SAT0292 INTEGRATIVE TRANSCRIPTOMIC AND FUNCTIONAL ANALYSIS REVEALS A ROLE OF DIMETHYL-Α-KETOGLUTARATE IN TGFΒ-DRIVEN CYTOSKELETON REGULATION AND MYOFIBROBLAST DIFFERENTIATION

Background: Systemic sclerosis has vascular, inflammatory and fibrotic components, which may be associated with different growth factors and cytokines. Platelet derived growth factor (PDGF) is associated with the vasculature, whereas tumor necrosis factor beta (TGFβ) is associated with inflammation and fibrosis. We have developed a fibroblast model system of dermal fibrosis for anti-fibrotic drugs testing, but the effect of the fibroblasts mechanistic properties are unknown. Objectives: We investigated different mechanical capacities of PDGF and TGFβ treated human healthy dermal fibroblasts in the SiaJ setting. Methods: Primary human healthy dermal fibroblasts were grown in DMEM medium containing 0.4% fetal calf serum, ficoll (to produce a crowded environment) and ascorbic acid for up to 17 days. A wound was induced by scratching the cells at 0, 1, 3 or 7 days after treatment initiation. Wound closure was followed for 3 days. Contraction capacity was tested by creating gels of human fibroblasts produced collagens containing dermal fibroblasts and contraction was assessed at day 2 by calculating the percentage of gel size to total well size. Collagen type I, III and VI formation (PRO-C1, PRO-C3 and PRO-C6) and fibronectin (FBN-C) were evaluated by validated ELISAs (Nordic Bioscience). Gene expression was analyzed after 2 days in culture. Statistical analyses included One-way ANOVA and student’s t-test. Results: Generally, PDGF closed the wound in half the time of w/o and TGFβ, when treatment and cells are added concurrently or scratched one day after treatment initiation. When treatments were added 3 or 7 days prior to scratch, the cells treated with PDGF had proliferated to a higher degree than w/o and TGFβ. A consequence of this, was that when cells were scratch the sheet of cells produced was lifted from the bottom and fold over itself, leaving a much greater scratch than in the other treatments. However, despite this increased gap the PDGF treated cells closed the wound at the same time as w/o and TGFβ, confirming the results of the cells scratched at day 0 and 1. Inhibition of contraction by ML-7 of otherwise untreated cells inhibited contraction significantly compared to untreated cells alone (p=0.0009). Contraction was increased in both TGFβ and PDGF treated cells compared to untreated cells (both p TGFβ increased the gene expression of most genes assessed, except Col6a1. PDGF increased the gene expression of Col3a1, Col5a1 and Col6a1 above the critical fold change of 2, but only significantly in Col5a1 and Col6a1 (both p Conclusion: This study demonstrates that TGFβ and PDGF have different mechanical capacities in human healthy dermal fibroblasts; TGFβ increased gene expression of ECM related genes, such as collagens and have increased FBN release in the supernatant already 2 days after initial treatment. PDGF has increased contraction, proliferation and migratory capacities and less expression of ECM related genes and proteins. Disclosure of Interests: Anne Sofie Siebuhr Employee of: Nordic Bioscience, Sofie Falkenlove Madsen: None declared, Morten Karsdal Shareholder of: Nordic Bioscience A/S., Employee of: Full time employee at Nordic Bioscience A/S., Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Full time employee at Nordic Bioscience A/S., Pernille Juhl Employee of: Nordic Bioscience