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

我们利用信号序列陷阱法从小鼠胚胎心脏中克隆了一种新的分泌蛋白DANCE。人类和大鼠的对应体也被鉴定出来。它是一种分泌蛋白，具有六个钙结合EGF结构域和一个RGD序列，通过它与整合素结合。通过FISH方法确定其染色体定位在人类染色体14q32.1上。到目前为止，尚无关于该位点遗传疾病的报告。我们进一步分析了DANCE在正常和患病动物中的表达谱。在小鼠发育过程中，首次在胚胎9.5天迁移的神经嵴细胞、其衍生物鳃弓间充质细胞和心包膜中发现其表达。在12.5天，它在心脏流出道和主动脉（内皮细胞和平滑肌细胞）、心内膜缓冲组织、头间质、间质组织和其他一些间质组织中表达。在14.5 dpc胚胎中，一些神经神经源性组织如头间质、心流出道和交感神经节继续表达DANCE，而其他神经嵴组织如肾上腺则不表达DANCE。在成人主动脉中，DANCE的表达明显减少。然而，在胸主动脉肋间分支点发现强烈的局灶性表达。这与动脉粥样硬化区域相吻合，分支区域血流动力学应力的变化与动脉粥样硬化的诱导有关。因此，我们用喂食高胆固醇饮食的低密度脂蛋白受体缺陷小鼠研究了DANCE在动脉粥样硬化血管中的表达。斑块上的内皮细胞与同一血管的正常区域相比，DANCE mRNA表达显著增加。我们还发现，球囊损伤后，DANCE mRNA在动脉中的表达显著增加，在第14天达到最高水平，与平滑肌细胞复制减少相一致。通过对损伤血管的原位杂交，内皮细胞和平滑肌细胞中均观察到DANCE mRNA的表达，在细胞从增殖状态恢复到静止状态的时空状态下，DANCE mRNA的表达量达到最大值，提示当细胞增殖停止时，DANCE可能以自分泌或旁分泌的方式影响细胞生长。因此DANCE作为一种全身适用形式的分泌蛋白，具有预防PTCA术后再狭窄等临床应用潜力。我们成功培育了DANCE基因敲除小鼠，并发现了异常表型，阐明其机制是我们目前研究的目标之一。少

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