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)

The contribution of VEGF (vascular endothelial growth factor) to angiogenesis and tumor aggressiveness has been shown in several tumor types, but no comparative data regarding the impact of the VEGF-subtypes on endothelial and tumor cell protection are available. Therefore, we analysed the cytoprotective effects of the two major soluble VEGF-subtypes, VEGF-121 and -165, on HUVEC cell cultures (human umbilical vein endothelial cells) and squamous cell carcinoma (SCC) cell lines after ionizing radiation. We performed clonogenic analyses and proliferation assays for evaluation of cell survival and proliferative activity. Experiments were performed employing different intensities up to 8 Gy with or without added recombinant VEGF-121 or -165 and compared to non-irradiated cultures. To evaluate a possible contribution of the VEGF-receptors, we performed immunohistochemical stainings of VEGF-R1 (flt), -R2 (KDR,flk), and Neuropilin-1. In the SCC cell lines and HUVEC cultures, cell survival and proliferative activity were reduced in a dosage-dependent manner. The addition of VEGF-121 yielded a significant increase of resistance from 1.2 to 2.7 Gy (+125%) for killing 50% of subjected cells, while VEGF-165 was less effective in this regard (+83%). Conversely, in HUVEC cultures, 50%-survival was increased more strongly by VEGF-165 compared to VEGF-121 (+100% vs. +43%). Accordingly, proliferation was more intensely stimulated in HUVECs by VEGF-165 than by VEGF-121. VEGF-receptors were expressed in all cell cultures analysed at comparable levels. We conclude that the two VEGF-subtypes differentially increase the survival of tumor and endothelial cells after irradiation in vitro. We hypothesise, that the release of VEGF by the tumor protects tumor cells and endothelium resulting in increased radiation resistance.
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PMID:VEGF-subtype specific protection of SCC and HUVECs from radiation induced cell death. 1558 41

There is increasing evidence that vascular endothelial growth factor (VEGF) has autocrine as well as paracrine functions in tumour biology. Vascular endothelial growth factor-mediated cell survival signalling occurs via the classical tyrosine kinase receptors Flt-1, KDR/Flk-1 and the more novel neuropilin (NP) receptors, NP-1 and NP-2. A 24-mer peptide, which binds to neuropilin-1, induced apoptosis of murine and human breast carcinoma cells, whereas a peptide directed against KDR had no effect. Both anti-NP1 and anti-KDR peptides induced endothelial cell apoptosis. Confocal microscopy using 5-(6)-carboxyfluorescein-labelled peptides showed that anti-NP1 bound to both tumour and endothelial cells, whereas anti-KDR bound endothelial cells only. This study demonstrates that NP-1 plays an essential role in autocrine antiapoptotic signalling by VEGF in tumour cells and that NP1-blockade induces tumour cell and endothelial cell apoptosis. Specific peptides can therefore be used to target both autocrine (tumour cells) and paracrine (endothelial cells) signalling by VEGF.
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PMID:A peptide corresponding to the neuropilin-1-binding site on VEGF(165) induces apoptosis of neuropilin-1-expressing breast tumour cells. 1565 56

A comprehensive, biophysically accurate, computational model of vascular endothelial growth factor (VEGF) family member interactions with endothelial cell surface receptors was developed to study angiogenesis. Neuropilin-1 (NRP1) and the signaling VEGF receptor, VEGFR2, do not interact directly but are bridged by one VEGF isoform, VEGF(165). Using the model and published experimental data, we estimated the kinetic rate of this VEGFR2-NRP1 coupling in vitro. With the use of this rate, our model gives predictions in good quantitative agreement with several independent in vitro experiments involving VEGF(121) and VEGF(165) isoforms, confirming that VEGFR2-NRP1 coupling through VEGF(165) can fully explain the observed differences in receptor binding and phosphorylation in response to these isoforms. Model predictions also determine the mechanism of action of a commonly used NRP1 antibody and predict the results of potential future experiments. This is the first model to include VEGF isoforms or NRPs, and it is a necessary step toward a quantitative molecular level description of VEGF that can be extended to in vivo situations. The model has applications for both proangiogenic and antiangiogenic therapies, such as for heart disease and cancer, as well as in tissue engineering.
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PMID:Differential binding of VEGF isoforms to VEGF receptor 2 in the presence of neuropilin-1: a computational model. 1570 57

Neuropilin-1 (Npn-1) is a cell surface receptor that binds vascular endothelial growth factor (VEGF), a potent mediator of endothelial permeability, chemotaxis, and proliferation. In vitro, Npn-1 can complex with VEGF receptor-2 (VEGFR2) to enhance VEGFR2-mediated endothelial cell chemotaxis and proliferation. To determine the role of Npn-1/VEGFR2 complexes in VEGF-induced endothelial barrier dysfunction, endothelial cells were stably transfected with Npn1 or VEGFR2 alone (PAE/Npn and PAE/KDR, respectively), or VEGFR2 and Npn-1 (PAE/KDR/Npn-1). Permeability, estimated by measurement of transendothelial electrical resistance (TER), of PAE/Npn and PAE/KDR cell lines was not altered by VEGF165. In contrast, TER of PAE/KDR/Npn-1 cells decreased in dose-dependent fashion following VEGF165 (10 to 200 ng/mL). Activation of VEGFR2, and 2 downstream signaling intermediates (p38 and ERK1/2 MAPK) involved in VEGF-mediated permeability, also increased in PAE/KDR/Npn-1. Consistent with these data, inhibition of Npn-1, but not VEGFR2, attenuated VEGF165-mediated permeability of human pulmonary artery endothelial cells (HPAE), and VEGF121 (which cannot ligate Npn-1) did not alter TER of HPAE. Npn-1 inhibition also attenuated both VEGF165-mediated pulmonary vascular leak and activation of VEGFR2, p38, and ERK1/2 MAPK, in inducible lung-specific VEGF transgenic mice. These data support a critical role for Npn-1 in regulating endothelial barrier dysfunction in response to VEGF and suggest that activation of distinct receptor complexes may determine specificity of cellular response to VEGF.
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PMID:Neuropilin-1 regulates vascular endothelial growth factor-mediated endothelial permeability. 1592 19

Vascular endothelial growth factor (VEGF) has been indicated to play a role during endochondral ossification by stimulation of blood vessel invasion into hypertrophic cartilage resulting in its replacement by trabecular bone. We could demonstrate a dose-dependent chemoattractive effect of VEGF-A and PlGF-1, but not VEGF-E or VEGF-C, on human mesenchymal progenitor cells. Quantitative realtime PCR revealed the expression of VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (Flt-4), which markedly declined during osteogenic differentiation. In addition, expression of neuropilin-1 and -2 was detected by RT-PCR. In an in vitro kinase assay, we could demonstrate activation of VEGFR-1 and VEGFR-2 upon stimulation with specific ligands. These findings are consistent with the idea that the chemotactic effect of VEGF-A on MPC is mediated via VEGFR-1, and that VEGF-A and PlGF-1, have a functional role for recruitment of osteoprogenitor cells in the course of endochondral bone formation or remodeling.
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PMID:VEGF-A and PlGF-1 stimulate chemotactic migration of human mesenchymal progenitor cells. 1600 48

Delta-like 4 (Dll4), a membrane-bound ligand for Notch1 and Notch4, is selectively expressed in the developing endothelium and in some tumor endothelium, and it is induced by vascular endothelial growth factor (VEGF)-A and hypoxia. Gene targeting studies have shown that Dll4 is required for normal embryonic vascular remodeling, but the mechanisms underlying Dll4 regulatory functions are currently not defined. In this study, we generated primary human endothelial cells that overexpress Dll4 protein to study Dll4 function and mechanism of action. Human umbilical vein endothelial cells retrovirally transduced with Dll4 displayed reduced proliferative and migratory responses selectively to VEGF-A. Expression of VEGF receptor-2, the principal signaling receptor for VEGF-A in endothelial cells, and coreceptor neuropilin-1 was significantly decreased in Dll4-transduced endothelial cells. Consistent with Dll4 signaling through Notch, expression of HEY2, one of the transcription factors that mediates Notch function, was significantly induced in Dll4-overexpressing endothelial cells. The gamma-secretase inhibitor L-685458 significantly reconstituted endothelial cell proliferation inhibited by immobilized extracellular Dll4 and reconstituted VEGFR2 expression in Dll4-overexpressing endothelial cells. These results identify the Notch ligand Dll4 as a selective inhibitor of VEGF-A biologic activities down-regulating 2 VEGF receptors expressed on endothelial cells and raise the possibility that Dll4 may be exploited therapeutically to modulate angiogenesis.
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PMID:Up-regulation of the Notch ligand Delta-like 4 inhibits VEGF-induced endothelial cell function. 1621 2

The molecular mechanisms by which capillary supply is maintained with advancing age remain to be elucidated. To help clarify these mechanisms, we investigated the gene expression levels of angiogenesis-related factors in young (2.5-month-old), adult (6-month-old), and old (22-month-old) mice. To assess the capillary supply, the capillary endothelium in frozen transverse sections was identified by staining for alkaline phosphatase. The mRNA levels for angiogenesis-related factors were analyzed using real-time RT-PCR. The capillary supply to individual muscle fibers, assessed as the number of capillaries around a muscle fiber, did not change with advancing age. Real-time RT-PCR analysis showed that (1) the level of mRNA for VEGF was lower in old animals than young animals; (2) the mRNA levels of Flt-1 and neuropilin-1 are lower in old animals than young animals, while that of KDR/Flk-1 remained unchanged with advancing age; and (3) the levels of mRNA for angiopoietin-1 and -2 remained unchanged, while the mRNA for Tie-2 was lower in old animals than young animals. These findings suggest that capillary supply is maintained irrespective of the down-regulation of several angiogenesis-related factors and that old animals possess the minimum levels of maintenance and reparative abilities needed to preserve the capillary supply.
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PMID:Effect of aging on expression of angiogenesis-related factors in mouse skeletal muscle. 1628 25

Prostate cancer frequently metastasizes to bone resulting in the formation of osteoblastic metastases through unknown mechanisms. Vascular endothelial growth factor (VEGF) has been shown recently to promote osteoblast activity. Accordingly, we tested if VEGF contributes to the ability of prostate cancer to induce osteoblast activity. PC-3, LNCaP, and C4-2B prostate cancer cell lines expressed both VEGF-165 and VEGF-189 mRNA isoforms and VEGF protein. Prostate cancer cells expressed the mRNA for VEGF receptor (VEGFR) neuropilin-1 but not the VEGFRs Flt-1 or KDR. In contrast, mouse pre-osteoblastic cells (MC3T3-E1) expressed Flt-1 and neuropilin-1 mRNA but not KDR. PTK787, a VEGFR tyrosine kinase inhibitor, inhibited the proliferation of human microvascular endothelial cells but not prostate cancer proliferation in vitro. C4-2B conditioned medium induced osteoblast differentiation as measured by production of alkaline phosphatase and osteocalcin and mineralization of MC3T3-E1. PTK787 blocked the C4-2B conditioned medium-induced osteoblastic activity. VEGF directly induced alkaline phosphatase and osteocalcin but not mineralization of MC3T3-E1. These results suggest that VEGF induces initial differentiation of osteoblasts but requires other factors, present in C4-2B, to induce mineralization. To determine if VEGF influences the ability of prostate cancer to develop osteoblastic lesions, we injected C4-2B cells into the tibia of mice and, after the tumors grew for 6 weeks, administered PTK787 for 4 weeks. PTK787 decreased both intratibial tumor burden and C4-2B-induced osteoblastic activity as measured by bone mineral density and serum osteocalcin. These results show that VEGF contributes to prostate cancer-induced osteoblastic activity in vivo.
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PMID:Vascular endothelial growth factor contributes to prostate cancer-mediated osteoblastic activity. 1632 39

Destruction of the neovasculature is essential for efficient tumor eradication by photodynamic therapy (PDT). Since the over-expression of receptors for vascular endothelial growth factor (VEGF) is correlated with tumor angiogenesis and subsequent growth, we conjugated a photosensitizer (5-(4-carboxyphenyl)-10,15,20-triphenyl-chlorin, TPC), via a spacer (6-aminohexanoic acid, Ahx), to a VEGF receptor-specific heptapeptide (ATWLPPR). ATWLPPR and TPC-Ahx-ATWLPPR bound exclusively to neuropilin-1 (NRP-1) recombinant chimeric protein (IC50=19 and 171 microM, respectively) but were devoid of affinity for VEGF receptor type 2 (VEGFR-2, KDR), to which ATWLPPR was initially thought to bind. TPC-Ahx-ATWLPPR was incorporated up to 25-fold more in human umbilical vein endothelial cells (HUVEC) than TPC over a 24-h period, and the addition of 8 mM ATWLPPR induced a significant decrease of this uptake (P<0.05), corroborating a receptor-mediated incorporation. Slightly less cytotoxic in the dark, TPC-Ahx-ATWLPPR exhibited enhanced in vitro photodynamic activity (10.4-fold), compared to TPC. Pharmacokinetic analysis in nude mice xenografted with U87 human malignant glioma cells revealed relevant tumor levels as soon as 1 h after intravenous injection of TPC-Ahx-ATWLPPR, and a rapid elimination from the blood compartment. Moreover, TPC-Ahx-ATWLPPR was not degraded in vivo up to 2 h after intravenous injection. Taken together, our results demonstrate that TPC-Ahx-ATWLPPR is a much more potent photosensitizer in vitro than TPC, in NRP-1-expressing cells. Thus, it may efficiently potentiate the vascular effect of PDT in vivo.
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PMID:A peptide competing with VEGF165 binding on neuropilin-1 mediates targeting of a chlorin-type photosensitizer and potentiates its photodynamic activity in human endothelial cells. 1642 22

Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-A165 (VEGF-A165) in endothelial cells. To define the role of NP-1 in the biological functions of VEGF, we developed a specific peptide antagonist of VEGF binding to NP-1 based on the NP-1 binding site located in the exon 7- and 8-encoded VEGF-A165 domain. The bicyclic peptide, EG3287, potently (K(i) 1.2 microM) and effectively (>95% inhibition at 100 microM) inhibited VEGF-A165 binding to porcine aortic endothelial cells expressing NP-1 (PAE/NP-1) and breast carcinoma cells expressing only NP-1 receptors for VEGF-A, but had no effect on binding to PAE/KDR or PAE/Flt-1. Molecular dynamics calculations, a nuclear magnetic resonance structure of EG3287, and determination of stability in media, indicated that it constitutes a stable subdomain very similar to the corresponding region of native VEGF-A165. The C terminus encoded by exon 8 and the three-dimensional structure were both critical for EG3287 inhibition of NP-1 binding, whereas modifications at the N terminus had little effect. Although EG3287 had no direct effect on VEGF-A165 binding to KDR receptors, it inhibited cross-linking of VEGF-A165 to KDR in human umbilical vein endothelial cells co-expressing NP-1, and inhibited stimulation of KDR and PLC-gamma tyrosine phosphorylation, activation of ERKs1/2 and prostanoid production. These findings characterize the first specific antagonist of VEGF-A165 binding to NP-1 and demonstrate that NP-1 is essential for optimum KDR activation and intracellular signaling. The results also identify a key role for the C-terminal exon 8 domain in VEGF-A165 binding to NP-1.
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PMID:Characterization of a bicyclic peptide neuropilin-1 (NP-1) antagonist (EG3287) reveals importance of vascular endothelial growth factor exon 8 for NP-1 binding and role of NP-1 in KDR signaling. 1651 43


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