VEGF is required for growth and survival in neonatal mice.
(1/1452)
We employed two independent approaches to inactivate the angiogenic protein VEGF in newborn mice: inducible, Cre-loxP- mediated gene targeting, or administration of mFlt(1-3)-IgG, a soluble VEGF receptor chimeric protein. Partial inhibition of VEGF achieved by inducible gene targeting resulted in increased mortality, stunted body growth and impaired organ development, most notably of the liver. Administration of mFlt(1-3)-IgG, which achieves a higher degree of VEGF inhibition, resulted in nearly complete growth arrest and lethality. Ultrastructural analysis documented alterations in endothelial and other cell types. Histological and biochemical changes consistent with liver and renal failure were observed. Endothelial cells isolated from the liver of mFlt(1-3)-IgG-treated neonates demonstrated an increased apoptotic index, indicating that VEGF is required not only for proliferation but also for survival of endothelial cells. However, such treatment resulted in less significant alterations as the animal matured, and the dependence on VEGF was eventually lost some time after the fourth postnatal week. Administration of mFlt(1-3)-IgG to juvenile mice failed to induce apoptosis in liver endothelial cells. Thus, VEGF is essential for growth and survival in early postnatal life. However, in the fully developed animal, VEGF is likely to be involved primarily in active angiogenesis processes such as corpus luteum development. (+info)
Role of alphavbeta3 integrin in the activation of vascular endothelial growth factor receptor-2.
(2/1452)
Interaction between integrin alphavbeta3 and extracellular matrix is crucial for endothelial cells sprouting from capillaries and for angiogenesis. Furthermore, integrin-mediated outside-in signals co-operate with growth factor receptors to promote cell proliferation and motility. To determine a potential regulation of angiogenic inducer receptors by the integrin system, we investigated the interaction between alphavbeta3 integrin and tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR-2) in human endothelial cells. We report that tyrosine-phosphorylated VEGFR-2 co-immunoprecipitated with beta3 integrin subunit, but not with beta1 or beta5, from cells stimulated with VEGF-A165. VEGFR-2 phosphorylation and mitogenicity induced by VEGF-A165 were enhanced in cells plated on the alphavbeta3 ligand, vitronectin, compared with cells plated on the alpha5beta1 ligand, fibronectin or the alpha2beta1 ligand, collagen. BV4 anti-beta3 integrin mAb, which does not interfere with endothelial cell adhesion to vitronectin, reduced (i) the tyrosine phosphorylation of VEGFR-2; (ii) the activation of downstream transductor phosphoinositide 3-OH kinase; and (iii) biological effects triggered by VEGF-A165. These results indicate a new role for alphavbeta3 integrin in the activation of an in vitro angiogenic program in endothelial cells. Besides being the most important survival system for nascent vessels by regulating cell adhesion to matrix, alphavbeta3 integrin participates in the full activation of VEGFR-2 triggered by VEGF-A, which is an important angiogenic inducer in tumors, inflammation and tissue regeneration. (+info)
Differential regulation of vascular endothelial growth factor and its receptor fms-like-tyrosine kinase is mediated by nitric oxide in rat renal mesangial cells.
(3/1452)
Under conditions associated with local and systemic inflammation, mesangial cells and invading immune cells are likely to be responsible for the release of large amounts of nitric oxide (NO) in the glomerulus. To further define the mechanisms of NO action in the glomerulus, we attempted to identify genes which are regulated by NO in rat glomerular mesangial cells. We identified vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase (FLT-1) to be under the regulatory control of exogenously applied NO in these cells. Using S-nitroso-glutathione (GSNO) as an NO-donating agent, VEGF expression was strongly induced, whereas expression of its FLT-1 receptor simultaneously decreased. Expressional regulation of VEGF and FLT-1 mRNA was transient and occurred rapidly within 1-3 h after GSNO treatment. Expression of a second VEGF-specific receptor, fetal liver kinase-1 (FLK-1/KDR), could not be detected. The inflammatory cytokine interleukin-1beta mediated a moderate increase in VEGF expression after 24 h and had no influence on FLT-1 expression. In contrast, platelet-derived growth factor-BB and basic fibroblast growth factor had no effect on VEGF expression, but strongly induced FLT-1 mRNA levels. Obviously, there is a differential regulation of VEGF and its receptor FLT-1 by NO, cytokines and growth factors in rat mesangial cells. (+info)
Vascular endothelial growth factor (VEGF) directly stimulates endothelial cell proliferation and migration via tyrosine kinase receptors of the split kinase domain family. It mediates vascular growth and angiogenesis in the embryo but also in the adult in a variety of physiological and pathological conditions. The potential binding site of VEGF with its receptor was identified using cellulose-bound overlapping peptides of the extracytosolic part of the human vascular endothelial growth factor receptor II (VEGFR II). Thus, a peptide originating from the third globular domain of the VEGFR II comprising residues 247RTELNVGIDFNWEYP261 was revealed as contiguous sequence stretch, which bound 125I-VEGF165. A systematic replacement with L-amino acids within the peptide representing the putative VEGF-binding site on VEGFR II indicates Asp255 as the hydrophilic key residue for binding. The dimerized peptide (RTELNVGIDFNWEYPAS)2K inhibits VEGF165 binding with an IC50 of 0.5 microM on extracellular VEGFR II fragments and 30 microM on human umbilical vein cells. VEGF165-stimulated autophosphorylation of VEGFR II as well as proliferation and migration of microvascular endothelial cells was inhibited by the monomeric peptide RTELNVGIDFNWEYPASK at a half-maximal concentration of 3-10, 0.1, and 0.1 microM, respectively. We conclude that transduction of the VEGF165 signal can be interrupted with a peptide derived from the third Ig-like domain of VEGFR II by blockade of VEGF165 binding to its receptor. (+info)
Early induction of angiogenetic signals in gliomas of GFAP-v-src transgenic mice.
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Angiogenesis is a prerequisite for solid tumor growth. Glioblastoma multiforme, the most common malignant brain tumor, is characterized by extensive vascular proliferation. We previously showed that transgenic mice expressing a GFAP-v-src fusion gene in astrocytes develop low-grade astrocytomas that progressively evolve into hypervascularized glioblastomas. Here, we examined whether tumor progression triggers angiogenetic signals. We found abundant transcription of vascular endothelial growth factor (VEGF) in neoplastic astrocytes at surprisingly early stages of tumorigenesis. VEGF and v-src expression patterns were not identical, suggesting that VEGF activation was not only dependent on v-src. Late-stage gliomas showed perinecrotic VEGF up-regulation similarly to human glioblastoma. Expression patterns of the endothelial angiogenic receptors flt-1, flk-1, tie-1, and tie-2 were similar to those described in human gliomas, but flt-1 was expressed also in neoplastic astrocytes, suggesting an autocrine role in tumor growth. In crossbreeding experiments, hemizygous ablation of the tumor suppressor genes Rb and p53 had no significant effect on the expression of VEGF, flt-1, flk-1, tie-1, and tie-2. Therefore, expression of angiogenic signals is an early event during progression of GFAP-v-src tumors and precedes hypervascularization. Given the close similarities in the progression pattern between GFAP-v-src and human gliomas, the present results suggest that these mice may provide a useful tool for antiangiogenic therapy research. (+info)
Vascular endothelial growth factor receptor KDR tyrosine kinase activity is increased by autophosphorylation of two activation loop tyrosine residues.
(6/1452)
Vascular endothelial growth factor is an important physiological regulator of angiogenesis. The function of this endothelial cell selective growth factor is mediated by two homologous tyrosine kinase receptors, fms-like tyrosine kinase 1 (Flt-1) and kinase domain receptor (KDR). Although the functional consequence of vascular endothelial growth factor binding to the Flt-1 receptor is not fully understood, it is well established that mitogenic signaling is mediated by KDR. Upon sequencing several independent cDNA clones spanning the cytoplasmic region of human KDR, we identified and confirmed the identity of a functionally required valine at position 848 in the ATP binding site, rather than the previously reported glutamic acid residue, which corresponds to an inactive tyrosine kinase. The cytoplasmic domain of recombinant native KDR, expressed as a glutathione S-transferase fusion protein, can undergo autophosphorylation in the presence of ATP. In addition, the kinase activity can be substantially increased by autophosphorylation at physiologic ATP concentrations. Mutation analysis indicates that both tyrosine residues 1054 and 1059 are required for activation, which is a consequence of an increased affinity for both ATP and the peptide substrate and has no effect on kcat, the intrinsic catalytic activity of the enzyme. KDR kinase catalyzes phosphotransfer by formation of a ternary complex with ATP and the peptide substrate. We demonstrate that tyrosine kinase antagonists can preferentially inhibit either the unactivated or activated form of the enzyme. (+info)
In vitro hematopoietic and endothelial potential of flk-1(-/-) embryonic stem cells and embryos.
(7/1452)
Mice deficient in the Flk-1 receptor tyrosine kinase are known to die in utero because of defective vascular and hematopoietic development. Here, we show that flk-1(-/-) embryonic stem cells are nevertheless able to differentiate into hematopoietic and endothelial cells in vitro, although they give rise to a greatly reduced number of blast colonies, a measure of hemangioblast potential. Furthermore, normal numbers of hematopoietic progenitors are found in 7.5-day postcoitum flk-1(-/-) embryos, even though 8. 5-day postcoitum flk-1(-/-) embryos are known to be deficient in such cells. Our results suggest that hematopoietic/endothelial progenitors arise independently of Flk-1, but that their subsequent migration and expansion require a Flk-1-mediated signal. (+info)
VEGF deprivation-induced apoptosis is a component of programmed capillary regression.
(8/1452)
The pupillary membrane (PM) is a transient ocular capillary network, which can serve as a model system in which to study the mechanism of capillary regression. Previous work has shown that there is a tight correlation between the cessation of blood flow in a capillary segment and the appearance of apoptotic capillary cells throughout the segment. This pattern of cell death is referred to as synchronous apoptosis (Lang, R. A., Lustig, M., Francois, F., Sellinger, M. and Plesken, H. (1994) Development 120, 3395-3404; Meeson, A., Palmer, M., Calfon, M. and Lang, R. A. (1996) Development 122, 3929-3938). In the present study, we have investigated whether the cause of synchronous apoptosis might be a segmental deficiency of either oxygen or a survival factor. Labeling with the compound EF5 in a normal PM indicated no segmental hypoxia; this argued that oxygen deprivation was unlikely to be the cause of synchronous apoptosis. When rat plasma was used as a source of survival factors in an in vitro PM explant assay, inhibition of vascular endothelial growth factor (VEGF) all but eliminated the activity of plasma in suppressing apoptosis. This argued that VEGF was an important plasma survival factor. Furthermore, inhibition of VEGF in vivo using fusion proteins of the human Flk-1/KDR receptor resulted in a significantly increased number of capillaries showing synchronous apoptosis. This provides evidence that VEGF is necessary for endothelial cell survival in this system and in addition, that VEGF deprivation mediated by flow cessation is a component of synchronous apoptosis. (+info)