Hsieh Y-C S, Hsieh S-J, Chang Y-S, Hsueh C-M, Hsu S-L
Graduate Institute of Chinese Pharmaceutical Sciences, China Medical University, Taichung, Taiwan, ROC.
Br J Pharmacol. 2007 Aug;151(8):1235-45. doi: 10.1038/sj.bjp.0707345. Epub 2007 Jun 25.
Endothelial cell proliferation, migration and adhesion are necessary for the formation of new blood vessels. We reported previously that baicalein strongly inhibited proliferation of rat heart endothelial cells and here we assess effects on migration and adhesion of these cells.
Effects of baicalein on endothelial migration and adhesion were determined by in vitro wound assays and in modified Boyden chambers. Protein expression and subcellular distribution in rat heart endothelial cells were analysed by immunoblots and immunofluorescence staining.
Pretreatment with baicalein for 48 h resulted in a concentration-dependent inhibition of endothelial migration, with an IC(50) of approximately 20 microM. Adhesion assays revealed that baicalein stimulated endothelial cell adhesion to fibronectin and vitronectin, effects blocked by the synthetic peptide Arg-Gly-Asp (RGD). Moreover, treatment with a blocking antibody against integrin alpha5beta1 drastically attenuated baicalein-mediated endothelial adhesion to fibronectin, but not to vitronectin. Furthermore, baicalein-mediated anti-migration effect and adhesion promotion could be partially reversed by the addition of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Western blot analysis indicated that baicalein increased expression levels of integrin-alpha5beta1, -alphavbeta3 and vinculin proteins. Immunofluorescence staining showed that baicalein induced a marked reorganization of actin stress fibres and the recruitment of vinculin and integrins to focal adhesion plaques, with consequently increased formation of focal adhesion contacts.
Baicalein markedly inhibited the migration and enhanced the adhesion of rat heart endothelial cells, possibly by up-regulation of the integrins (alpha5beta1 and alphavbeta3) and vinculin and by promotion of actin reorganization and focal adhesion contact formation.
内皮细胞的增殖、迁移和黏附是新血管形成所必需的。我们之前报道过黄芩苷能强烈抑制大鼠心脏内皮细胞的增殖,在此我们评估其对这些细胞迁移和黏附的影响。
通过体外伤口试验和改良的博伊登小室测定黄芩苷对内皮细胞迁移和黏附的影响。采用免疫印迹和免疫荧光染色分析大鼠心脏内皮细胞中的蛋白表达和亚细胞分布。
黄芩苷预处理48小时导致内皮细胞迁移受到浓度依赖性抑制,半数抑制浓度(IC50)约为μM。黏附试验显示,黄芩苷刺激内皮细胞黏附于纤连蛋白和玻连蛋白,这些作用可被合成肽精氨酸 - 甘氨酸 - 天冬氨酸(RGD)阻断。此外,用抗整合素α5β1的阻断抗体处理可显著减弱黄芩苷介导的内皮细胞对纤连蛋白的黏附,但对玻连蛋白的黏附无影响。此外,添加12(S)-羟基二十碳四烯酸(12(S)-HETE)可部分逆转黄芩苷介导的抗迁移作用和黏附促进作用。蛋白质印迹分析表明,黄芩苷增加了整合素α5β1、αvβ3和纽蛋白的表达水平。免疫荧光染色显示,黄芩苷诱导肌动蛋白应力纤维明显重排,并使纽蛋白和整合素募集到黏着斑,从而增加黏着斑接触的形成。
黄芩苷显著抑制大鼠心脏内皮细胞的迁移并增强其黏附,可能是通过上调整合素(α5β1和αvβ3)和纽蛋白,以及促进肌动蛋白重排和黏着斑接触形成来实现的。