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Acidic Fibroblast Growth Factor-Pseudomonas Exotoxin Chimeric Protein Elicits Antiangiogenic Effects on Endothelial Cells

Molecular and Cellular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut [J. R. M., M. J. L., C. B. S.], Pathology Department, Yale School of Medicine, New Haven, Connecticut [J. R. M., J. A. M.], and Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, Maryland [I. P.]

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-PE66^4GluKDEL, 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 >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 ß₁, aFGF-PE, or a combination of both. Transforming growth factor ß₁ elicits in vitro angiogenesis in these 3-dimensional cultures which consist of rapid formation of complex tubular structures. Transforming growth factor ß₁-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.

¹ To whom requests for reprints should be addressed, at TargeTech Inc., 290 Pratt Street, Meriden, CT 06450 (J. R. M.) or Bristol-Myers Squibb, 3005 First Avenue, Seattle, WA 98121 (C. B. S.).

Received 3/ 2/92. Accepted 7/ 8/92.