Hsieh H J, Cheng C C, Wu S T, Chiu J J, Wung B S, Wang D L
Department of Chemical Engineering, National Taiwan University, Taipei, Republic of China.
J Cell Physiol. 1998 May;175(2):156-62. doi: 10.1002/(SICI)1097-4652(199805)175:2<156::AID-JCP5>3.0.CO;2-N.
Intracellular reactive oxygen species (ROS) may participate in cellular responses to various stimuli including hemodynamic forces and act as signal transduction messengers. Human umbilical vein endothelial cells (ECs) were subjected to laminar shear flow with shear stress of 15, 25, or 40 dynes/cm2 in a parallel plate flow chamber to demonstrate the potential role of ROS in shear-induced cellular response. The use of 2',7'-dichlorofluorescin diacetate (DCFH-DA) to measure ROS levels in ECs indicated that shear flow for 15 minutes resulted in a 0.5- to 1.5-fold increase in intracellular ROS. The levels remained elevated under shear flow conditions for 2 hours when compared to unsheared controls. The shear-induced elevation of ROS was blocked by either antioxidant N-acetyl-cysteine (NAC) or catalase. An iron chelator, deferoxamine mesylate, also significantly reduced the ROS elevation. A similar inhibitory effect was seen with a hydroxyl radical (.OH) scavenger, 1,3-dimethyl-2-thiourea (DMTU), suggesting that hydrogen peroxide (H202), .OH, and possibly other ROS molecules in ECs were modulated by shear flow. Concomitantly, a 1.3-fold increase of decomposition of exogenously added H2O2 was observed in extracts from ECs sheared for 60 minutes. This antioxidant activity, abolished by a catalase inhibitor (3-amino-1,2,4-triazole), was primarily due to the catalase. The effect of ROS on intracellular events was examined in c-fos gene expression which was previously shown to be shear inducible. Decreasing ROS levels by antioxidant (NAC or catalase) significantly reduced the induction of c-fos expression in sheared ECs. We demonstrate for the first time that shear force can modulate intracellular ROS levels and antioxidant activity in ECs. Furthermore, the ROS generation is involved in mediating shear-induced c-fos expression. Our study illustrates the importance of ROS in the response and adaptation of ECs to shear flow.
细胞内活性氧（ROS）可能参与细胞对包括血流动力学力在内的各种刺激的反应，并作为信号转导信使。在平行板流动腔中，对人脐静脉内皮细胞（ECs）施加剪切应力分别为15、25或40达因/平方厘米的层流切应力，以证明ROS在剪切诱导的细胞反应中的潜在作用。使用2',7'-二氯荧光素二乙酸酯（DCFH-DA）测量ECs中的ROS水平表明，15分钟的剪切流导致细胞内ROS增加0.5至1.5倍。与未剪切的对照相比，在剪切流条件下2小时内ROS水平保持升高。抗氧化剂N-乙酰半胱氨酸（NAC）或过氧化氢酶可阻断剪切诱导产生ROS的升高。铁螯合剂甲磺酸去铁胺也显著降低了ROS的升高。羟基自由基（·OH）清除剂1,3-二甲基-2-硫脲（DMTU）也有类似的抑制作用，表明ECs中的过氧化氢（H2O2）、·OH以及可能的其他ROS分子受到剪切流的调节。同时，在剪切60分钟的ECs提取物中观察到外源添加的H2O2分解增加了1.3倍。这种抗氧化活性被过氧化氢酶抑制剂（3-氨基-1,2,4-三唑）消除，主要是由于过氧化氢酶。在先前已证明可被剪切诱导的c-fos基因表达中检测了ROS对细胞内事件的影响。通过抗氧化剂（NAC或过氧化氢酶）降低ROS水平可显著降低剪切的ECs中c-fos表达的诱导。我们首次证明剪切力可调节ECs中的细胞内ROS水平和抗氧化活性。此外，ROS的产生参与介导剪切诱导的c-fos表达。我们的研究说明了ROS在ECs对剪切流的反应和适应中的重要性。
