NCT02426229) were analyzed by RNA sequencing, real-time quantitative reverse transcription polymerase chain reaction (qRT)-PCR, and immunofluorescent staining.

Results: Thrombin inhibition for six-months by oral dabigatran resulted in significantly decreased expression of 708 lung fibroblast genes as compared to basal levels before dabigatran treatment. Using Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, we determined that thrombin-inhibition by dabigatran primarily affected extracellular matrix (ECM) and ECM-related genes. Fibrosis-associated genes, including smooth muscle alpha-actin (SMA, ACTA2), tenascin C, collagen 1 alpha 1 (COL1A1), collagen 3 alpha1 (COL3A1), collagen 8 alpha 2 (COL8A2), collagen 10 alpha 1 (COL10A1), collagen 5 alpha 1 (COL5A1), fibronectin 1, connective tissue growth factor (CTGF), and procollagen-lysine-2-oxoglutarate-5-dioxygenase-2 (PLOD2) were all significantly down regulated following thrombin inhibition by dabigatran treatment. Real-time qRT-PCR and immunofluorescent staining confirmed significant downregulation of the selected genes at the mRNA and protein levels.

Conclusion: Inhibition of thrombin in this SSc-ILD patient treated with low-dose dabigatran etexilate downregulated profibrotic proteins in lung fibroblasts, providing further support for the use of thrombin inhibitors as a therapeutic approach for the treatment of patients with SSc-ILD.