INDUCTION OF COLLAGEN CHAIN FORMATION IN RESPONSE TO FIBROTIC FACTORS IN DERMAL AND PULMONARY FIBROBLASTS

BackgroundSystemic sclerosis (SSc) patients who develop pulmonary fibrosis, have an increased mortality rate1. Excessively activated fibroblasts deposit extracellular matrix (ECM), which leads to fibrosis resulting in stiffening of the tissue in both skin and lung. Currently there is no cure for fibrosis in SSc, only drugs which can slow the fibrotic process, and there is therefore, a medical need for further understanding of the pathogenesis. Fibrosis is associated with growth factors, including tumor growth factor beta 1 (TGF-β1) and platelet derived growth factor-ab (PDGF-ab).ObjectivesWe investigated how TGF-β1 and PDGF-ab stimulation affected the gene and protein expression of specific collagen chains of type I, III and VI collagens in primary healthy human dermal (DF) and pulmonary fibroblasts (PF).MethodsThe fibroblasts were grown in 0.4% fetal calf serum DMEM, ficoll (to produce a crowded environment) and ascorbic acid for up to 12 days. They were stimulated with TGF-β [0.01 nM], PDGF-ab [3 nM] or a combination of TGF-β [0.01 nM] and PDGF-ab [3 nM], while non-stimulated fibroblasts served as control. ECM protein formation was assessed in supernatant from day 0, 4, 8 and 12, by ELISAs which detects the N-terminal of the pro-collagen of type I and III collagen, and the C5 domain of the α3 chain of type VI collagen. Gene expression was analyzed after 4 days and the relative gene expression was calculated based on multiple reference genes2. Statistical analysis includes one-way and two-way ANOVA.ResultsTGF-β increased the gene expression of Col1α1 in DF (p<0.0001) and Col1α2, Col3α1 and Col6α3 in both DF (p<0.01, p<0.001, p<0.05, respectively) and PF (p<0.0001) compared to control. PDGF-ab showed no difference in gene expression of the DF but decreased multiple genes in PF (p<0.01). The combination of TGF-β and PDGF-ab increased the gene expression of Col1α1 in DF (p<0.01), Col3α1 in PF (p<0.0001) Col1α2 and Col6α3 in both DF (p<0.05 and p<0.01) and PF (p<0.0001) compared to control. None of the stimulations lead to an increase in the Col6α1 and Col6α2. The TGF-β induced gene expression corresponded with increased ECM formation of type I and VI collagen from day 4 (p<0.01), and type III collagen from day 8 (p<0.05) in both DF and PF. PDGF-ab stimulation led to an increased ECM formation of type I and VI collagen in both DF and PF (p<0.01), and type III collagen in DF (p<0.01). The combination stimulation with TGF-β and PDGF-ab induced a corresponding increase in both gene expression and ECM formation of type I and VI collagen in both DF and PF (p<0.01). While the combination increased the ECM formation of type III collagen in both fibroblast types, the gene expression of Col3α1 were only increased in PF (p<0.0001).ConclusionThis study demonstrates that TGF-β stimulation alone and in combination with PDGF-ab results in increased gene and protein expression of type I and VI collagen in both DF and PF, and additionally type III collagen in PF. However, there was a disconnect between the gene and protein expression profiles after PDGF-ab stimulation, which have to be investigated further. This study may provide new insights to the differences between fibroblast of different origin and their response to fibrotic factors.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]Vandesompele, J. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol.3, research0034.1 (2002).Disclosure of InterestsSofie Falkenløve Madsen: None declared, Pernille Juhl Employee of: Nordic Bioscience, Jannie Marie Bülow Sand Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, Morten Karsdal Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience, Christian Thudium Shareholder of: Nordic Bioscience, Employee of: Nordic Bioscience