We also demonstrate that addition of exogenous PDGF-BB, which could overwhelm the periendothelial endogenous gradient of PDGF-BB, impairs the mural cell recruitment induced by co-stimulation with VEGF-A and FGF-2 in vitro and also functional neovascular formation in vivo. Mural cells with FGF-2-upregulated PDGFRβ could detect the periendothelial PDGF-BB gradient induced by VEGF-A and be recruited to the endothelium. Since endothelium-derived PDGF-BB protein has affinity to extracellular matrix (ECM) molecules, it is located in the vicinity of endothelial cells and may form a steep gradient in the periendothelial compartment ( Abramsson et al., 2003). In addition to these direct effects, we demonstrate here that the co-stimulation with VEGF-A and FGF-2 induces efficient mural cell recruitment to nascent vessels through enhancement of endogenous PDGF-B–PDGFRβ signaling in concert: VEGF-A enhances PDGF-BB secretion by the endothelium, and FGF-2 enhances PDGFRβ expression in mural cells. Stimulation with VEGF-A alone directly promotes the proliferation of endothelial cells, while stimulation with FGF-2 alone directly promotes the proliferation of endothelial as well as mural cells. In the present study, we examined the mechanism of angiogenic synergism between VEGF-A and FGF-2. The roles of FGF signaling in vascular formation have recently been reported in mouse embryo and tissue explant models using dominant-negative FGF receptor 1 (FGFR1) ( Lee et al., 2000 Rousseau et al., 2003), and in embryonic stem cells lacking FGFR1 ( Magnusson et al., 2004), although they have not been clarified by studies using genetically engineered mice lacking FGF ligands ( Miller et al., 2000) or FGF receptors (FGFRs) ( Arman et al., 1998 Deng et al., 1994 Yamaguchi et al., 1994). FGF-2, by contrast, has been reported to act as a mitogen for both endothelial and mural cells ( D'Amore and Smith, 1993 Bikfalvi et al., 1997). VEGF-A was originally identified as a specific mitogen for endothelial cells, and the importance of VEGF-A in vascular development in vivo has been established by studies investigating VEGF-A knockout mice ( Carmeliet et al., 1996 Ferrara et al., 1996). The growth factors used for therapeutic angiogenesis in previous studies were principally vascular endothelial growth factor A (VEGF-A) and fibroblast growth factor 2 (FGF-2), both of which are well-investigated pro-angiogenic molecules. Therapeutic angiogenesis, a strategy in which angiogenic growth factors improve revascularization of ischemic tissues, has been extensively investigated ( Ylä-Herttuala and Alitalo, 2003). The findings provide insights into the mechanisms underlying the effects of co-stimulation by growth factors, which could lead to rational design of therapeutic angiogenic strategies. Thus, we demonstrated that the directional enhancement of endogenous PDGF-B–PDGFRβ signaling is indispensable for the synergistic effect of VEGF-A and FGF-2 on neoangiogenesis in adults. These findings indicated the importance of preservation of the periendothelial PDGF-BB gradient. These effects were abrogated not only by anti-PDGFRβ neutralizing antibody, but also by exogenous PDGF-BB, which could overwhelm the endogenous PDGF-BB distribution. Co-stimulation with VEGF-A and FGF-2 caused significant mural cell recruitment in vitro and formation of functional neovasculature in vivo, compared with single-agent stimulation. Experiments in vitro revealed that, in addition to having direct mitogenic effects, these molecules enhance intercellular PDGF-B signaling in a cell-type specific manner: VEGF-A enhances endothelial PDGF-B expression, whereas FGF-2 enhances mural PDGF receptor β (PDGFRβ ) expression. In the present study, we investigated the mechanism of synergism between VEGF-A and FGF-2 by using Matrigel plug assay in vivo and embryonic stem cell (ESC)-derived VEGF receptor 2 (VEGFR2)-positive cells in vitro. Combined stimulation with VEGF-A, FGF-2, or PDGF-BB has emerged as a potent strategy for therapeutic angiogenesis, although the mechanisms underlying the synergism of these factors are not well understood.
0 kommentar(er)
