Characterization of the extracellular domain in vascular endothelial growth factor receptor-1 (Flt-1 tyrosine kinase)

Flt-1 tyrosine kinase, vascular endothelial growth factor (VEGF) receptor-1, binds VEGF and a new VEGF-related ligand, placenta growth factor, but KDR/Flk-1 (VEGF receptor-2) binds only VEGF. To characterize the functional regions in the Flt-1 extracellular domain such as the ligand binding region and the dimer formation of the receptor, we constructed a series of mutants of the Flt-1 extracellular domain as soluble forms in a baculovirus system. We found that a region carrying the N-terminal 1st to 3rd immunoglobulin (Ig)-like domains of Flt-1 binds both ligands with high affinity. However, for dimer formation of soluble Flt-1, a region further downstream in the Flt-1 extracellular domain was required. Mutant Flt-1 receptors expressed in COS cells confirmed the requirement of the 4th to 7th Ig region for the activation of Flt-1 tyrosine kinase. Soluble Flt-1 carrying the N-terminal 1st to 3rd Ig region suppressed VEGF-dependent endothelial proliferation in vitro to the same level as the larger forms of soluble Flt-1, suggesting that the binding of one soluble Flt-1 molecule to one subunit of the VEGF homodimer may be sufficient to block the VEGF activity.     

Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice

Receptor tyrosine kinases Flt-1 and Flk-1/KDR, and their ligand, the vascular endothelial growth factor (VEGF), were shown to be essential for angiogenesis in the mouse embryo by gene targeting. Flk-1/KDR null mutant mice exhibited impaired endothelial and hematopoietic cell development. On the other hand, Flt-1 null mutation resulted in early embryonic death at embryonic day 8.5, showing disorganization of blood vessels, such as overgrowth of endothelial cells. Flt-1 differs from Flk-1 in that it displays a higher affinity for VEGF but lower kinase activity, suggesting the importance of its extracellular domain. To examine the biological role of Flt-1 in embryonic development and vascular formation, we deleted the kinase domain without affecting the ligand binding region. Flt-1 tyrosine kinase-deficient homozygous mice (flt-1(TK-/-)) developed normal vessels and survived. However, VEGF-induced macrophage migration was strongly suppressed in flt-1(TK-/-) mice. These results indicate that Flt-1 without tyrosine kinase domain is sufficient to allow embryonic development with normal angiogenesis, and that a receptor tyrosine kinase plays a main biological role as a ligand-binding molecule.     

A novel type of vascular endothelial growth factor, VEGF-E (NZ-7 VEGF), preferentially utilizes KDR/Flk-1 receptor and carries a potent mitotic activity without heparin-binding domain

Vascular endothelial growth factor (VEGF) mediates endothelial cell proliferation, angiogenesis, and vascular permeability via the endothelial cell receptors, KDR/Flk-1 and Flt-1. Recently, a gene encoding a polypeptide with about 25% amino acid identity to mammalian VEGF was identified in the genome of Orf virus (OV), a parapoxvirus that affects sheep and goats and occasionally, humans, to generate lesions with angiogenesis. In this study, we examined the biological activities and receptor of OV-derived NZ-7 VEGF (VEGF-E). VEGF-E was found to be a dimer of about 20 kDa with no basic domain nor affinity for heparin column, similar to VEGF121 subtype. VEGF121 has 10-100-fold less endothelial cell mitotic activity than VEGF165 due to lack of a heparin-binding basic region. Interestingly, however, VEGF-E showed almost equal levels of mitotic activity on primary endothelial cells and vascular permeability activity as VEGF165. Furthermore, VEGF-E bound KDR/Flk-1 (VEGFR-2) and induced its autophosphorylation to almost the same extent as VEGF165, but did not bind Flt-1 (VEGFR-1) nor induce autophosphorylation of Flt-1. These results indicate that VEGF-E is a novel type of endothelial growth factor, utilizing only one of the VEGF receptors, and carrying a potent mitogenic activity without affinity to heparin.     

Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, induces endothelial proliferation in vitro and vascular permeability in vivo. The human transmembrane c-fms-like tyrosine kinase Flt-1 has recently been identified as a VEGF receptor. Flt-1 kinase has seven immunoglobulin-like extracellular domains and a kinase insert sequence, features shared by two other human gene-encoded proteins, kinase insert domain-containing receptor (KDR) and FLT-4. In this study we show that the mouse homologue of KDR, Flk-1, is a second functional VEGF receptor. Flk-1 binds VEGF with high affinity, undergoes autophosphorylation, and mediates VEGF-dependent Ca2+ efflux in Xenopus oocytes injected with Flk-1 mRNA. We also demonstrate by in situ hybridization that Flk-1 protein expression in the mouse embryo is restricted to the vascular endothelium and the umbilical cord stroma. VEGF and its receptors Flk-1/KDR and Flt-1 may play a role in vascular development and regulation of vascular permeability.     

Increased vascular endothelial growth factor production in the lungs of rats with hypoxia-induced pulmonary hypertension

Vascular endothelial growth factor (VEGF) is a potent mitogenic and permeability factor targeting predominantly endothelial cells. At least two tyrosine kinase receptors, Flk-1 and Flt-1, mediate its action and are mostly expressed by endothelial cells. VEGF and VEGF receptor expression are upregulated by hypoxia in vivo and the role of VEGF in hypoxia-induced angiogenesis has been extensively studied in a variety of disease entities. Although VEGF and its receptors are abundantly expressed in the lung, their role in hypoxic pulmonary hypertension and the accompanying vascular remodeling are incompletely understood. We report in this in vivo study that hypoxia increases mRNA levels for both VEGF and Flk-1 in the rat lung. The kinetics of the hypoxic response differ between receptor and ligand: Flk-1 mRNA showed a biphasic response to hypoxia with a significant, but transient, rise in mRNA levels observed after 9-15 h of hypoxic exposure and the highest levels noted after 3 wk. In contrast, VEGF mRNA levels did not show a significant increase with acute hypoxia, but increased progressively after 1-3 wk of hypoxia. By in situ hybridization, VEGF mRNA was localized predominantly in alveolar epithelial cells with increased signal in the lungs of hypoxic animals compared with controls. Immunohistochemical staining with anti-VEGF antibodies localized VEGF peptide throughout the lung parenchyma and was increased in hypoxic compared with normoxic animals. Furthermore, hypoxic animals had significantly higher circulating VEGF concentrations compared with normoxic controls. Lung vascular permeability as measured by extravasation of Evans Blue dye was not significantly different between normoxic and hypoxic animals, although a tendency for increased permeability was seen in the hypoxic animals. These findings suggest a possible role for VEGF in the pulmonary response to hypoxia.     

SCCA1 and SCCA2 are proteinase inhibitors that map to the serpin cluster at 18q21.3

Vascular endothelial growth factor (VEGF) is an essential molecule in the development and formation of mammalian blood vessels in health and disease. VEGF is also increasingly implicated in other biological processes including renal development and pathophysiology. The biological activities of VEGF in vivo and in its target cells in culture are mediated through two receptor protein tyrosine kinases, KDR/Flk-1 and Flt-1. KDR/Flk-1 is able to mediate the tyrosine phosphorylation of several cellular components as well as the generation of second messengers. Recent findings have revealed novel signaling mechanisms which may mediate the biological actions of VEGF. In contrast, the signal transduction mechanisms triggered by Flt-1 remain largely unknown.     

Comparative studies on the in vitro decidualization process in the baboon (Papio anubis) and human

Blood supply is essential for the maintenance of epididymal function. Since there is no considerable neovascularization in the epididymis, this tissue could represent a suitable model to study the vascular endothelial growth factor (VEGF) effect for vascular permeability. We studied the expression and function of VEGF and its receptors fms-like tyrosine kinase (Flt-1) and fetal liver kinase (designated as kinase insert domain-containing receptor, KDR in the human) in the human epididymis. VEGF and VEGF receptors mRNA were detected in the human epididymal tissue. VEGF protein was localized in peritubular and in ciliated cells of efferent ducts as well as in peritubular and basal cells of the epididymal duct. Vascular endothelial cells did not express VEGF. Flt-1 protein was localized in ciliated cells of efferent ducts and in lymphatic vessels. Vascular endothelial cells were negative for Flt-1 but positive for KDR. In vitro VEGF165 treatment of epididymal tissue induced endothelial fenestrations and opening of interendothelial junctions. Additionally, we observed for the first time that VEGF could induce transendothelial gaps. We conclude that these gaps might be of importance not only for molecular transport but also for cell passage across the vessel wall, which may be significant for tumor metastasis. VEGF may act as a paracrine effector to influence the permeability of lymphatic vessels via Flt-1, and of blood vessels via KDR.     

Role of VEGF receptor-1 (Flt-1) in mediating calcium-dependent nitric oxide release and limiting DNA synthesis in human trophoblast cells

Vascular endothelial growth factor (VEGF) receptor KDR (kinase-insert-domain-containing receptor) is linked to endothelial cell proliferation, and VEGF receptor Flt-1 (fms-like tyrosine kinase) is essential for the organization of embryonic vasculature. Flt-1 is also known to be expressed on adult endothelial and trophoblast cells, although its function has not yet been established. Herein we report that human trophoblast and endothelial cells contain functional Flt-1 receptors for VEGF that trigger the synthesis and release of nitric oxide (NO) by the activation of constitutive NO synthase (cNOS). In first-trimester human trophoblast cells isolated by chorionic villous sampling, VEGF165 stimulated NO release in a concentration- and time-dependent manner, with a maximal increase of 60% (in comparison to basal release levels) occurring within 30 minutes (basal: 1342 pmol/ml; VEGF (10 ng/ml): 2162 pmol/ml; p < 0.001), as measured by an NO chemiluminescence analyzer. VEGF20, a peptide fragment that is composed of the first 20 amino acids at N-terminus, displayed properties of a partial agonist. VEGF165- and VEGF20-mediated NO biosynthesis was attenuated by NG-nitro-L-arginine in a concentration-dependent fashion, indicating NOS activation. VEGF-neutralizing anti-VEGF monoclonal antibody significantly inhibited VEGF-mediated NO release (p < 0.001), and the addition of a neutralizing anti-Flt-1 antibody inhibited the response by 79.6% +/- 7.59%, an effect found to be reversible with higher concentrations of VEGF. In contrast, anti-KDR antibody had no significant inhibitory effect. RT-PCR confirmed the presence of mRNA encoding the Flt-1 and KDR receptors as well as the endothelial form of cNOS in trophoblast cells. VEGF165-stimulated NO release was inhibited by genistein (5 microM; p < 0.001) as well as by the removal of calcium from the extracellular environment (p < 0.001), which suggests the contingency of this process on tyrosine phosphorylation and extracellular calcium, respectively. Addition of sodium nitroprusside, an NO donor, inhibited trophoblast DNA synthesis in a concentration-dependent manner, as measured by [3H]thymidine incorporation, without affecting cell viability. VEGF under maximal NO production had no mitogenic activity, suggesting that trophoblast-derived NO may limit trophoblast proliferation. Endogenous trophoblast DNA synthesis increased 3-fold in the presence of anti-Flt-1 antibody but not in the presence of anti-KDR antibody, suggesting that Flt-1 functions as a growth suppressive receptor to counteract the proliferative actions of KDR. Levels of immunoreactive endothelial cNOS were markedly increased in growth-restricted placentae (n = 4) in comparison to those of normal (n = 5) placentae, which may account for the relatively small-sized placentae associated with intrauterine growth restriction. VEGF165 stimulated NO release via phosphorylation of the Flt-1 receptor, indicating that VEGF may be an autocrine regulator of NO biosynthesis by aiding trophoblast penetration into spinal arterioles during the first trimester and preventing platelet aggregation within the placenta. Finally, the activation of Flt-1 receptor suppressed trophoblast DNA synthesis within the placenta via NO.     

Intraventricular brain-derived neurotrophic factor reduces infarct size after focal cerebral ischemia in rats

Brain-derived neurotrophic factor (BDNF), acting through the high-affinity receptor tyrosine kinase (TrkB), is widely distributed throughout the central nervous system and displays in vitro trophic effects on a wide range of neuronal cells, including hippocampal, cerebellar, and cortical neurons. In vivo, BDNF rescues motorneurons, hippocampal, and substantia nigral dopaminergic cells from traumatic and toxic brain injury. After transient middle cerebral artery occlusion (MCAO), upregulation of BDNF-mRNA in cortical neurons suggests that BDNF potentially plays a neuroprotective role in focal cerebral ischemia. In the current study, BDNF (2.1 micrograms/d) in vehicle or vehicle alone (controls) was delivered intraventricularly for 8 days, beginning 24 hours before permanent middle cerebral artery occlusion by intraluminal suture in Wistar rats (n = 13 per group). There were no differences in physiological variables recorded during surgery for the two groups. Neurological deficit (0 to 4 scale), which was assessed on a daily basis, improved in BDNF-treated animals compared with controls (P < 0.05; analysis of variance and Scheffe's test). There were no significant differences in weight in BDNF-treated animals and controls during the experiment. After elective killing on day 7 after MCAO, brains underwent 2,3,5-triphenyltetrazolium chloride staining for calculation of the infarct volume and for histology (hematoxylin and eosin and glial fibrillary acid protein). The mean total infarct volume was 83.1 +/- 27.1 mm3 in BDNF-treated animals and 139.2 +/- 56.4 mm3 in controls (mean +/- SD; P < 0.01, unpaired, two-tailed t-test). The cortical infarct volume was 10.8 +/- 7.1 mm3 in BDNF-treated animals and 37.9 +/- 19.8 mm3 in controls (mean +/- SD; P < 0.05; unpaired, two-tailed t-test), whereas ischemic lesion volume in caudoputaminal infarction was not significantly different. These results show that pretreatment with intraventricular BDNF reduces infarct size after focal cerebral ischemia in rats and support the hypothesis of a neuroprotective role for BDNF in stoke.     

Endothelin-like immunoreactivity in hippocampus following transient global cerebral ischemia. I. Neuronal and glial cells

The effect of transient, global ischemia on endothelin (ET) localization in CA1 area of hippocampus of the rats was investigated using post-embedding immunogold method. This paper provides immunocytochemical evidence that cerebral ischemia is accompanied by the increase of production of endothelin in CA1 area of the rat. This study focuses on the analysis ET-like immunoreactive neurons and glial cells in hippocampal CA1 area after long-term survival after ischemia: 1 week-12 months. One and two weeks after ischemia appearance of shrunken neurons and significant increase in ET-like immunoreactivity in astroglial cells as well as their proliferation was observed. Six and twelve months--marked immunoreactivity of endothelin in shrunken neurons and in perineuronal microglial macrophages was observed. The data suggest the possibility that the increased intracellular content of endothelin has a role in the development of neuronal death even following a long-term period after ischemia.     

Comparative effects of the chelators sodium 4,5-dihydroxybenzene-1,3-disulfonate (Tiron) and diethylenetriaminepentaacetic acid (DTPA) on acute uranium nephrotoxicity in rats

The effect of transient, global cerebral ischemia on the distribution of endothelin (ET) in blood-brain barrier (BBB) in CA1 area of hippocampus long-time after ischemia was estimated using post-embedding immunogold technique. ET-like immunoreactivity as a gold particles was localized in all compartments of the blood-brain barrier e.g. in endothelial cells, in pericytes, in periendothelial space including basement membrane, and in astroglial processes. In control animal the density of labelling in all elements of BBB in CA1 area of hippocampus was moderate. ET-like immunoreactivity (ET-like IR) was estimated 1 week-12 months after ischemia. Intense ET-like IR in all elements of BBB was noted 2 and 6 months after ischemia. A potential pathophysiological role of endothelin in cerebral vasospasm in long-time after ischemia is well documented.     

Vascular permeability factor/vascular endothelial growth factor-mediated signaling in mouse mesentery vascular endothelium

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a multifunctional cytokine and growth factor that has important roles in both pathological and physiological angiogenesis. VPF/VEGF induces vascular hyperpermeability, cell division, and other activities by interacting with two specific receptor tyrosine kinases, KDR/Flk-1 and Flt-1, that are selectively expressed on vascular endothelium. The signaling cascade that follows VPF/VEGF interaction with cultured endothelium is only partially understood but is known to result in increased intracellular calcium, activation of protein kinase C, and tyrosine phosphorylations of both receptors, phospholipase C-gamma (PLC-gamma) and phosphatidylinositol 3'-kinase. For many reasons, signaling events elicited in cultured endothelium may not mimic mediator effects on intact normal or tumor-induced microvessels in vivo. Therefore, we developed a system that would allow measurement of VPF/VEGF-induced signaling on intact microvessels. We used mouse mesentery, a tissue whose numerous microvessels are highly responsive to VPF/VEGF and that we found to express Flk-1 and Flt-1 selectively. At intervals after injecting VPF/VEGF i.p., mesenteries were harvested, extracted, and immunoprecipitated. Immunoblots confirmed that VPF/VEGF induced tyrosine phosphorylation of several proteins in mesenteric microvessels as in cultured endothelium: Flk-1; PLC-gamma; and mitogen-activated protein kinase. Similar phosphorylations were observed when mesentery was exposed to VPF/VEGF in vitro, or when mesenteries were harvested from mice bearing the mouse ovarian tumor ascites tumor, which itself secretes abundant VPF/VEGF. Other experiments further elucidated the VPF/VEGF signaling pathway, demonstrating phosphorylation of both PYK2 and focal adhesion kinase, activation of c-jun-NH2-kinase with phosphorylation of c-Jun, and an association between Flk-1 and PLC-gamma. In addition, we demonstrated translocation of mitogen-activated protein kinase to the cell nucleus in cultured endothelium. Taken together, these experiments describe a new model system with the potential for investigating signaling events in response to diverse mediators on intact microvessels in vivo and have further elucidated the VPF/VEGF signaling cascade.     

Inflammation and glial responses in ischemic brain lesions

Focal cerebral ischemia elicits a strong inflammatory response involving early recruitment of granulocytes and delayed infiltration of ischemic areas and the boundary zones by T cells and macrophages. Infiltration of hematogenous leukocytes is facilitated by an upregulation of the cellular adhesion molecules P-selectin, intercellular adhesion molecule-1 and vascular adhesion molecule-1 on endothelial cells. Blocking of the leukocyte/endothelial cell adhesion process significantly reduces stroke volume after transient, but not permanent middle cerebral artery occlusion. In the infarct region microglia are activated within hours and within days transform into phagocytes. Astrocytes upregulate intermediate filaments, synthesize neurotrophins and form glial scars. Local microglia and infiltrating macrophages demarcate infarcts and rapidly remove debris. Remote from the lesion no cellular infiltration occurs, but astroglia and microglia are transiently activated. Astrocytic activation is induced by spreading depression. In focal ischemia neurons die acutely by necrosis and in a delayed fashion by programmed cell death, apoptosis. Proinflammatory cytokines such as tumor necrosis factor-alpha and interleukin-1 beta are upregulated within hours in ischemic brain lesions. Either directly or via induction of neurotoxic mediators such as nitric oxide, cytokines may contribute to infarct progression in the post-ischemic period. On the other hand, inflammation is tightly linked with rapid removal of debris and repair processes. At present it is unclear whether detrimental effects of inflammation outweigh neuroprotective mechanisms or vice versa. In global ischemia inflammatory responses are limited, but micro- and astroglia are also strongly activated. Glial responses significantly differ between brain regions with selective neuronal death and neighbouring areas that are more resistent to ischemic damage.     

Deficiency of intercellular adhesion molecule 1 attenuates microcirculatory disturbance and infarction size in focal cerebral ischemia

Recent evidence has shown crucial roles for cell-adhesion molecules in inflammation-induced rolling, adhesion, and accumulation of neutrophils in tissue. Intercellular adhesion molecule-1 (ICAM-1) is one of these adhesion molecules. Previous studies have shown marked reduction in the size of infarction after focal cerebral ischemia by depletion of granulocytes and administration of the antibody against ICAM-1. In the present study we investigated the role of ICAM-1 in the size of ischemic lesions, accumulation of granulocytes, and microcirculatory compromise in focal cerebral ischemia by using ICAM-1-knockout mice. Ischemic lesions were significantly mitigated in knockout mice after permanent and transient focal ischemia, even though the number of granulocytes in the infarcted tissue was almost the same between knockout and wild-type mice. Depletion of granulocytes further decreased the size of ischemic lesions after transient focal ischemia in ICAM-1-knockout mice. Microcirculation was reduced after focal ischemia, but it was better preserved in the cerebral cortex of knockout mice than that of wild-type mice. The present study demonstrated that ICAM-1 played a role in microcirculatory failure and subsequent development and expansion of infarction after focal cerebral ischemia. However, it is highly unlikely that ICAM-1 played a key role in accumulation of granulocytes after focal cerebral ischemia.     

Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis

Vascular endothelial growth factor (VEGF) expression in various cell types is induced by hypoxia and other stimuli. VEGF mediates endothelial cell proliferation, angiogenesis, vascular growth, and vascular permeability via the endothelial cell receptors, kinase insert domain-containing receptor (KDR)/fetal liver kinase 1 (Flk-1) and FLT-1. Alanine-scanning mutagenesis was used to identify a positively charged surface in VEGF that mediates binding to KDR/Flk-1. Arg82, Lys84 and His86, located in a hairpin loop, were found to be critical for binding KDR/Flk-1, while negatively charged residues, Asp63, Glu64, and Glu67, were associated with FLT-1 binding. A VEGF model based on PDGFb indicated these positively and negatively charged regions are distal in the monomer but are spatially close in the dimer. Mutations within the KDR site had minimal effect on FLT-1 binding, and mutants deficient in FLT-1 binding did not affect KDR binding. Endothelial cell mitogenesis was abolished in mutants lacking KDR affinity; however, FLT-1 deficient mutants induced normal proliferation. These results suggest dual sets of determinants in the VEGF dimer that cross-link cell surface receptors, triggering endothelial cell growth and angiogenesis. Furthermore, this mutational analysis implicates KDR, but not FLT-1, in VEGF induction of endothelial cell proliferation.     

Effect of naftidrofuryl (LS129) on axonal growth

Wistar rats, eight days old, were subjected to permanent bilateral forebrain ischemia, followed by hypoxia for 15 minutes. A cerebral infarct, mainly involving the cerebral neocortex, hippocampus, amygdala, striatum and subcortical white matter was produced. Neurons and glia showing punctate chromatin condensation and karyorrhectic cells were observed 12 hours after hypoxia-ischemia. Their number increased during the first two days and recruitment of cells with degenerating nuclei occurred until day five. In situ labeling of nuclear DNA fragmentation stained many normal-appearing nuclei, as well as punctate chromatin condensations and nuclear fragments in karyorrhectic cells. Delayed neuronal death in the CA1 area of the hippocampus was observed after 20 minutes of transient forebrain ischemia in the adult gerbil. In situ labeling of nuclear DNA fragmentation demonstrated stained punctate chromatin condensation in a few degenerating cells at 48 hours post-ischemia. Substantial labeling of CA1 neurons occurred in the fourth day. Agarose gel electrophoresis of extracted brain DNA from ischemic infant rats and adult gerbils showed a ladder-type pattern which is typical of nuclear DNA fragmentation into oligonucleosomal fragments (internucleosomal cleavage). These findings suggest that endonuclease(s) activation may play a role in cell death induced by different forms of hypoxia-ischemia.     

Focal cerebral ischemia in the mouse: description of a model and effects of permanent and temporary occlusion

Our study is to demonstrate the advantages and disadvantages of middle cerebral artery occlusion (MCAO) model in the mouse. CD-1 mice had permanent MCAO for 24 h, or temporary occlusion for either 1 h followed by 23 h of reperfusion or 2 h of occlusion with 22 h of reperfusion. The infarct volume and blood-brain barrier disruption were smaller in the 1-h/23-h temporary occlusion than in either the 24-h permanent occlusion group or the 2-h/22-h temporary occlusion group (p<0.05). Our study demonstrates that blood flow, infarct volume, and blood-brain barrier disruption remain important markers of focal cerebral ischemia.     

Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant

Angiogenesis, the sprouting of capillaries from pre-existing blood vessels, is a fundamental process in the formation of the vascular system during embryonic development. In adulthood, angiogenesis takes place during corpus luteum formation and in pathological conditions such as wound healing, diabetic retinopathy, and tumor-igenesis. Vascularization is essential for solid tumour growth and is thought to be regulated by tumour cell-produced factors, which have a chemotactic and mitogenic effect on endothelial cells. Vascular endothelial growth factor (VEGF), a homodimeric glycoprotein of relative molecular mass 45,000, is the only mitogen, however, that specifically acts on endothelial cells, and it may be a major regulator of tumour angiogenesis in vivo. Its expression has been shown to be upregulated by hypoxia, and its cell-surface receptor, Flk-1, is exclusively expressed in endothelial cells. Here we investigate the biological relevance of the VEGF/Flk-1 receptor/ligand system for angiogenesis using a retrovirus encoding a dominant-negative mutant of the Flk-1/VEGF receptor to infect endothelial target cells in vivo, and find that tumour growth is prevented in nude mice. Our results emphasize the central role of the Flk-1/VEGF system in angiogenesis in general and in the development of solid tumours in particular.     

Identification of the extracellular domains of Flt-1 that mediate ligand interactions

Vascular Endothelial Growth Factor (VEGF) mediates its actions through the Flt-1 and KDR(Flk-1) receptor tyrosine kinases. To localize the extracellular region of Flt-1 that is involved in ligand interactions, we prepared secreted fusion proteins between various combinations of its seven extracellular IgG-like folds. Ligand binding studies show that in combination, domains one and two (amino acids 1-234) are sufficient to achieve VEGF165 interactions. Either domain alone is insufficient to achieve this effect. However, Scatchard analysis reveals that despite the binding capabilities of this construct, the Kd is five fold lower than ligand binding to the full extracellular domain. We find that addition of domain three to this minimal site restores high affinity receptor binding. Further, we show that domains one and two are sufficient to achieve interactions of Flt-1 with Placental Growth Factor (PIGF-1).