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

Fibrin glue (FG) has been used as a delivery system for bioactive agents on grafts and angioplasty sites. Reports from two different institutions suggest that heparin concentrations of 500 U/mL in FG inhibit smooth muscle cell (SMC) proliferation, but do not effect endothelial cell (EC) proliferation. The purposes of this study were to (1) quantify the diffusive release of fibroblast growth factor-1 (FGF-1) and heparin from FG; (2) determine the effect of heparin and FGF-1 on SMC proliferation when the cells are immediately plated on the FG; and (3) by means of the diffusive release data, design a new in vitro model that may differentiate the effect of FG-incorporated FGF-1 and heparin, rather than the released, solubilized components of these two factors, on SMC and EC proliferation.

125I-FGF-1 or 3H-heparin release from FG into the overlying media 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 containing 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.

In the first 24 hours, 70% +/- 1% of the FGF-1 and 59% +/- 2% of the heparin in the FG was released into the overlying media, with minimal release occurring thereafter. The cell type or absence of cells did not affect 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 plating because of a diffusive release primarily in the first 24 hours. A heparin concentration of 500 U/mL inhibited SMC proliferation, as compared with 5 U/mL heparin, only when immediate plating of SMCs was used. Comparing immediate versus delayed SMC plating, at equivalent FGF-1 and heparin doses, immediate plating induced greater proliferation than delayed plating; this was 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 growth was inhibited at 50 U/mL heparin, as compared with 5 U/mL heparin.

The differences in SMC proliferation when comparing immediate versus 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 system that minimizes the effects of soluble factors. This is an important distinction, 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 within the FG.