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Home > Medical Professionals > Research Initiatives Conference > Poster Abstracts Accepted > Moya Design and Characterization of a More Potent

Design and Characterization of a More Potent and Longer Lasting FGF-1 Based Protein

Monica L Moya1, Eric M Brey1, Luke P Brewster2, Vicki Husak3, Joan Ellinger3, Wilson H Burgess4, Howard P Greisler2
¹Illinois Institue of Technology, Chicago, IL;²Loyola University Medical Center, Chicago, IL;³Hines Veteran's Hospital, Chicago, IL;⁴Clearant Inc, Gaithersburg, MD

Objective: Therapeutic stimulation of neovascularization has been investigated for tissue engineering and for the treatment of ischemic tissues. Strategies using growth factors have shown promise in pre-clinical models, but translation to human application has met with limited success partly because of the short half-lives of injected proteins and their dependence on additional factors (such as heparin) for maximum therapeutic response. We have previously shown that a heparin binding growth associated molecule (HBGAM)- fibroblast growth factor 1 (FGF-1) chimeric protein retains FGF-1’s normal mitogenic properties with enhanced EC specificity but without the requirement of exogenous heparin. Separately we have shown that by replacing the arginine with lysine at residue 136 (R136K mutant) in FGF-1’s primary thrombin cleavage site we were able to enhance the half-life of FGF-1. The goal of this research was to generate an R136K-HBGAM chimera, a protein with both heparin independence and thrombin resistance properties. Methods: Canine jugular vein ECs and carotid artery SMCs were grown in 96 well plates to approximately 80% confluence. After growth arrest in serum free media for 24 hours, equal molar concentrations of FGF-1, R136K, HBGAM-FGF-1 and HBGAM-R136K were added to the media with and without 5 U/ml of heparin. Cell proliferation was assessed using Tritiated-thymidine incorporation Thrombin degradation of the proteins was measured using sandwich ELISAs developed in our lab. Thrombin was added to the proteins and incubated at 37°C for specified time points and amount of non-degraded proteins was measured at each. Results Unlike FGF-1 or R136K, HBGAM-FGF-1 and HBGAM-R136K induced heparin independent proliferation of ECs and SMCs. HBGAM-R136K induced less proliferation of both ECs and SMCs than HBGAM-FGF-1 at low concentrations, with a delay in maximum stimulation. In the absence of heparin HBGAM-R136K was able to achieve the maximum potency of FGF-1 plus heparin. Degradation assays are currently ongoing. Conclusions By fusing HBGAM to our R136K mutant we have generated a growth factor that stimulates heparin independent proliferation of both ECs and SMCs. The mitogenicity of HBGAM-R136K for both ECs and SMCs achieved the maximum stimulation of FGF-1 without addition of exogenous heparin. The HBGAM-R136K mutant chimera may offer unique advantages over currently available angiogens for vascular tissue engineering and therapeutic angiogenesis.