Ushio-Fukai Masuko, Tang Yan, Fukai Tohru, Dikalov Sergey I, Ma Yuxian, Fujimoto Mitsuaki, Quinn Mark T, Pagano Patrick J, Johnson Chad, Alexander R Wayne
Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Ga 30322, USA.
Circ Res. 2002 Dec 13;91(12):1160-7. doi: 10.1161/01.res.0000046227.65158.f8.
Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell proliferation and migration, primarily through the receptor tyrosine kinase VEGF receptor2 (Flk1/KDR). Reactive oxygen species (ROS) derived from NAD(P)H oxidase are critically important in many aspects of vascular cell regulation, and both the small GTPase Rac1 and gp91(phox) are critical components of the endothelial NAD(P)H oxidase complex. A role of NAD(P)H oxidase in VEGF-induced angiogenesis, however, has not been defined. In the present study, electron spin resonance spectroscopy is utilized to demonstrate that VEGF stimulates O2*- production, which is inhibited by the NAD(P)H oxidase inhibitor, diphenylene iodonium, as well as by overexpression of dominant-negative Rac1 (N17Rac1) and transfection of gp91(phox) antisense oligonucleotides in human umbilical vein endothelial cells (ECs). Antioxidants, including N-acetylcysteine (NAC), various NAD(P)H oxidase inhibitors, and N17Rac1 significantly attenuate not only VEGF-induced KDR tyrosine phosphorylation but also proliferation and migration of ECs. Importantly, these effects of VEGF are dramatically inhibited in cells transfected with gp91(phox) antisense oligonucleotides. By contrast, ROS are not involved in mediating these effects of sphingosine 1-phosphate (S1P) on ECs. Sponge implant assays demonstrate that VEGF-, but not S1P-, induced angiogenesis is significantly reduced in wild-type mice treated with NAC and in gp91(phox-/-) mice, suggesting that ROS derived from gp91(phox)-containing NAD(P)H oxidase play an important role in angiogenesis in vivo. These studies indicate that VEGF-induced endothelial cell signaling and angiogenesis is tightly controlled by the reduction/oxidation environment at the level of VEGF receptor and provide novel insights into the NAD(P)H oxidase as a potential therapeutic target for angiogenesis-dependent diseases.
血管内皮生长因子（VEGF）主要通过受体酪氨酸激酶VEGF受体2（Flk1/KDR）刺激内皮细胞增殖和迁移来诱导血管生成。源自NAD（P）H氧化酶的活性氧（ROS）在血管细胞调节的许多方面都至关重要，小GTP酶Rac1和gp91（phox）都是内皮NAD（P）H氧化酶复合物的关键成分。然而，NAD（P）H氧化酶在VEGF诱导的血管生成中的作用尚未明确。在本研究中，利用电子自旋共振光谱证明VEGF刺激超氧阴离子（O2* - ）生成，该生成受到NAD（P）H氧化酶抑制剂二苯碘鎓、显性负性Rac1（N17Rac1）的过表达以及人脐静脉内皮细胞（ECs）中gp91（phox）反义寡核苷酸转染的抑制。抗氧化剂，包括N-乙酰半胱氨酸（NAC）、各种NAD（P）H氧化酶抑制剂和N17Rac1，不仅显著减弱VEGF诱导的KDR酪氨酸磷酸化，还减弱ECs的增殖和迁移。重要的是，VEGF的这些作用在用gp91（phox）反义寡核苷酸转染的细胞中受到显著抑制。相比之下，ROS不参与介导1-磷酸鞘氨醇（S1P）对ECs的这些作用。海绵植入试验表明，在用NAC处理的野生型小鼠和gp91（phox - / - ）小鼠中，VEGF诱导的血管生成显著减少，而S1P诱导的血管生成未减少，这表明源自含gp91（phox）的NAD（P）H氧化酶的ROS在体内血管生成中起重要作用。这些研究表明，VEGF诱导内皮细胞信号传导和血管生成受到VEGF受体水平的还原/氧化环境的严格控制，并为NAD（P）H氧化酶作为血管生成依赖性疾病的潜在治疗靶点提供了新的见解。
