Yue T L, Wang X, Louden C S, Gupta S, Pillarisetti K, Gu J L, Hart T K, Lysko P G, Feuerstein G Z
Department of Cardiovascular Phamacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
Mol Pharmacol. 1997 Jun;51(6):951-62. doi: 10.1124/mol.51.6.951.
2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol-17beta and the oral contraceptive agent 17-ethylestradiol. 2-ME was recently reported to inhibit endothelial cell proliferation. The current study was undertaken to explore the mechanism of 2-ME effects on endothelial cells, especially whether 2-ME induces apoptosis, a prime mechanism in tissue remodeling and angiogenesis. Cultured bovine pulmonary artery endothelial cells (BPAEC) exposed to 2-ME showed morphological (including ultrastructural) features characteristic of apoptosis: cell shrinkage, cytoplasmic and nuclear condensation, and cell blebbing. 2-ME-induced apoptosis in BPAEC was a time- and concentration-dependent process (EC50 = 0.45 +/- 0.09 microM, n = 8). Nucleosomal DNA fragmentation in BPAEC treated with 2-ME was identified by agarose gel electrophoresis (DNA ladder) as well as in situ nick end labeling. Under the same experimental conditions, estradiol-17beta and two of its other metabolites, estriol and 2-methoxyestriol (< or =10 microM), did not have an apoptotic effect on BPAEC. 2-ME activated stress-activated protein kinase (SAPK)/c-Jun amino-terminal protein kinase in BPAEC in a concentration-dependent manner. The activity of SAPK was increased by 170 +/- 27% and 314 +/- 22% over the basal level in the presence of 0.4 and 2 microM 2-ME (n = 3-6), respectively. The activation of SAPK was detected at 10 min, peaked at 20 min, and returned to basal levels at 60 min after exposure to 2-ME. Inhibition of SAPK/c-Jun amino-terminal protein kinase activation by basic fibroblast growth factor, insulin-like growth factor, or forskolin reduced 2-ME-induced apoptosis. Immunohistochemical analysis of BPAEC indicated that 2-ME up-regulated expression of both Fas and Bcl-2. In addition, 2-ME inhibited BPAEC migration (IC50 = 0.71 +/- 0.11 microM, n = 4) and basic fibroblast growth factor-induced angiogenesis in the chick chorioallantoic membrane model. Taken together, these results suggest that promotion of endothelial cell apoptosis, thereby inhibiting endothelial cell proliferation and migration, may be a major mechanism by which 2-ME inhibits angiogenesis.
2-甲氧基雌二醇（2-ME）是雌二醇-17β和口服避孕药17-乙基雌二醇的内源性代谢产物。最近有报道称2-ME可抑制内皮细胞增殖。本研究旨在探讨2-ME对内皮细胞作用的机制，尤其是2-ME是否诱导凋亡，这是组织重塑和血管生成中的一个主要机制。暴露于2-ME的培养牛肺动脉内皮细胞（BPAEC）呈现出凋亡的形态学（包括超微结构）特征：细胞皱缩、细胞质和细胞核浓缩以及细胞出泡。2-ME诱导BPAEC凋亡是一个时间和浓度依赖性过程（半数有效浓度[EC50]=0.45±0.09微摩尔，n=8）。通过琼脂糖凝胶电泳（DNA梯带）以及原位缺口末端标记鉴定了用2-ME处理的BPAEC中的核小体DNA片段化。在相同实验条件下，雌二醇-17β及其另外两种代谢产物雌三醇和2-甲氧基雌三醇（≤10微摩尔）对BPAEC没有凋亡作用。2-ME以浓度依赖性方式激活BPAEC中的应激激活蛋白激酶（SAPK）/c-Jun氨基末端蛋白激酶。在存在0.4和2微摩尔2-ME的情况下，SAPK的活性分别比基础水平增加了170±27%和314±22%（n=3-6）。在暴露于2-ME后10分钟检测到SAPK的激活，在20分钟达到峰值，并在60分钟恢复到基础水平。碱性成纤维细胞生长因子、胰岛素样生长因子或福斯可林对SAPK/c-Jun氨基末端蛋白激酶激活的抑制减少了2-ME诱导的凋亡。对BPAEC的免疫组织化学分析表明，2-ME上调了Fas和Bcl-2的表达。此外，2-ME抑制BPAEC迁移（IC50=0.71±0.11微摩尔，n=4）以及在鸡胚绒毛尿囊膜模型中碱性成纤维细胞生长因子诱导的血管生成。综上所述，这些结果表明促进内皮细胞凋亡，从而抑制内皮细胞增殖和迁移，可能是2-ME抑制血管生成的主要机制。
