Lu G, Greene E L, Nagai T, Egan B M
Department of Pharmacology, Medical University of South Carolina, Charleston, USA.
Hypertension. 1998 Dec;32(6):1003-10. doi: 10.1161/01.hyp.32.6.1003.
Obese hypertensive patients with cardiovascular risk factor clustering have increased plasma nonesterified fatty acid levels and are at high risk for atherosclerotic events. Our previous studies demonstrated that oleic acid induces a mitogenic response in rat aortic smooth muscle cells (RASMCs) through protein kinase C (PKC)- and extracellular signal-regulated kinase (ERK)-dependent pathways. In the present study we investigated the possibility that the generation of reactive oxygen species (ROS) constitutes a critical component of the oleic acid-induced mitogenic signaling pathway in RASMCs. We studied the effect(s) of oleic acid on the generation of ROS using the oxidant-sensitive fluoroprobe 2',7'-dichlorofluorescin diacetate. Relative fluorescence intensity and fluorescent images were obtained with laser confocal scanning microscopy from 1 to 5 minutes, since preliminary studies demonstrated that the peak fluorescence intensity occurred within 5 minutes. Oleic acid (100 micromol/L) induced a time-dependent increase of cell fluorescence that was >8-fold of that seen in control cells at 5 minutes. This was blocked by catalase, which suggests that H2O2 was the principal ROS. The oleic acid-induced increases in H2O2 were blocked when PKC was inhibited with the use of bisindolylmaleimide and when PKC activity was downregulated by exposing RASMCs to phorbol 12-myristate 13-acetate for 24 hours. Stearic and elaidic acids, which are weak PKC activators, did not significantly increase H2O2 production. The increase of H2O2 in response to oleic acid was inhibited by the antioxidant N-acetylcysteine. N-Acetylcysteine also completely blocked ERK activation and the increase of thymidine incorporation in response to oleic acid. The data suggest that generation of H2O2 in RASMCs exposed to oleic acid is PKC dependent. Moreover, H2O2 production emerges as a critical intermediary event in the oleic acid-mediated mitogenic signaling pathway between the activation of PKC and ERK. These observations raise the possibility that the elevated plasma nonesterified fatty acids, including oleic acid, in obese hypertensive patients contribute to vascular growth and remodeling by a PKC-dependent mechanism to generate ROS that subsequently activate ERK.
伴有心血管危险因素聚集的肥胖高血压患者血浆非酯化脂肪酸水平升高，发生动脉粥样硬化事件的风险很高。我们之前的研究表明，油酸通过蛋白激酶C（PKC）和细胞外信号调节激酶（ERK）依赖性途径诱导大鼠主动脉平滑肌细胞（RASMCs）发生促有丝分裂反应。在本研究中，我们探讨了活性氧（ROS）的生成是否是油酸诱导RASMCs促有丝分裂信号通路的关键组成部分。我们使用对氧化剂敏感的荧光探针2',7'-二氯荧光素二乙酸酯研究了油酸对ROS生成的影响。由于初步研究表明峰值荧光强度在5分钟内出现，因此在1至5分钟内用激光共聚焦扫描显微镜获得相对荧光强度和荧光图像。油酸（100微摩尔/升）诱导细胞荧光随时间增加，在5分钟时比对照细胞高8倍以上。这被过氧化氢酶阻断，这表明H2O2是主要的ROS。当使用双吲哚马来酰胺抑制PKC以及通过将RASMCs暴露于佛波醇12-肉豆蔻酸酯13-乙酸酯24小时下调PKC活性时，油酸诱导的H2O2增加被阻断。硬脂酸和反油酸是弱PKC激活剂，它们不会显著增加H2O2的产生。抗氧化剂N-乙酰半胱氨酸抑制了油酸诱导的H2O2增加。N-乙酰半胱氨酸还完全阻断了ERK激活以及油酸诱导的胸苷掺入增加。数据表明，暴露于油酸的RASMCs中H2O2的生成是PKC依赖性的。此外，H2O2的产生是油酸介导的促有丝分裂信号通路中PKC激活和ERK激活之间的关键中间事件。这些观察结果增加了一种可能性，即肥胖高血压患者血浆中升高的非酯化脂肪酸，包括油酸，通过PKC依赖性机制产生ROS，随后激活ERK，从而促进血管生长和重塑。
