title: Acidic fibroblast growth factor-Pseudomonas exotoxin chimeric protein elicits antiangiogenic effects on endothelial cells.
abstract: It has recently been shown that chimeric toxins composed of acidic fibroblast growth factor fused to mutant forms of Pseudomonas exotoxin (aFGF-PE) are cytotoxic to a variety of tumor cell lines with FGF receptors. Although aFGF-PE might be considered as a possible chemotherapeutic toxin, limited knowledge is available concerning its effect on endothelia. This study investigates whether one of the aFGF-PE fusion proteins, aFGF-PE664GluKDEL, can function as an anti-angiogenic agent. Protein synthesis studies using rat epididymal fat pad microvascular endothelial cells (RFCs) indicated that after 24 h in culture, aFGF-PE had a significant inhibitory effect on protein synthesis at concentrations greater than 100 ng/ml. In cultures incubated with 1000 ng/ml aFGF-PE, RFC protein synthesis was inhibited as much as 83%. RFCs were also cultured in a 3-dimensional type I collagen gel and incubated with either transforming growth factor beta 1, aFGF-PE, or a combination of both. Transforming growth factor beta 1 elicits in vitro angiogenesis in these 3-dimensional cultures which consist of rapid formation of complex tubular structures. Transforming growth factor beta 1-treated RFCs incubated with aFGF-PE were unable to produce this angiogenic response, nor were they able to migrate out of the 3-dimensional culture to form a monolayer as shown by controls. Cell viability analyses showed that aFGF-PE produced a dose-dependent toxic effect which ranged from 10 to 90% cell death. Competition assays in which the chimeric toxin was preincubated with antibodies to aFGF resulted in an 89% reversal of the inhibitory effects of aFGF-PE on endothelial cells. Acidic FGF-PE with a mutation in the ADP ribosylation domain of PE was inactive in both 2-dimensional and 3-dimensional cultures. These data show that aFGF-PE has specific in vitro cytotoxic, antiangiogenic, and antimigratory effects on microvascular endothelia.
authors.affiliation: Molecular and Cellular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut.
authors.forename: J R
authors.initials: JR
authors.lastname: Merwin
authors.forename: M J
authors.initials: MJ
authors.lastname: Lynch
authors.forename: J A
authors.initials: JA
authors.lastname: Madri
authors.forename: I
authors.initials: I
authors.lastname: Pastan
authors.forename: C B
authors.initials: CB
authors.lastname: Siegall
mesh_terms: D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects*; D005098:Exotoxins / Q000008:administration & dosage* / Q000737:chemistry; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009389:Neovascularization, Pathologic; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000633:toxicity; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D016220:Fibroblast Growth Factor 1; D011956:Receptors, Cell Surface; D017468:Receptors, Fibroblast Growth Factor; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A