Modulation of vascular cell growth kinetics by local cytokine delivery from fibrin glue suspensions

Purpose: Fibrin glue (FG) has been used as a delivery system for bioactive agents on grafts and angioplasty sites. Reports from two different institut ions suggest that heparin concentrations of 500 U/mL in FG inhibit smooth m uscle cell (SMC) proliferation, but do not effect endothelial cell (EC) pro liferation. The purposes of this study were to (1) quantify the diffusive r elease of fibroblast growth factor-1 (FGF-1) and heparin from FG; (2) deter mine the effect of heparin and FGF-1 on SMC proliferation when the cells ar e immediately plated on the EG; and (3) by means of the diffusive release d ata, design a new in vitro model that may differentiate the effect of FG-in corporated FGF-1 and heparin, rather than the released, solubilized compone nts of these two factors, on SMC and EC proliferation. Methods: I-125-FGF-1 or H-3-heparin release from FG into the overlying medi a was measured serially in a 96-hour period, either with or without cells. SMCs were immediately plated on FG containing various concentrations of FGF -1 and heparin. SMCs or ECs were plated on identical groups of FG containin g FGF-1 and heparin 24 hours after the FG was made to exclude the effect on cell growth of the initial release of FGF-1 into the media. Results: In the first 24 hours, 70% +/- 1% of the FGF-1 and 59% +/- 2% of t he heparin in the FG was released into the overlying media, with minimal re lease occurring thereafter. The cell type or absence of cells did not affec t release, but there was five times more FGF-1 and four times more heparin in the media at 72 hours for the immediate plating versus the delayed plati ng because of a diffusive release primarily in the first 24 hours. A hepari n concentration of 500 U/mL inhibited SMC proliferation, as compared with 5 U/mL heparin, only when immediate plating of SMCs was used. Comparing imme diate versus delayed SMC plating, at equivalent FGF-1 and heparin doses, im mediate plating induced greater proliferation than delayed plating; this wa s likely caused by the higher soluble FGF-1 concentration. Heparin doses as high as 500 U/mL had little effect on SMC proliferation. In contrast, ECs died with delayed plating on FG containing 500 U/mL, heparin, and their gro wth was inhibited at 50 U/mL heparin, as compared with 5 U/mL heparin, Conclusion: The differences in SMC proliferation when comparing immediate v ersus delayed plating are likely caused by diffusive release of heparin and FGF-1 into the media. Our ongoing work uses an optimized in vitro FG syste m that minimizes the effects of soluble factors. This is an important disti nction, because the cytokines that are released in vivo will be removed by blood flow and, thus, may not exert an effect unless they are contained wit hin the EG.