title: Fibroblast growth factor-2-toxin induced cytotoxicity: differential sensitivity of co-cultured vascular smooth muscle cells and endothelial cells.
abstract: Recombinant FGF-2-SAP is a mitotoxin consisting of the plant-derived ribosome-inactivating toxin saporin (SAP) fused to basic fibroblast growth factor (FGF-2). FGF-2-SAP targets and kills cells bearing upregulated FGF receptors. In vivo, FGF-2-SAP inhibits smooth muscle cell hyperplasia in models of restenosis. The present study examined the potential for a differential effect of FGF-2-SAP on canine vascular endothelial cells (EC) and smooth muscle cells (SMC) separately as well as in a novel co-culture model. Canine vascular SMC and EC cultures were established separately and made quiescent once cells reached 80% confluence. Following the release from growth arrest, both cell types were treated with FGF-2-SAP, or FGF-2, or SAP alone for 48 h. [3H]TdR incorporation 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 either side of a microporous 13 microm thick polyester membrane insert. Both cell types were grown to 80% confluence and independently made quiescent. Following the release from growth arrest, cells were treated with FGF-2-SAP, or FGF-2, or SAP alone. Negative and positive control groups were untreated wells containing phosphate buffered saline and complete growth media, respectively. After 48 h, both [3H]TdR incorporation and total DNA content, by fluorometric measurement, were quantitated in SMC and EC independently. FGF-2-SAP showed a concentration-dependent cytotoxicity in both canine SMC and EC but cytotoxicity for EC required substantially higher concentrations. In co-cultured SMC, FGF-2-SAP significantly decreased both [3H]TdR uptake and total DNA content at 0.5, 5, 50, and 500 ng/ml (0.01-10 nM) compared to positive controls. In co-cultured EC, FGF-2-SAP decreased [3H]TdR uptake at 50 and 500 ng/ml and total DNA content at 500 ng/ml compared to positive controls. Neither SAP alone nor FGF-2 alone showed a significant effect on [3H]TdR uptake or DNA content of either SMC or EC. In this unique co-culture model, which better replicates the relationship between SMC and EC in vivo, we demonstrated a dose-response range of FGF-2-SAP at which both the proliferation and total cell number of SMC, but not EC, is significantly reduced. These data suggest that FGF-2-SAP may have therapeutic utility in inhibiting myointimal hyperplasia in the absence of a deleterious effect on regenerating endothelium following vascular reconstructions.
authors.affiliation: Department of Surgery, The Chicago Medical School, North Chicago, IL, USA.
authors.forename: P H
authors.initials: PH
authors.lastname: Lin
authors.forename: D
authors.initials: D
authors.lastname: Ren
authors.forename: M K
authors.initials: MK
authors.lastname: Hirko
authors.forename: S S
authors.initials: SS
authors.lastname: Kang
authors.forename: G F
authors.initials: GF
authors.lastname: Pierce
authors.forename: H P
authors.initials: HP
authors.lastname: Greisler
mesh_terms: D000818:Animals; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D004247:DNA / Q000096:biosynthesis; D004285:Dogs; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D016222:Fibroblast Growth Factor 2 / Q000633:toxicity*; D006965:Hyperplasia; D000922:Immunotoxins / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects*; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000633:toxicity*; D011993:Recombinant Fusion Proteins / Q000633:toxicity; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins; D013050:Spectrometry, Fluorescence; D013936:Thymidine
chemical_list: D000922:Immunotoxins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D016222:Fibroblast Growth Factor 2; D004247:DNA; D009699:N-Glycosyl Hydrolases; D000076985:Saporins; D013936:Thymidine