FIBROBLAST GROWTH FACTOR-2 TOXIN-INDUCED CYTOTOXICITY - DIFFERENTIAL SENSITIVITY OF COCULTURED VASCULAR SMOOTH-MUSCLE CELLS AND ENDOTHELIAL-CELLS

Recombinant FGF-2-SAP is a mitotoxin consisting of the plant-derived r ibosome-inactivating toxin saporin (SAP) fused to basic fibroblast gro wth factor (FGF-2). FGF-2-SAP targets and kills cells bearing upregula ted FGF receptors. In vivo, FGF-2-SAP inhibits smooth muscle cell hype rplasia in models of restenosis. The present study examined the potent ial for a differential effect of FGF-2-SAP on canine vascular endothel ial cells (EC) and smooth muscle cells (SMC) separately as well as in a novel co-culture model. Canine vascular SMC and EC cultures were est ablished separately and made quiescent once cells reached 80% confluen ce. Following the release from growth arrest, both cell types were tre ated with FGF-2-SAP, or FGF-2, or SAP alone for 48 h. [H-3]TdR incorpo ration was used to determine the growth response of SMC and EC. The co -culture system was created by plating canine vascular SMC and EC on e ither side of a microporous 13 mu m thick polyester membrane insert. B oth cell types were grown to 80% confluence and independently-made qui escent. Following the release from growth arrest, cells were treated w ith FGF-2-SAP, or FGF-2, or SAP alone. Negative and positive control g roups were untreated wells containing phosphate buffered saline and co mplete growth media, respectively. After 48 h, both [H-3]TdR incorpora tion and total DNA content, by fluorometric measurement, were quantita ted in SMC and EC independently. FGF-2-SAP showed a concentration-depe ndent cytotoxicity in both canine SMC and EC but cytotoxicity for EC r equired substantially higher concentrations. In co-cultured SMC, FGF-2 -SAP significantly decreased both [H-3]TdR uptake and total DNA conten t at 0.5, 5, 50, and 500 ng/ml (0.01-10 nM) compared to positive contr ols. In co-cultured EC, FGF-2-SAP decreased [H-3]TdR uptake at 50 and 500 ng/ml and total DNA content at 500 ng/ml compared to positive cont rols. Neither SAP alone nor FGF-2 alone showed a significant effect on [H-3]TdR uptake or DNA content of either SMC or EC. In this unique co -culture model, which better replicates the relationship between SMC a nd EC in vivo, we demonstrated a dose-response range of FGF-2-SAP at w hich both the proliferation and total cell number of SMC, but not EC, is significantly reduced. These data suggest that FGF-2-SAP may have t herapeutic utility in inhibiting myointimal hyperplasia in the absence of a deleterious effect on regenerating endothelium following vascula r reconstructions. (C) 1998 Elsevier Science Ireland Ltd. All rights r eserved.