Thromboxane A₂ stimulates vascular smooth muscle hypertrophy by up- regulating the synthesis and release of endogenous basic fibroblast growth factor

We have shown previously that thromboxane A₂ stimulates hypertrophy of cultured rat aortic smooth muscle cells defined as protooncogene expression and protein synthesis without DNA synthesis or cellular proliferation (Dorn, G. W., II, Becker, M. W., Davis, M. G. (1992) J. Biol. Chem. 267, 24897- 24905). Since endogenous growth modulators could possibly regulate vascular smooth muscle growth to this vasoconstrictor, we tested the hypothesis that thromboxane-stimulated vascular smooth muscle hypertrophy was due to increased expression of endogenously produced basic fibroblast growth factor (bFGF). The thromboxane mimetic (15S)-hydroxy-11α,9α-(epoxymethano)prosta- 5Z,13E-dienoic acid (U46619) (1 μM) increased cultured rat aorta derived smooth muscle cell immunoreactive bFGF content by 331 ± 40% over untreated controls after 24 h. Co-incubation of vascular smooth muscle cells with a specific antisense oligodeoxynucleotide (AS) against codon 60 of bFGF coding sequence reduced thromboxane-stimulated bFGF expression by 72 ± 5% and prevented thromboxane-stimulated hypertrophy (nonsense oligonucleotide had no effects). Addition of exogenous bFGF (20 ng/ml) restored growth to AS- treated/thromboxane-stimulated vascular smooth muscle cells. Furthermore, addition to the culture medium of neutralizing antibody against bFGF inhibited U46619-stimulated vascular smooth muscle hypertrophy by 69 ± 17%, whereas nonimmune IgG had no effect. Since protein tyrosine phosphorylation is a cell signal associated with growth, thromboxane-stimulated tyrosine phosphorylation was also examined. Exposure to 1 μM U46619 for 10 min increased vascular smooth muscle immunoreactive phosphotyrosine content of 130-144-, 86-, 80-, 75-, and 58-kDa proteins. The tyrosine kinase inhibitor herbimycin A (5 μM) prevented thromboxane-stimulated tyrosine phosphorylation, but not thromboxane-stimulated hypertrophy, suggesting that tyrosine phosphorylation was not required for thromboxane-stimulated vascular smooth muscle growth. These results indicate that increased expression and release of endogenous bFGF, but not direct tyrosine phosphorylation, mediates the hypertrophic vascular smooth muscle response to thromboxane.