VEGF and VEGF Receptors

Among the proangiogenic factors VEGF-A has been the best characterized. VEGF-A is a potent mitogen for endothelial cells and can elicit a pronounced an-giogenic response in vivo. VEGF-A is also a survival factor for endothelial cells both in vitro and in vivo (Ferrara 2001; Leung et al. 1989). There are numerous isoforms of VEGF-A that are generated by alternative exon splicing that include VEGF-A121, VEGF-A165, VEGF-A189, and VEGF-A206 (Clauss 2000; Ferrara 2001). In addition to the different VEGF-A isoforms, there is a family of VEGF-related angiogenic growth factors VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PlGF; Clauss 2000; Ferrara 2001). The precise function of many of these VEGF-related ligands is currently not known or ambiguous.

VEGF-A is nearly ubiquitously expressed by both neuroblastoma primary tumors and cell lines, with the predominate isoform VEGF-A165 (Eggert et al. 2000; Fakhari et al. 2002; Komuro et al. 2001; Meister et al. 1999; Ribatti et al. 1998; Rossler et al. 1999). High expression ofVEGF-A has been shown to significantly correlate with advanced stage in several studies (Eggert et al. 2000; Fakhari et al. 2002; Komuro et al. 2001). Elevated serum levels of VEGF-A have also been observed in patients with stage-III tumors (Fakhari et al. 2002). Eggert and co-workers reported that the level of expression of other proangiogenic factors including VEGF-B, VEGF-C, bFGF, angiopoi-etin-2, transforming growth factor-a (TGF-a), and PDGF-A, was significantly higher in stage-3 and stage-4 neuroblastomas compared to stage-1,stage-2, or stage-4S tumors (Eggert et al. 2000). In this study, high levels of PDGF-A expression was also significantly associated with decreased survival; however, no correlation between VEGF-C or bFGF and stage was seen in a series of tumors analyzed by Komuro and colleagues (2001). In addition, while Fakhari et al. were able to show correlations between high levels of VEGF-A, VEGF-B and VEGF-C mRNA and advanced stage by real-time RT-PCR, only VEGF-A was detectable in tumor material by Western blotting (Fakhari et al. 2002). These observations suggest that VEGF-A is the major ligand of the VEGF family regulating angiogenesis in neuroblastoma. The role of the other VEGF ligands remains to be elucidated.

VEGF family members bind with differential affinity to three signaling VEGF receptors, VEGFR-1 (Flt-1), VEGFR-2 (flk-1/KDR), and VEGFR-3 (FLT4). VEGF binds to both VEGFR-1 and VEGFR-2, while PlGF and VEGF-B bind exclusively to VEGFR-1. VEGF-C and VEGF-D bind to VEGFR-2 and VEGFR-3, and are mitogens for both vascular and lymphatic endothelial cells. VEGFR-2 appears to be the principal receptor on endothelial cells by which VEGF exerts its angiogenic effects (Ferrara 2001). The role of VEGFR-1 is more complex. This receptor may act not only as a ligand-binding molecule but also aid in the recruitment of bone marrow-derived endothelial precursor cells to newly formed tumor vasculature (Lyden et al. 2001), and in promoting metastases by induction of metalloprotease MMP9 (Hiratsuka et al. 2002). VEGF isoforms that have a heparin-binding site can also bind to the semaphorin receptors neu-ropilin-1 and neuropilin-2 (Neufeld et al. 2002). Their exact role in angiogenesis is not clear,but neuropilins may modulate binding to VEGFR-2 and subsequent bioactivity (Soker et al. 1998). Recently, expression of neuropilin-1 and neuropilin-2 has been detected in vascular endothelial cells of primary neuroblastoma tumors, but their function remains to be determined (Fakhari et al. 2002).

Expression of VEGFR-2 and to a lesser degree VEGFR-1 in primary tumors has been reported in several studies, consistent with the importance of VEGFR-2 in vascular endothelium (Fakhari et al. 2002; Fukuzawa et al. 2002; Langer et al. 2000; Meister et al. 1999); however, the expression of VEGFR-2 in neuroblastoma tumor cells remains unresolved. VEGFR-2 was detected by RT-PCR in either none (Rossler et al. 1999), few (Langer et al. 2000), or all (Meister et al. 1999) of the neuroblastoma cell lines tested. In four cell lines in which VEGFR-2 was expressed, neutralizing antibody to VEGF did not result in inhibition of proliferation of neuroblastoma cells (Meister et al. 1999). In primary tumors,VEGFR-2 has been detected in tumor cells by immunohistochem-istry and in situ hybridization (Fukuzawa et al. 2002); however, VEGFR-2 could not be detected in Western blot analysis of tumor lysates suggesting either a limited expression of VEGFR-2 (e.g., in the vasculature) or a very low/minimal expression in the tumor cells.

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