Program in Clinical and Experimental Therapeutics, University of Georgia, USA.
J Cell Sci. 2012 Oct 15;125(Pt 20):4751-60. doi: 10.1242/jcs.103481. Epub 2012 Aug 1.
Although promising, the ability to regulate angiogenesis through delivery of VEGF remains an unrealized goal. We have shown previously that physiological levels of peroxynitrite (1 µM) are required for a VEGF-mediated angiogenic response, yet the redox-regulated mechanisms that govern the VEGF signal remain unexplored. We assessed the impact of VEGF and peroxynitrite on modifying redox-state, the level of reduced-glutathione (GSH) and S-glutathionylation on regulation of the low molecular weight protein tyrosine phosphatase (LMW-PTP) and focal adhesion kinase (FAK), which are key mediators of VEGF-mediated cell migration. Stimulation of human microvascular endothelial (HME) cells with VEGF (20 ng/ml) or peroxynitrite (1 µM) caused an immediate and reversible negative-shift in the cellular redox-state and thiol oxidation of LMW-PTP, which culminated in cell migration. VEGF causes reversible S-glutathionylation of LMW-PTP, which inhibits its phosphorylation and activity, and causes the transient activation of FAK. Modulating the redox-state using decomposing peroxynitrite (FeTPPS, 2.5 µM) or the GSH-precursor [N-acetylcysteine (NAC), 1 mM] caused a positive-shift of the redox-state and prevented VEGF-mediated S-glutathionylation and oxidative inhibition of LMW-PTP. NAC and FeTPPS prevented the activation of FAK, its association with LMW-PTP and cell migration. Inhibiting LMW-PTP expression markedly enhanced FAK activation and cell migration. Although mild oxidative stress achieved by combining VEGF with 0.1-0.2 mM peroxynitrite augmented cell migration, an acute shift to oxidative stress achieved by combining VEGF with 0.5 mM peroxynitrite induced and sustained FAK activation, and LMW-PTP S-glutathionylation, resulting in LMW-PTP inactivation and inhibited cell migration. In conclusion, our findings demonstrate that a balanced redox-state is required for VEGF to facilitate reversible S-glutathionylation of LMW-PTP, FAK activation and endothelial cell migration. Shifting the redox-state to reductive stress or oxidative stress inhibited the VEGF-mediated angiogenic response.
虽然有前景，但通过输送 VEGF 来调节血管生成仍然是一个尚未实现的目标。我们之前已经表明，生理水平的过氧亚硝酸盐（1µM）是 VEGF 介导的血管生成反应所必需的，但是调节 VEGF 信号的氧化还原调控机制仍未得到探索。我们评估了 VEGF 和过氧亚硝酸盐对改变氧化还原状态、还原型谷胱甘肽 (GSH) 水平和低分子量蛋白酪氨酸磷酸酶 (LMW-PTP) 和粘着斑激酶 (FAK) 的 S-谷胱甘肽化的影响，这些都是 VEGF 介导的细胞迁移的关键介质。用 VEGF（20ng/ml）或过氧亚硝酸盐（1µM）刺激人微血管内皮（HME）细胞会导致细胞氧化还原状态和 LMW-PTP 巯基氧化的立即和可逆负移，最终导致细胞迁移。VEGF 引起 LMW-PTP 的可逆 S-谷胱甘肽化，抑制其磷酸化和活性，并导致 FAK 的瞬时激活。使用分解过氧亚硝酸盐（FeTPPS，2.5µM）或 GSH 前体[N-乙酰半胱氨酸（NAC），1mM]调节氧化还原状态会导致氧化还原状态的正移，并防止 VEGF 介导的 LMW-PTP 的 S-谷胱甘肽化和氧化抑制。NAC 和 FeTPPS 阻止了 FAK 的激活、它与 LMW-PTP 的结合和细胞迁移。抑制 LMW-PTP 的表达显著增强了 FAK 的激活和细胞迁移。虽然通过将 VEGF 与 0.1-0.2mM 过氧亚硝酸盐结合来实现轻度氧化应激会增强细胞迁移，但通过将 VEGF 与 0.5mM 过氧亚硝酸盐结合来实现急性氧化应激会诱导并维持 FAK 的激活和 LMW-PTP 的 S-谷胱甘肽化，从而导致 LMW-PTP 失活和抑制细胞迁移。总之，我们的发现表明，平衡的氧化还原状态是 VEGF 促进 LMW-PTP 的可逆 S-谷胱甘肽化、FAK 激活和内皮细胞迁移所必需的。将氧化还原状态转移到还原性应激或氧化性应激会抑制 VEGF 介导的血管生成反应。
