Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Angiogenesis is important not only in normal embryogenesis, tissue organization and its maintenance but also in pathological processes such as ocular disease in diabetes mellitus and rapid growth of tumors in vivo. Recently, endothelial cell-specific growth factor (VEGF) and its receptors (Flt family) has been characterized, and this ligand-tyrosine kinase receptor is considered to be one of the most important systems involved in angiogenesis. VEGF is induced by a variety of normal or tumor cells under conditions such as hypoxia and hypoglycemia and in the presence of substances such as hormones and growth factors. On the other hand, receptors of the Flt family (Flt-1, KDR/Flk-1, Flt-4) are basically strictly expressed only on vascular endothelial cells with a rare exception. Thus, the stimulation of VEGF-Flt towards angiogenesis is through a paracrine mechanism. A direct involvement of Flt-1 and KDR/Flk-1 in vasculogenesis/angiogenesis has recently been demonstrated by gene targetting studies. Blocking of this system might be a useful tool for suppression of solid tumors in vivo.
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PMID:[Involvement of the VEGF-Flt-receptor family in angiogenesis]. 872 85

A number of growth factor receptor tyrosine kinases have been implicated in angiogenesis, including epidermal growth factor receptor, fibroblast growth factor receptor, platelet-derived growth factor receptor, Flk-1/KDR, Flt-1, Tie-1, and Tek/Tie-2. Flk-1/KDR, a receptor for vascular endothelial growth factor (VEGF), is expressed exclusively in endothelial cells. Using dominant-negative methods, Flk-1 was shown to play a role in angiogenesis and the growth of a variety of tumor types. Because of this, a drug discovery effort was established to identify Flk-1 kinase inhibitors. For initial screening, an ELISA in, a 96-well format was used to measure VEGF-induced Flk-1 tyrosine phosphorylation in whole cells. Compounds that inhibited ligand-induced receptor autophosphorylation were confirmed by antiphosphotyrosine immunoblotting. Inhibition of VEGF-stimulated DNA synthesis in human endothelial cells was also assessed. Inhibitors were further evaluated for their effects on vessel formation using the chorioallantoic membrane assay. Using these methods, antiangiogenesis compounds that inhibit Flk-1 tyrosine kinase activity, endothelial cell mitogenesis, and blood vessel formation in the chorioallantoic membrane assay have been found.
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PMID:Flk-1 as a target for tumor growth inhibition. 875 24

Placenta Growth Factor (PIGF) is a new member of vascular endothelial growth factor (VEGF) family. Although VEGF binds Flt family Flt-1 and KDR/Flk-1 tyrosine kinases at high affinity for signal transduction, biological activities and the receptors of PIGF have not been extensively studied. Reverse transcription-PCR showed that PIGF-2, a subtype of PIGF-1 that bears a basic amino acid-rich domain, is more abundant than PIGF-1 and thus is the major subtype in human placenta. Using antibodies specific to PIGF-1 or -2 as markers, we obtained large amounts of PIGFs in the baculovirus expression system. PIGF-2 had growth-stimulatory activity on human umbilical vein endothelial cells and vascular permeability activity in the Miles assay at levels about 10-fold lower than those of VEGF. All PIGF-1 activities were lower than those of PIGF-2. Both PIGFs competed for the binding of 125I-labeled VEGF to Flt-1 receptor but not to KDR/Flk-1 expressed on NIH3T3 cells. Furthermore, 125I-labeled PIGF bound to Flt-1 at high affinity but not to KDR/Flk-1. Supporting the notion that PIGF can use only Flt-1 as a receptor, PIGF activated Flt-1 to autophosphorylate, whereas PIGF could not generate signals from KDR/Flk-1. These results indicate that Flt-1, but not KDR/Flk-1, is a receptor for PIGF, suggesting that the weak biological activities of PIGF are due to its use of only part of the available VEGF signaling. These mild characteristics of PIGF may be important for the appropriate development and maintenance of normal placental tissue.
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PMID:Flt-1 but not KDR/Flk-1 tyrosine kinase is a receptor for placenta growth factor, which is related to vascular endothelial growth factor. 882 5

Angiogenesis, the development of new blood vessels, is an important process in tissue development and wound healing but becomes pathologic when associated with solid tumor growth, proliferative retinopathies, and rheumatoid arthritis. To date, there has not been a physiologically relevant in vitro model for human angiogenesis that can be used to screen for enhancers and inhibitors of human angiogenesis and allow further investigation of this process. Initially, culture conditions were established for the induction of human angiogenesis in vitro using fragments of human placental blood vessel. Once the assay was validated, it was examined for its ability to detect known inhibitors and enhancers of angiogenesis. The role of endogenous acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) in the angiogenic response was also assessed by performing RT-PCR on both the parent vessel and microvessel outgrowths. In addition, neutralizing antibodies against the three growth factors were used to quantify the relative importance of each growth factor in the angiogenic response. A fragment of human placental blood vessel was embedded in a fibrin gel in microculture plates and was found to give rise to a complex network of microvessels during a period of 7 to 21 days in culture. The response did not require the addition of exogenous growth factors, and thus provides a convenient system for testing substances for their ability to stimulate or inhibit a human in vitro angiogenic response. The ability of the well known angiogenesis antagonist, hydrocortisone, in the presence and absence of heparin, and suramin to significantly inhibit the angiogenic response indicated that the model could be used as an efficient in vitro assay for screening inhibitors of human angiogenesis. The presence of mRNA for aFGF, bFGF, and three isoforms of VEGF, as well as their receptors, FGFR1, FGFR2, Flt-1, and KDR, in vessel outgrowths and the parent vessel, as identified by RT-PCR, strongly implicated aFGF, bFGF, and VEGF as having an important role in this neovascularization response. This was further confirmed by the ability of neutralizing antibodies to aFGF, bFGF, and VEGF to inhibit the angiogenic response to varying extent. Furthermore, the response could be enhanced by the addition of these growth factors in serum-starved cultures. Finally, a stimulatory effect was observed when matrigel was incorporated into the fibrin gel, which indicates that components of the extracellular matrix also play an important role in governing the strength of the angiogenic response. A physiologic angiogenic response relevant to wound healing can be generated by culturing fragments of human placental blood vessels in fibrin gels. The growth factors aFGF, bFGF, and VEGF were shown to play an important role in stimulating this spontaneous angiogenic response. This assay, which can be performed in microcultures, was also shown to be an excellent method for screening for potential inhibitors and enhancers of human angiogenesis.
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PMID:A novel in vitro assay for human angiogenesis. 887 85

Vascular endothelial growth factor (VEGF) is an angiogenic inducer that mediates its effects through two high affinity receptor tyrosine kinases, Flt-1 and KDR. Flt-1 is required for endothelial cell morphogenesis whereas KDR is involved primarily in mitogenesis. Flt-1 has an alternative ligand, placenta growth factor (PlGF). Both Flt-1 and KDR have seven immunoglobulin (Ig)-like domains in the extracellular domain. The significance and function of these domains for ligand binding and receptor activation are unknown. Here we show that deletion of the second domain of Flt-1 completely abolishes the binding of VEGF. Introduction of the second domain of KDR into an Flt-1 mutant lacking the homologous domain restored VEGF binding. However, the ligand specificity was characteristic of the KDR receptor. We then created chimeric receptors where the first three or just the second Ig-like domains of Flt-1 replaced the corresponding domains in Flt-4, a receptor that does not bind VEGF, and analyzed their ability to bind VEGF. Both swaps conferred upon Flt-4 the ability to bind VEGF with an affinity nearly identical to that of wild-type Flt-1. Furthermore, transfected cells expressing these chimeric Flt-4 receptors exhibited increased DNA synthesis in response to VEGF or PlGF. These results demonstrate that a single Ig-like domain is the major determinant for VEGF-PlGF interaction and that binding to this domain may initiate a signal transduction cascade.
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PMID:The second immunoglobulin-like domain of the VEGF tyrosine kinase receptor Flt-1 determines ligand binding and may initiate a signal transduction cascade. 889 Jan 65

Kaposi sarcoma (KS) is the most common tumor associated with HIV-1 infection and develops in nearly 30% of cases. The principal features of this tumor are abnormal vascularization and the proliferation of endothelial cells and spindle (tumor) cells. KS-derived spindle cells induce vascular lesions and display enhanced vascular permeability when inoculated subcutaneously in the nude mouse. This finding suggests that angiogenesis and capillary permeability play a central role in the development and progression of KS. In this study, we show that AIDS-KS cell lines express higher levels of vascular endothelial growth factor/vascular permeability factor (VEGF/VGF) than either human umbilical vein endothelial cells or human aortic smooth muscle cells. AIDS-KS cells and primary tumor tissues also expressed high levels of Flt-1 and KDR, the receptors for VEGF, while the normal skin of the same patients did not show any expression. We further demonstrate that VEGF antisense oligonucleotides AS-1 and AS-3 specifically block VEGF mRNA and protein production and inhibit KS cell growth in a dose-dependent manner. Furthermore, growth of KS cells in nude mice was specifically inhibited by VEGF antisense oligonucleotides. These results show that VEGF is an autocrine growth factor for AIDS-KS cells. To our knowledge, this is the first report that shows that VEGF acts as a growth stimulator in a human tumor. Inhibitors of VEGF or its cognate receptors may thus be candidates for therapeutic intervention.
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PMID:Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma. 902 68

Tissue hypoxia is a characteristic feature of malignant tumors and healing wounds, conditions that are associated with angiogenesis and with increased expression of vascular permeability factor (VPF; also called vascular endothelial growth factor, VEGF), a selective endothelial cell mitogen inducing microvascular hyperpermeability in vivo. We investigated the regulation of VPF/VEGF and its receptors by tissue hypoxia in normal human skin explants and in cultured skin cells in vitro. VPF/VEGF mRNA expression was dramatically upregulated in epidermal keratinocytes, dermal fibroblasts, and dermal microvessels after 24 h of skin organ culture. Hypoxia also enhanced the expression of VPF/VEGF in cultured epidermal keratinocytes and dermal microvascular endothelial cells (predominantly VPF/VEGF121 and VPF/VEGF165) and in dermal fibroblasts (additional upregulation of VPF/VEGF189). The expression of the VPF/VEGF receptor Flt-1 was selectively induced on dermal microvessels in skin explant cultures and in dermal endothelial cell monolayer cultures under hypoxic conditions. In contrast, the KDR receptor was downregulated by hypoxia. These results suggest that hypoxia likely regulates cutaneous angiogenesis and microvascular permeability by two distinct mechanisms: (i) Induction of VPF/VEGF in epithelial and mesenchymal cells, including endothelial cells. (ii) Differential modulation of VPF/VEGF receptor expression by microvascular endothelial cells. These mechanisms may be of importance in the pathogenesis of healing wounds and some malignant tumors that are commonly characterized by hypoxia and overexpression of VPF/VEGF.
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PMID:Hypoxia regulates the expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and its receptors in human skin. 903 22

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).
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PMID:Identification of the extracellular domains of Flt-1 that mediate ligand interactions. 907 Aug 53

Vascular endothelial growth factor (VEGF) stimulated the tyrosine phosphorylation of multiple components in confluent human umbilical vein endothelial cells (HUVECs) including bands of Mr 205,000, corresponding to the VEGF receptors Flt-1 and KDR, and Mr 145,000, 120,000, 97,000, and 65,000-70,000. VEGF caused a striking and transient increase in mitogen-activated protein (MAP) kinase activity and stimulated phospholipase C-gamma tyrosine phosphorylation, but it had no effect on phosphatidylinositol 3'-kinase activity. VEGF caused a marked increase in tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)), which was both rapid and concentration-dependent. VEGF produced similar effects on p125(FAK) in the endothelial cell line ECV.304. VEGF stimulated tyrosine phosphorylation of the 68-kDa focal adhesion-associated component, paxillin, with similar kinetics and concentration dependence to that for p125(FAK). Thrombin and the phorbol ester, phorbol 12-myristate 13-acetate, also increased p125(FAK) tyrosine phosphorylation in HUVECs. The effect of VEGF on p125(FAK) tyrosine phosphorylation was completely inhibited by the actin filament-disrupting agent cytochalasin D and was partially inhibited by the protein kinase C inhibitor GF109203X. Inhibition of the MAP kinase pathway using a specific inhibitor of MAP kinase kinase had no effect on p125(FAK) tyrosine phosphorylation. VEGF stimulated migration and actin stress fiber formation in confluent HUVEC, and VEGF-induced p125(FAK)/paxillin tyrosine phosphorylation was accompanied by increased immunofluorescent staining of p125(FAK), paxillin, and phosphotyrosine in focal adhesions in confluent cultures of HUVECs. These findings identify p125(FAK) and paxillin as components in a VEGF-stimulated signaling pathway and suggest a novel mechanism for VEGF regulation of endothelial cell functions.
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PMID:Vascular endothelial growth factor stimulates tyrosine phosphorylation and recruitment to new focal adhesions of focal adhesion kinase and paxillin in endothelial cells. 918 76

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.
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PMID:Role of VEGF receptor-1 (Flt-1) in mediating calcium-dependent nitric oxide release and limiting DNA synthesis in human trophoblast cells. 919 54


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