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James W Foster, Yassine Daoud, Walter J. Stark, Laura Kallay, John R Hassell, Albert S Jun, Shukti Chakravarti; Cultured stromal cells from keratoconus corneas show reduced fibrillar collagen deposition with no changes in overall collagen production. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1931.
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© ARVO (1962-2015); The Authors (2016-present)
The corneal thinning disease, keratoconus is the leading cause for corneal transplants. We have recently shown altered Transforming growth factor β (TGFβ) signalling in human keratoconic patients within our in vitro disease cell culture model (Foster et. al., PLoS ONE 9, e106556, 2014). As TGFβ signalling regulates extracellular matrix (ECM) production and turnover we investigated this in stromal cells of multiple keratoconus (KC) and donor (DN) corneas.
7 DN and 7 KC keratocytes were extracted through collagenase digestion and cultured in DMEM:F12 supplemented with 5% FBS to encourage activation and proliferation. Fibroblasts were then seeded and cultured for up to 21 days in low-glucose serum-free media to encourage reversion to a keratocyte-like phenotype. The activation state of keratocytes was monitored using bright-field microscopy in conjunction with SMA, Thy-1, CD34, ALDH3A1 and Keratocan expression via western blot and qRT-PCR. Total collagen in the media and cell layer quantified using the hydroxyproline assay. ECM production was followed using the markers: keratocan, lumican, collagen I, collagen V. Two-tailed student’s t test was used for statistical analysis.
In their comparable quiescent keratocyte-like state (low Thy-1/SMA) KC and DN cells had similar hydroxyproline and thus overall collagen content. However, in KC, secreted Collagen type I was higher, and Collagen type V was lower in the culture media. In the cell layer extract both Collagen V and I were decreased in the KC samples. Both media and cell layer associated keratocan, a critical corneal ECM proteoglycan, was decreased 30% and 57% respectively (p=0.132 and p=0.0026), while ALDH3A1 in cell extracts was ~30% (p=0.0276) reduced in KC relative to DN.
Defects in ECM assembly and deposition are suggested in KC as Collagen V, with a known role in fibrillar collagen deposition was decreased, while fibrillar Collagen I was decreased in the cell layer but increased in the media. Reduced corneal crystallins suggest altered cellular phenotype, and an altered TGFβ signalling network underlying ECM defects as suggested in our previous study.
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