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

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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