Medizinische Klinik und Poliklinik II, Universitätsklinikum Bonn, Sigmund-Freud-Strasse 25, Bonn, Germany.
Basic Res Cardiol. 2011 Jun;106(4):563-75. doi: 10.1007/s00395-011-0174-z. Epub 2011 Apr 12.
The mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) and the zinc finger transcription factor Kruppel-like factor-4 (KLF4) are involved in the regulation of redox homeostasis, apoptosis and cell proliferation. We have shown that estrogen exerts antioxidative actions via induction of MnSOD in cultured rat aortic vascular smooth muscle cells (VSMC). The purpose of the present study was to investigate whether estrogen inhibits VSMC proliferation via alteration of KLF4 and MnSOD expression. In cultured rat aortic VSMC, estrogen binding to estrogen receptor-alpha led to rapid increase in KLF4 expression and reduction of cell proliferation by 50%. Protein separation revealed that KLF4 was shifted to the nucleus when VSMC were treated with estrogen. Estrogen-mediated induction of KLF4 and the antiproliferative effect involved activation of PI-3 kinase, Akt phosphorylation and induction of NO synthase activity. Experiments in freshly isolated denuded aortic segments revealed an increase in KLF4 abundance after estrogen treatment and demonstrated that eNOS is expressed in the media at low levels. Transfection experiments showed that estrogen-induced overexpression of MnSOD required KLF4 and that both KLF4 and MnSOD were indispensable for the observed antiproliferative effect of estrogen in VSMC. To confirm these data in vivo, we investigated neointima formation after carotid artery injury in wild-type (WT) and MnSOD+/- mice. Estrogen deficiency led to enhanced neointima formation and higher numbers of Ki67-positive proliferating cells in the neointima of ovariectomized WT and MnSOD+/- mice. Moreover, MnSOD+/- mice showed more extensive neointima formation and Ki67 immunostaining. Interestingly, estrogen replacement prevented neointima formation in WT mice but failed to completely inhibit neointima formation in MnSOD+/- mice. Cultured VSMC derived from MnSOD+/- mice showed enhanced proliferation as compared to WT VSMC, and estrogen treatment failed to inhibit proliferation in MnSOD+/- VSMC. In conclusion, these data demonstrate the importance of MnSOD and KLF4 for proliferation control in VSMC. Our results provide novel insights into how proliferation of VSMC is regulated by estrogen and may help to identify novel targets for the treatment of vascular diseases such as restenosis.
线粒体抗氧化酶锰超氧化物歧化酶（MnSOD）和锌指转录因子 Krüppel 样因子-4（KLF4）参与调节氧化还原平衡、细胞凋亡和细胞增殖。我们已经表明，雌激素通过诱导培养的大鼠主动脉血管平滑肌细胞（VSMC）中的 MnSOD 发挥抗氧化作用。本研究的目的是研究雌激素是否通过改变 KLF4 和 MnSOD 的表达来抑制 VSMC 的增殖。在培养的大鼠主动脉 VSMC 中，雌激素与雌激素受体-α结合导致 KLF4 表达迅速增加，细胞增殖减少 50%。蛋白质分离表明，当 VSMC 用雌激素处理时，KLF4 转移到核内。雌激素介导的 KLF4 诱导和抗增殖作用涉及 PI-3 激酶的激活、Akt 磷酸化和诱导一氧化氮合酶活性。在新鲜分离的无被主动脉段进行的实验表明，雌激素处理后 KLF4 丰度增加，并表明 eNOS 在低水平表达于中膜。转染实验表明，雌激素诱导的 MnSOD 过表达需要 KLF4，并且 KLF4 和 MnSOD 对于雌激素在 VSMC 中观察到的抗增殖作用都是必不可少的。为了在体内证实这些数据，我们研究了颈动脉损伤后野生型（WT）和 MnSOD+/- 小鼠的新生内膜形成。雌激素缺乏导致去卵巢 WT 和 MnSOD+/- 小鼠新生内膜中新生内膜形成增强和 Ki67 阳性增殖细胞增多。此外，MnSOD+/- 小鼠显示出更广泛的新生内膜形成和 Ki67 免疫染色。有趣的是，雌激素替代预防了 WT 小鼠的新生内膜形成，但未能完全抑制 MnSOD+/- 小鼠的新生内膜形成。与 WT VSMC 相比，源自 MnSOD+/- 小鼠的培养 VSMC 显示出增强的增殖，并且雌激素处理未能抑制 MnSOD+/- VSMC 的增殖。总之，这些数据表明 MnSOD 和 KLF4 对 VSMC 增殖的控制很重要。我们的研究结果为雌激素调节 VSMC 增殖的机制提供了新的见解，并可能有助于确定血管疾病（如再狭窄）治疗的新靶点。
