- Email: [email protected]
The role of fibroblast growth factors in the central nervous system
the FGFs, have also been implicated
The Role of Fibroblast Growth Factors in the Central Nervous System
in
other developmental processes. FGFs may be important at all stages of CNS development, from tissue induction and proliferation of stem cells in early embryogenesis through the stabilization of the differentiated state of neuronal cells and their survival.
Ann Logan FGF in Early Embryogenesis
The fibroblast growth factors are well-characterized mitogens that are found in the central nervous system (CNS). Their physiological roles are not yet known, but increasing evidence suggests their involvement in CNS development, injury responses, and possibly oncogenesis.
Growth factors are regulatory proteins that control cell proliferation and many other cellular responses by receptormediated mechanisms similar to those used by hormones. Most tissues produce and respond to growth factors, and the CNS is no exception. Despite the early identification of growth factors in the CNS and their recent rigorous characterization, their functions within the CNS are only just beginning to be understood. The fibroblast growth factors (FGFs) are a family of closely related proteins. They have been purified to homogeneity, their primary structure has been determined, and their cDNA cloned and sequenced. The family can be subdivided into acidic forms (aFGF, with an isoelectric point of 5-6) or basic forms (bFGF, with an isoelectric point of 9.6) and now encompasses substances prepared from a variety of cell types and tissues. All share a structural homology (-55% between acidic and basic forms) and a characteristic high affinity for the sulfated mucopolysaccharide heparin. The two molecular forms of FGF are coded for by distinct genes, the aFGF gene being located on human chromosome 5 and the bFGF gene on human chromosome 4 (Mergia et al. 1986). Whereas bFGF is widespread, aFGF is more restricted, occurring predominantly in the CNS. Within the CNS aFGF and bFGF have been immunocytochemically localized in neurons (Pettmann Ann Logan is at the Department of Physiology, The Medical School, Birmingham B15 2TJ. UK.
TEM JanuarylFehmary
1986), but bFGF is also found in astrocytes (Ferrara et al. 1987; Hatten et al. 1988). Equivalent levels of FGF bioactivity are found throughout the CNS (Logan and Logan 1986), indicating that here the FGFs are ubiquitous. Both forms of FGF interact with common cell surface receptors (Neufeld and Gospodarowicz 1986) that have yet to be sequenced and cloned. They stimulate the proliferation and differentiation of cultured cells of embryonic mesodermal and neuroectodermal origin. They also regulate cell activities other than growth, and are therefore multifunctional. Their recently defined activity as potent angiogenic factors is clearly relevant to a number of physiological responses in many tissue systems. This review summarizes current evidence concerning the roles of the FGFs, specifically in the CNS. Of special interest is their potential importance in embryogenesis (as neurotrophic agents in CNS development), injury responses, and oncogenesis. Several excellent, comprehensive reviews describe the present knowledge of the structural properties, mechanisms of action and molecular biology of the FGFs (Baird et al. 1986; Gospodarowicz 1988; Gospodarowicz et al. 1986 and 1987; Haynes 1988; Thomas 1987).
??
FGF in Development
Growth factors and their receptors evidently have important roles in development. These may include the control of cell proliferation in specific tissues, but a number of growth factors, including
0 1990, Else&x
Science
Publishing
In early embryonic development, the basic body plan arises as cells in different regions become programmed to follow a particular developmental path. The animal and vegetal poles of the egg thereby get directed along different developmental pathways (Figure 1). Mesoderm is induced from the animal segment by signals originating from cells in the vegetal region. Signals from the cells of the dorsovegetal region lead to development of a small organizer zone that induces dorsal-type mesoderm structures to develop, such as the notochord. Signals from the cells of the ventrovegetal region induce the formation of ventral-type mesoderm, which will comprise blood cells, mesenchyme, and mesothelium (Slack et al. 1987). This pattern of formation and tissue induction seems to be under the control of specific gens .
inducing
factors
or morpho-
Interest in this subject was stimulated by the report that bFGF, together with another factor, transforming growth factor-p (TGF-P), mimics the biological effects of the vegetalizing factor in early amphibian embryos and acts as a ventrovegetal morphogen for ventral-type mesoderm (Slack et al. 1987 and 1988; Kimelman and Kirschner 1987). The presence of mRNA of the bFGF type was also demonstrated in early amphibian embryos (Kimelman and Kirschner 1987). Hence, FGF may be one of the morphogens responsible for inducing mesoderm. FGF has also been implicated in the very early embryonic development of higher vertebrates. A gene that codes for an FGF-like protein, int-2 (Dickson and Peters 1987), may play a direct role in the development of the gastrulating and early somite stage mouse embryo, regulating cell migration, and tissue induction (Wilkinson et al. 1989). These early influences of FGF-related proteins in embryogenesis may explain the origin of the wide range of FGF-
Co., Inc. 1043-2760/90/$2.00
149
VENTRAL
tion is taking place. Although transcrip-
4 VENTRAL
tion of the aFGF and bFGF genes has yet to be demonstrated within the developing mammalian CNS, expression of the FGF-related gene, int-2, has been found in the CNS of the fetal mouse (Wilkinson et al. 1989). Transcripts of the int-2 gene are in differentiating Purkinje cells and in cells of the neuroblastic layer of the retina. There is increasing in vitro and in vivo evidence to suggest a neurotrophic role in CNS development for FGF.
4 VENTRAL
In vitro Evidence
VEN t RAL
Figure 1. The diagram depicts an amphibian blastula, divided into animal (A) and vegetal (V) segments, with the dorsal side at the bottom. Trophic signals from the dorsovegetal (DV) and ventrovegetal (VV) cells induce cells of the animal segment to differentiate into mesoderm (M) and a small organizer zone (0). Signals (FGF) from the ventrovegetal cells induce the formation of ventral mesoderm (VM), comprising blood cells, mesenthyme, and mesothelium. Signals from the organizer zone induce the formation of dorsal mesoderm (DM) from which the notochord develops.
cells in adult tissue systems. FGF-related proteins also may have other roles in later fetal development, particularly of the CNS.
responsive
FGF in CNS Development Normal CNS development involves neuroblast proliferation and migration followed by the selective death of many of the developing neurons. Trophic factors may play some part in this process, and among these the FGFs are known neurotrophic factors promoting the proliferation, differentiation, and/or survival of specific neurons (for review, see Davies 1988). Bioactive FGF has been demonstrated in the brains of rat embryos (Logan et al. 1985) as early as embryonic day 15, when widespread neuronal differentia-
150
The stabilization and functional maintenance of cultured, differentiated neuronal cells by FGF is now widely reported. In cultures of central and peripheral neurons of the rat and chick (Unsicker et al. 1987) and in cells of the PC12 neuronal cell line (Rydel and Greene 1987), aFGF and bFGF promote survival and enhance neurite outgrowth and differentiated cell function. Enriched neuronal populations derived from early embryonic rat brain proliferate in response to bFGF and later express cholinergic differentiation (Gensburger et al. 1987). bFGF also promotes both the survival and differentiation of embryonic nerve cells in cultures derived from the hippocampal region or the cortex of the rat (Morrison et al. 1986; Walicke et al. 1986). In addition, bFGF stimulates neurite extension in cultures of cerebellar granule cells from newborn mice (Hatten et al. 1988).
In vivo Evidence The in vivo administration of bFGF prevents the normally occurring death of some primary sensory neurons in quail embryos (Hofer and Barde 1988). bFGF promotes the survival of adult rat retinal ganglion cells after transection of the optic nerve and protects adult sensory neurons from lesion-induced death (Sievers et al. 1987). In the adult rat CNS, bFGF also prevents the death of cholinergic neurons in the medial septum and diagonal band of Broca following transection of their axons (Anderson et al. 1988). Thus, FGF may play a role in the normal support of basal forebrain cholinergic neurons. These results suggest that FGFs exert a neurotrophic influence by acting directly upon neurons in the developing
0 1990, Elsevier Science Publishing
and adult CNS to promote their proliferation, differentiation, functional maintenance, and survival. FGFs also have pronounced effects on the proliferation and differentiation of glial cell populations (Pettmann et al. 1982; Gospodarowicz et al. 1987). Glial cells are important in the developing CNS as providers of the physical and trophic environment that promotes neural growth and differentiation. The FGFs may therefore also influence CNS development by modulation of glial neurotrophic properties. Furthermore, FGF may influence CNS development through its angiogenic properties, since FGF may be responsible for capillary ingrowth into the developing brain (Risau 1986). The source of the FGFs acting in the developing CNS remains to be determined. Whether the neurons or the astrocytes are their source, it is not yet clear how these growth factors are packaged and released from them. The FGFs lack a clear signal peptide sequence to direct their secretion, but they may be liberated from cells undergoing developmentally regulated death. This programmed local release of FGFs may contribute to the neurotrophic environment which allows successful growth of selected neurons. Future studies to elucidate the exact neurotrophic role of the FGFs in the developing CNS are important. The trophic environment of the embryonic CNS may hold vital clues for attempts to construct a postinjury environment in the adult that facilitates successful regeneration of lesioned axons.
??
FCF and CNS Injury
Responses
Complete lesions of neural pathways in the adult mammalian CNS are rarely followed by significant functional recovery. Injury damages local vasculature, disrupts the interactions between neural cells, and destroys both neurons and their connections. Soon after the lesion, repair responses such as neovascularization, glial proliferation and differentiation, axonal sprouting, and reactive synaptogenesis are in evidence. In most cases, however, the initial attempt of the axons to regenerate is aborted, as a dense, permanent scar is laid down to seal off the damaged area (Berry et al. 1983). We know very little about the interre-
Co., Inc. 1043.2760/90/$2,00
TEM JanuarylFebruury
lations of these events and how the injury response is initiated. The apparent incapacity for successful regeneration of adult mammalian CNS neurons clearly does not reflect an innate inability of axons to regenerate (Aguayo 1985), nor is the scar an impenetrable barrier to regenerating axons, although it must markedly hinder their growth. It seems that a major factor preventing functional recovery is the absence of an appropriate complement of environmental cues that facilitates successful regrowth and reconnection of lesioned nerve pathways. These cues are clearly present in the developing brain, which can regenerate very well after injury (Berry et al. 1983). There is now strong evidence that, during development or after injury, trophic factors are important in determining the success or failure of growth and regeneration (Bjorklund and Stenevi 1977 and 1981; Gage et al. 1984; Kromer et al. 1981a and b; NietoSampedro and Cotman 1985). What combination of trophic molecules provides the environmental cues for successful regeneration within the CNS is not yet known, but an increasing number of regulatory growth factors have been identified in the mature and developing CNS, and some of these have been implicated in postinjury responses (Berry et al. 1983; Bjorklund et al. 1979; Logan 1988a; Logan et al. 1985; Manthorpe et al. 1983; Nieto-Sampedro et al. 1982,1983,1984, and 1985). The presence of neurotrophic FGFs in developing and adult nervous tissue suggests their potential involvement in neural regeneration processes, including those following injury (Berry et al. 1983; Logan 1988a).
A Role for FGF in Repair of the Injured CNS? If FGFs are released locally into the wound from dead or damaged cells and also from injury-responsive cells, then one of their important functions could be to promote repair of injured CNS tissue (Figure 2). The hypothesis that the FGFs play a role in responses to wounding was initially based on the localization of FGF within the CNS and on observations that these factors stimulate all the cell types involved in such responses (Berry et al. 1983). Specifically, the FGFs are potent mitogens for capillary endothelial cells and vascular
TEM JanuayylFebruary
Figure 2. Following a penetrating injury to the CNS, FGFs are released into the lesion site from damaged or dying cells and from invading mesodermal cells. The biologically active FGFs initiate the regeneration and scarring responses by their actions upon the neural, glial, and mesenchymal elements involved.
smooth muscle cells (whose activity underlies the FGFs’ angiogenic effects), astrocytes, oligodendrocytes, and fibroblasts. They are also chemoattractants for astrocytes and neurotrophic agents (for review of target cell specificity, see Gospodarowicz 1988). In addition to aFGF and bFGF being present in neurons (Janet et al. 1987; Pettmann et al. 1986), from which they can be released when axons are severed, bFGF has been detected in astrocytes (Ferrara et al. 1988) and also in the macrophages which invade the injury site (Baird et al. 1986). Thus, FGF produced from CNS cells (endogenous) or from elsewhere (exogenous) could act within the CNS after injury in an autocrine and/or paracrine fashion to promote the initial axon sprouting and the organization of the mature gliabcollagen scar. The relative contributions of exogenous and endoge-
nous FGFs and of various molecular forms of FGF remain to be established. FGFs React to CNS Injury Growing evidence supports the hypothesis (Berry et al. 1983) that FGFs are important trophic factors regulating some of the postinjury responses seen in the CNS. Following a penetrating injury to the CNS of the rat, the levels of aFGF and bFGF mRNA rise significantly and in parallel with immunoreactive and bioactive protein levels of a- and bFGF within the injured tissues (Logan et al. 1988a and b). Others (Nieto-Sampedro et al. 1988) have also measured increases in aFGF immunoreactivity in the wound cavity of lesioned rat cortex. They suggested that this growth factor was endogenous and may have arisen by active synthesis and secretion, but have identi-
0 1990, Elsevier Science Publishing Co., Inc. 1043-2760/901$2.00
1st
fied neither the cell type nor the possible mechanisms involved. Finklestein et al. (1988) have also demonstrated an increase in the levels of immunoreactive bFGF in lesioned CNS tissue and localized this to cells which resemble reactive astrocytes. Ferrara et al. (1988) have demonstrated the expression of bFGF mRNA by astrocytes. Other studies (Logan 1988a and b) suggest that the time course of the FGF mRNA and protein response to injury observed in the rat closely follows that of the reactive gliosis and neuronal sprouting observed histologically (Berry et al. 1983). These observations provide compelling evidence that a- and bFGF may be synthesized and/or released and/or activated in injured CNS tissue.
FGFs Are Neurotrophic Injured CNS
Factors
in the
Other in vivo experiments also suggest a role for the FGFs in CNS injury responses and indicates their potential use as therapeutic agents to promote neuron survival following injury. Local application of bFGF prevents neuron losses in dorsal root ganglia following sciatic nerve transection in the rat (Otto et al. 1987). Administration of bFGF to the wound site promotes the survival of retinal ganglion cells following transection of the optic nerve (Sievers et al. 1987; Bahr et al. 1989). Infusion of bFGF into the right ventricle of the rat also reduces the death following axotomy of cholinergic neurons that project to the hippocampal formation (Anderson et al. 1988).
Neuron Regeneration
vs Scar Formation
Recent evidence that hippocampal and other CNS neurons bear high-affinity receptors for bFGF (Walicke et al. 1989) indicates that FGF may exert its neurotrophic influences directly upon neurons by receptor-mediated mechanisms. This suggests that the FGFs are physiologically relevant neurotrophic factors. To date, there is no clear experimental evidence to suggest whether FGFs are involved in producing the glial/collagen scar. If FGF is responsible for initiating scar formation in addition to neuronal sprouting following injury, its neurotrophic benefits could be outweighed by the functionally deleterious consequences of producing a scar. Specula-
1.52
tion (Walicke et al. 1989) that responses to CNS injury result from competition by the neuronal and mesenchymal elements involved in regeneration and scarring for the FGF released is, at present, unsubstantiated. Separation of the two injury responses on the basis of the molecular forms of FGF and the receptors responsible for their initiation could clearly have important clinical implications. The Development CNS Injury
of Novel Therapies for
The ability to manipulate the CNS lesion environment into one that reduces scar formation and promotes regeneration would be an important step towards functional repair. If cell-specific analogues of the different FGFs can be produced, together with specific antagonists of their activities, these might be clinically applicable. The development of treatments to aid repair of both acute and chronic damage to the CNS based on the activities of the FGFs on neuronal and mesenchymal cells is an important goal.
??
FGF and CNS Oncogenesis
Advances in molecular biology have enabled new insights into the mechanisms underlying transformation of cells. Recent research has shown that aberrant expression of growth factors, their receptors, or components of their signaling pathways is associated with many different types of tumors. FGF-Related
Oncogenes
Expression of FGF-related oncogenes (transforming genes) has been demonstrated in a number of different nonneural tumors, and this suggests the potential importance of FGF-like proteins in tumor growth. These oncogenes include int-2, which has been implicated in virus-induced mouse mammary carcinogenesis (Dickson et al. 1984), and hsti KS3, FGF-5, and FGF-6, which were detected as oncogenes in tumor DNA that could transform NIH 3T3 murine fibroblasts (Delli-Bovi et al. 1987; Yoshida et al. 1987; Zhan et al. 1988). Expression
of FGF in CNS Tumors
Most CNS tissue contains intrinsically low levels of FGF mRNA, raising specu-
0 1990, Elsevier Science
Publishing
lation that the mRNA is unstable 01‘ tightly regulated under normal conditions (Logan 1988a). Evidence is emerging that some CNS tumors do express increased levels of FGF. Early reports of high levels of bioactive FGF in a number of diverse CNS tumors (Logan et al. 1984) are supported by more recent evidence. An astrocytoma cell line derived from a neural tumor was shown to exhibit a high level of bFGF mRNA expression and to produce biologically active bFGF that remained cell associated (Murphy et al. 1988). These workers have also shown a tumor-specific elevation in bFGF transcript levels and FGFlike proteins in tumors of Schwann cell origin such as acoustic neuromas (Murphy et al. 1989). They could lind no evidence of gene amplification or rearrangement in these tumors, and suggest that the raised levels are due to increased transcription or stabilization of mRNA. Similarly, cells derived from a human retinoblastoma were shown to express the bFGF gene and produce bFGF protein (Schweigerer et al. 1987). This bFGF protein stimulates the proliferation of capillary endothelial cells. Cell lines derived from human gliomas expressed aFGF mRNA and produced and responded to aFGF protein (Libermann et al. 1987).
Autocrine
vs. Paracrine Activity
The roles of the FGFs within these tumors remain to be established, but their influence could be autocrine or paracrine. Inappropriate production of the FGFs, related proteins, or their receptors may directly affect proliferation of tumor cells. Furthermore, FGFs may also increase expression of plasminogen activators, collagenases, and other proteolytic enzymes within tumor cells, and this might mediate remodeling and aid tumor metastasis. Acting as a paracrine factor, FGFs’ angiogenic activity may also be of direct relevance to tumor growth.
Tumor Angiogenesis Solid tumors are dependent upon angiogenesis, since increases in cell number must be supported by coincident increases in capillary blood supply. Thus, FGFs could be responsible for the increased vascular supply that delivers oxygen and nutrients and removes waste
Co., Inc. 1043-2760/90/$2.00
TEM JanuarylFebmary
products in actively growing tumors. Furthermore, the increased blood supply may facilitate tumor metastasis. In a recent paper, Folkman et al. (1989) confirmed a correlation between the presence of factors that stimulate tumor growth and angiogenesis. They suggest that angiogenesis is a secondary event in oncogenesis and, although necessary for tumor progression, is not one of the primary oncogenic events that initiate transformation. Thus, transforming cells acquire the ability to express angiogenic growth factor genes early in tumor development which, in turn, induce neovascularization, thereby facilitating tumor progression.
Clinical Implications The immediate and future clinical applications of these findings are potentially important but ill-defined. Understanding the mechanisms of oncogenesis and tumor progression must form the basis of clinical management of patients with CNS tumors. Identification of the specific genes responsible for transformation and progression of tumors may enable diagnoses and prognostic forecasts to be made and early treatment to be initiated. Therapeutic strategies involving FGFs and the pathophysiological systems in which they may participate are potentially diverse. Gene expression may provide a marker for tumor localization or a quantitative method of assessing tumor activity. FGF release remains poorly understood, but may be amenable to modulation. The major means whereby FGF systems may be influenced, however, is through their specific receptors, and once a receptor (or family of receptors) has been characterized, agonists and antagonists may prove an important means of inducing tumor regression. Moreover, angiogenesis may itself be a process amenable to direct inhibition and thereby allow limitation of tumor growth. The use of interferon to inhibit the abnormal proliferation of capillary blood vessels in a case of pulmonary capillary hemangiomatosis (White et al. 1989) may have broader clinical implications. The suggestion that interferon may act directly to inhibit the FGF-producing cells present in neovascular lesions may point the way for the development of antiangiogenic therapies.
TEM JanuatylFehruaty
??
Future Directions
One of the most important questions still to be resolved concerning the FGFs is that of their physiological and pathophysiological roles within the CNS. In particular, their importance as mitogenie, differentiation, and maintenance factors for the neuronal, glial, and mesenchymal cells involved within the CNS in development, injury responses, and oncogenesis demands continued investigation. Differentiation of the in vivo roles of aFGF vs bFGF remains to be determined. The separation of their functional activities at the cellular level, their molecular, cellular and tissue interactions, and the control and interrelationships of their gene expression needs particular attention. Critical studies on FGFs physiological roles await the characterization of the FGF receptors and the cloning of their cDNA. Definition of FGF-responsive cells within the CNS by analysis of FGF receptor expression and action depends on the availability of receptor probes. The potential importance of this information to the development of novel therapies for the treatment of CNS tumors or injury is clear. The recent molecular characterization of the FGF proteins and the cloning and mapping of their genes has made available recombinant proteins, antibodies, and cDNA probes. These are now enabling molecular biological techniques to be applied to studies into all of these aspects of FGF activity. The possibility of their development as human therapeutic agents and prognostic markers may then be objectively analyzed.
??
Acknowledgment
Ann Logan is at present a Research Fellow supported by the Medical Research Council and the International Spinal Research Trust.
References Aguayo AJ: Axonal regeneration from injured neurones in the adult central nervous system. In Cotman CW, ed. Synaptic Plasticity. New York, Guildford, 1985, p 457. Anderson KJ, Dam D, Lee S, Cotman DW: Basic fibroblast growth factor prevents death of lesioned cholinergic neurones invivo. Nature 1988; 332:360.
0 1990, Elsevier Science Publishing
Bahr M, Vanselow J, Thanos S: Ability of adult rat ganglion cells to regrow axons in vitro can be influenced by fibroblast growth factor and gangliosides. Neurosci Lett 1989; 96:197. Baird A, Esch F, Mormede P, et al.: Molecular characterisation of fibroblast growth factor: distribution and biological activities in various tissues. Ret Prog Horm Res 1986; 42:142. Berry M, Maxwell W, Logan A, Mathewson A, McConnell P, Ashhurst D, Thomas GH: Deposition of scar tissue in the central nervous system. Acta Neurochir Suppl 1983; 32:31. Bjorklund A, Stenevi U: Experimental reinnervation of the rat hippocampus by grafted sympathetic ganglia. I. Axonal regeneration along the hippocampal fimbria. Brain Res 1977; 138:259. Bjorklund A, Stenevi U: In vivo evidence for a hippocampal andrenergic neurotrophic factor specifically released on septal deafferentation. Brain Res 1981; 229:403. Bjorklund A, Kromer LF, Stenevi U: Cholinergic reinnervation of the rat hippocampus by septal implant is stimulated by perforant path lesion. Brain Res 1979; 173:57. Davies A: Role of neurotrophic factors in development. Trends Genet 1988; 4:139. Delli-Bovi P, Curatola AM, Kern FG, Greco A, Ittmann M, Basilic0 C: An oncogene isolated by transfection of Kaposi’s sarcoma DNA encodes a growth factor that is a member of the FGF family. Cell 1987; 50:729. Dickson C, Smith R, Brookes S, Peters G: Tumorigenesis by mouse mammary tumor virus: proviral activation of a cellular gene in the common integration region int-2. Cell 1984; 37:529. Dickson C, Peters G: Potential oncogene product related to growth factors. Nature 1987; 326:833. Ferrara N, Ousley F, Gospodarowicz D: Bovine brain astrocytes express basic fibroblast growth factor, a neurotrophic and angiogenic mitogen. Brain Res 1988; 462:223. Finklestein SP, Apostolides PJ, Caday CG, Prosser J, Philips MF, Klagsbnm M: Increased basic fibroblast growth factor (bFGF) immunoreactivity at the site of focal brain wounds. Brain Res 1988; 460:253. Folkman J, Watson K, Ingber D, Hanahan D: Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 1989; 339:58. Gage FH, Bjorklund A, Stenevi U: Denervation releases a neuronal survival factor in adult rat hippocampi. Nature 1984; 308:637. Gensburger C, Labourdette G, Sensenbrenner M: Brain tibroblast growth factor stimulates the proliferation of rat neuronal precursor cells in vitro. FEBS Lett 1987; 217: 1. Gospodarowicz D: Fibroblast growth ISI Atl Sci Biochem 1988; 101.
Co., Inc. 1043-2760/901$2.00
factor.
153
Gospodarowicz D, Neufeld G, Schweigerer L: Fibroblast growth factor. Mol Cell Endocrinol 1986; 46: 187. Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G: Structural characterisation and biological functions of fibroblast growth factor. Endocr Rev 1987; 8:95. Haynes LW: Fibroblast (heparin-binding) growing factors in neuronal development and repair. Mol Neurobiol 1988; 2: 1. Hatten ME, Lynch M, Rydel RE, Sanchez J, Joseph-Silverstein J, Moscatelli D, R&in DB: In vitro neurite extension by granule neurones is dependent upon astroglialderived fibroblast growth factor. Dev Biol 1988; 125:280. Hofer MM, Barde Y-A: Brain-derived neurotrophic factor prevents neuronal death in vivo. Nature 1988; 331:261.
Morrison RS, Sharma A, de Vellis J, Bradshaw RA: Basic fibroblast growth factor supports the survival of cerebral cortical neurones in primary culture. Proc Nat1 Acad Sci USA 1986; 8317537.
Rydel RE. Greene LA: Acidic and basic fibroblast growth factors promote stable neurite outgrowth and neuronal diffcrentiation in cultures of PC12 cells. J Neurosci 1987; 7:3639.
Murphy PR, Sato R, Sato Y, Friesen HG; Fibroblast growth factor messenger ribonucleic acid expression in a human astrocytoma cell line: regulation by serum and cell density. Mol Endocrinol 1988; 2:591.
Schweigerer L, Neufeld G, Gospodarowicz D: Basic fibroblast growth factor is present in cultured human retinoblastoma cells. Invest Ophthalmol Vis Sci 1987; 28:1838.
Murphy PR, Myal Y, Sato Y, Sato R, West M, Friesen HG: Elevated expression of basic fibroblast growth factor messenger ribonucleic acid in acoustic neuromas. Mol Endocrinol 1989; 3:225. Nieto-Sampedro M, Cotman CW: Growth factor induction and temporal order in central nervous system repair. In Cotman CW, ed. Synaptic plasticity. New York, Guildford, 1985, p 407.
Kimelman D, Kirschner M: Synergistic induction of mesoderm by FGF and TGF-/3 and the identification of an mRNA coding for FGF in the early Xenopus embryo. Cell 1987; 51:869.
Nieto-Sampedro M, Lewis ER, Cotman CW, et al.: Brain injury causes a time-dependent increase in neuronotrophic activity at the lesion site. Science 1982; 217:860.
Kromer LF, Bjorklund A, Stenevi U: Innervation of embryonic hippocampal implants by regenerating axons of cholinergic septal neurones in the adult rat. Brain Res 1981a; 210:153.
Nieto-Sampedro M, Manthorpe M, Barbin G, Varon S, Cotman CW: Injury-induced neuronotrophic activity in adult rat brain: correlation with survival of delayed implants in the wound cavity. J Neurosci 1983; 3:2219.
Kromer ation adult onic Brain
LF, Bjorklund A, Stenevi U: Regenerof the septo-hippocampal pathway in rats is promoted by utilizing embryhippocampal implants as bridges. Res 1981b; 210:173.
Nieto-Sampedro M, Whittemore SR, Needels DL, Larson J, Cotman CW: The survival of brain transplants is enhanced by extracts from injured brain. Proc Nat1 Acad Sci USA 1984; 81:6250.
Libermann TA, Friesel R, Jaye M, et al.: An angiogenic growth factor is expressed in human glioma cells. EMBO J 1987; 6:1627.
Nieto-Sampedro M, Saneto RP, de Vellis J, Cotman CW: The control of glial populations in brain: changes in astrocyte mitogenie and morphogenic factors in response to injury. Brain Res 1985; 343:320.
Logan A: Oligodeoxynucleotide probe hybridisation to rat brain fibroblast growth factor mRNA. Neurosci Lett Suppl 1988b; 32:S64.
Nieto-Sampedro M, Lim R, Hicklin DJ, Cotman CW: Early release of glia maturation factor and acidic fibroblast growth factor after rat brain injury. Neurosci Lett 1988; 86:36 1.
Logan A, Logan SD: Distribution of fibroblast growth factor in the central and peripheral nervous systems of various mammals. Neurosci Lett 1986; 69:162.
Neufeld G, Gospodarowicz acidic Iibroblast growth with the same cell surface Chem 1986; 261:5631.
Logan A, Logan SD, Hitchcock ER: Fibroblast growth factor-like activity in human brain tumours. J Endocrinol 1984; 102:30.
Otto D, Unsicker K, Grothe C: Pharmacological effects of nerve growth factor and fibroblast growth factor applied to the transectioned sciatic nerve on neurone death in adult rat dorsal root ganglia. Neurosci Lett 1987; 83:156.
Logan A, Berry M, Thomas GH, Gregson NA, Logan SD: Identification and partial purification of fibroblast growth factor from the brains of developing rats and leucodystrophic mice. Neuroscience 1985; 15:1239. Manthorpe M, Nieto-Sampedro M, Skaper SD, et al.: Neurotrophic activity in brain wounds of the developing rat: correlation with implant survival in the wound cavity. Brain Res 1983; 267:47.
154
1n ncu-
Risau W: Developing brain produces an angiogenesis factor. Proc Natl Acad Sci USA 1986; 83:3855.
Janet T, Miehe B, Pettmann B, Labourdette G, Sensenbrenner M: Ultrastructural localisation of fibroblast growth factor in neurones of rat brain. Neurosci Lett 1987; 80:153.
Logan A: Increased levels of acidic fibroblast growth factor mRNA in the lesioned rat brain. Mol Cell Endocrinol 1988a; 58:275.
growth factor (FGF) is localiscd rones. Neurosci Lett 1986; 69:175.
Mergia A, Eddy R, Abraham JA, Fiddes JC, Shows TB: The genes for basic and acidic fibroblast growth factors are on different human chromosomes. Biochem Biophys Res Commun 1986; 138:644.
D: Basic and factor interact receptor. J Biol
Pettmann B, Weibel M, Daune G, Sensenbrenner M, Labourdette G: Stimulation of proliferation and maturation of rat astroblasts in serum-free culture by an astroglial growth factor. J Neurosci Res 1982; 8:463. Pettmann B, Labourdette Sensenbrenner M: The
G, Weibel M, brain fibroblast
Sievers J, Haussmann B, Unsicker K, Berry M: Fibroblast growth factors promote the survival of adult rat retinal ganglion cells after transection of the optic nerve. Neurosci Lett 1987; 76:157. Slack JMW, Darlington BG, Heath JK, Godsave SF: Mesoderm induction in earlyxenopus embryos by heparin-binding growth factors. Nature 1987; 326: 197. Slack JMW, Darlington BF, Health HK, Godsave SF: Heparin binding growth factors as agents of mesoderm induction in early Xerzopus embryo. Nature 1988; 326:297. Thomas FASEB
KA: Fibroblast J 1987; 1:434.
growth
factors.
Unsicker K, Reichert-Preibsch H, Schmidt R, Pettmann B, Labourdette G, Sensenbrenner M: Astroglial and fibroblast growth factors have neurotrophic functions for cultured peripheral and central nervous system neurones. Proc Nat1 Acad Sci USA, 1987; 84:5459. Walicke P, Cowan WM, Ueno K, Baird A, Guillemin R: Fibroblast growth factor promotes survival of dissociated hippocampal neurones and enhances neurite extension. Proc Nat1 Acad Sci USA 1986; 83:3012. Walicke PA, Fiege J-J, Baird A: Characterisation of the neuronal receptor for basic fibroblast growth factor and comparison to receptors on mesenchymal cells. J Biol Chem 1989; 264:4120. White CW, Sondheimer HM, Crouch ER, Wilson H, Fan LL: Treatment of pulmonary hemangiomatosis with recombinant interferon alpha-2a. N Engl J Med 1989; 320:1197 Wilkinson DG, Bhatt S, McMahon AP: Expression pattern of the FGF-related protooncogene int-2 suggests multiple roles in foetal development. Development 1989; 105:131. Yoshida T, Miyagawa K, Odagiri H, Sakamoto H, Little PFR, Terada M, Sugimura T: Genomic sequence of hst, a transforming gene encoding a protein homologous to fibroblast growth factors and the int-2encoded protein. Proc Nat1 Acad Sci USA 1987; 84:7305. Zhan XI, Bates B, Hu X, Goldfarb M: The human FGF-5 oncogene encodes a novel protein related to libroblast growth factors. TEM Mol Cell Biol 1988; 8:3487.
0 1990, Elsevier Science Publishing Co., Inc. 1043.2760/90/$2.00
TEM January/February
The Role of Fibroblast Growth Factors in the Central Nervous System
in
other developmental processes. FGFs may be important at all stages of CNS development, from tissue induction and proliferation of stem cells in early embryogenesis through the stabilization of the differentiated state of neuronal cells and their survival.
Ann Logan FGF in Early Embryogenesis
The fibroblast growth factors are well-characterized mitogens that are found in the central nervous system (CNS). Their physiological roles are not yet known, but increasing evidence suggests their involvement in CNS development, injury responses, and possibly oncogenesis.
Growth factors are regulatory proteins that control cell proliferation and many other cellular responses by receptormediated mechanisms similar to those used by hormones. Most tissues produce and respond to growth factors, and the CNS is no exception. Despite the early identification of growth factors in the CNS and their recent rigorous characterization, their functions within the CNS are only just beginning to be understood. The fibroblast growth factors (FGFs) are a family of closely related proteins. They have been purified to homogeneity, their primary structure has been determined, and their cDNA cloned and sequenced. The family can be subdivided into acidic forms (aFGF, with an isoelectric point of 5-6) or basic forms (bFGF, with an isoelectric point of 9.6) and now encompasses substances prepared from a variety of cell types and tissues. All share a structural homology (-55% between acidic and basic forms) and a characteristic high affinity for the sulfated mucopolysaccharide heparin. The two molecular forms of FGF are coded for by distinct genes, the aFGF gene being located on human chromosome 5 and the bFGF gene on human chromosome 4 (Mergia et al. 1986). Whereas bFGF is widespread, aFGF is more restricted, occurring predominantly in the CNS. Within the CNS aFGF and bFGF have been immunocytochemically localized in neurons (Pettmann Ann Logan is at the Department of Physiology, The Medical School, Birmingham B15 2TJ. UK.
TEM JanuarylFehmary
1986), but bFGF is also found in astrocytes (Ferrara et al. 1987; Hatten et al. 1988). Equivalent levels of FGF bioactivity are found throughout the CNS (Logan and Logan 1986), indicating that here the FGFs are ubiquitous. Both forms of FGF interact with common cell surface receptors (Neufeld and Gospodarowicz 1986) that have yet to be sequenced and cloned. They stimulate the proliferation and differentiation of cultured cells of embryonic mesodermal and neuroectodermal origin. They also regulate cell activities other than growth, and are therefore multifunctional. Their recently defined activity as potent angiogenic factors is clearly relevant to a number of physiological responses in many tissue systems. This review summarizes current evidence concerning the roles of the FGFs, specifically in the CNS. Of special interest is their potential importance in embryogenesis (as neurotrophic agents in CNS development), injury responses, and oncogenesis. Several excellent, comprehensive reviews describe the present knowledge of the structural properties, mechanisms of action and molecular biology of the FGFs (Baird et al. 1986; Gospodarowicz 1988; Gospodarowicz et al. 1986 and 1987; Haynes 1988; Thomas 1987).
??
FGF in Development
Growth factors and their receptors evidently have important roles in development. These may include the control of cell proliferation in specific tissues, but a number of growth factors, including
0 1990, Else&x
Science
Publishing
In early embryonic development, the basic body plan arises as cells in different regions become programmed to follow a particular developmental path. The animal and vegetal poles of the egg thereby get directed along different developmental pathways (Figure 1). Mesoderm is induced from the animal segment by signals originating from cells in the vegetal region. Signals from the cells of the dorsovegetal region lead to development of a small organizer zone that induces dorsal-type mesoderm structures to develop, such as the notochord. Signals from the cells of the ventrovegetal region induce the formation of ventral-type mesoderm, which will comprise blood cells, mesenchyme, and mesothelium (Slack et al. 1987). This pattern of formation and tissue induction seems to be under the control of specific gens .
inducing
factors
or morpho-
Interest in this subject was stimulated by the report that bFGF, together with another factor, transforming growth factor-p (TGF-P), mimics the biological effects of the vegetalizing factor in early amphibian embryos and acts as a ventrovegetal morphogen for ventral-type mesoderm (Slack et al. 1987 and 1988; Kimelman and Kirschner 1987). The presence of mRNA of the bFGF type was also demonstrated in early amphibian embryos (Kimelman and Kirschner 1987). Hence, FGF may be one of the morphogens responsible for inducing mesoderm. FGF has also been implicated in the very early embryonic development of higher vertebrates. A gene that codes for an FGF-like protein, int-2 (Dickson and Peters 1987), may play a direct role in the development of the gastrulating and early somite stage mouse embryo, regulating cell migration, and tissue induction (Wilkinson et al. 1989). These early influences of FGF-related proteins in embryogenesis may explain the origin of the wide range of FGF-
Co., Inc. 1043-2760/90/$2.00
149
VENTRAL
tion is taking place. Although transcrip-
4 VENTRAL
tion of the aFGF and bFGF genes has yet to be demonstrated within the developing mammalian CNS, expression of the FGF-related gene, int-2, has been found in the CNS of the fetal mouse (Wilkinson et al. 1989). Transcripts of the int-2 gene are in differentiating Purkinje cells and in cells of the neuroblastic layer of the retina. There is increasing in vitro and in vivo evidence to suggest a neurotrophic role in CNS development for FGF.
4 VENTRAL
In vitro Evidence
VEN t RAL
Figure 1. The diagram depicts an amphibian blastula, divided into animal (A) and vegetal (V) segments, with the dorsal side at the bottom. Trophic signals from the dorsovegetal (DV) and ventrovegetal (VV) cells induce cells of the animal segment to differentiate into mesoderm (M) and a small organizer zone (0). Signals (FGF) from the ventrovegetal cells induce the formation of ventral mesoderm (VM), comprising blood cells, mesenthyme, and mesothelium. Signals from the organizer zone induce the formation of dorsal mesoderm (DM) from which the notochord develops.
cells in adult tissue systems. FGF-related proteins also may have other roles in later fetal development, particularly of the CNS.
responsive
FGF in CNS Development Normal CNS development involves neuroblast proliferation and migration followed by the selective death of many of the developing neurons. Trophic factors may play some part in this process, and among these the FGFs are known neurotrophic factors promoting the proliferation, differentiation, and/or survival of specific neurons (for review, see Davies 1988). Bioactive FGF has been demonstrated in the brains of rat embryos (Logan et al. 1985) as early as embryonic day 15, when widespread neuronal differentia-
150
The stabilization and functional maintenance of cultured, differentiated neuronal cells by FGF is now widely reported. In cultures of central and peripheral neurons of the rat and chick (Unsicker et al. 1987) and in cells of the PC12 neuronal cell line (Rydel and Greene 1987), aFGF and bFGF promote survival and enhance neurite outgrowth and differentiated cell function. Enriched neuronal populations derived from early embryonic rat brain proliferate in response to bFGF and later express cholinergic differentiation (Gensburger et al. 1987). bFGF also promotes both the survival and differentiation of embryonic nerve cells in cultures derived from the hippocampal region or the cortex of the rat (Morrison et al. 1986; Walicke et al. 1986). In addition, bFGF stimulates neurite extension in cultures of cerebellar granule cells from newborn mice (Hatten et al. 1988).
In vivo Evidence The in vivo administration of bFGF prevents the normally occurring death of some primary sensory neurons in quail embryos (Hofer and Barde 1988). bFGF promotes the survival of adult rat retinal ganglion cells after transection of the optic nerve and protects adult sensory neurons from lesion-induced death (Sievers et al. 1987). In the adult rat CNS, bFGF also prevents the death of cholinergic neurons in the medial septum and diagonal band of Broca following transection of their axons (Anderson et al. 1988). Thus, FGF may play a role in the normal support of basal forebrain cholinergic neurons. These results suggest that FGFs exert a neurotrophic influence by acting directly upon neurons in the developing
0 1990, Elsevier Science Publishing
and adult CNS to promote their proliferation, differentiation, functional maintenance, and survival. FGFs also have pronounced effects on the proliferation and differentiation of glial cell populations (Pettmann et al. 1982; Gospodarowicz et al. 1987). Glial cells are important in the developing CNS as providers of the physical and trophic environment that promotes neural growth and differentiation. The FGFs may therefore also influence CNS development by modulation of glial neurotrophic properties. Furthermore, FGF may influence CNS development through its angiogenic properties, since FGF may be responsible for capillary ingrowth into the developing brain (Risau 1986). The source of the FGFs acting in the developing CNS remains to be determined. Whether the neurons or the astrocytes are their source, it is not yet clear how these growth factors are packaged and released from them. The FGFs lack a clear signal peptide sequence to direct their secretion, but they may be liberated from cells undergoing developmentally regulated death. This programmed local release of FGFs may contribute to the neurotrophic environment which allows successful growth of selected neurons. Future studies to elucidate the exact neurotrophic role of the FGFs in the developing CNS are important. The trophic environment of the embryonic CNS may hold vital clues for attempts to construct a postinjury environment in the adult that facilitates successful regeneration of lesioned axons.
??
FCF and CNS Injury
Responses
Complete lesions of neural pathways in the adult mammalian CNS are rarely followed by significant functional recovery. Injury damages local vasculature, disrupts the interactions between neural cells, and destroys both neurons and their connections. Soon after the lesion, repair responses such as neovascularization, glial proliferation and differentiation, axonal sprouting, and reactive synaptogenesis are in evidence. In most cases, however, the initial attempt of the axons to regenerate is aborted, as a dense, permanent scar is laid down to seal off the damaged area (Berry et al. 1983). We know very little about the interre-
Co., Inc. 1043.2760/90/$2,00
TEM JanuarylFebruury
lations of these events and how the injury response is initiated. The apparent incapacity for successful regeneration of adult mammalian CNS neurons clearly does not reflect an innate inability of axons to regenerate (Aguayo 1985), nor is the scar an impenetrable barrier to regenerating axons, although it must markedly hinder their growth. It seems that a major factor preventing functional recovery is the absence of an appropriate complement of environmental cues that facilitates successful regrowth and reconnection of lesioned nerve pathways. These cues are clearly present in the developing brain, which can regenerate very well after injury (Berry et al. 1983). There is now strong evidence that, during development or after injury, trophic factors are important in determining the success or failure of growth and regeneration (Bjorklund and Stenevi 1977 and 1981; Gage et al. 1984; Kromer et al. 1981a and b; NietoSampedro and Cotman 1985). What combination of trophic molecules provides the environmental cues for successful regeneration within the CNS is not yet known, but an increasing number of regulatory growth factors have been identified in the mature and developing CNS, and some of these have been implicated in postinjury responses (Berry et al. 1983; Bjorklund et al. 1979; Logan 1988a; Logan et al. 1985; Manthorpe et al. 1983; Nieto-Sampedro et al. 1982,1983,1984, and 1985). The presence of neurotrophic FGFs in developing and adult nervous tissue suggests their potential involvement in neural regeneration processes, including those following injury (Berry et al. 1983; Logan 1988a).
A Role for FGF in Repair of the Injured CNS? If FGFs are released locally into the wound from dead or damaged cells and also from injury-responsive cells, then one of their important functions could be to promote repair of injured CNS tissue (Figure 2). The hypothesis that the FGFs play a role in responses to wounding was initially based on the localization of FGF within the CNS and on observations that these factors stimulate all the cell types involved in such responses (Berry et al. 1983). Specifically, the FGFs are potent mitogens for capillary endothelial cells and vascular
TEM JanuayylFebruary
Figure 2. Following a penetrating injury to the CNS, FGFs are released into the lesion site from damaged or dying cells and from invading mesodermal cells. The biologically active FGFs initiate the regeneration and scarring responses by their actions upon the neural, glial, and mesenchymal elements involved.
smooth muscle cells (whose activity underlies the FGFs’ angiogenic effects), astrocytes, oligodendrocytes, and fibroblasts. They are also chemoattractants for astrocytes and neurotrophic agents (for review of target cell specificity, see Gospodarowicz 1988). In addition to aFGF and bFGF being present in neurons (Janet et al. 1987; Pettmann et al. 1986), from which they can be released when axons are severed, bFGF has been detected in astrocytes (Ferrara et al. 1988) and also in the macrophages which invade the injury site (Baird et al. 1986). Thus, FGF produced from CNS cells (endogenous) or from elsewhere (exogenous) could act within the CNS after injury in an autocrine and/or paracrine fashion to promote the initial axon sprouting and the organization of the mature gliabcollagen scar. The relative contributions of exogenous and endoge-
nous FGFs and of various molecular forms of FGF remain to be established. FGFs React to CNS Injury Growing evidence supports the hypothesis (Berry et al. 1983) that FGFs are important trophic factors regulating some of the postinjury responses seen in the CNS. Following a penetrating injury to the CNS of the rat, the levels of aFGF and bFGF mRNA rise significantly and in parallel with immunoreactive and bioactive protein levels of a- and bFGF within the injured tissues (Logan et al. 1988a and b). Others (Nieto-Sampedro et al. 1988) have also measured increases in aFGF immunoreactivity in the wound cavity of lesioned rat cortex. They suggested that this growth factor was endogenous and may have arisen by active synthesis and secretion, but have identi-
0 1990, Elsevier Science Publishing Co., Inc. 1043-2760/901$2.00
1st
fied neither the cell type nor the possible mechanisms involved. Finklestein et al. (1988) have also demonstrated an increase in the levels of immunoreactive bFGF in lesioned CNS tissue and localized this to cells which resemble reactive astrocytes. Ferrara et al. (1988) have demonstrated the expression of bFGF mRNA by astrocytes. Other studies (Logan 1988a and b) suggest that the time course of the FGF mRNA and protein response to injury observed in the rat closely follows that of the reactive gliosis and neuronal sprouting observed histologically (Berry et al. 1983). These observations provide compelling evidence that a- and bFGF may be synthesized and/or released and/or activated in injured CNS tissue.
FGFs Are Neurotrophic Injured CNS
Factors
in the
Other in vivo experiments also suggest a role for the FGFs in CNS injury responses and indicates their potential use as therapeutic agents to promote neuron survival following injury. Local application of bFGF prevents neuron losses in dorsal root ganglia following sciatic nerve transection in the rat (Otto et al. 1987). Administration of bFGF to the wound site promotes the survival of retinal ganglion cells following transection of the optic nerve (Sievers et al. 1987; Bahr et al. 1989). Infusion of bFGF into the right ventricle of the rat also reduces the death following axotomy of cholinergic neurons that project to the hippocampal formation (Anderson et al. 1988).
Neuron Regeneration
vs Scar Formation
Recent evidence that hippocampal and other CNS neurons bear high-affinity receptors for bFGF (Walicke et al. 1989) indicates that FGF may exert its neurotrophic influences directly upon neurons by receptor-mediated mechanisms. This suggests that the FGFs are physiologically relevant neurotrophic factors. To date, there is no clear experimental evidence to suggest whether FGFs are involved in producing the glial/collagen scar. If FGF is responsible for initiating scar formation in addition to neuronal sprouting following injury, its neurotrophic benefits could be outweighed by the functionally deleterious consequences of producing a scar. Specula-
1.52
tion (Walicke et al. 1989) that responses to CNS injury result from competition by the neuronal and mesenchymal elements involved in regeneration and scarring for the FGF released is, at present, unsubstantiated. Separation of the two injury responses on the basis of the molecular forms of FGF and the receptors responsible for their initiation could clearly have important clinical implications. The Development CNS Injury
of Novel Therapies for
The ability to manipulate the CNS lesion environment into one that reduces scar formation and promotes regeneration would be an important step towards functional repair. If cell-specific analogues of the different FGFs can be produced, together with specific antagonists of their activities, these might be clinically applicable. The development of treatments to aid repair of both acute and chronic damage to the CNS based on the activities of the FGFs on neuronal and mesenchymal cells is an important goal.
??
FGF and CNS Oncogenesis
Advances in molecular biology have enabled new insights into the mechanisms underlying transformation of cells. Recent research has shown that aberrant expression of growth factors, their receptors, or components of their signaling pathways is associated with many different types of tumors. FGF-Related
Oncogenes
Expression of FGF-related oncogenes (transforming genes) has been demonstrated in a number of different nonneural tumors, and this suggests the potential importance of FGF-like proteins in tumor growth. These oncogenes include int-2, which has been implicated in virus-induced mouse mammary carcinogenesis (Dickson et al. 1984), and hsti KS3, FGF-5, and FGF-6, which were detected as oncogenes in tumor DNA that could transform NIH 3T3 murine fibroblasts (Delli-Bovi et al. 1987; Yoshida et al. 1987; Zhan et al. 1988). Expression
of FGF in CNS Tumors
Most CNS tissue contains intrinsically low levels of FGF mRNA, raising specu-
0 1990, Elsevier Science
Publishing
lation that the mRNA is unstable 01‘ tightly regulated under normal conditions (Logan 1988a). Evidence is emerging that some CNS tumors do express increased levels of FGF. Early reports of high levels of bioactive FGF in a number of diverse CNS tumors (Logan et al. 1984) are supported by more recent evidence. An astrocytoma cell line derived from a neural tumor was shown to exhibit a high level of bFGF mRNA expression and to produce biologically active bFGF that remained cell associated (Murphy et al. 1988). These workers have also shown a tumor-specific elevation in bFGF transcript levels and FGFlike proteins in tumors of Schwann cell origin such as acoustic neuromas (Murphy et al. 1989). They could lind no evidence of gene amplification or rearrangement in these tumors, and suggest that the raised levels are due to increased transcription or stabilization of mRNA. Similarly, cells derived from a human retinoblastoma were shown to express the bFGF gene and produce bFGF protein (Schweigerer et al. 1987). This bFGF protein stimulates the proliferation of capillary endothelial cells. Cell lines derived from human gliomas expressed aFGF mRNA and produced and responded to aFGF protein (Libermann et al. 1987).
Autocrine
vs. Paracrine Activity
The roles of the FGFs within these tumors remain to be established, but their influence could be autocrine or paracrine. Inappropriate production of the FGFs, related proteins, or their receptors may directly affect proliferation of tumor cells. Furthermore, FGFs may also increase expression of plasminogen activators, collagenases, and other proteolytic enzymes within tumor cells, and this might mediate remodeling and aid tumor metastasis. Acting as a paracrine factor, FGFs’ angiogenic activity may also be of direct relevance to tumor growth.
Tumor Angiogenesis Solid tumors are dependent upon angiogenesis, since increases in cell number must be supported by coincident increases in capillary blood supply. Thus, FGFs could be responsible for the increased vascular supply that delivers oxygen and nutrients and removes waste
Co., Inc. 1043-2760/90/$2.00
TEM JanuarylFebmary
products in actively growing tumors. Furthermore, the increased blood supply may facilitate tumor metastasis. In a recent paper, Folkman et al. (1989) confirmed a correlation between the presence of factors that stimulate tumor growth and angiogenesis. They suggest that angiogenesis is a secondary event in oncogenesis and, although necessary for tumor progression, is not one of the primary oncogenic events that initiate transformation. Thus, transforming cells acquire the ability to express angiogenic growth factor genes early in tumor development which, in turn, induce neovascularization, thereby facilitating tumor progression.
Clinical Implications The immediate and future clinical applications of these findings are potentially important but ill-defined. Understanding the mechanisms of oncogenesis and tumor progression must form the basis of clinical management of patients with CNS tumors. Identification of the specific genes responsible for transformation and progression of tumors may enable diagnoses and prognostic forecasts to be made and early treatment to be initiated. Therapeutic strategies involving FGFs and the pathophysiological systems in which they may participate are potentially diverse. Gene expression may provide a marker for tumor localization or a quantitative method of assessing tumor activity. FGF release remains poorly understood, but may be amenable to modulation. The major means whereby FGF systems may be influenced, however, is through their specific receptors, and once a receptor (or family of receptors) has been characterized, agonists and antagonists may prove an important means of inducing tumor regression. Moreover, angiogenesis may itself be a process amenable to direct inhibition and thereby allow limitation of tumor growth. The use of interferon to inhibit the abnormal proliferation of capillary blood vessels in a case of pulmonary capillary hemangiomatosis (White et al. 1989) may have broader clinical implications. The suggestion that interferon may act directly to inhibit the FGF-producing cells present in neovascular lesions may point the way for the development of antiangiogenic therapies.
TEM JanuatylFehruaty
??
Future Directions
One of the most important questions still to be resolved concerning the FGFs is that of their physiological and pathophysiological roles within the CNS. In particular, their importance as mitogenie, differentiation, and maintenance factors for the neuronal, glial, and mesenchymal cells involved within the CNS in development, injury responses, and oncogenesis demands continued investigation. Differentiation of the in vivo roles of aFGF vs bFGF remains to be determined. The separation of their functional activities at the cellular level, their molecular, cellular and tissue interactions, and the control and interrelationships of their gene expression needs particular attention. Critical studies on FGFs physiological roles await the characterization of the FGF receptors and the cloning of their cDNA. Definition of FGF-responsive cells within the CNS by analysis of FGF receptor expression and action depends on the availability of receptor probes. The potential importance of this information to the development of novel therapies for the treatment of CNS tumors or injury is clear. The recent molecular characterization of the FGF proteins and the cloning and mapping of their genes has made available recombinant proteins, antibodies, and cDNA probes. These are now enabling molecular biological techniques to be applied to studies into all of these aspects of FGF activity. The possibility of their development as human therapeutic agents and prognostic markers may then be objectively analyzed.
??
Acknowledgment
Ann Logan is at present a Research Fellow supported by the Medical Research Council and the International Spinal Research Trust.
References Aguayo AJ: Axonal regeneration from injured neurones in the adult central nervous system. In Cotman CW, ed. Synaptic Plasticity. New York, Guildford, 1985, p 457. Anderson KJ, Dam D, Lee S, Cotman DW: Basic fibroblast growth factor prevents death of lesioned cholinergic neurones invivo. Nature 1988; 332:360.
0 1990, Elsevier Science Publishing
Bahr M, Vanselow J, Thanos S: Ability of adult rat ganglion cells to regrow axons in vitro can be influenced by fibroblast growth factor and gangliosides. Neurosci Lett 1989; 96:197. Baird A, Esch F, Mormede P, et al.: Molecular characterisation of fibroblast growth factor: distribution and biological activities in various tissues. Ret Prog Horm Res 1986; 42:142. Berry M, Maxwell W, Logan A, Mathewson A, McConnell P, Ashhurst D, Thomas GH: Deposition of scar tissue in the central nervous system. Acta Neurochir Suppl 1983; 32:31. Bjorklund A, Stenevi U: Experimental reinnervation of the rat hippocampus by grafted sympathetic ganglia. I. Axonal regeneration along the hippocampal fimbria. Brain Res 1977; 138:259. Bjorklund A, Stenevi U: In vivo evidence for a hippocampal andrenergic neurotrophic factor specifically released on septal deafferentation. Brain Res 1981; 229:403. Bjorklund A, Kromer LF, Stenevi U: Cholinergic reinnervation of the rat hippocampus by septal implant is stimulated by perforant path lesion. Brain Res 1979; 173:57. Davies A: Role of neurotrophic factors in development. Trends Genet 1988; 4:139. Delli-Bovi P, Curatola AM, Kern FG, Greco A, Ittmann M, Basilic0 C: An oncogene isolated by transfection of Kaposi’s sarcoma DNA encodes a growth factor that is a member of the FGF family. Cell 1987; 50:729. Dickson C, Smith R, Brookes S, Peters G: Tumorigenesis by mouse mammary tumor virus: proviral activation of a cellular gene in the common integration region int-2. Cell 1984; 37:529. Dickson C, Peters G: Potential oncogene product related to growth factors. Nature 1987; 326:833. Ferrara N, Ousley F, Gospodarowicz D: Bovine brain astrocytes express basic fibroblast growth factor, a neurotrophic and angiogenic mitogen. Brain Res 1988; 462:223. Finklestein SP, Apostolides PJ, Caday CG, Prosser J, Philips MF, Klagsbnm M: Increased basic fibroblast growth factor (bFGF) immunoreactivity at the site of focal brain wounds. Brain Res 1988; 460:253. Folkman J, Watson K, Ingber D, Hanahan D: Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 1989; 339:58. Gage FH, Bjorklund A, Stenevi U: Denervation releases a neuronal survival factor in adult rat hippocampi. Nature 1984; 308:637. Gensburger C, Labourdette G, Sensenbrenner M: Brain tibroblast growth factor stimulates the proliferation of rat neuronal precursor cells in vitro. FEBS Lett 1987; 217: 1. Gospodarowicz D: Fibroblast growth ISI Atl Sci Biochem 1988; 101.
Co., Inc. 1043-2760/901$2.00
factor.
153
Gospodarowicz D, Neufeld G, Schweigerer L: Fibroblast growth factor. Mol Cell Endocrinol 1986; 46: 187. Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G: Structural characterisation and biological functions of fibroblast growth factor. Endocr Rev 1987; 8:95. Haynes LW: Fibroblast (heparin-binding) growing factors in neuronal development and repair. Mol Neurobiol 1988; 2: 1. Hatten ME, Lynch M, Rydel RE, Sanchez J, Joseph-Silverstein J, Moscatelli D, R&in DB: In vitro neurite extension by granule neurones is dependent upon astroglialderived fibroblast growth factor. Dev Biol 1988; 125:280. Hofer MM, Barde Y-A: Brain-derived neurotrophic factor prevents neuronal death in vivo. Nature 1988; 331:261.
Morrison RS, Sharma A, de Vellis J, Bradshaw RA: Basic fibroblast growth factor supports the survival of cerebral cortical neurones in primary culture. Proc Nat1 Acad Sci USA 1986; 8317537.
Rydel RE. Greene LA: Acidic and basic fibroblast growth factors promote stable neurite outgrowth and neuronal diffcrentiation in cultures of PC12 cells. J Neurosci 1987; 7:3639.
Murphy PR, Sato R, Sato Y, Friesen HG; Fibroblast growth factor messenger ribonucleic acid expression in a human astrocytoma cell line: regulation by serum and cell density. Mol Endocrinol 1988; 2:591.
Schweigerer L, Neufeld G, Gospodarowicz D: Basic fibroblast growth factor is present in cultured human retinoblastoma cells. Invest Ophthalmol Vis Sci 1987; 28:1838.
Murphy PR, Myal Y, Sato Y, Sato R, West M, Friesen HG: Elevated expression of basic fibroblast growth factor messenger ribonucleic acid in acoustic neuromas. Mol Endocrinol 1989; 3:225. Nieto-Sampedro M, Cotman CW: Growth factor induction and temporal order in central nervous system repair. In Cotman CW, ed. Synaptic plasticity. New York, Guildford, 1985, p 407.
Kimelman D, Kirschner M: Synergistic induction of mesoderm by FGF and TGF-/3 and the identification of an mRNA coding for FGF in the early Xenopus embryo. Cell 1987; 51:869.
Nieto-Sampedro M, Lewis ER, Cotman CW, et al.: Brain injury causes a time-dependent increase in neuronotrophic activity at the lesion site. Science 1982; 217:860.
Kromer LF, Bjorklund A, Stenevi U: Innervation of embryonic hippocampal implants by regenerating axons of cholinergic septal neurones in the adult rat. Brain Res 1981a; 210:153.
Nieto-Sampedro M, Manthorpe M, Barbin G, Varon S, Cotman CW: Injury-induced neuronotrophic activity in adult rat brain: correlation with survival of delayed implants in the wound cavity. J Neurosci 1983; 3:2219.
Kromer ation adult onic Brain
LF, Bjorklund A, Stenevi U: Regenerof the septo-hippocampal pathway in rats is promoted by utilizing embryhippocampal implants as bridges. Res 1981b; 210:173.
Nieto-Sampedro M, Whittemore SR, Needels DL, Larson J, Cotman CW: The survival of brain transplants is enhanced by extracts from injured brain. Proc Nat1 Acad Sci USA 1984; 81:6250.
Libermann TA, Friesel R, Jaye M, et al.: An angiogenic growth factor is expressed in human glioma cells. EMBO J 1987; 6:1627.
Nieto-Sampedro M, Saneto RP, de Vellis J, Cotman CW: The control of glial populations in brain: changes in astrocyte mitogenie and morphogenic factors in response to injury. Brain Res 1985; 343:320.
Logan A: Oligodeoxynucleotide probe hybridisation to rat brain fibroblast growth factor mRNA. Neurosci Lett Suppl 1988b; 32:S64.
Nieto-Sampedro M, Lim R, Hicklin DJ, Cotman CW: Early release of glia maturation factor and acidic fibroblast growth factor after rat brain injury. Neurosci Lett 1988; 86:36 1.
Logan A, Logan SD: Distribution of fibroblast growth factor in the central and peripheral nervous systems of various mammals. Neurosci Lett 1986; 69:162.
Neufeld G, Gospodarowicz acidic Iibroblast growth with the same cell surface Chem 1986; 261:5631.
Logan A, Logan SD, Hitchcock ER: Fibroblast growth factor-like activity in human brain tumours. J Endocrinol 1984; 102:30.
Otto D, Unsicker K, Grothe C: Pharmacological effects of nerve growth factor and fibroblast growth factor applied to the transectioned sciatic nerve on neurone death in adult rat dorsal root ganglia. Neurosci Lett 1987; 83:156.
Logan A, Berry M, Thomas GH, Gregson NA, Logan SD: Identification and partial purification of fibroblast growth factor from the brains of developing rats and leucodystrophic mice. Neuroscience 1985; 15:1239. Manthorpe M, Nieto-Sampedro M, Skaper SD, et al.: Neurotrophic activity in brain wounds of the developing rat: correlation with implant survival in the wound cavity. Brain Res 1983; 267:47.
154
1n ncu-
Risau W: Developing brain produces an angiogenesis factor. Proc Natl Acad Sci USA 1986; 83:3855.
Janet T, Miehe B, Pettmann B, Labourdette G, Sensenbrenner M: Ultrastructural localisation of fibroblast growth factor in neurones of rat brain. Neurosci Lett 1987; 80:153.
Logan A: Increased levels of acidic fibroblast growth factor mRNA in the lesioned rat brain. Mol Cell Endocrinol 1988a; 58:275.
growth factor (FGF) is localiscd rones. Neurosci Lett 1986; 69:175.
Mergia A, Eddy R, Abraham JA, Fiddes JC, Shows TB: The genes for basic and acidic fibroblast growth factors are on different human chromosomes. Biochem Biophys Res Commun 1986; 138:644.
D: Basic and factor interact receptor. J Biol
Pettmann B, Weibel M, Daune G, Sensenbrenner M, Labourdette G: Stimulation of proliferation and maturation of rat astroblasts in serum-free culture by an astroglial growth factor. J Neurosci Res 1982; 8:463. Pettmann B, Labourdette Sensenbrenner M: The
G, Weibel M, brain fibroblast
Sievers J, Haussmann B, Unsicker K, Berry M: Fibroblast growth factors promote the survival of adult rat retinal ganglion cells after transection of the optic nerve. Neurosci Lett 1987; 76:157. Slack JMW, Darlington BG, Heath JK, Godsave SF: Mesoderm induction in earlyxenopus embryos by heparin-binding growth factors. Nature 1987; 326: 197. Slack JMW, Darlington BF, Health HK, Godsave SF: Heparin binding growth factors as agents of mesoderm induction in early Xerzopus embryo. Nature 1988; 326:297. Thomas FASEB
KA: Fibroblast J 1987; 1:434.
growth
factors.
Unsicker K, Reichert-Preibsch H, Schmidt R, Pettmann B, Labourdette G, Sensenbrenner M: Astroglial and fibroblast growth factors have neurotrophic functions for cultured peripheral and central nervous system neurones. Proc Nat1 Acad Sci USA, 1987; 84:5459. Walicke P, Cowan WM, Ueno K, Baird A, Guillemin R: Fibroblast growth factor promotes survival of dissociated hippocampal neurones and enhances neurite extension. Proc Nat1 Acad Sci USA 1986; 83:3012. Walicke PA, Fiege J-J, Baird A: Characterisation of the neuronal receptor for basic fibroblast growth factor and comparison to receptors on mesenchymal cells. J Biol Chem 1989; 264:4120. White CW, Sondheimer HM, Crouch ER, Wilson H, Fan LL: Treatment of pulmonary hemangiomatosis with recombinant interferon alpha-2a. N Engl J Med 1989; 320:1197 Wilkinson DG, Bhatt S, McMahon AP: Expression pattern of the FGF-related protooncogene int-2 suggests multiple roles in foetal development. Development 1989; 105:131. Yoshida T, Miyagawa K, Odagiri H, Sakamoto H, Little PFR, Terada M, Sugimura T: Genomic sequence of hst, a transforming gene encoding a protein homologous to fibroblast growth factors and the int-2encoded protein. Proc Nat1 Acad Sci USA 1987; 84:7305. Zhan XI, Bates B, Hu X, Goldfarb M: The human FGF-5 oncogene encodes a novel protein related to libroblast growth factors. TEM Mol Cell Biol 1988; 8:3487.
0 1990, Elsevier Science Publishing Co., Inc. 1043.2760/90/$2.00
TEM January/February