Naito Yuji, Shimozawa Makoto, Manabe Hikori, Nakabe Nami, Katada Kazuhiro, Kokura Satoshi, Yoshida Norimasa, Ichikawa Hiroshi, Kon Tatsuya, Yoshikawa Toshikazu
Department of Medical Proteomics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kyoto 602-8566, Japan.
Eur J Pharmacol. 2006 Sep 28;546(1-3):11-8. doi: 10.1016/j.ejphar.2006.07.030. Epub 2006 Jul 25.
Oxidized low-density lipoprotein (ox-LDL) plays an important in the development of atherosclerosis by stimulating the production of reactive oxygen species in endothelial cells, and thereby up-regulating vascular cell adhesion molecule-1 (VCAM-1). The objectives of the present study were to determine the effects of azelnidipine, a new calcium channel blocker, on the expression of VCAM-1 induced by 7-ketocholesterol, components of ox-LDL, and tumor necrosis factor-alpha (TNF-alpha). The scavenging activities of azelnidipine against superoxide, hydroxyl, and carbon-centered radicals were determined by electron spin resonance assay. The levels of intracellular reactive oxygen species were determined fluorometrically with the use of dichlorodihydrofluorescein diacetate (H(2)DCF-DA). Human aortic endothelial cells and U937 were used as endothelial cells and monocytic cells, respectively. The surface expression and mRNA levels of VCAM-1 were determined by enzyme immunoassay and RT-PCR performed on endothelial cell monolayers stimulated with 7-ketocholesterol or TNF-alpha. The numbers of monocytic cells adhering on the stimulated endothelial cells were counted in the microscopic fields. Translocation of p65 protein to the nucleus was estimated by fluorescence microscopy. Azelnidipine, but not nifedipine, reduced the signal intensity of 1,1-diphenyl-2-picrylhydrazyl radicals. Azelnidipine scavenged hydroxyl radicals, but not superoxide radicals. Intracellular levels of reactive oxygen species and RelA (p65) nuclear translocation in stimulated endothelial cells were reduced by azelnidipine. Azelnidipine significantly inhibited the expression of protein and mRNA of VCAM-1, and prevented the U937 cell adhesion to endothelial cells treated with 7-ketocholesterol or TNF-alpha. These results suggest that azelnidipine works as an anti-atherogenic agent by inhibiting the reactive oxygen species-dependent expression of VCAM-1 induced by 7-ketocholesterol and TNF-alpha.
氧化型低密度脂蛋白（ox-LDL）通过刺激内皮细胞中活性氧的产生，进而上调血管细胞黏附分子-1（VCAM-1），在动脉粥样硬化的发展过程中起重要作用。本研究的目的是确定新型钙通道阻滞剂阿折地平对7-酮胆固醇、ox-LDL成分和肿瘤坏死因子-α（TNF-α）诱导的VCAM-1表达的影响。通过电子自旋共振分析法测定阿折地平对超氧阴离子、羟基和碳中心自由基的清除活性。使用二氯二氢荧光素二乙酸酯（H(2)DCF-DA）通过荧光法测定细胞内活性氧水平。分别用人主动脉内皮细胞和U937作为内皮细胞和单核细胞。通过酶免疫测定法和对用7-酮胆固醇或TNF-α刺激的内皮细胞单层进行RT-PCR来测定VCAM-1的表面表达和mRNA水平。在显微镜视野中计数黏附在受刺激内皮细胞上的单核细胞数量。通过荧光显微镜估计p65蛋白向细胞核的转位。阿折地平而非硝苯地平降低了1,1-二苯基-2-苦基肼自由基的信号强度。阿折地平清除羟基自由基，但不清除超氧阴离子自由基。阿折地平降低了受刺激内皮细胞中细胞内活性氧水平和RelA（p65）核转位。阿折地平显著抑制VCAM-1的蛋白质和mRNA表达，并阻止U937细胞黏附于用7-酮胆固醇或TNF-α处理的内皮细胞。这些结果表明，阿折地平通过抑制7-酮胆固醇和TNF-α诱导的依赖活性氧的VCAM-1表达而发挥抗动脉粥样硬化作用。
