Improving endothelial healing with novel chimeric mitogens.

BACKGROUND: Chimeric proteins may be used to direct cell-specific activity. Heparin-binding growth-associated molecule (HBGAM) binds to cell receptors that are relatively more robust on endothelial cells, and it may confer endothelial cell selectivity to potent angiogens such as fibroblast growth factor-1 (FGF-1). METHODS: By ligating fibroblast growth factor or its potent mutant, S130K, to HBGAM, we tested their effect on re-endothelialization after angioplasty injury by using a canine model. RESULTS: Both HBGAM/S130K- and HBGAM/FGF-1-treated arteries had increased neointimal mitotic index and re-endothelialization rates at 30 days compared with control arteries without inducing a significant increase in the neointimal thickness or the ratio of neointimal to medial thickness between treatment and control groups. CONCLUSION: HBGAM/S130K and HBGAM/FGF-1 facilitates endothelial healing without myointimal thickening after canine carotid artery balloon angioplasty injury. Application of these growth factors in fibrin glue may improve endothelialization clinically after angioplasty or endarterectomy.

Silyl-heparin bonding improves the patency and in vivo thromboresistance of carbon-coated polytetrafluoroethylene vascular grafts.

OBJECTIVES: Our purpose was to improve the performance of carbon-coated expanded polytetrafluoroethylene vascular grafts by bonding the grafts with silyl-heparin, a biologically active heparin analog, using polyethylene glycol as a cross-linking agent. Material and method Silyl-heparin-bonded carbon-coated expanded polytetrafluoroethylene vascular grafts (Bard Peripheral Vascular, Tempe, Ariz), were evaluated for patency and platelet deposition 2 hours, 7 days, and 30 days after graft implantation in a canine bilateral aortoiliac artery model. Platelet deposition was determined by injection of autologous, (111)Indium-radiolabeled platelets, followed by a 2-hour circulation period prior to graft explantation. Histologic studies were performed on a 2-mm longitudinal strip of each graft (7-day and 30-day groups). Heparin activity of the explanted silyl-heparin grafts was determined by using an antithrombin-III based thrombin binding assay. RESULTS: Overall chronic graft patency (7-day and 30-day groups) was 100% for the silyl-heparin bonded (16/16) grafts versus 68.75% for control (11/16) grafts (P =.043). Acute 2-hour graft patency was 100% for the silyl-heparin bonded (6/6) grafts versus 83.3% for control (5/6) grafts. Radiolabeled platelet deposition studies revealed a significantly lower amount of platelets deposited on the silyl-heparin grafts as compared with control grafts in the 30-day group (13.8 +/- 7.18 vs 28.4 +/- 9.73, CPM per cm2 per million platelets, mean +/- SD, P =.0451, Wilcoxon rank sum test). In the 2-hour group of dogs, a trend towards a lower deposition of platelets on the silyl-heparin grafts was observed. There was no significant difference in platelet deposition between the two grafts in the 7-day group. Histologic studies revealed a significant reduction in intraluminal graft thrombus present on the silyl-heparin grafts as compared with control grafts in the 30-day group of animals. In contrast, there was no difference in amount of graft thrombus present on both graft types in the 7-day group of dogs. Pre-implant heparin activity on the silyl-heparin bonded grafts was 2.0 IU/cm(2) (international units[IU]/cm(2)). Heparin activity remained present on the silyl-heparin grafts after explantation at all 3 time points (2 hours: above upper limit of assay, upper limit = 0.57, n = 6; 7 days: 0.106 +/- 0.015, n = 5; 30 days: 0.007 +/- 0.001, n = 5; mean +/- SD, IU/cm(2)). CONCLUSION: Silyl-heparin bonding onto carbon-coated expanded polytetrafluoroethylene vascular grafts resulted in (1) improved graft patency, (2) increased in vivo graft thromboresistance, and (3) a significant reduction in intraluminal graft thrombus. This graft may prove to be useful in the clinical setting.

Silyl-heparin adsorption improves the in vivo thromboresistance of carbon-coated polytetrafluoroethylene vascular grafts.

BACKGROUND: Expanded polytetrafluoroethylene (ePTFE) remains the most commonly utilized synthetic graft material for infrainguinal arterial reconstruction. However, patency rates of ePTFE bypass grafts are inferior to those observed with autogenous vein grafts. Modification of the luminal surface of ePTFE grafts such as coating with carbon or heparin, may prevent early graft failures and improve overall patency rates. We now report our results with a silyl-heparin adsorbed carbon-coated ePTFE graft. METHODS: Silyl-heparin was adsorbed onto carbon-coated ePTFE vascular grafts (Bard Peripheral Vascular, Tempe, Arizona), which were then evaluated for patency and platelet deposition acutely (2 hours after implantation) and 7 days after graft implantation in mongrel dogs. Dogs underwent bilateral aortoiliac grafting where one heparin adsorbed carbon-coated graft and one carbon-coated graft (control) were placed on either (alternating) side. Platelet deposition was determined by injection of autologous (111)Indium radiolabeled platelets followed by a 2-hour circulation period prior to explantation of grafts. Heparin activity of the silyl-heparin grafts (at preimplantation and explantation) was determined using an antithrombin-III based thrombin binding assay. RESULTS: Graft patency was 100% for both heparin coated (5 of 5) grafts and control (5 of 5) grafts in the acute group of dogs. In the 7-day group, patency was 87.5% for heparin coated (7 of 8) grafts and 50% for control (4 of 8) grafts (P = 0.28, Fisher's exact test). Radiolabeled platelet studies revealed a significantly lower deposition of platelets on heparin coated grafts compared with control grafts in the acute group (17.3 +/- 13.5 versus 35.2 +/- 17.9 counts per minute, per cm(2) per million platelets, mean +/- SEM; n = 5, P <0.05, paired Student t test). In the 7-day group of dogs with bilaterally patent grafts (4 of 8), a trend toward a lower deposition of platelets on heparin coated grafts compared with control grafts was observed (1.55 +/- 0.409 versus 2.14 +/- 1.13 counts per minute, per cm(2) per million platelets, mean +/- SEM; n = 4, P = 0.52, paired Student t test). Eight percent of the preimplantation heparin activity remained on the explanted silyl-heparin grafts after 2 hours and only 2% after 7 days. CONCLUSIONS: Silyl-heparin adsorption onto carbon-coated ePTFE vascular grafts resulted in improved acute thromboresistance in a canine bilateral aortoiliac model. Ongoing laboratory efforts are aimed at improving the silyl-heparin retention on vascular grafts. This graft may prove to be useful in the clinical setting.

R136K fibroblast growth factor-1 mutant induces heparin-independent migration of endothelial cells through fibrin glue.

OBJECTIVES: 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. CONCLUSION: 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.