THU0335 FIBROSIS IS NOT JUST FIBROSIS – TGF-î’ AND PDGF INDUCE DIFFERENT EXTRACELLULAR MATRIX PROFILE: A MODEL OF DERMAL FIBROBLASTS WHICH MIMICS SYSTEMIC SCLEROSIS POTENTIAL FOR EVALUATION OF ANTI-FIBROTIC COMPOUNDS WITH TRANSLATIONAL BIOMARKERS
Authors: Pernille Juhl, Sandie Bomdesen, Anne-Christine Bay-Jensen, Morten Karsdal, Michael Jonathan Davies, Anne Sofie Siebuhr
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> Fibroblasts are responsible for the excessive extracellular matrix formation (ECM) observed in systemic sclerosis (SSc). These cells are activated by stimulants from both the vascular and immune systems, including platelet derived growth factor (PDGF) and transforming growth factor beta (TGF-β). While both TGF-β and PDGF induces extracellular matrix (ECM) formation, there is a need to understand the quantitative differences to allow for more optimal drug development and patient targeted therapies. This may be accomplished by using translational tools in cell culture systems for the evaluation of potential anti-fibrotic compounds in SSc. <h3>Objectives:</h3> This study investigated the ability of dermal fibroblast to produce ECM that mimics SSc though the use of clinical validated biomarkers. <h3>Methods:</h3> Primary human dermal fibroblasts were grown in DMEM media containing 0.4% fetal calf serum, FICOLL (to produce a crowded environment) and ascorbic acid for 14 days. The cells were stimulated with PDGF [0.04-4 nM], TGF-β [0.01-4 nM], a combination of interleukin 6 (IL-6) [3 nM] and the soluble IL-6 Receptor (sIL-6R) [0.3 nM], or a combination of TGF-β [1 nM], IL-6 [3 nM], and sIL-6R [0.3 nM]. An ALK-5 (TGF-β receptor) inhibitor (iALK-5, [1 nM-10 µM]) was added together with 1 nM TGF-β with or without 7 days pretreatment and stimulated for 17 days in total. Media were changed twice a week. Unstimulated cells (w/o) were used as controls. Type I, III and VI collagen formation (PRO-C1, PRO-C3, PRO-C6) and fibronectin (FBN-C) were evaluated by ELISAs in the supernatant collected at each media change. Staining with hematoxylin and eosin (H&amp;E) together with immunohistochemical staining were performed. Kruskal-Wallis tests were used examine the difference between treatments at different days. <h3>Results:</h3> Stimulation with TGF-β induced type I collagen formation from day 3, with a 4-fold change (FC) detected on day 10 compared to w/o (P&lt;0.0001, Figure 1), and type III collagen formation in a dose dependent manner from day 10 (4.4-FC, P&lt;0.0001). TGF-β did not induce type VI collagen formation. PDGF induced type III collagen formation from day 6, with an 8.3-FC by day 10 (P&lt;0.0001) and type VI collagen formation from day 3 with a 6.0-FC by 10 (P=0.0006). PDGF did not induce type I collagen formation. Media alone did not induce any collagen formation. H&amp;E staining showed that fibroblasts treated with PDGF and TGF-β were more confluent and disorganized compared to untreated cells. Visual examination showed type I collagen attachment to the bottom of the wells and this increased in response to stimulants. Stimulation with IL-6 decreased fibronectin levels compared to w/o, whereas TGF-β increased this. A combination of both cytokines resulted in a further increase in fibronectin levels (IL-6: 44 ng/ml, TGF-β: 308 ng/ml, IL-6+TGF-β: 368 ng/ml, P&lt;0.0001) (Figure 2). Treatment with iALK-5 (≥1 µM) decreased type I collagen formation levels to those of the vehicle, while 100 nM showed a tendency to be lower (TGF-β: 129 ng/ml, TGF-β+100 nM iALK-5: 94 ng/ml, not significant). IALK-5 at 1 µM or above, could rescue cells after 7 days pretreatment with 1 nM TGF-β (TGF-β: 129 ng/ml, TGF-β+1 µM iALK-5: 37 ng/ml, TGF-β+10 µM iALK-5: 14 ng/ml). Levels were decreased from day 10 (Figure 3). <h3>Conclusion:</h3> TGF-β and PDGF induced very different ECM compositions, with PDGF inducing much more type VI collagen formation as compared to TGF-β. In contract, TGF-β induce a highly type I collage dependent ECM. This model system and biomarkers may be used to test novel anti-fibrotic compounds, and allow for identification of patient with the endotype of fibrosis that may responds the best to treatment. <h3>Disclosure of Interests:</h3> Pernille Juhl: None declared, Sandie Bomdesen: None declared, Anne-Christine Bay-Jensen Shareholder of: I own shares of Nordic Bioscience, Employee of: I am a full-time employee in Nordic Bioscience, Morten Karsdal Shareholder of: I own shares of Nordic Bioscience, Employee of: I am a full-time employee in Nordic Bioscience, Michael Jonathan Davies: None declared, Anne Sofie Siebuhr Employee of: I am a full-time employee in Nordic Bioscience