Fibroblasts Are Not Just Fibroblasts - Clear Differences In Ecm Production Between Dermal And Lung Fibroblasts In Response To Growth Factors

Background:Many systemic sclerosis (SSc) patients develop lung fibrosis, which contribute significantly to increased mortality1. Activated and proliferating fibroblasts are responsible for the excessive extracellular matrix (ECM) formation and stiffening of the connective tissue leading to skin and lung fibrosis. There is currently no effective treatment for the fibrosis in SSc and there is therefore a medical need for further understanding the pathogenesis of fibrosis. Fibrosis is associated with different growth factors, including tumor growth factor beta 1 (TGF-β1) and platelet derived growth factor-ab (PDGF-ab)2.Objectives:We investigated how stimulation with TGF-β1 and PDGF-ab affected the migration capacity and the ECM production using translational biomarkers of type I, III and VI collagens in healthy human dermal and lung fibroblasts.Methods:The fibroblasts were grown in DMEM media containing 0.4% fetal calf serum, ficoll (to produce a crowded environment) and ascorbic acid for up to 12 days. The cells were stimulated with TGF-β1 [0.04-1 nM] or PDGF-ab [3 nM] at treatment initiation and changed twice a week. Non-stimulated fibroblasts (w/o) were used as control. A wound was induced by scratching the cells at day 1 after treatment initiation and the migration was followed for 2 days. Type I, III and VI collagen formation (PRO-C1, PRO-C3 and PRO-C6, respectively) were evaluated by validated ELISAs (Nordic Bioscience) in supernatant from day 0, 4, 8 and 12. Statistical analysis included 2-way ANOVA and Dunnett’s test.Results:The PDGF-ab stimulated dermal fibroblasts migrated significantly more than the non-stimulated (p<0.0001) and TGF-β1 stimulated (p<0.001) dermal fibroblasts 48 hours after the scratch (migration app. 70%, 30% and 30% respectively). There was no difference between the migration of the non-stimulated, TGF-β1 and PDGF-ab stimulated lung fibroblasts after 48 hours, as all migrated to approximately 70%.TGF-β1 stimulation led to a significant increase in type I collagen formation (PRO-C1) in both dermal and lung fibroblasts from day 4 and onwards compared to w/o (p<0.0001). TGF-β1 also lead to a significant increase in type III collagen formation (PRO-C3) from day 8 in lung fibroblasts compared to w/o (p<0.0001). PDGF-ab stimulation led to a significant increase in type III collagen formation in dermal fibroblasts from day 8 compared to w/o (p<0.0001). PDGF-ab stimulation led to a significant increase in type VI collagen formation (PRO-C6) in both dermal and lung fibroblasts from day 4 and onwards compared to w/o (p<0.0001).Conclusion:PDGF-ab increased the migration activity of the dermal fibroblasts, where the lung fibroblasts had a general high migration activity. The dermal and lung fibroblasts showcase the same ECM production within both type I and type VI collagen formation. The two fibroblasts types did however react opposite each other regarding the type III collagen formation: the dermal fibroblasts responded to PDGF-ab stimulation, where the lung fibroblasts responded to the TGF-β1 stimulation. The clear differences in the ECM production between the dermal and lung fibroblasts can be important in the search for an effective treatment for fibrosis in SSc and related lung fibrosis.References:[1]McNearney, T. A. et al. Pulmonary involvement in systemic sclerosis: Associations with genetic, serologic, sociodemographic, and behavioral factors. Arthritis Care Res.57, 318–326 (2007).[2]Wynn, T. A. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J. Clin. Invest.117, 524–529 (2007).Disclosure of Interests:Sofie Falkenløve Madsen Employee of: Nordic Bioscience and University of Copenhagen, Anne Sofie Siebuhr Employee of: Nordic Bioscience A/S, Henrik Jessen: None declared, Jannie Marie Bülow Sand Employee of: Nordic Bioscience A/S, Morten Karsdal Shareholder of: Nordic Bioscience A/S, Employee of: Nordic Bioscience A/S, Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Nordic Bioscience A/S