A peptide has been isolated from ovine hypothalamus which, at 1 x 10(-9)M, inhibits secretion in vitro of immunoreactive rat or human growth hormones and is similarly active in vivo in rats. Its structure is H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH The synthetic replicate is biologically active.

Fibroblast growth factors (FGFs) are potent mitogens for vascular and capillary endothelial cells in vitro and can stimulate the formation of blood capillaries (angiogenesis) in vivo. A crucial event in this process is the invasion of the perivascular extraceflular matrix by sprouting endothelial cells. Using a recently developed in vitro model of angiogenesis, we show here that highly purified basic pituitary FGF can induce capillary endothelial cells to invade a three-dimensional collagen matrix and to organize themselves to form characteristic tubules that resemble blood capillaries. We also show that basic FGF concomitantly stimulates endothelial cells to produce a urokinase-type plasminogen activator, a protease that has been implicated in the neovascular response. The results demonstrate that basic FGF can stimulate processes that are characteristic of angiogenesis in vivo, including endothelial cell migration, invasion, and production of plasminogen activator.The formation of new blood capillaries (angiogenesis) occurs in a wide range of important biological processes in response to angiogenic factors released by either normal or tumoral cells (1). A crucial step in the sequence of events that leads to the angiogenic response is the invasion of the perivascular extracellular matrix by sprouting endothelial cells (2). The process includes endothelial cell migration, proliferation, and production of enzymes capable of modifying the extracellular matrix. We have recently shown that the invasiveness of capillary endothelial cells can be induced experimentally in vitro by well-defined chemical signals (3). Cells grown on three-dimensional collagen gels and treated with the tumor promoter 4/3-phorbol 12-myristate 13-acetate (PMA) infiltrate the underlying collagen matrix and organize into vessellike tubular structures (3). Although phorbol esters are not physiologically occurring substances, they have bedn shown to mimic, in many instances, the effects of endogenous mediators, such as hormones or growth factors (4-7). It was therefore important to establish whether phenomena similar to those induced by PMA could also be triggered by physiological angiogenic factors.In this study, we have examined the effect of highly purified basic fibroblast growth factor (FGF) on MATERIALS AND METHODSIsolation of FGF. Basic FGF was purified to homogeneity from bovine pituitaries by successive steps ofammonium sulfate precipitation, ion exchange chromatography, and heparin-Sepharose affinity chromatography (8). Purity of the growth factor was established by reverse-phase high-performance liquid chromatography, amino acid analyses, NaDodSO4/ polyacrylamide gel electrophoresis, and amino-terminal sequence analyses (10).Cell Culture. Three-dimensional gels of reconstituted collagen fibrils were prepared as described (3). Cloned capillary endothelial cells derived from the bovine adrenal cortex (14) were a generous gift of M. B. Furie and S. C. Silverstein (Columbia University, New York). The cells were routinely subcul...

A 44 amino acid peptide with growth hormone-releasing activity has been isolated from a human tumor of the pancreas that had caused acromegaly. The primary structure of the tumor-derived peptide is H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala- Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly -Ala-Arg-Ala-Arg-Leu-NH2. The synthetic replicate has full biological activity in vitro and in vivo specifically to stimulate the secretion of immunoreactive growth hormone. The tumor-derived peptide is identical in biological activity and similar in physiochemical properties to the still uncharacterized growth hormone-releasing factor present in extracts of hypothalamic tissues.

The two major mitogenic polypeptides for endothelial cells have been purified to homogeneity. The complete primary structure of bovine pituitary basic fibroblast growth factor (FGF) and the amino-terminal amino acid sequence of bovine brain acidic FGF have been established by gas-phase sequence analyses. Homogeneous preparations of these polypeptides are potent mitogens (basic FGF, ED50 60 pg/ml; acidic FGF ED50 6000 pg/ml) for many diverse cell types including capillary endothelial cells, vascular smooth muscle cells, and adrenocortical and granulosa cells; in vivo, basic FGF is a powerful angiogenic agent in the chick chorioallantoic membrane assay. The available protein sequence data demonstrate the existence of significant structural homology between the two polypeptides.

Basic fibroblast growth factor (FGF) has been found to increase neuronal survival and neurite e on in a highly purified population of fetal rat hippocampal neurons under well-defined serum-free cell culture conditions. In the presence of FGF, neuronal survival after 7 days in culture on a simple plastic substrate is increased 4-fold, to 54% of the Initial population. Survival is increased 2-fold to 40% on polyornithine-laminin. When FGF was bound to plastic or heparin substrates, neurite outgrowth was sgnflcantiy increased to lengths comparable to those seenwith lamin; however, FGF produced no further increase in neurite outgrowth on laminin. Half-maximal survival was observed at FGF concentrations of about 15 pg/nl (1 pM); half-maximal process outgrowth occurred at about 375 pg/ml (20 pM). The responsive cells were identified as neurons by their labeling with tetanus toxin and by antibodies to neurofilAments and to the neuron-speciflc enolase. Astrocytes, identified by the presence of glial fibrillary acidic protein, constituted about 10% of cells present at 1 week both in the presence and in the absence of FGF. These results strongly suggest that, in addition to its known mitogenic effects on nonneuronal cells, FGF possesses neurotrophic activity for hippocampal neurons.The discovery that during normal development substantial numbers of nerve cells in most regions of the central and peripheral nervous systems die and that the proportion of neurons that survive in any given region is closely related to the size of their target field has led to the hypothesis that all nerve cells are dependent for their growth and maintenance on the availability of specific trophic (growth) factors (1-3). The first neurotrophic factor to be identified was "nerve growth factor" (NGF), and the critical role it plays in supporting the survival and growth of sympathetic and certain sensory neurons has been well documented (4,5 Until recently very few systems have been described in which it is possible to reproducibly obtain adequate cultures of virtually pure neuronal (as opposed to mixed neuronal and glial) cultures. One such system involves the use of dissociated hippocampal neurons from late fetal rats (9-12). The main advantage of this system is that the cell population is unusually homogeneous, the overwhelming majority of the cells at embryonic day 18 being pyramidal neurons (9), and with appropriate controls the proportion of nonneuronal cells in the cultures can be kept to less than 10%1 (10,11). Previous studies have established that virtually all the neurons degenerate within 72-96 hr ifthey are maintained at low density and in a chemically defined medium (10-12), but they can be maintained for periods of several weeks in the presence of explants of hippocampal tissue or a feeder layer of astrocytes, or in medium conditioned by astrocytes (9, 10, 12). We have used this system to examine the effects of highly purified basic fibroblast growth factor (FGF) on the survival of the isolated neurons and on their capacit...

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Two functional domains in the primary structure of basic fibroblast growth factor (FGF) have been identified on the basis of their ability to interact with the FGF receptor, bind radiolabeled heparin, and modulate the cellular response to FGF. Peptides derived from these two functional domains can act as partial agonists and antagonists in biological assays of FGF activity. Peptides related to the sequences of FGF-(24-68)-NH2 and FGF-(106-115)-NH2 inhibit thymidine incorporation into 3T3 fibroblasts when they are stimulated by FGF but have no effect when the cells are treated with either platelet-derived growth factor or epidermal growth factor. They also possess partial agonist activity and can stimulate DNA synthesis when tested in the absence of exogenous FGF. The active peptides have no effect on the binding of epidermal growth factor to its receptor on A431 cells and they can modulate the effects of FGF, but not fibronectin, on endothelial cell adhesion. The results suggest the possibility of designing specific analogs of FGF that are capable of inhibiting the biological effects of FGF.The molecular characterization of basic and acidic fibroblast growth factors (FGFs) has confirmed the existence of two classes of closely related angiogenic factors and helped establish the identity of an unusual family of mitogens (1-7). Acidic and basic FGFs exist in several molecular forms that are products of proteolytic processing at homologous sites (1)(2)(3)8). Both growth factors stimulate a wide spectrum of target cells derived from the primary and secondary mesenchyme as well as from the neural crest. The growth factors bind immobilized heparin with an unusually high affinity, a characteristic that has been exploited for their isolation and characterization from many tissues (4, 9). Because both FGFs also have the capacity to stimulate neovascularization (1-4), their physiological functions have been associated with reproduction, growth, and development. The possible identity of FGF-related proteins with the tumor angiogenic factor activities described by Folkman (10) has suggested that this family of mitogens may also play a critical role in several pathophysiological processes including the growth of tumors, diabetic proliferative retinopathies, and the woundhealing response. The ability of basic FGF to stimulate nerve regeneration (11) and to induce a neovascular response in the chicken chorioallantoic membrane (12), the rat brain (13), the kidney capsule (14), and the carotid artery (unpublished work) has suggested a possible therapeutic application of FGFs in stimulating tissue regeneration, the recovery from episodes of ischemia, and tissue transplantation.The identification of specific functional domains in the primary structure of basic FGF was examined as a first step in the design of recombinant analogs of basic FGF that have modified heparin-binding activity, increased biological activity, and/or antagonist activity. The results presented here identify two peptide sequences of interest (Fig. 1). M...

Inhibin is a gonadal protein that specifically inhibits the secretion of pituitary follicle-stimulating hormone (FSH). Two forms of inhibin (A and B) have been purified from porcine follicular fluid and characterized as heterodimers of relative molecular mass (Mr) 32,000 (ref. 2). Each inhibin is comprised of an identical alpha-subunit of Mr 18,000 and a distinct but related beta-subunit of Mr 13,800-14,700 linked by interchain disulphide bond(s). Throughout the purification of inhibins, we consistently observed two fractions which stimulated the secretion of pituitary FSH. We report here the isolation of one of the FSH-releasing proteins; it has a Mr of 24,000 and its N-terminal sequences up to residue 32 are identical to those of each beta-subunit of inhibins A and B. In the presence of reducing agents, SDS-polyacrylamide gel electrophoresis resolves the FSH-releasing substance into two subunits which are identical in their migration behaviour to the reduced beta-subunits of inhibins A and B. Based on the N-terminal sequence data and Mr of the intact and reduced molecules, we propose that the FSH-releasing substance, which is active in picomolar concentrations, is a heterodimeric protein composed of the two beta-subunits of inhibins A and B linked by interchain disulphide bond(s). The structural organization of the FSH-releasing substance is homologous to that of transforming growth factor-beta (TGF-beta), which also possesses FSH-releasing activity in the same bioassay. We suggest that the substance be called activin to signify the fact that it has opposite biological effects to inhibin.