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Exploration of blood flow-sensing molecules in vascular endothelial cells

血管内皮细胞血流传感分子的探索

基本信息

批准号：
13308052
负责人：
ANDO Joji
金额：
$ 26.54万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13308052/
关键词：
endothelial cells intracellular signal transduction calcium signaling purinoceptor transcription factor Sp1 calcium ion channel fluid shear stress blood vessel 細胞内情報伝イオンチャンネル ATP 細胞内情報伝達

项目摘要

Our previous study demonstrated that a subtype of P2X purinoceptor, P2X4, is predominantly expressed in human vascular ECs and plays a central role in blood flow-related Ca^<2+> signaling. In the current study, we investigated the effect of shear stress on P2X4 receptor expression in ECs. When Cultured human umbilical vein ECs (HUVECs) were exposed to physiological levels of laminar shear stress in a flow-loading apparatus, the P2X4 mRNA levels began to decrease as early as 1 h after the onset of flow and decreased progressively with time, reaching around 60% of the static control at 24 h. The P2X4 protein levels also decreased significantly. Functional analysis of the 1.9-kb P2X4 5'promoter indicated that a region containing a consensus binding site for the Sp1 transcription factor between bp -67 and -58 was required for the shear stress responsiveness. Mutations of the Sp1 site abolished the response of the P2X4 promoter to shear stress. Electrophoretic mobility shift assays showed a marked decrease in binding of Sp1 to the Sp1 consensus site in shear stressed cells, suggesting that Sp1 mediates the shear stress-induced down regulation of P2X4 gene transcription. Superoxide dismutase (SOD) and a peroxide-scavenging enzyme catalase significantly inhibited the shear stress-induced decrease in the P2X4 mRNA levels, indicating that reactive oxygen species play a role, at least partially, in shear stress-mediated regulation of P2X4 gene expression in ECs. We also show that shear-induced changes in purinoceptor expression attenuate the sensitivity of ECs to ATP causing Ca^<2+> responses. Thus there may be a feedback system in our body, which controls the sensitivity of ECs to shear stress via blood flow-mediated attenuation of purinoceptor expression to maintain vasucular homeostasis.

我们以前的研究表明，P2X嘌呤受体的一个亚型，即P2X4，主要表达于人的血管内皮细胞，并在血流相关的钙信号转导中发挥中心作用。在本研究中，我们研究了切应力对内皮细胞中P2X4受体表达的影响。将培养的人脐静脉内皮细胞(HUVECs)置于生理水平的层流切应力作用下，其P2X4基因表达水平在血流开始后1h就开始下降，并随着时间的延长逐渐下降，24h时达到静态对照的60%左右，其蛋白表达水平也显著下降。对1.9kb的P2X4 5‘启动子的功能分析表明，剪切应力响应需要一个包含Sp1转录因子在BP-67和-58之间的共同结合位点的区域。Sp1位点的突变消除了P2X4启动子对剪切力的反应。凝胶迁移率改变分析显示，在剪切力作用下，Sp1与Sp1共同位点的结合显著减少，提示Sp1介导了剪切力诱导的P2X4基因转录下调。超氧化物歧化酶(SOD)和过氧化氢清除酶过氧化氢酶(CAT)能显著抑制切应力诱导的细胞内P2X4基因表达水平的降低，说明在切应力诱导的内皮细胞中，活性氧至少部分参与了对P2X4基因表达的调控。我们还表明，剪切诱导的嘌呤受体表达的变化减弱了内皮细胞对三磷酸腺苷的敏感性，导致钙离子反应。因此，在我们的体内可能有一个反馈系统，通过血流介导的嘌呤受体表达的减弱来控制内皮细胞对剪切力的敏感性，以维持血管的动态平衡。