Milovanova Tatyana, Manevich Yefim, Haddad Alex, Chatterjee Shampa, Moore Jonni S, Fisher Aron B
Institute For Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104-6068, USA.
Antioxid Redox Signal. 2004 Apr;6(2):245-58. doi: 10.1089/152308604322899314.
We have shown previously that flow-adapted endothelial cells respond to cessation of flow with cell membrane depolarization and increased production of reactive oxygen species, resulting in activation of transcription factors and increased DNA synthesis. This study utilized flow cytometry to evaluate cellular proliferation with ischemia and to determine the role of reactive oxygen species and apoptosis. PKH26-labeled rat pulmonary microvascular endothelial cells were seeded in an artificial capillary system and subjected to flow at 5 dynes/cm(2) for 96 h or for 72 h followed by 24 h of simulated "ischemia." Ischemia resulted in a 2.5-fold increase in the cellular proliferation index. Cell-cycle analysis showed G0/G1 arrest and decreased S plus G2/M during flow adaptation, whereas ischemia resulted in a three-fold increase of cells in S plus G2/M phases. Apoptotic cells as detected by TUNEL and annexin V binding assays were ~5% of total cells with no differences between static, flow-adapted, and "ischemic" groups. Reactive oxygen species production during a 1-h period following onset of ischemia was confirmed by oxidation of the fluorophore, dichlorofluorescein, and was inhibited by cromakalim, a K(ATP) channel agonist, or diphenyleneiodonium, a flavoprotein inhibitor. Cromakalim and diphenyleneiodonium also markedly inhibited cell proliferation in the flow-adapted ischemic cells, but had no effect on subconfluent cells cultured under static conditions. These results indicate reactive oxygen species-dependent endothelial cell proliferation in flow-adapted microvascular endothelial cells as a response to ischemia and indicate that this response is not a consequence of apoptosis.
我们之前已经表明，适应血流的内皮细胞会对血流停止作出反应，表现为细胞膜去极化和活性氧生成增加，从而导致转录因子激活和DNA合成增加。本研究利用流式细胞术评估缺血时的细胞增殖情况，并确定活性氧和细胞凋亡的作用。将PKH26标记的大鼠肺微血管内皮细胞接种到人工毛细血管系统中，使其在5达因/平方厘米的血流条件下流动96小时，或先流动72小时，然后进行24小时的模拟“缺血”。缺血导致细胞增殖指数增加2.5倍。细胞周期分析显示，在血流适应过程中细胞停滞于G0/G1期，S期和G2/M期细胞减少，而缺血导致S期和G2/M期细胞增加了三倍。通过TUNEL和膜联蛋白V结合试验检测到的凋亡细胞约占总细胞的5%，静态组、血流适应组和“缺血”组之间无差异。缺血开始后1小时内活性氧的生成通过荧光团二氯荧光素的氧化得到证实，并被K(ATP)通道激动剂克罗卡林或黄素蛋白抑制剂二苯碘鎓抑制。克罗卡林和二苯碘鎓也显著抑制了血流适应的缺血细胞中的细胞增殖，但对静态培养的亚汇合细胞没有影响。这些结果表明，在适应血流的微血管内皮细胞中，活性氧依赖性的内皮细胞增殖是对缺血的一种反应，并且表明这种反应不是细胞凋亡的结果。
