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L. S. Etheredge, B. Kane, J. Hassell; Growth Factor Dependent Changes in the Secretome of Keratocytes. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4039.
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Fibrillar collagens and keratan sulfate proteoglycans are the major components of the cornea stroma and must be replaced by keratocytes during wound healing to ensure corneal strength and transparency. We evaluated the effect of IGF-I, TGF-ß, FGF-2, and PDGF on morphology, rate of proliferation, and ECM synthesis by primary cultured bovine keratocytes.
Keratocytes were collagenase-isolated from bovine corneas and plated at 20,000 cells/cm2 in DMEM/F12 to allow cell attachment, and then cultured with either DMEM/F12 (control), or DMEM/F12 supplemented with either 10ng IGF-I, 2ng TGF-ß, 10ng FGF-2, or 10ng PDGF/ml for 10 days, all with ascorbate. The growth of the cells was determined by DNA content (cyquant assay) and the morphological appearance by phase-contrast microscopy. The level of extracellular matrix (ECM) components synthesized by cells and secreted into the media was determined by (1) 3H glycine incorporation into papain sensitive protein; (2) 3H glycine incorporation collagenase sensitive protein; (3) analysis of pepsin resistant fibrillar collagen by SDS/PAGE and simply blue staining; (4) western blots with protein-specific antibodies to procollagen I and III, αSMA, fibronectin, and keratocan.
Compared to control cultures, IGF-I stimulated cell number by 0.7 fold, total protein by 11.0 fold, fibrillar collagen by 10.4 fold, and keratocan by 16.6 fold; TGF-β stimulated cell number by 1.8 fold, total protein by 6.7 fold, fibrillar collagen by 4.3 fold, but inhibited keratocan by 80%; FGF-2 stimulated cell number by 3.5 fold, but inhibited both fibrillar collagen and keratocan by 60%, and total protein by 30%; PDGF stimulated cell number by 2.4 fold, total protein by 6.6 fold, fibrillar collagen by 5.2 fold, and keratocan by 3.7 fold. Procollagen I was stimulated by IGF-I, TGF-β, and PDGF and inhibited by FGF-2. TGF-β stimulated procollagen III, had the highest levels of fibronectin, and expressed αSMA. Cells in IGF-I and PDGF appeared dendritic while those in FGF-2 and TGF-β appeared fibroblastic and myofibroblastic respectively.
The results of this study show that over half of total protein synthesis stimulated by IGF-I, TGF-β, and PDGF was fibrillar collagen. In addition, only IGF-I and PDGF enhanced the production of keratocan while TGF-β induced the highest synthesis of fibronectin. In contrast, FGF-2 inhibited synthesis of total protein, collagen, and keratocan. This suggests that the levels of synthesis of individual ECM components are growth factor dependent and that IGF-I and PDGF may be useful in restoring normal matrix synthesis by keratocytes during corneal wound healing.
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