Analysis of novel proteins produced by vascular tissues and their clinical application

血管组织产生的新型蛋白质分析及其临床应用

• 批准号: 11557052
• 负责人: SASAYAMA Shigetake
• 金额: $ 8.19万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557052/
• 关键词: atherosclerosis; PTCA; restenosis; vascular endothelial cell; vascular smooth muscle cell; neural crest cell; embryonic development; integrin; リモデリング; 細胞増殖因子; 冠動脈形成術

We cloned a novel secreted protein, DANCE, from mouse embryonic hearts by the signal sequence-trap method. Its human and rat counterparts were also identified. It has shown to be a secreted protein with six calcium-binding EGF domains and one RGD sequence through which it binds to integrins. Its chromosomal localization was determined to be on human chromosome 14q32.1 by FISH method. There is to date no report of inherited diseases in this locus. We proceeded to analyze the expression profiles of DANCE both in normal and diseased animals. On the development of mouse, its expression is first identified on migrating nerual crest cells, their derivative branchial arch mesenchymal cells and the pericardium at embronic day 9.5. Atday 12.5-dpc, it is expressed in the cardiac outflow tract and aorta (both in endothelial cells and in smooth muscle cells), endocardial cushion tissues, head mesenchyme, intersomitic tissues and several other mesenchymal tissues. In 14.5-dpc embryos, some neural c … More rest-derived tissues such as head mesenchyme, cardiac outflow tract, and symphathetic ganglia continue to express DANCE, whereas other neural crest tissues such as adrenal gland do not. In adult aorta, DANCE expression is largely diminished. However, intense focal expression is found at intercostal branching points in the thoracic aorta. This coinsides with atherogenic region where alternation of hemodynamic stress at branching regions is implicated on the induction of atherogenesis. Accordingly, we studied DANCE expression in atherosclerotic vessels using LDL receptor-deficient mice fed with a high cholesterol diet. Endothelial cells overlying the plaques exhibited a significant increase in DANCE mRNA expression compared with normal regions of the same vessel. We could also find that DANCE mRNA expression is markedly increased in arteries following balloon injury with the highest levels seen at 14 days, coinciding with decreasing smooth muscle cell replication. By in situ hybridization of injured vessels, DANCE mRNA is observed in both endothelial cells and smooth muscle cells, with their maximal expression found at the spatial and temporal condition of returning from cell-proliferation to quiescence, suggesting that DANCE may affect cell growth as a "brake" in autocrine or paracrine manner when proliferation should stop. Thus DANCE, as a secreted protein of systemic applicable form, harbors a potential of clinical application like the prevention of restenosis after PTCA.We have succeeded in generating knockout mice of DANCE and found the abnormal phenotype, the elucidation of whose mechanism is one of the present targets of our study. Less

我们用信号序列捕获法从小鼠胚胎心脏中克隆了一个新的分泌蛋白DANCE。它的人类和大鼠对应物也被确定。它已被证明是一种分泌蛋白，具有6个钙结合EGF结构域和一个RGD序列，通过该序列它与整联蛋白结合。经FISH检测其染色体定位于人染色体14q32.1。迄今为止，没有关于该地区遗传性疾病的报告。我们继续分析DANCE在正常和患病动物中的表达谱。在小鼠的发育过程中，其表达首先在胚胎9.5天的迁移神经嵴细胞、其衍生的鳃弓间充质细胞和心包上被鉴定。在12.5-dpc时，它在心脏流出道和主动脉（在内皮细胞和平滑肌细胞中）、内膜垫组织、头部间充质、体间组织和一些其它间充质组织中表达。在14.5-dpc胚胎中，一些神经c ...更多信息 静息来源的组织如头部间充质、心脏流出道和交感神经节继续表达DANCE，而其它神经嵴组织如肾上腺则不表达。在成人主动脉中，DANCE表达大大减少。然而，在胸主动脉的肋间分支点发现了强烈的局灶性表达。这与动脉粥样硬化形成区域相一致，其中分支区域的血流动力学应力的改变与动脉粥样硬化形成的诱导有关。因此，我们研究了DANCE的表达在动脉粥样硬化血管使用LDL受体缺陷小鼠喂养高胆固醇饮食。与同一血管的正常区域相比，斑块上的内皮细胞表现出DANCE mRNA表达的显著增加。我们还发现，DANCE mRNA表达在球囊损伤后动脉中显著增加，在14天时观察到最高水平，与平滑肌细胞复制减少一致。损伤血管的原位杂交结果显示，DANCE mRNA在内皮细胞和平滑肌细胞中均有表达，且在细胞从增殖状态恢复到静止状态的时空条件下表达最高，提示DANCE可能以自分泌或旁分泌的方式影响细胞的生长。因此，DANCE作为一种分泌型的全身应用蛋白，具有预防PTCA术后再狭窄等临床应用的潜力。我们成功地建立了DANCE基因敲除小鼠，发现了DANCE基因的异常表型，阐明其机制是目前的研究目标之一。少

项目成果

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