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Acidic fibroblast growth factor-Pseudomonas exotoxin chimeric protein elicits antiangiogenic effects on endothelial
J R Merwin1, M J Lynch, J A Madri
1Molecular and Cellular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut.
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.
Insights
Acidic fibroblast growth factor fused to Pseudomonas exotoxin (aFGF-PE) shows anti-angiogenic and cytotoxic effects on microvascular endothelial cells. This chimeric toxin inhibits protein synthesis and migration, demonstrating potential as an anti-cancer agent.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Chimeric toxins like acidic fibroblast growth factor fused to mutant Pseudomonas exotoxin (aFGF-PE) show tumor cell cytotoxicity.
- Limited data exists on the effects of aFGF-PE on endothelial cells, crucial for understanding its therapeutic potential.
Purpose of the Study:
- To investigate the anti-angiogenic potential of aFGF-PE664GluKDEL, a specific aFGF-PE fusion protein.
- To evaluate the effects of aFGF-PE on microvascular endothelial cell (RFC) protein synthesis, angiogenesis, and migration.
Main Methods:
- Protein synthesis inhibition assays using RFCs.
- 3-dimensional collagen gel cultures to assess angiogenesis and cell migration, with or without transforming growth factor beta 1.
- Cell viability analysis and competition assays using anti-aFGF antibodies.
Main Results:
- aFGF-PE significantly inhibited RFC protein synthesis at concentrations >100 ng/ml, with up to 83% inhibition at 1000 ng/ml.
- aFGF-PE blocked transforming growth factor beta 1-induced in vitro angiogenesis and prevented RFC migration in 3D cultures.
- Cell viability assays revealed a dose-dependent toxic effect of aFGF-PE (10-90% cell death), and antibody competition reversed inhibitory effects by 89%.
Conclusions:
- aFGF-PE exhibits specific in vitro cytotoxic, anti-angiogenic, and antimigratory effects on microvascular endothelia.
- These findings support the potential of aFGF-PE as an anti-angiogenic agent for cancer therapy.