c-fos-induced growth factor/vascular endothelial growth factor D induces angiogenesis in vivo and in vitro.
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c-fos-induced growth factor/vascular endothelial growth factor D (Figf/Vegf-D) is a secreted factor of the VEGF family that binds to the vessel and lymphatic receptors VEGFR-2 and VEGFR-3. Here we report that Figf/Vegf-D is a potent angiogenic factor in rabbit cornea in vivo in a dose-dependent manner. In vitro Figf/Vegf-D induces tyrosine phosphorylation of VEGFR-2 and VEGFR-3 in primary human umbilical cord vein endothelial cells (HUVECs) and in an immortal cell line derived from Kaposi's sarcoma lesion (KS-IMM). The treatment of HUVECs with Figf/Vegf-D induces dose-dependent cell growth. Figf/VEGF-D also induces HUVEC elongation and branching to form an extensive network of capillary-like cords in three-dimensional matrix. In KS-IMM cells Figf/Vegf-D treatment results in dose-dependent mitogenic and motogenic activities. Taken together with the previous observations that Figf/Vegf-D expression is under the control of the nuclear oncogene c-fos, our data uncover a link between a nuclear oncogene and angiogenesis, suggesting that Figf/Vegf-D may play a critical role in tumor cell growth and invasion. (+info)
Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers.
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Vascular endothelial growth factor-D (VEGF-D) binds and activates the endothelial cell tyrosine kinase receptors VEGF receptor-2 (VEGFR-2) and VEGF receptor-3 (VEGFR-3), is mitogenic for endothelial cells, and shares structural homology and receptor specificity with VEGF-C. The primary translation product of VEGF-D has long N- and C-terminal polypeptide extensions in addition to a central VEGF homology domain (VHD). The VHD of VEGF-D is sufficient to bind and activate VEGFR-2 and VEGFR-3. Here we report that VEGF-D is proteolytically processed to release the VHD. Studies in 293EBNA cells demonstrated that VEGF-D undergoes N- and C-terminal cleavage events to produce numerous secreted polypeptides including a fully processed form of M(r) approximately 21,000 consisting only of the VHD, which is predominantly a non-covalent dimer. Biosensor analysis demonstrated that the VHD has approximately 290- and approximately 40-fold greater affinity for VEGFR-2 and VEGFR-3, respectively, compared with unprocessed VEGF-D. In situ hybridization demonstrated that embryonic lung is a major site of expression of the VEGF-D gene. Processed forms of VEGF-D were detected in embryonic lung indicating that VEGF-D is proteolytically processed in vivo. (+info)
A mutant form of vascular endothelial growth factor (VEGF) that lacks VEGF receptor-2 activation retains the ability to induce vascular permeability.
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Vascular endothelial growth factor (VEGF) is a major mediator of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to stimulate endothelial cell mitogenesis, and the potent induction of vascular permeability. These activities are at least in part mediated by binding to two high affinity receptors, VEGFR-1 and VEGFR-2. In this study we have made mutations of mouse VEGF in order to define the regions that are required for VEGFR-2-mediated functions. Development of a bioassay, which responds only to signals generated by cross-linking of VEGFR-2, has allowed evaluation of these mutants for their ability to activate VEGFR-2. One mutant (VEGF0), which had amino acids 83-89 of VEGF substituted with the analogous region of the related placenta growth factor, demonstrated significantly reduced VEGFR-2 binding compared with wild type VEGF, indicating that this region was required for VEGF-VEGFR-2 interaction. Intriguingly, when this mutant was evaluated in a Miles assay for its ability to induce vascular permeability, no difference was found when compared with wild type VEGF. In addition we have shown that the VEGF homology domain of the structurally related growth factor VEGF-D is capable of binding to and activating VEGFR-2 but has no vascular permeability activity, indicating that VEGFR-2 binding does not correlate with permeability activity for all VEGF family members. These data suggest different mechanisms for VEGF-mediated mitogenesis and vascular permeability and raise the possibility of an alternative receptor mediating vascular permeability. (+info)
Monoclonal antibodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3.
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Vascular endothelial growth factor-D (VEGF-D), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activates VEGF receptor-2 (VEGFR-2/Flk1/KDR) and VEGFR-3 (Flt4). These receptor tyrosine kinases, localized on vascular and lymphatic endothelial cells, signal for angiogenesis and lymphangiogenesis. VEGF-D consists of a central receptor-binding VEGF homology domain (VHD) and N-terminal and C-terminal propeptides that are cleaved from the VHD to generate a mature, bioactive form consisting of dimers of the VHD. Here we report characterization of mAbs raised to the VHD of human VEGF-D in order to generate VEGF-D antagonists. The mAbs bind the fully processed VHD with high affinity and also bind unprocessed VEGF-D. We demonstrate, using bioassays for the binding and cross-linking of VEGFR-2 and VEGFR-3 and biosensor analysis with immobilized receptors, that one of the mAbs, designated VD1, is able to compete potently with mature VEGF-D for binding to both VEGFR-2 and VEGFR-3 for binding to mature VEGF-D. This indicates that the binding epitopes on VEGF-D for these two receptors may be in close proximity. Furthermore, VD1 blocks the mitogenic response of human microvascular endothelial cells to VEGF-D. The anti-(VEGF-D) mAbs raised to the bioactive region of this growth factor will be powerful tools for analysis of the biological functions of VEGF-D. (+info)
Expression of vascular endothelial growth factors A, B, C, and D and their relationships to lymph node status in lung adenocarcinoma.
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Vascular endothelial growth factors (VEGFs) C and D are novel members of the VEGF family that show some selectivity toward lymphatic endothelial cells. Recent studies suggest that VEGF-C may be involved in lymphangiogenesis and spread of cancer cells via lymphatic vessels. However, whether other VEGF family members play a role in lymph node metastasis is largely unknown. The aim of the present study was to explore whether expressions of VEGF-A, VEGF-B, VEGF-C, and VEGF-D are correlated with lymph node status in lung adenocarcinoma. Total RNA was isolated from 60 surgical specimens of lung adenocarcinoma with (n = 27) or without (n = 33) lymph node metastasis. The relative mRNA abundance of VEGF-A, VEGF-B, VEGF-C, and VEGF-D was measured by real-time reverse transcription-PCR analysis based on TaqMan fluorescence methodology. We found that, as single factors, expression of none of the four VEGF family members clearly correlated with the presence of lymph node metastasis. The only tendency noted was for higher VEGF-B and VEGF-C and lower VEGF-D levels in the node-positive group. However, two-way scatterplot analysis revealed that tumors with lymph node metastasis were associated with a pattern of low VEGF-D and high VEGF-A, VEGF-B, or VEGF-C, such that the ratios of VEGF-D:VEGF-A, VEGF-D:VEGF-B, or VEGF-D:VEGF-C were significantly lower in the node-positive group. Strikingly, none of the 11 tumors with high VEGF-D levels metastasized to lymph nodes. Furthermore, a low VEGF-D:VEGF-C ratio correlated with the presence of lymphatic invasion, and six of seven tumors with a pattern of very high expression of VEGF-C and low expression of VEGF-D displayed lymph vessel invasion that extended along the bronchovascular tree beyond the main tumor. Finally, levels of VEGF-A, but not VEGF-B or VEGF-C, were higher in tumors with large nodal metastasis (> or = 1 cm) than in those with small (< 1 cm) nodal metastasis. These results support the hypothesis that two VEGF family members are involved in lymph node metastasis at two distinct steps; VEGF-C facilitates entry of cancer cells into the lymph vasculature, whereas VEGF-A promotes the growth of metastatic tumor through angiogenesis. The results also suggest that the balance between VEGF-C and VEGF-D could be important rather than the level of VEGF-C alone. Whether a low VEGF-D level plays a causative role in lymph node metastasis requires further investigation. (+info)
VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues.
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Recently, vascular endothelial growth factor receptor 3 (VEGFR-3) has been shown to provide a specific marker for lymphatic endothelia in certain human tissues. In this study, we have investigated the expression of VEGFR-3 and its ligands VEGF-C and VEGF-D in fetal and adult tissues. VEGFR-3 was consistently detected in the endothelium of lymphatic vessels such as the thoracic duct, but fenestrated capillaries of several organs including the bone marrow, splenic and hepatic sinusoids, kidney glomeruli and endocrine glands also expressed this receptor. VEGF-C and VEGF-D, which bind both VEGFR-2 and VEGFR-3 were expressed in vascular smooth muscle cells. In addition, intense cytoplasmic staining for VEGF-C was observed in neuroendocrine cells such as the alpha cells of the islets of Langerhans, prolactin secreting cells of the anterior pituitary, adrenal medullary cells, and dispersed neuroendocrine cells of the gastrointestinal tract. VEGF-D was observed in the innermost zone of the adrenal cortex and in certain dispersed neuroendocrine cells. These results suggest that VEGF-C and VEGF-D have a paracrine function and perhaps a role in peptide release from secretory granules of certain neuroendocrine cells to surrounding capillaries. (+info)
In fibroblasts Vegf-D expression is induced by cell-cell contact mediated by cadherin-11.
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Vascular endothelial growth factors (VEGFs) are a highly conserved family of growth factors all angiogenic in vivo with mitogenic and chemotactic activity on endothelial cells. VEGFs are expressed in fibroblasts either in hypoxia or in response to growth factors. Here we report that, differently from the other members of the family, Vegf-D is induced by cell-cell contact. By in situ hybridization we demonstrated that noninteracting fibroblasts express low levels of Vegf-D mRNA, whereas contacting cells express high levels of Vegf-D transcripts. By immunostaining we observed that the surface protein cadherin-11 is localized at the opposite sites of interacting cell surfaces. Ca(2+) deprivation from the culture medium determined the loss of cadherin-11 from the cell surfaces and down-regulation of Vegf-D mRNA. Moreover, a cadherin-11 antisense RNA construct inhibited Vegf-D expression in confluent BALB/c fibroblasts, whereas in NIH 3T3 cells, which express low levels of cadherin-11, Vegf-D induction could be obtained by overexpression of cadherin-11. This suggests that cell interaction mediated by cadherin-11 induces the expression of the angiogenic factor Vegf-D in fibroblasts. (+info)
Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice.
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Vascular endothelial growth factor receptor-3 (VEGFR-3) has an essential role in the development of embryonic blood vessels; however, after midgestation its expression becomes restricted mainly to the developing lymphatic vessels. The VEGFR-3 ligand VEGF-C stimulates lymphangiogenesis in transgenic mice and in chick chorioallantoic membrane. As VEGF-C also binds VEGFR-2, which is expressed in lymphatic endothelia, it is not clear which receptors are responsible for the lymphangiogenic effects of VEGF-C. VEGF-D, which binds to the same receptors, has been reported to induce angiogenesis, but its lymphangiogenic potential is not known. In order to define the lymphangiogenic signalling pathway we have created transgenic mice overexpressing a VEGFR-3-specific mutant of VEGF-C (VEGF-C156S) or VEGF-D in epidermal keratinocytes under the keratin 14 promoter. Both transgenes induced the growth of lymphatic vessels in the skin, whereas the blood vessel architecture was not affected. Evidence was also obtained that these growth factors act in a paracrine manner in vivo. These results demonstrate that stimulation of the VEGFR-3 signal transduction pathway is sufficient to induce specifically lymphangiogenesis in vivo. (+info)