Unterluggauer Hermann, Hampel Barbara, Zwerschke Werner, Jansen-Dürr Pidder
Abteilung Molekular- und Zellbiologie, Institut f. Biomedizinische Alternsforschung der Osterreichischen Akademie der Wissenschaften, Rennweg 10, A-6020, Innsbruck, Austria.
Exp Gerontol. 2003 Oct;38(10):1149-60. doi: 10.1016/j.exger.2003.08.007.
Replicative senescence of human endothelial cells was analyzed, using primary endothelial cells from the human umbilical vein endothelial cells (HUVEC) as an experimental model system. We had shown before that senescent HUVEC arrest in the G1 phase of the cell cycle and that a subpopulation of the senescent cells undergoes cell death. We now demonstrate that cell death occurs by apoptosis, characterized by activation of caspase 3. Using the redox-sensitive dye dihydrorhodamine 123, a significant accumulation of reactive oxygen species is detected in senescent but not young endothelial cells. To determine if increased oxidative stress may contribute to the senescent phenotype, cells were treated with tert-butyl hydroperoxide (tBHP), which is known to increase oxidative stress by decreasing the intracellular glutathione levels. We show here that mild tBHP stress induces a phenotype of premature senescence in a subpopulation of the treated cells, which closely resembles the phenotype of naturally senescent HUVEC, including growth arrest, senescence-associated beta-gal activity, and apoptotic cell death. These results establish a model of premature senescence for human endothelial cells, which will be suitable to analyze mechanisms of age-associated cell death.
利用人脐静脉内皮细胞（HUVEC）的原代内皮细胞作为实验模型系统，对人内皮细胞的复制性衰老进行了分析。我们之前已经表明，衰老的HUVEC停滞在细胞周期的G1期，并且衰老细胞的一个亚群会发生细胞死亡。我们现在证明细胞死亡是通过凋亡发生的，其特征是半胱天冬酶3的激活。使用对氧化还原敏感的染料二氢罗丹明123，在衰老的而非年轻的内皮细胞中检测到活性氧的显著积累。为了确定氧化应激增加是否可能导致衰老表型，用叔丁基过氧化氢（tBHP）处理细胞，已知tBHP通过降低细胞内谷胱甘肽水平来增加氧化应激。我们在此表明，轻度tBHP应激在处理过的细胞亚群中诱导出早衰表型，这与自然衰老的HUVEC的表型非常相似，包括生长停滞、衰老相关的β-半乳糖苷酶活性和凋亡细胞死亡。这些结果建立了人内皮细胞早衰模型，该模型将适用于分析与年龄相关的细胞死亡机制。
