Covalently conjugated VEGF–fibrin matrices for endothelialization

• Published in: Journal of controlled release 2001-05, Vol.72 (1), p.101-113
• Main Authors: Zisch, Andreas H., Schenk, Ursula, Schense, Jason C., Sakiyama-Elbert, Shelly E., Hubbell, Jeffrey A.
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Blood. Blood coagulation. Reticuloendothelial system; Cell Division - drug effects; Cloning, Molecular; Coagulants - pharmacology; Cross-Linking Reagents; Culture Techniques; Drug Carriers; Endothelial Growth Factors - administration & dosage; Endothelial Growth Factors - pharmacology; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; Endothelium, Vascular - growth & development; Escherichia coli; Fibrin - chemistry; General pharmacology; Humans; Hydrogels; Lymphokines - administration & dosage; Lymphokines - pharmacology; Medical sciences; Mitogens - administration & dosage; Mitogens - pharmacology; Neovascularization, Physiologic - drug effects; Pharmaceutical technology. Pharmaceutical industry; Pharmacology. Drug treatments; Recombinant Fusion Proteins - pharmacology; Transglutaminases - pharmacology; vascular endothelial growth factor; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors
• ISSN: 0168-3659; 1873-4995
• DOI: 10.1016/S0168-3659(01)00266-8

Vascular endothelial growth factor (VEGF) is a key factor in endothelial cell biology and blood vessel formation and a candidate therapeutic for the stimulation of angiogenesis-dependent tissue regeneration. The objective of this study was to confer the angiogenic activity of VEGF 121 upon the biomaterial fibrin, a natural substrate for endothelial cell growth and clinically accepted as ‘fibrin glue’. To achieve this, we engineered fibrin-based hydrogels that were covalently modified with VEGF 121. Our laboratory has recently developed novel methodology that allows the covalent incorporation of exogenous bioactive peptides by the transglutaminase activity of factor XIIIa into fibrin during coagulation. Here, this ability of factor XIIIa to crosslink additional proteins within fibrin was employed to covalently incorporate VEGF 121. By recombinant DNA methodology, a mutant VEGF 121 variant, α 2-PI 1–8-VEGF 121, which contains an additional factor XIIIa substrate sequence NQEQVSPL at the aminoterminus, was expressed in E. coli. In soluble form, the mutant protein fully retained its mitogenic activity for endothelial cells. Using 125I-labeled α 2-PI 1–8-VEGF 121, its covalent incorporation and the efficiency of incorporation into fibrin was demonstrated and characterized. The immobilized, fibrin-conjugated VEGF 121 protein remained an active and very efficient mitogen for human endothelial cells grown on two-dimensional VEGF 121-modified fibrin surfaces, and the incorporation of increasing amounts of α 2-PI 1–8-VEGF 121 resulted in dose-dependent enhancement of endothelial cell growth. The VEGF-modified fibrin matrices can be formed as injectable gels in a single-step reaction under physiological conditions in vivo. When used as a ingrowth matrix, such VEGF incorporating materials could be useful in a variety of clinical situations that require an angiogenic response into an ischemic region or inplant.