Robert Friesel

Molecular mechanisms of angiogenesis: fibroblast growth factor signal transduction.

The fibroblast growth factors are a family of structurally related polypeptides that are mitogenic for a broad range of cell types as well as mediators of a wide spectrum of developmental and pathophysiological processes in vivo and in vitro. The fibroblast growth factor family presently consists of nine distinct members. Indeed, the FGF prototypes FGF‐1 (acidic) and FGF‐2 (basic) are well described as modifiers of angiogenesis. The absence of a signal sequence to direct their secretion and their ability to traffic to the nucleus are unique structural features that may be relevant to the regulation of their activities. The FGF receptor family consists of four trans‐membrane receptor tyrosine kinases. Each of these receptors give rise to multiple isoforms as a result of alternative splicing of their mRNAs. The significance of these multiple isoforms is not fully understood; however it is known that alternative splicing in the extracellular domain of these receptors results in altered ligand binding specificities. In addition, alternative splicing in the cytoplasmic domain results in isoforms with increased oncogenic potential. This review7 will describe recent insights into the pathways used for the regulation of FGF secretion and cellular trafficking as well as signaling by FGFRs.—Friesel, R. H., Maciag, T. Molecular mechanisms of angiogenesis: fibroblast growth factor signal transduction. FASEBJ. 9, 919‐925

Identification of Sef, a novel modulator of FGF signalling

Fibroblast growth factors (FGFs) are members of a family of some 30 secreted proteins important in the regulation of cellular proliferation, migration, differentiation and survival. Here we report the identification of a novel modulator of FGF signal transduction, sef, isolated from a zebrafish embryo library through an in situ hybridization screen. The sef gene encodes a transmembrane protein, and belongs to the synexpression group that includes some of the fgf genes. Sef expression is positively regulated by FGF, and ectopic expression of sef in zebrafish or Xenopus laevis embryos specifically inhibits FGF signalling. In co-immunoprecipitation assays, the intracellular domain of Sef interacts with FGF receptors, FGFR1 and FGFR2. Injection of antisense sef morpholino oligos mimicked the phenotypes observed by ectopic fgf8 expression, suggesting that Sef is required to limit FGF signalling during development.

The Role of Fibroblast Growth Factors in Tumor Growth

Biological processes that drive cell growth are exciting targets for cancer therapy. The fibroblast growth factor (FGF) signaling network plays a ubiquitous role in normal cell growth, survival, differentiation, and angiogenesis, but has also been implicated in tumor development. Elucidation of the roles and relationships within the diverse FGF family and of their links to tumor growth and progression will be critical in designing new drug therapies to target FGF receptor (FGFR) pathways. Recent studies have shown that FGF can act synergistically with vascular endothelial growth factor (VEGF) to amplify tumor angiogenesis, highlighting that targeting of both the FGF and VEGF pathways may be more efficient in suppressing tumor growth and angiogenesis than targeting either factor alone. In addition, through inducing tumor cell survival, FGF has the potential to overcome chemotherapy resistance highlighting that chemotherapy may be more effective when used in combination with FGF inhibitor therapy. Furthermore, FGFRs have variable activity in promoting angiogenesis, with the FGFR-1 subgroup being associated with tumor progression and the FGFR-2 subgroup being associated with either early tumor development or decreased tumor progression. This review highlights the growing knowledge of FGFs in tumor cell growth and survival, including an overview of FGF intracellular signaling pathways, the role of FGFs in angiogenesis, patterns of FGF and FGFR expression in various tumor types, and the role of FGFs in tumor progression.

Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase.

Heparin-binding growth factors (HBGFs) bind to high-affinity cell surface receptors which possess intrinsic tyrosine kinase activity. A Mr 150,000 protein phosphorylated on tyrosine in response to class 1 HBGF (HBGF-1) was purified and partially sequenced. On the basis of this sequence, cDNA clones were isolated from a human endothelial cell library and identified as encoding phospholipase C-gamma. Phosphorylation of phospholipase C-gamma in intact cells treated with HBGF-1 was directly demonstrated by using antiphospholipase C-gamma antibodies. Thus, HBGF-1 joins epidermal growth factor and platelet-derived growth factor, whose receptor activation leads to tyrosine phosphorylation and probable activation of phospholipase C-gamma.

Constitutive Activation of Fibroblast Growth Factor Receptor-2 by a Point Mutation Associated with Crouzon Syndrome

The fibroblast growth factor receptors (FGFRs) are a family of ligand-activated, membrane-spanning tyrosine kinases. Mutations in several human FGFR genes have been identified as playing a role in certain disorders of bone growth and development. One of these, Crouzon syndrome, an autosomal dominant disorder causing craniosynostosis, has been associated with mutations in the human FGFR-2 gene. We report here that microinjection of Xenopus embryos with RNA encoding an FGFR-2 protein bearing a Cys Tyr mutation (FGFR-2CS) found in Crouzon syndrome results in fibroblast growth factor (FGF)-independent induction of mesoderm in animal pole explants. Wild-type FGFR-2 did not induce mesoderm when injected at similar doses. The effects of the mutant receptor were blocked by co-expression of dominant negative mutants of either Raf or Ras. Analysis of the mutant receptor protein expressed in Xenopus oocytes indicates that it forms covalent homodimers, does not bind radiolabeled FGF, and has increased tyrosine phosphorylation. These results indicate that FGFR-2CS forms an intermolecular disulfide bond resulting in receptor dimerization and ligand-independent activation that may play a role in the etiology of Crouzon syndrome.

Ligand-independent Activation of Fibroblast Growth Factor Receptors by Point Mutations in the Extracellular, Transmembrane, and Kinase Domains

The fibroblast growth factor receptors (FGFRs) are a family of receptor protein tyrosine kinases that have been shown to mediate a variety of cellular processes including angiogenesis, wound healing, tumorigenesis, and embryonic development. Distinct FGFR mutations in individuals with autosomal dominant disorders of bone growth and development provide a unique opportunity to determine the function of FGFRs during embryonic development. To determine the consequences of these mutations on receptor function, we have made mutations in Xenopus FGFR1 (XFGFR1) and FGFR2 (XFGFR2) that correspond to several of the mutations identified in these dysmorphic syndromes. Analysis of mutant receptor proteins expressed in Xenopus oocytes indicates that all but one have elevated tyrosine kinase activity relative to their wild-type counterparts. Those mutations that give an unpaired cysteine residue in the extracellular domain result in intermolecular disulfide bond formation and covalent receptor dimerization. Microinjection of Xenopus embryos with RNA encoding mutant receptors with elevated tyrosine kinase activity results in ligand-independent induction of mesoderm in animal pole explants. Wild-type XFGFR1 and XFGFR2 do not induce mesoderm when injected at similar doses. Co-injection of RNA encoding a dominant negative FGF receptor, lacking the tyrosine kinase domain, together with RNA encoding various activated FGFRs inhibits mesoderm induction by a receptor activated by a transmembrane domain mutation or extracellular mutations that introduce an unpaired cysteine residue into the extracellular domain but does not inhibit mesoderm induction by receptors bearing a tyrosine kinase domain mutation. These results indicate that different point mutations may activate FGFRs by distinct mechanisms and that ligand-independent FGFR activation may be a feature in common to many skeletal disorders.

Heparin protects heparin-binding growth factor-I from proteolytic inactivation in vitro

Heparin inhibits proteolytic digestion of heparin-binding growth factor-I (HBGF-I) by trypsin, plasmin and other proteases. This property is lost after thermal denaturation of HBGF-I, suggesting that a heparin:HBGF-I structural interaction rather than a heparin:trypsin interaction is responsible for the resistance of HBGF-I to digestion with trypsin. Heparin is also able to partially protect HBGF-I from thermal denaturation as demonstrated by the ability of heparin to protect HBGF-I from trypsin digestion. The protective effect of heparin is dependent upon the concentration of heparin as well as temperature and duration of denaturation. Autoradiography of 125I-HBGF-I incubated with human umbilical vein endothelial cells demonstrates near complete protection of HBGF-I from proteolytic modification when the incubation is performed in the presence of heparin. These data suggest that (i) the mechanism of the heparin-induced increase in human endothelial cell number at confluence involves the protection of HBGF-I by heparin against proteolytic inactivation and (ii) heparin provides conformational stability to the proteolytic growth factor which reduces the susceptibility of HBGF-I to denaturation.

Vascular Injury Induces Expression of Periostin Implications for Vascular Cell Differentiation and Migration

Objective— Periostin mRNA is among the most strongly upregulated transcripts in rat carotid arteries after balloon injury. The goal of the present study was to gain insight into the significance of periostin in the vasculature. Methods and Results— Periostin expression after injury was localized to smooth muscle cells of the neointima and the adventitia. The expression of periostin in smooth muscle cells in vitro was not regulated by cytokines such as fibroblast growth factor-2 (FGF-2). In contrast, stimulation of MC3T3-E1 osteoblastic cells, NIH3T3 fibroblasts, or mesenchymal C3H10T1/2 cells with FGF-2 reduced periostin mRNA levels to <5% of controls, whereas conversely bone morphogenetic protein-2 (BMP-2) increased periostin mRNA levels. Periostin expression was induced and maintained during retinoic acid-induced smooth muscle cell differentiation in A404 cells. In addition, overexpression of periostin in C3H10T1/2 cells caused an increase in cell migration that could be blocked with an anti-periostin antibody. Conclusions— Periostin expression is associated with smooth muscle cell differentiation in vitro and promotes cell migration. Unlike other mesenchymally derived cell lines, periostin expression is not regulated by FGF-2 in smooth muscle cells. This distinction may be useful in discriminating smooth muscle and fibroblast lineages.

An angiogenic growth factor is expressed in human glioma cells

Progression to increased malignancy frequently occurs in human brain tumors of glial origin and usually involves neovascularization–a massive proliferation of endothelial cells into the tumor tissue. We have shown previously that subversion of a normal growth factor‐related pathway is frequently associated with human gliomas. Here we show that human glioma cell lines express the gene encoding the angiogenic peptide endothelial cell growth factor (ECGF) or acidic fibroblast growth factor (a‐FGF) and that an ECGF‐like polypeptide is produced by these cells. The glioma‐derived growth factor was partially purified from cell extracts by heparin‐Sepharose affinity chromatography where it eluted at 1.5 M sodium chloride. On reversed‐phase h.p.l.c., growth factor activity for endothelial cells was eluted at the same concentration of acetonitrile as found for bovine brain‐ECGF, also a potent mitogen for endothelial cells. Moreover, human glioma cells possess specific cell surface receptors for ECGF and are mitogenically stimulated by exogenous addition of this growth factor. Glioma derived‐ECGF may therefore have a dual influence: first, by autocrine growth‐stimulation of human gliomas and, second, by paracrine‐stimulation of endothelial cell proliferation which results in neovascularization of the tumor tissue.

Collagen Triple Helix Repeat Containing 1, a Novel Secreted Protein in Injured and Diseased Arteries, Inhibits Collagen Expression and Promotes Cell Migration

Collagen triple helix repeat containing 1 (Cthrc1) was identified in a screen for differentially expressed sequences in balloon-injured versus normal arteries. Cthrc1 expression was not detectable in normal arteries. However, on injury it was transiently expressed by fibroblasts of the remodeling adventitia and by smooth muscle cells of the neointima. It was also found in the matrix of calcifying human atherosclerotic plaques. CTHRC1 is a secreted 28-kDa protein that is glycosylated and highly conserved from lower chordates to mammals. A short collagen motif with 12 Gly-X-Y repeats appears to be responsible for trimerization of the protein and this renders the molecule susceptible to cleavage by collagenase. Cthrc1 mRNA expression levels are increased in response to transforming growth factor-&bgr; and bone morphogenetic protein-4. Cell migration assays performed with CTHRC1-overexpressing fibroblasts and smooth muscle cells demonstrate that increased CTHRC1 levels are associated with enhanced migratory ability. Furthermore, CTHRC1 overexpression caused a dramatic reduction in collagen type I mRNA and protein levels. Our data indicate that the novel molecule CTHRC1 is transiently expressed in the arterial wall in response to injury where it may contribute to vascular remodeling by limiting collagen matrix deposition and promoting cell migration.