Basic Fibroblast Growth Factor-induced Activation of Latent Transforming Growth Factor/3 in Endothelial Cells: Regulation of Plasminogen Activator Activity

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/3 (TGF-/3) 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-/3 neutralized the inhibitory activity of this conditioned medium, indicating that the medium contained active TGF-~. Northern blot analysis and quantitation of acid activatable latent TGF-/3 in conditioned medium demonstrated that bFGF exposure did not increase the amount of transcription or secretion of latent TGF-/3 by the endothelial cells. Both aprotinin, an inhibitor of plasmin, and anti-urokinase type PA IgG blocked the generation of active TGF-/~ in cultures exposed to bFGE These results demonstrated that plasmin generated by uPA activity is required for the activation of latent TGF-/3 in endothelial cell cultures treated with bFGE Activation of TGF-/3 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-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. T rIE importance of complex interactions between peptide growth factors in mediating cellular physiology is becoming increasingly evident. Angiogenesis, a strictly regulated process that occurs in a variety of developmental, physiological, and pathological settings (Folkman, 1984; Furcht, 1986), can be stimulated by a number of growth factors (Folkman and Klagsburn, 1987). The initial stages of angiogenesis are characterized by local degradation of the basal lamina, followed by migration and proliferation of endothelial cells (Ausprunk and Folkman, 1977). The effects of angiogenic factors on endothelial cell proliferation, migration, and protease synthesis have been studied in an effort to characterize the contribution of these molecules to neovascularization. Two proteins synthesized by almost all cell types, including endothelial cells, and believed to contribute Correspondence should br addressed to Daniel B. Rifkin, Department of Cell Biology and Kaplan Cancer Center, New York University Medical Center, and Raymond and Beverly Sackler Foundation Laboratory, New York 10016. to angiogenesis are basic FGF (bFGF) and transforming growth factor-B (TGF-B). ~ Basic FGF, an 18-kD peptide, induces angiogenesis in a variety of in vivo assays (Shing et al., 1985; Folkman and Klagsburn, 1987). In vitro, bFGF stimulates endothelial cell proliferation (Gospodarowicz et al., 1985), migration (Sato and Rifkin, 1988), plasminogen activator (PA) and collagenase synthesis (Moscatelli et al., 1986), invasion into the amnion membrane (Mignatti et al., 1989), and formation of patent capillaries in collagen and fibrin gels (Montesano et al., 1986). Yet, whereas an increase in protease synthesis is necessary for the invasion of endothelial ceils into a basement membrane (Mignatti et al., 1986) and subsequent tube formation (Montesano et al., 1986), uncontrolled dissolution of matrix interferes with in vitro angiogenesis (Montesano et al., 1987, 1990). Furthermore, endothelial ceils undergoing normal angiogenesis do not remain indefinitely 1. Abbreviations used in this paper: BAE, bovine aortic endothelial; bFGE basic FGF; TGF-/3, transforming growth factor/3; PA, plasminogen activator; uPA, urokinase. 9 The Rockefeller University Press, 0021-9525/92/08/901/9 $2.00 The Journal of Cell Biology, Volume 118, Number 4, August 1992 901-909 901 on July 9, 2017 jcb.rress.org D ow nladed fom invasive. In the final steps of capillary formation, endothelial cell migration stops and new basement membrane is formed. Thus, if bFGF contributes to angiogenesis in vivo, a mechanism must exist for modulating the invasive phenotype that it induces. TGF-/3 is a 25-kD molecule that is a potent inhibitor of endothelial cell proliferation, migration, and protease synthesis (Heimark et al., 1986; Muller et al., 1987; Saksela et al., 1987; Frater-Schroder et al., 1986). It is secreted constitutively by endothelial cells as part of a high molecular weight complex (180-210 kD) (Flaumenhaft, R., M. Abe, Y. Sato, K. Miyazono, C. H. Heldin, and D. B. Rifldn, manuscript submitted for publication) consisting of mature TGF-/3 associated through noncovalent interactions with a 75-kD latency-associated peptide (Derynck et al., 1985) that is disulfide-linked to a 125-190-kD latent TGF-/~ binding protein (Miyazono et al., 1988). Mature TGF-B must be released from this complex to bind to its cell surface receptor and to elicit a biological response (Lawrence et al., 1985; Pircher et al., 1986). Activation of latent TGF-/3 occurs in co-cultures of endothelial cells and smooth muscle cells (AntonelliOrlidge et ell., 1989; Sato and Rifldn, 1989) and is mediated by plasmin cleavage of the aminoterminal propeptide (Lyons et al., 1988, 1990; Sato and Rifkin, 1989; Sato et al., 1990). TGF-/3 inhibits endothelial cell proliferation, migration, and protease synthesis in cell cultures, and it induces tube formation when added to endothelial cells grown in three-dimensional collagen gels (Madri et al., 1988; Merwin et al., 1990). In addition, "I'GF-/3 is angiogenic when injected subcutaneously into newborn mice (Roberts et al., 1986) or introduced into the chick chorioallantoic membrane (Yang and Moses, 1990). However, since TGF-/~ is a potent chemoattractant for macrophages and fibroblasts, it might induce angiogenesis indirectly by attracting cells that secrete factors capable of initiating angiogenesis. Although TGF-/3 may not act directly on endothelial cells during the invasive stage of angiogenesis, it may be involved in resolving the angiogenic process. TGF-/~ increases the production of the type 1 plasminogen activator inhibitor (Laiho et al., 1986; Lund et al., 1987; Saksela et al., 1987) and the tissue inhibitor of metalloproteinases (Edwards et al., 1987; Overall et al., 1989). This results in the suppression of extracellular proteolytic activity and blockade of endothelial cell invasion (Mignatti et al., 1989; Pepper et al., 1990). TGF-/3 has also been shown to stimulate production of extracellular matrix components and the formation of cell-cell junctions in endothelial cells (Madri et al., 1989; Merwin et al., 1990; Newton et al., 1990). If TGF-/~ is involved in reversing the invasive stage of angiogenesis and mediating the formation of new capillaries, it must be converted from the latent TGF-/~ secreted by endothelial cells to its active form. Thus, we have examined the effect of bFGF on the capacity of endothelial cells to activate latent TGF-/3. We have also tried to determine whether the activation of TGF-/3 has a role in regulating the bFGF-stimulated induction of PA activity. The results demonstrate that active TGF-/3 is generated by endothelial cells exposed to bFGF. The generation of mature TGF-/3 is preceded by an increase in PA activity and elicits a decrease in PA levels at later times. Inhibition of plasmin or PA activity prevents the appearance of active TGF-/3 in endothelial cells exposed to bFGF, showing that bFGF-induced PA is required for the activation of TGF-/3 via the formation of plasmin. The inclusion of neutralizing antibodies to TGF-/3 in bFGF-treated cultures blocks the normally observed decrease in PA activity. Thus bFGF activity may be controlled by a negative feedback loop in which bFGF stimulation of PA production causes the activation of latent TGF-/3, which, in turn, limits the degree and duration of the PAinducing activity of bFGE On the basis of these findings, a model of angiogenesis is presented that involves the coordinated activities of bFGF and TGF-/3, and that may have implications regarding the extracellular regulation of growth factor activity. Materials and Methods