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N. Sundarraj, E. DeGarmo, J. Funderburgh, L. DiCesare, Y. Sado; Phenotypic changes associated with corneal keratocytes activation in vitro: expression of type IV collagen isoforms . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3777.
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Purpose: : Corneal stromal cells (keratocytes) are quiescent in the adult cornea and are activated to differentiate to fibroblasts and myofibroblasts in response to an injury. Activation of keratocytes has been reported to result in the changes in the expression of collagen IV isoforms in vivo. This study evaluated whether similar changes occur upon activation of keratocytes in vitro. Methods: Rabbit corneal stromal cells were cultured in: 1,DMEM/F12 medium; 2, DMEM/F12 supplemented with Albumax (Invitrogen), insulin, trnasferrin and selenium (ITS); 3, DMEM/F12 with 5% fetal bovine serum (FBS), bFGF and heparin and 4, DMEM/F12 with 1% FBS and TGFß1. The expression of type IV collagen isoforms and keratan sulfate proteoglycan(s) (KSPG) was monitored immunocytochemically and by western blot analysis. Results: Corneal stromal cells grown in serum free DMEM/F12 morphologically resembled the quiescent keratocytes in vivo, did not proliferate and expressed alpha 1, 2 and 3 (IV) collagen chains and KSPG. ITS promoted cell division without affecting the above phenotypic characteristics. However, stromal cells activated with FBS and bFGF (fibroblasts) or with TGFß1(myofibroblasts), did not express KSPGs and alpha 3(IV) collagen but continued to express alpha 1 and 2(IV) collagen chains. These phenotypic changes in the activated cells did not revert back upon depriving the cells of FBS, bFGF or TGFß1 Conclusions: The present studies indicate that keratocytes in culture can be induced to undergo a limited number of cell division by insulin and/or transferrin and retain some of the phenotypic characteristics, including the expression of KSPG and the alpha 3 isoform of collagen IV, which are lost upon their activation to fibroblasts or myofibroblasts
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