Faculty Bibliography

Angiogenesis factors have been recently purified from both tumors and nonneoplastic tissues. The biological properties shown by these factors in vivo and on cultured endothelial cells will be discussed

The role of endothelial cell growth factors in the maintenance of the blood vessel wall is, as we have described here, much more complex than merely stimulating the mitogenesis of endothelial cells. The FGFs are capable of eliciting an array of responses in endothelial cells, some, or all, of which are important for neovascularization and the control of clot dissolution. These endothelial cell responses include protease elaboration, chemotaxis, and mitogenesis. That these growth factors seem neither to be constitutively released into the medium of cultured cells that synthesize bFGF, nor released into the bloodstream in vivo suggests that the temporal and local control of neovascularization may involve the regulation of growth factor release from cells such as endothelial cells, fibroblasts, and macrophages. Although there is no known example of this for bFGF, it is well known that both thrombin and Factor Xa stimulate the release of a mitogenic protein from endothelial cells and that low oxygen tension stimulates the release of macrophage-derived angiogenesis factor. In addition, both TGF beta and heparin alone appear to play a role in wound healing and vessel wall maintenance. The work of Roberts et al suggests that TGF beta is not only angiogenic, but also stimulates the growth of fibrotic tissue as well. Studies on mast cells demonstrated that released heparin is chemotactic for endothelial cells and can potentiate tumor angiogenesis. An attractive hypothesis is that these molecules not only act as FGF potentiators or inhibitors but that they also may exert their angiogenic effects by inducing FGF release from cells. Perhaps angiogenin, an angiogenic molecule with no mitogenic activity, works in this way. However, no evidence as yet exists concerning this point. A second level of control of neovascularization may involve the interaction of FGF with other molecules released into the same microenvironment. For example, thrombin and TGF beta released from platelets, as well as heparin released from mast cells, have all been demonstrated to affect bFGF activity in vitro and may act as modifiers of FGF activity in vivo. Since bFGF can modulate fibrinolytic activity, one could imagine that its release into a wound region of the vasculature could have detrimental effects on clot formation and subsequent wound healing. Thus, the transient inhibition of bFGF activity by TGF beta would allow clot formation before the induction of neovascularization by bFGF, TGF beta thereby playing a role in the regulation of the sequence in which events occur.(ABSTRACT TRUNCATED AT 400 WORDS)

A Mr 25,000 form of basic fibroblast growth factor (bFGF) has been isolated from guinea pig brain along with the typical Mr 18,000 form. Both forms were purified to homogeneity by a combination of heparin-affinity chromatography and ion-exchange chromatography on an FPLC Mono S column. The Mr 25,000 form, like the Mr 18,000 form, was not eluted from the heparin-affinity column with 0.95 M NaCl, but was eluted with 2 M NaCl. The Mr 25,000 guinea pig protein stimulated plasminogen activator production by cultured bovine capillary endothelial cells in a dose-dependent manner at concentrations of 0.1-10 ng/ml, the same range that was effective for guinea pig and human Mr 18,000 bFGFs. The binding of human 125I-labeled bFGF to baby hamster kidney cells is inhibited equally by the Mr 25,000 guinea pig protein and the Mr 18,000 guinea pig and human bFGFs. Polyclonal antibodies raised against human bFGF recognize both the Mr 25,000 and 18,000 guinea pig proteins in an immunoblot analysis. In a radioimmunoassay, both the Mr 25,000 and Mr 18,000 guinea pig proteins compete equally well with iodinated human bFGF for binding to the anti-human bFGF antibodies. When treated with low concentrations of trypsin, the Mr 25,000 guinea pig bFGF was converted to a Mr 18,000 protein. These results show that the two molecules are closely related and suggest that the Mr 25,000 protein shares substantial homology with the Mr 18,000 bFGF

Basic fibroblast growth factor (bFGF), a potent inducer of angiogenesis in vivo, stimulates the production of both urokinase- and tissue-type plasminogen activators (PAs) in cultured bovine capillary endothelial cells. The observed increase in proteolytic activity induced by bFGF was effectively diminished by picogram amounts of transforming growth factor beta (TGF beta), but could not be abolished by increasing the amount of TGF beta. However, the inhibition by TGF beta was greatly enhanced if the cells were pretreated with TGF beta before addition of bFGF. After prolonged incubation of cultures treated simultaneously with bFGF and TGF beta, the inhibitory effect of TGF beta diminished and the stimulatory effect of the added bFGF dominated as assayed by PA levels. TGF beta did not alter the receptor binding of labeled bFGF, nor did a 6-h pretreatment with TGF beta reduce the amount of bFGF bound. The major difference between the effects of bFGF and TGF beta was that while bFGF effectively enhanced PA activity expressed by the cells, TGF beta decreased the amounts of both cell-associated and secreted PA activity by decreasing enzyme production. Both bFGF and TGF beta increased the secretion of the endothelial-type plasminogen activator inhibitor

The amino acid sequence of a human placental bFGF was determined by a combination of protein and cDNA sequencing. The placental bFGF consists of 157 amino acid residues with a calculated molecular weight of 17,464 and is highly homologous to bovine pituitary bFGF. The human protein contains an amino terminal extension when compared to the sequence established for bovine bFGF (Esch et al., 1985) and to the sequence of the predicted translation product based on human bFGF cDNA clones (Abraham et al., 1986)

To understand the role of proteinases in tumor invasion, the effects of inhibitors of metallo-, serine-, and cysteine-proteinases on this process were studied using 125I-iododeoxyuridine-labeled B16/BL6 cells grown on human amnion basement membrane. Cellular invasion was quantitated by measuring the radioactivity associated with the amniotic membrane after the B16/BL6 cells on the basement membrane were removed by lysis followed by scraping. The results obtained with proteinase inhibitors showed that inhibitors of collagenase and plasmin prevented invasion of the amnion. Tissue invasion was also blocked by antiurokinase antibodies. On the contrary, cysteine-proteinase inhibitors and anti-tissue plasminogen activator antiserum were ineffective. Mersalyl, a compound known to activate collagenase, stimulated invasion under conditions where plasmin formation or activity were inhibited. Evidence for the role of a plasminogen activator-plasmin-collagenase activation cascade in B16 invasion is provided

A 17,500-dalton protein which stimulates plasminogen activator production in cultured bovine capillary endothelial cells has been purified from a SK-Hep-1 human hepatoma cell lysate by using heparin affinity chromatography and fast protein-liquid ion exchange chromatography. The purified molecule stimulated plasminogen activator production in a dose-dependent manner between 0.01 and 1 ng/ml. It also stimulated collagenase synthesis, DNA synthesis, and motility in capillary endothelial cells in the same concentration range. This molecule was identified as a basic fibroblast growth factor-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with a polyclonal antibody raised against the human placental basic fibroblast growth factor

We have previously purified from human placenta a basic fibroblast growth factor (FGF)-like molecule which stimulates the production of plasminogen activator (PA) and collagenase, induces DNA synthesis, produces an increase in motility in cultured bovine capillary endothelial (BCE) cells, and induces angiogenesis in vivo. The ability of basic FGF to stimulate PA production in BCE cells was used as an assay for the presence of basic FGF-like molecules in extracts of both normal and tumor-derived cultured cells. The identity of the PA-stimulatory activity with basic FGF was confirmed by its high affinity for heparin and by its cross-reactivity with antibodies to human placental basic FGF. Basic FGF-like molecules were identified in eight of ten cell lines tested, and the amount of FGF-like activity present in these cells bore no relation to their origin from normal or tumor tissue. The test cells, BCE cells, had one of the highest levels of FGF-like activity, suggesting that it may have an autocrine role in these cells