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James W Foster, Ricardo Martins Gouveia, Che John Connon, ; Recovery of the keratocyte phenotype in vitro is mediated through glucose response signalling. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5142.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the role of glucose availability on the phenotype of human corneal stromal fibroblasts in vitro in serum free conditions. Serum free conditions have been utilised to partially restore the quiescent phenotype of keratocytes that had been expanded in serum containing culture conditions. However, the resulting phenotype differs from in vivo as the cells do not possess the dendritic morphology and lack several key markers. We hypothesise that glucose concentration can further restore the native phenotype in serum free conditions.
Human corneal keratocytes were extracted from normal corneal limbal rings and expanded in DMEM:F12 containing 5% foetal bovine serum for 2 passages to activate the keratocytes. Fibroblasts were then maintained in serum free media of different glucose concentration for 3 weeks and their phenotype monitored through immunohistochemistry, quantitative PCR and immuno-blotting. The phosphodiesterase inhibitor IBMX and the PKCζ inhibitor ZIP was used to probe the effects of cAMP on keratocyte phenotype.
Cells maintained in low-glucose, serum-free conditions reverted to a dendritic phenotype, stained positive for ALDH1A1 and had a condensed ‘bean shaped’ nucleus with weak F-actin staining over 3 weeks. Immunoblotting demonstrated these cells were also positive for keratocan, lumican and CD34 whilst only weak expression of smooth muscle actin was detected. SMA expression was shown to be dose dependent with increasing glucose concentration eliciting increased expression, the reverse was demonstrated for ALDH1A1 with a 10 fold increase in expression relative to day 0 in low glucose media (n=5). CD34 transcription increased 40 fold relative to day 0 (n=5), and showed increased post transcriptional modification. Increased intracellular cAMP increased CD34 expression 2 fold over 7 days (n=5), whilst inhibition of PKCζ reduced the expression of ALDH1A1 and CD34 to basal levels.
The quiescent phenotype of the native human corneal keratocyte is rapidly lost when placed in standard tissue culture conditions. This study demonstrates the ability to modulate the phenotype of keratocytes in vitro by controlling glucose concentrations within the media. The ability of low glucose concentrations’ to revert keratocyte phenotype back to a more native type suggests a role for glucose availability on corneal keratocyte phenotype mediated through cAMP and PKCζ.
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