The promoter region of VEGF contains hypoxia-responsive elements in addition to several potential Ap-1, Ap-2 and Sp-1 binding sites, indicating that VEGF transcription can be enhanced in response to multiple stimuli (Tisher et al., 1991 Levy et al., 1995 Liu et al., 1995). The other VEGF isoforms show increased affinity for heparan sulfate proteoglycans: VEGF 165 is soluble although a fraction can remain bound to the extracellular matrix VEGF 189 and VEGF 206 are almost exclusively sequestered in the extracellular matrix ( Ferrara et al., 1992 Houck et al., 1992 Park et al., 1993). VEGF 165, a 46-kD homodimeric glycoprotein, is the predominant isoform produced by a variety of normal and transformed cells. VEGF exists in five molecular species of 121, 145, 165, 189, and 206 amino acids (VEGF 121, VEGF 145, VEGF 165, VEGF 189, VEGF 206) that derive from exon splicing of a single gene ( Houck et al., 1991 Keck et al., 1989 Leung et al., 1991 Tisher et al., 1991 Poltorak et al., 1997). VEGF, the prototype member of a family of four structurally related growth factors, is a potent mitogen for micro- and macrovascular endothelial cells but lacks appreciable mitogenic activity for other cell types ( Ferrara and Davis-Smyth, 1997 Senger et al., 1993). This hypothesis is supported by the observation that FGF-2 has an autocrine effect on several cell functions required for angiogenesis, including proliferation, migration, proteinase production, and integrin expression ( Sato and Rifkin, 1988 Mignatti et al., 1991 Sato et al., 1991 Peverali et al., 1994 Bikfalvi et al., 1995 Klein et al., 1996). 1990 Schulze-Osthoff et al., 1990), suggesting that endothelial cell-derived FGF-2 may mediate angiogenesis with an autocrine mode of action. In vivo, FGF-2 has been detected in the basal lamina of blood capillaries, primarily at sites of vessel branching, and in the endothelium of the capillaries of some tumors ( Folkman et al., 1988 DiMario et al., 1989 Cordon et al. Because of its high affinity for heparin and heparan sulfate glycosaminoglycans, significant amounts of FGF-2 are associated with the extracellular matrix of in vitro cell cultures ( Vlodavski et al., 1987 Bashkin et al., 1989 Flaumenhaft et al., 1989 Rojeli et al., 1989).
1991, 1992), FGF-2 is found extracellularly and modulates several cell functions in an autocrine manner ( Sato and Rifkin, 1988 Mignatti et al., 1991 Sato et al., 1991 Peverali et al., 1994 Bikfalvi et al., 1995 Klein et al., 1996). Although the mechanism(s) by which FGF-2 is released from cells is/are still unknown ( Mignatti and Rifkin, 1991 Mignatti et al. However, FGF-2 lacks a signal peptide that directs secretion through the classical secretory pathway ( Abraham et al., 1986). The biological activity of FGF-2 is mediated through a dual receptor system consisting of four high-affinity, tyrosine kinase receptors and low-affinity, heparan sulfate proteoglycans located at the cell surface ( Moscatelli, 1987 Flaumenhaft et al., 1989 Lee et al., 1989 Dionne et al., 1990 Keegan et al., 1991 Partanen et al., 1991).
Thus, angiogenesis in vivo can be modulated by a novel mechanism that involves the autocrine action of vascular endothelial cell-derived FGF-2 and VEGF. Because occasional fibroblasts or other cell types present in the corneal stroma show no significant expression of VEGF mRNA, these findings demonstrate that endothelial cell-derived VEGF is an important autocrine mediator of FGF-2-induced angiogenesis. Systemic administration of neutralizing VEGF antibody dramatically reduces FGF-2-induced angiogenesis. During angiogenesis induced by FGF-2 in the mouse cornea, the endothelial cells of forming capillaries express VEGF mRNA and protein. Endogenous 18-kD FGF-2 production upregulates VEGF expression through extracellular interaction with cell membrane receptors high- M r FGF-2 (22–24-kD) acts via intracellular mechanism(s). Neutralizing monoclonal antibody to VEGF inhibits FGF-2–induced endothelial cell proliferation. Addition of recombinant FGF-2 to cultured endothelial cells or upregulation of endogenous FGF-2 results in increased VEGF expression. Here we show that FGF-2 induces VEGF expression in vascular endothelial cells through autocrine and paracrine mechanisms. FGF-2 and VEGF are potent angiogenesis inducers in vivo and in vitro.
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