title: R136K fibroblast growth factor-1 mutant induces heparin-independent migration of endothelial cells through fibrin glue.
abstract: OBJECTIVE
  R136K is a mutation of fibroblast growth factor-1 (FGF-1) in which arginine replaces lysine at the primary thrombin cleavage site. This may be important in vivo in inducing endothelial cell (EC) migration and coverage of arterial injury sites by allowing R136K to be used in a fibrin glue delivery system, without thrombin-induced degradation, in the absence of heparin. The objectives of this study were to determine whether R136K, with and without heparin, can induce migration of EC and smooth muscle cells (SMC) through fibrin glue, and to compare these results with those of wild-type FGF-1; and to determine the resistance of R136K to thrombin-induced degradation versus FGF-1.
  
  
  METHODS
  The dose-response migration through fibrin glue induced by wild-type FGF-1 and the R136K mutant in the presence and absence of heparin was tested with EC and SMC. Migration was tested with 50, 100, and 200 ng/mL of both FGF-1 and R136K, either with or without 5 U/mL of heparin. Migration of EC was also assessed after growth inhibition with mitomycin C. A novel modified Boyden chamber-type migration assay using fibrin glue on the upper surface of the chamber filter was used to test migration. The fluorescent marker calcein was used to identify those cells that had migrated through the fibrin glue and were embedded in the filter. Molecular degradation by thrombin was assessed with sodium dodecylsulfate polyacrylamide gel electrophoresis.
  
  
  RESULTS
  For EC, R136K in the absence of heparin induced significantly more migration than did FGF-1 at 50 (P <.002), 100 (P <.0001), and 200 (P <.0001) ng/mL. In the presence of heparin, a chemotactic response of EC to cytokine was seen at all doses, with no significant difference between FGF-1 and R136K. A dose-dependent difference was noted in this group between the 100 and 200 ng/mL concentrations of cytokine (for FGF-1, P <.0001; for R136K, P <.0001). SMC showed no difference in migration with FGF-1, R136K, or negative control at any dose in the presence or absence of heparin. Gel electrophoresis demonstrated that R136K was more resistant to thrombin degradation than was FGF-1.
  
  
  CONCLUSIONS
  Site-directed mutagenesis of FGF-1 to R136K enables induction of heparin-independent migration of EC through fibrin glue at an optimal concentration of 100 ng/mL. Neither FGF-1 nor R136K elicits SMC migration through fibrin glue. The ability of R136K to induce EC migration through fibrin glue in the absence of heparin may prove useful in vivo by inducing EC migration and coverage of arterial injury sites, thus potentially reducing thrombogenicity and intimal hyperplasia.
authors.affiliation: Department of Surgery, Hines VA Hospital, IL, USA.
authors.forename: Victor Z
authors.initials: VZ
authors.lastname: Erzurum
authors.forename: Jie-Fang
authors.initials: JF
authors.lastname: Bian
authors.forename: Vicki A
authors.initials: VA
authors.lastname: Husak
authors.forename: Joan
authors.initials: J
authors.lastname: Ellinger
authors.forename: Lian
authors.initials: L
authors.lastname: Xue
authors.forename: Wilson H
authors.initials: WH
authors.lastname: Burgess
authors.forename: Howard P
authors.initials: HP
authors.lastname: Greisler
mesh_terms: D002455:Cell Division / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D016207:Cytokines / Q000008:administration & dosage; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects*; D015718:Fibrin Tissue Adhesive / Q000008:administration & dosage*; D005343:Fibrinolytic Agents / Q000008:administration & dosage*; D016220:Fibroblast Growth Factor 1 / Q000008:administration & dosage*; D006493:Heparin / Q000008:administration & dosage*; D006801:Humans; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects; D013917:Thrombin / Q000187:drug effects / Q000378:metabolism; D014014:Tissue Adhesives / Q000008:administration & dosage*
chemical_list: D016207:Cytokines; D015718:Fibrin Tissue Adhesive; D005343:Fibrinolytic Agents; D014014:Tissue Adhesives; D016220:Fibroblast Growth Factor 1; D006493:Heparin; D013917:Thrombin