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Thomas Stahnke, Marian Löbler, Andreas Wree, Oliver Stachs, Klaus-Peter Schmitz, Rudolf Guthoff; Characterization of human ocular fibroblast-subpopulations to prevent fibrosis following fistulating glaucoma surgeries. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2139.
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© ARVO (1962-2015); The Authors (2016-present)
Fibrotic re-stenosis following fistulating glaucoma surgeries is the major factor for a postoperative decrease in liquid drain. Hence, one of the most important clinical goals is to develop strategies for fibrosis prevention. This study aimed at characterization of fibrotic potential of different primary human ocular fibroblast-subpopulations. Particularly, we focused on the TGF-b pathway. As this signaling pathway is involved in fibrosis, we investigated its implication in extracellular matrix (ECM) component synthesis and secretion in different ocular fibroblast-subpopulations.
Human primary fibroblasts from choroidea (hCF), sclera (hSF), Tenon capsule (hTF), and orbital fat tissue (hOF) were isolated and cultured. For immunocytochemical analysis cells were cultured on coverslips and fixed. Incubation with primary antibodies directed against TGF-b pathway components and ECM proteins was carried out followed by incubation with Cy2 and Cy3 conjugated secondary antibodies. Coverslips were mounted with DAPI-containing mounting medium. To analyze protein lysates 1-D-SDS-PAGE and Western blotting were performed according to the standard protocols. Proteins were transferred to PVDF-membranes and antibodies were used to detect TGF-b pathway components. Targets were analyzed by enhanced chemiluminescence (ECL) detection method followed antibody incubation.
Cytokine TGF-b1 and the relevant receptors TGF-b-receptor 1 and 2 expression was demonstrated in every fibroblast-subpopulation. Moreover, TGF-b pathway activation could be observed by phosphorylation specific translocation of the downstream mediators (smad 2 and 3) into the nuclei. However, the level of activation and ECM protein synthesis was different between fibroblast-subpopulations with lowest levels in hCF cultures.
Cell culture model of primary ocular fibroblast-subpopulations pave the way for comparative analyzing of fibrotic processes. Moreover, tissue-specific inhibition of cell proliferation, ECM synthesis or transformation into fibrotic active myofibroblasts could be possible. Exploring the influence of TGF-b pathway inhibitors between different fibroblast-subpopulations opens up specific possibilities in cell population dependent prevention of postoperative scarring processes and fibrosis after fistulating glaucoma surgeries.
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