Mitogenicity and release of vascular endothelial growth factor with and without heparin from fibrin glue

Purpose: Fibrin glue (FG) has been used for local cytokine delivery on both vascular grafts and angioplasty sites. We measured the diffusive release o f vascular endothelial growth factor (VEGF) and heparin from FG and the mit ogenic activity of VEGF with and without heparin in FG on canine endothelia l cells (ECs) and smooth muscle cells (SMCs). Methods: Release of VEGF labeled with iodine 125 and tritiated heparin from FG into the overlying media was serially measured over 96 hours, and the d ata are reported as the mean percent released +/- SD. Proliferation assays measuring tritiated thymidine incorporation were performed for ECs and SMCs plated in media with 10% serum on FG containing various concentrations of VEGF and heparin. Media was placed on the FG for 24 hours and removed befor e plating cells to minimize the effect of the released, soluble VEGF and he parin. Results: At 24 hams, 54% +/- 1% and 58% +/- 1% of the radioactive VEGF and heparin were released, respectively, with minimal release thereafter (58% /- 1% and 66% +/- 1% at 96 hours). The ECs, SMCs, or media only (no cells) was plated an FG containing radioactive VEGF in an immediate or 24-hour del ayed fashion for 72 hours to determine the percent release of VEGF into the media with the two different methods of plating. Cell type and the presenc e or absence of cells did not affect VEGF release, but there was three time s more VEGF in the media for the immediate versus delayed plating (P < .001 ). Without heparin, VEGF at 100 ng/mL, or more in the FG was needed to indu ce EC proliferation. Heparin at 5 U/mL, enhanced EC proliferation at the VE GF dose of 100 ng/mL, as compared with no heparin (P < .001), but not at th e VEGF dose of 1000 ng/mL, which Likely represents a maximal response. With heparin at 500 U/mL, the ECs died. In contrast, VEGF, in the presence or a bsence of heparin, did not affect SMC proliferation. Conclusions: We conclude that FG with VEGF at 1000 ng/mL, and heparin at 5 U/mL is the optimal concentration for in vivo use because this may encourag e EC, but not SMC, proliferation. The VEGF at 1000 ng/mL, should leave mito genic concentrations of VEGF intact after the initial, diffusive loss, and the addition of heparin at 5 U/mL may enhance VEGF mitogenic activity.