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TD Blalock, H MacArthur, GR Grotendorst, MH Goldstein, GS Schultz; Detection of Connective Tissue Growth Factor Binding in Human Corneal Fibroblasts and in Rat Corneas Following Phototherapeutic Keratectomy . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4213.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To biochemically characterize specific binding of connective tissue growth factor (CTGF) to cultured human corneal fibroblasts and to measure levels of CTGF binding in rat corneas during healing of excimer laser ablation wounds. Methods: Recombinant human CTGF was labeled with 125I using Iodogen beads and purified using G-25 gel filtration chromatography. Time course of CTGF binding to cultures of human corneal fibroblasts was determined by incubating cultures at 4° C or 37° C with 125I-CTGF alone or in the presence of 1 ug/ml unlabeled CTGF for 30 min, 1 hr, 2 hr, 4 hr, and 16 hr then the cell cultures were washed, solubilized, and bound 125I-CTGF was measured by gamma scintillation counting. Specificity of CTGF binding was established by incubating cultures with 125I-CTGF alone or in the presence of 1 ug/ml unlabeled CTGF or other unlabeled growth factors (TGF-b, TGF-a, EGF, bFGF, PDGF-BB, insulin). Equilibrium binding constants were calculated by Scatchard analysis of specific CTGF binding data. Cell surface binding proteins were covalently cross-linked with 125I-CTGF, solubilized, separated by polyacrylamide gel electrophoresis, and detected by exposure to X-ray film. To assess changes in CTGF binding during corneal healing, corneas of 21 rats were ablated 160 microns by excimer laser, and on days 0, 1, 3, 5, 7, 14, and 21 following surgery, 4 mm corneal punches were harvested and specific binding of 125I-CTGF measured. Results: Approximately 55% of total 125I-CTGF binding to human corneal fibroblasts was competed for by 1 ug/ml unlabeled CTGF but not by other unlabeled proteins. Specific binding 125I-CTGF reached a plateau by 2 hr at 4° C and by 30 minutes at 37° C. Scatchard analysis indicated two classes of binding sites with Kd's of 5 nM and 166 nM. 125I-CTGF was cross-linked to a binding protein with a molecular weight of about 250 kDa, which was competed for by unlabeled CTGF. CTGF binding to rat corneas increased over 50-fold on day 5 following excimer ablation. Conclusion: Specific, high affinity binding of CTGF was detected in human corneal fibroblasts and in rat corneas after PRK. Since CTGF promotes fibrosis, CTGF and its receptor may play a crucial role in the process of corneal wound healing and both make excellent target candidates for therapies to reduce scarring.
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