title: Vascular endothelial growth factor-induced endothelial cell proliferation is regulated by interaction between VEGFR-2, SH-PTP1 and eNOS.
abstract: VEGF receptor-2 plays a critical role in endothelial cell proliferation during angiogenesis. However, regulation of receptor activity remains incompletely explained. Here, we demonstrate that VEGF stimulates microvascular endothelial cell proliferation in a dose-dependent manner with VEGF-induced proliferation being greatest at 5 and 100 ng/ml and significantly reduced at intermediate concentrations (>50% at 20 ng/ml). Neutralization studies confirmed that signaling occurs via VEGFR-2. In a similar fashion, ERK/MAPK is strongly activated in response to VEGF stimulation as demonstrated by its phosphorylation, but with a decrease in phosphoryation at 20 ng/ml VEGF. Immunoblotting analysis revealed that VEGF did not cause a dose-dependent change in expression of VEGFR-2 but instead resulted in reduced phosphorylation of VEGFR-2 when cells were exposed to 10 and 20 ng/ml of VEGF. VEGFR-2 dephosphorylation was associated with an increase in the protein tyrosine phosphatase, SH-PTP1, and endothelial nitric oxide synthase (eNOS). Immunoprecipitation and selective immunoblotting confirmed the association between VEGFR-2 dephosphorylation and the upregulation of SH-PTP1 and eNOS. Transfection of endothelial cells with antisense oligonucleotide against VEGFR-2 completely abolished VEGF-induced proliferation, whereas anti SH-PTP1 dramatically increased VEGF-induced proliferation by 1 and 5-fold at 10 and 200 ng/ml VEGF, respectively. Suppression of eNOS expression only abolished endothelial cell proliferation at VEGF concentrations above 20 ng/ml. Taken together, these results indicate that activation of VEGFR-2 by VEGF enhances SH-PTP1 activity and eNOS expression, which in turn lead to two diverse events: one is that SH-PTP1 dephosphorylates VEGFR-2 and ERK/MAPK, which weaken VEGF mitogenic activity, and the other is that eNOS increases nitric oxide production which in turn lowers SH-PTP1 activity via S-nitrosylation.
authors.affiliation: Cell and Molecular Biology Group, School of Optometry and Vision Sciences, Cardiff University, Cardiff CF10 3NB, UK.
authors.forename: Jun
authors.initials: J
authors.lastname: Cai
authors.forename: Wen G
authors.initials: WG
authors.lastname: Jiang
authors.forename: Asif
authors.initials: A
authors.lastname: Ahmed
authors.forename: Mike
authors.initials: M
authors.lastname: Boulton
mesh_terms: D000818:Animals; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008954:Models, Biological; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D012160:Retina / Q000166:cytology; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D016376:Oligonucleotides, Antisense; D042461:Vascular Endothelial Growth Factor A; D052250:Nitric Oxide Synthase Type III; D040301:Vascular Endothelial Growth Factor Receptor-2; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases