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Xanthi Koufomichali, Julie Steinbrink, Yuki Morizane, Demetrios Vavvas; The Role Of AMP-dependent Kinase (AMPK) In The Function Of Fibroblasts Under Various Glucose Concentrations. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5933.
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© ARVO (1962-2015); The Authors (2016-present)
AMP-activate protein kinase (AMPK) is the major energy sensor of the cell. It gets activated under altered energy conditions such as hypoxia, stress, exercise and low glucose. Tissue repair involves coordinated interaction of numerous cell types with fibroblast having a central role. It's both a synthetic and a signaling cell (as it deposits collagen-rich matrix and secrets growth factors). Fibroblasts are known to be important in the development of diabetic retinopathy. Here we wanted to investigate the role of AMPK in an in vitro experimental model of fibroblast function and wound healing under different levels of glucose concentration.
Wild type (WT) and AMPKa12 knockout (AMPKKO) mouse embryonic fibroblasts (MEFs), were cultured at different glucose levels (0mM, 5mM, 25mM, 30mM) and two different cultured media (M199 and DMEM). Cell proliferation was checked with a modified MTT assay. Levels of several growth factors (VEGF, TGF-β, PDGF-AA, PDGF-BB, and MMP-9) were checked with ELISA. Migration was assessed with a modified scratch wound assay.
AMPKKO MEFs showed decreased growth rate at early time points compared to WT MEFs. At later time points there were minimal differences. Serum Starvation led to more cytotoxicity of the AMPKKO MEFs. Lower glucose concentrations showed diminished proliferation rates for both WT and KO MEFs. Modified Scratch wound assay showed a trend towards enhanced migration of the AMPKKO MEFS compared to WT. Evaluation by ELISAs showed a statistically increased level of VEGF, PDGF-AA and MMP-9 production by the knockout MEFs. The secretion levels were highest in the two groups with 0mM glucose, and decreased in elevated glucose concentrations. The levels of TGFβ-1 secretion were not statistically different between WT and AMPKKO MEFs at the various glucose concentrations. PDGF-BB was not of a detectable level. When we used medium M199 (5mM glucose), both WT and AMPKKO grew in a slower pace than with medium DMEM (25mM glucose). AMPKKO were affected in a greater extend from the use of medium M199 than WT MEFs.
AMPK seems to play an important role in the proliferation, migration and cytokine secretion of fibroblasts. Absence of AMPK enhanced the production of key fibroblast cytokines used in proliferation and wound repair under all glucose concentrations tested. This has implications for diabetic retinopathy.
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