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Abirami Santhanam, Andre Torricelli, Jiahui Wu, Steven E Wilson, ; The epithelial basement membrane component perlecan is produced by stromal cells in vitro. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4902.
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To investigate the production of the corneal epithelial basement membrane (BM) component perlecan by cultured stromal cells.
Keratocytes were isolated from fresh rabbit corneal stroma treated with hyaluronidase and collagenase for 24 hours. Keratocytes were then grown in different serum conditions 1%, 5% and 10% FBS with or without the growth factors FGF-2 (40ng/ml) and heparin sulfate (HS) (5ug/ml) or TGF-β (2ng/ml) for 60-72 hours. Different culture condition effects were analyzed by real time PCR, immunostaining and western blots for the cell specific markers keratocan, lumican and alpha-smooth muscle actin (SMA). Perlecan mRNA synthesis was analyzed by QPCR and protein production by western blotting, immunostaining and ELISA.
Keratocytes grown in serum-free medium expressed high levels of keratocan and lumican (keratocan+, lumican+ and SMA-) while those grown in 10% FBS with FGF-2 expressed low levels and were corneal fibroblasts (keratocan- lumican- and SMA-). As the serum concentration increased, the cultured cells became less keratocyte and more corneal fibroblast in phenotype. Analysis of α-SMA revealed that cells grown in 1% FBS with TGF-β expressed high levels of α-SMA and were myofibroblasts (keratocan- lumican- and SMA+). Keratocytes produced more perlecan protein than corneal fibroblasts or myofibroblasts (Fig.1).
Keratocytes grown in vitro produce perlecan that likely contributes to epithelial basement membrane regeneration in vivo. Corneal fibroblasts and myofibroblasts also produce perlecan, albeit at lower levels than keratocytes.
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