Binding, internalization, and degradation of basic fibroblast growth factor in human microvascular endothelial cells (original) (raw)
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Journal of Clinical Investigation, 1992
Vascular endothelial growth factor (VEGF) is a secreted heparin-binding mitogen; its growth-promoting activity is limited to vascular endothelial cells in vitro and VEGF also stimulates angiogenesis in vivo. To identify target cells for VEGF and investigate the potential physiological role of this factor, iodinated recombinant human VEGF ('"I-rhVEGF) was used for in vitro ligand autoradiography on tissue sections from adult rats. "2'I-rhVEGF exhibited saturable, displaceable binding to a single class of sites with high affinity and low capacity in all tissues and organs examined. Colocalization of 25I-rhVEGF binding with Factor VIII-like immunoreactivity demonstrated binding sites associated with vascular endothelial cells of both fenestrated and nonfenestrated microvessels and the endothelium of large vessels, while no displaceable binding was evident on nonendothelial cells. Specific binding was associated with quiescent as well as proliferating vessels. These findings support the hypothesis that VEGF plays a specific role in both the maintenance and in the induction of growth of vascular endothelial cells.
Biochemistry, 1989
Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of 12%bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 X 10l2 binding sites/mm2 E C M with an apparent kD of 610 nM. F G F binds to heparan sulfate (HS) in E C M as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 pg/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 pg/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. F G F released from ECM was biologically active, as indicated by its stimulation of cell proliferation and D N A synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descemet's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound F G F may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response.
Journal of Cellular Physiology, 1989
Vahbbina 7 9, 25 124 Brescia, /ta/y Normal FBAE AG 7680 cells and chemically transformed FBAE GM 7373 cells were compared for their capacity to produce and to respond to bFGF. Normal FBAE cells showed higher levels of bFGF protein and of poly(A)+ bFGF mRNA than transformed GM 7373 cells, indicating that chemical transformation in FBAE cells is paralleled by a decrease of bFGF gene expression. Basic FGF induced cell proliferation in both normal and transformed FBAE cells. However, bFGF appeared to be much more potent in transformed than in normal cells. No differences in bFGF membrane receptors were observed between normal and transformed FBAE cells in terms of apparent molecular weight, number per cell, dissociation constant, and kinetic of downregulation. In respect to normal cells, however, transformed G M 7373 cells showed higher basal levels of PKC activity. This kinase is activated by bFGF and is involved in mediating the mitogenic activity of bFGF, as shown by the capacity of the PKC inhibitor H-7 to abolish the mitogenic activity of bFGF both in normal and transformed FBAE cells. Like bFGF, the PKC activators DAG and TPA exerted a stronger mitogenic activity in transformed than in normal FBAE cells. Thus, the different susceptibility of normal and transformed FBAE cells to bFGF appears to depend on differences in the postreceptor signal transduction mediated by PKC rather than on differences in bFCF receptors. The results indicate that chemical (ransformation causes significant modifications of bFGF physiology in FBAE cells. The relevance of these modifications to the genesis of tumors of vascular origin deserves further investigation. FGFs represent a family of mitogens characterized by their affinity for heparin (Gospodarowicz et al., 1987a, b). These mitogens are the product of two distinct genes that codify for two different proteins: acidic FGF and bFGF. Both factors, which share approximately 50% homology in amino acid sequence, induce cell proliferation, chemotaxis, and protease production in cultured endothelial cells (Moscatelli et al., 1986a; Presta et al., 1986; Gospodarowicz et al., 1987a, b) and are angiogenic in Vi VO (Esch et al., 1985; Thomas et al., suggest that bFGF might be involved in a n autocrine regulation of endothelial cell growth in vitro and, POSsibly, in vivo. An increasing body of evidence indicates that cancer cells might show alterations in the production of autocrine peptide growth factors, of their receptors, or of the mechanisms of postreceptor signal transduction (for a
Journal of Cellular Physiology, 1989
Basic fibroblast growth factor (bFGF) binds to heparin-like molecules present in the extracellular matrix (ECM) of transformed fetal bovine aortic endothelial G M 7373 cells. Binding of bFGF to ECM can be competed by heparin or heparan sulfate, and ECM-bound bFGF can be released by treating the cells with heparinase or heparatinase. After binding to ECM, bFGF is slowly released into the medium in a biologically active form, as shown by its capacity to induce an increase of cell-associated plasminogen activator activity and cell proliferation. The increase is prevented upon removal of ECM-bound bFGF by a neutral 2 M NaCl wash. Soluble heparin and heparan sulfate reduce the amount of ECMbound bFGF released into the medium, possibly competing with ECM polysaccharides for heparinase-like enzymes produced by endothelial cells, suggesting that these enzymes are involved in the mobilization of ECM-bound bFGF. University of Florence, Italy. Both heparinase and heparitinase were free of protease activity, as assayed using casein as a substrate and both were highly specific for heparin and heparan sulfate, respectively (M. Del Rosso, personal communication).