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David Eveleth, Xue Xia, Ken Thomas, Michael Blaber; Engineered FGF-1 derivatives stimulate proliferation of corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1169.
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© ARVO (1962-2015); The Authors (2016-present)
Fibroblast growth factors (FGFs) are potent stimulators of corneal endothelial cell growth in vitro and in vivo, but their therapeutic value is compromised due to poor pharmaceutical properties. We sought to improve the stability and potency of FGF-1 via site-directed mutagenesis and evaluate the potency and suitability of these modified compounds for stimulation of corneal endothelial cell growth.
Site-directed mutagenesis was used to generate engineered FGF-1s (eFGF-1s) with enhanced stability and improved pharmaceutical properties. Biophysical and structural properties were measured using isothermal equilibrium denaturation and X-ray crystallography. Pharmacokinetics were evaluated in rabbits using ELISA-based quantification of FGFs. Mitogenic activity was evaluated using primary cultures of rabbit and human corneal endothelial cells.
Substitutions at positions 12, 117 and 134 resulted in an increase in melting temperature of 19K and a 33-fold increase in activity in the absence of added heparin. Substitutions of the cysteines at positions 83 and 117 are thermodynamically destabilizing but can be compensated for by stabilizing mutations at positions 44 and 132. eFGF-1s exhibit longer half-lives in rabbits and stimulate mitogenesis of primary rabbit and human corneal endothelial cells at EC50 values substantially lower than wtFGF-1, even in the absence of heparin. Cultures maintained a morphology consistent with endothelial cells and did not undergo fibroblastic transition in response to FGFs.
Engineered FGF-1s with superior pharmaceutical properties stimulate corneal endothelial cell growth.
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