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<0.0001). TGFβ+ ML-7 inhibited the contraction to the level of w/o (p=0.0024), which was only 35% of ML-7 alone. In the contraction study the cells were terminated after 2 days of culture, thus prior to when biomarker of ECM remodeling is usually assessed. However, FBN-C was detectable and a significant release of fibronectin by TGFβ and PDGF compared to w/o was found in the supernatant (both p<0.0001). The gene expression of FBN was only increased with TGFβ (p<0.05) and not PDGF. ML-7 alone tended to decrease FBN-C and in combination with TGFβ the FBN level was significantly decreased compared to TGFβ alone (p<0.0001). However, the level of TGFβ+ML-7 was significantly higher than ML-7 alone (p=0.038).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<0.05).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 Falkenløve 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
OsSLI1, a Homeodomain Containing Transcription Activator, Involves Abscisic Acid Related Stress Response in Rice (Oryza sativaL.)