title: Construction and characterization of a thrombin-resistant designer FGF-based collagen binding domain angiogen.
abstract: Humans demonstrate limited spontaneous endothelialization of prosthetic bypass grafts. However the local application of growth factors to prosthetic grafts or to injured blood vessels can provide an immediate effect on endothelialization. Novel chimeric proteins combining potent angiogens with extracellular matrix binding domains may localize to exposed matrices and provide sustained activity to promote endothelial regeneration after vascular interventions. We have ligated a thrombin-resistant mutant of fibroblast growth factor (FGF)-1 (R136K) with a collagen binding domain (CBD) in order to direct this growth factor to sites of exposed vascular collagen or selected bioengineered scaffolds. While FGF-1 and R136K are readily attracted to a variety of matrix proteins, R136K-CBD demonstrated selective and avid binding to collagen approximately 4x that of FGF-1 or R136K alone (P<0.05). The molecular stability of R136K-CBD was superior to FGF-1 and R136K. Its chemotactic activity was superior to R136K and FGF-1 (11+/-1% vs. 6+/-2% and 4+/-1%; P<0.01). Its angiogenic activity was similar to R136K and significantly greater than control by day 2 (P<0.01). After day 3, FGF-1-treated endothelial cell's (EC) sprouts had regressed back to levels insignificant compared to the control group (P=0.17), while both R136K and R136K-CBD continued to demonstrate greater sprout lengthening as compared to control (P<0.0002). The mitogenic activity of all growth factors was greater than control groups (20% PBS); in all comparisons (P<0.0001). This dual functioning angiogen provides proof of concept for the application of designer angiogens to matrix binding proteins to intelligently promote endothelial regeneration of selected matrices.
authors.affiliation: Department of Surgery, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA.
authors.forename: Luke P
authors.initials: LP
authors.lastname: Brewster
authors.forename: Cicely
authors.initials: C
authors.lastname: Washington
authors.forename: Eric M
authors.initials: EM
authors.lastname: Brey
authors.forename: Andrew
authors.initials: A
authors.lastname: Gassman
authors.forename: Anu
authors.initials: A
authors.lastname: Subramanian
authors.forename: Jen
authors.initials: J
authors.lastname: Calceterra
authors.forename: William
authors.initials: W
authors.lastname: Wolf
authors.forename: Connie L
authors.initials: CL
authors.lastname: Hall
authors.forename: William H
authors.initials: WH
authors.lastname: Velander
authors.forename: Wilson H
authors.initials: WH
authors.lastname: Burgess
authors.forename: Howard P
authors.initials: HP
authors.lastname: Greisler
mesh_terms: D001241:Aspirin / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects; D003094:Collagen / Q000235:genetics / Q000378:metabolism*; D004338:Drug Combinations; D004789:Enzyme Activation / Q000187:drug effects; D005337:Fibrin / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000378:metabolism*; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008938:Mitosis; D011485:Protein Binding; D012680:Sensitivity and Specificity; D019802:Succinic Acid / Q000378:metabolism* / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism*
chemical_list: D004338:Drug Combinations; C469372:aspirin, succinic acid drug combination; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D005346:Fibroblast Growth Factors; D005337:Fibrin; D003094:Collagen; D019802:Succinic Acid; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D013917:Thrombin; D001241:Aspirin