Abstract
Exposure of bovine aortic or capillary endothelial cells to basic FGF (bFGF) for 1 h resulted in an approximately sixfold increase in plasminogen activator (PA) activity by 18 h that returned nearly to basal levels by 36 h. We hypothesized that the decrease in PA activity following bFGF stimulation was mediated by transforming growth factor beta (TGF-beta) formed from its inactive precursor. Conditioned medium collected from endothelial cells 36 h after a 1-h exposure to bFGF, but not control medium, inhibited basal levels of PA activity when transferred to confluent monolayers of bovine aortic endothelial cells. Antibody to TGF-beta neutralized the inhibitory activity of this conditioned medium, indicating that the medium contained active TGF-beta. Northern blot analysis and quantitation of acid activatable latent TGF-beta in conditioned medium demonstrated that bFGF exposure did not increase the amount of transcription or secretion of latent TGF-beta by the endothelial cells. Both aprotinin, an inhibitor of plasmin, and anti-urokinase type PA IgG blocked the generation of active TGF-beta in cultures exposed to bFGF. These results demonstrated that plasmin generated by uPA activity is required for the activation of latent TGF-beta in endothelial cell cultures treated with bFGF. Activation of TGF-beta by endothelial cells exposed to bFGF appears to limit both the degree and duration of PA stimulation. Thus, in bFGF-stimulated endothelial cell cultures, PA levels are controlled by a negative feedback loop: PA, whose expression is stimulated by bFGF, contributes to the formation of TGF-beta, which in turn opposes the effects of bFGF by limiting PA synthesis and activity. These studies suggest a role for TGF-beta in reversing the invasive stage of angiogenesis and contributing to the formation of quiescent capillaries.
Highlights
Exposure of bovine aortic or capillary endothelial cells to basic FGF for 1 h resulted in an approximately sixfold increase in plasminogen activator (PA) activity by 18 h that returned nearly to basal levels by 36 h
A 1-h exposure to basic FGF (bFGF) resulted in an increase in PA activity,followed by a marked decline to basal levels after 36 h in both Bovine aortic endothelial (BAE) (Fig. 1) and BCE cells
The observation that bFGF is a potent inducer of proliferation, migration, and protease synthesis in cultured endothelial cells (Gospodarowicz et al, 1985; Moscatelli et al, 1986; Sato and Rifkin, 1988) is consistent with the role of bFGF as an initiator of angiogenesis (Shing et al, 1985; Foikman and Klagsbrun, 1987)
Summary
Exposure of bovine aortic or capillary endothelial cells to basic FGF (bFGF) for 1 h resulted in an approximately sixfold increase in plasminogen activator (PA) activity by 18 h that returned nearly to basal levels by 36 h. In bFGF-stimulated endothelial cell cultures, PA levels are controlled by a negative feedback loop: PA, whose expression is stimulated by bFGF, contributes to the formation of TGF-fl, which in turn opposes the effects of bFGF by limiting PA synthesis and activity. These studies suggest a role for TGF-fl in reversing the invasive stage of angiogenesis and contributing to the formation of quiescent capillaries. If bFGF contributes to angiogenesis in vivo, a mechanism must exist for modulating the invasive phenotype that it induces
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