Cai Jun, Jiang Wen G, Ahmed Asif, Boulton Mike
Cell and Molecular Biology Group, School of Optometry and Vision Sciences, Cardiff University, Cardiff CF10 3NB, UK.
Microvasc Res. 2006 Jan;71(1):20-31. doi: 10.1016/j.mvr.2005.10.004. Epub 2005 Dec 9.
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.
血管内皮生长因子受体-2（VEGF receptor-2）在血管生成过程中的内皮细胞增殖中起关键作用。然而，受体活性的调节机制仍未完全阐明。在此，我们证明VEGF以剂量依赖性方式刺激微血管内皮细胞增殖，VEGF诱导的增殖在5和100 ng/ml时最大，而在中间浓度（20 ng/ml时>50%）时显著降低。中和研究证实信号通过VEGFR-2传导。同样，ERK/MAPK在VEGF刺激下因磷酸化而被强烈激活，但在20 ng/ml VEGF时磷酸化水平降低。免疫印迹分析显示，VEGF不会导致VEGFR-2表达的剂量依赖性变化，而是当细胞暴露于10和20 ng/ml VEGF时导致VEGFR-2磷酸化减少。VEGFR-2去磷酸化与蛋白酪氨酸磷酸酶SH-PTP1和内皮型一氧化氮合酶（eNOS）的增加有关。免疫沉淀和选择性免疫印迹证实了VEGFR-2去磷酸化与SH-PTP1和eNOS上调之间的关联。用针对VEGFR-2的反义寡核苷酸转染内皮细胞完全消除了VEGF诱导的增殖，而抗SH-PTP1在10和200 ng/ml VEGF时分别使VEGF诱导的增殖显著增加了1倍和5倍。抑制eNOS表达仅在VEGF浓度高于20 ng/ml时消除内皮细胞增殖。综上所述，这些结果表明VEGF对VEGFR-2的激活增强了SH-PTP1活性和eNOS表达，进而导致两个不同的事件：一是SH-PTP1使VEGFR-2和ERK/MAPK去磷酸化，从而削弱VEGF的促有丝分裂活性；另一个是eNOS增加一氧化氮生成，进而通过S-亚硝基化降低SH-PTP1活性。
