Analysis of abnormal differentiation of smooth muscles in arterial lesions and an in vitro trial to induce smooth muscle cell differentiation

增殖和去分化都涉及动脉病变的形成，如动脉粥样硬化或冠状动脉成形术后发生的再狭窄。血管平滑肌在增殖过程中从收缩（成人型）到合成状态（胚胎型）改变其表型。在这个项目中，我们研究了何时以及如何平滑肌的表型调制发生，并参与血管病变的形成，在动物模型和人类从收缩蛋白的基因表达的观点。我们首先分离了三种类型的平滑肌肌球蛋白重链（MHC）亚型（SM 1，SM 2和SMemb）的cDNA克隆，并开发了敏感的免疫组织学。在球囊损伤的兔髂动脉中，发现新生内膜细胞在损伤后2-3周由具有胚胎表型的平滑肌组成，尽管它们在4周内保持再分化为成人表型的能力。在人类冠状动脉中， 关于我们 在第一个十年开始异常增厚，到第四个十年可能达到介质的3-5倍厚。人冠状动脉中平滑肌的表型调节是内膜-中膜边缘区域的SM 2和SM 1消失，随后是巨噬细胞的积累，因此表明人冠状动脉粥样硬化以与动物模型中发现的完全不同的方式发展。然而，在冠状动脉再狭窄病变中，我们发现具有胚胎表型的平滑肌细胞剧烈增殖。这些结果表明，使用抗平滑肌MHC异构体的免疫组织化学是非常有用的研究动脉粥样硬化和动脉硬化的过程中。SMemb基因的5 '启动子区没有TATA盒。然而，一个新的顺式元件在约100 bp的似乎在该基因的激活中起关键作用。而SM 1基因则含有TATA盒和多个与肌肉发育和分化有关的顺式元件。最后，我们尝试用P19小鼠胚胎癌细胞系在体外诱导平滑肌细胞分化。实验结果表明，在维甲酸的诱导下，P19可以被诱导为平滑肌细胞，因为在该方案下，SM 1 MHC呈阳性。少

Both proliferation and dedifferentiation involve the formation of arterial lesions, like arterio- and atherosclerosis or restenosis occurring following coronary angioplasy. Vascular smooth muscles change their phenotype from the contractile (adult type) to the synthetic state (embryonic type) during proliferation. In this project we examined when and how the phenotypic modulation of smooth muscles occurs and participates in the formation of vascular lesions in both animal models and human from the standpoint of gene expression of contractile proteins. We first isolated cDNA clones for three types of smooth muscle myosin heavy chain (MHC) isoforms (SM1, SM2 and SMemb) and developed sensitive immunohistology. In the ballooning-injured rabbit aortas, neointimal cells were found to be composed of smooth muscles with the embryonic phentotype at 2-3 weeks after injury, although they maintained the ability to re-differentiate to the adult phenotype in 4 weeks. In human coronary arteries, inti … More mal thickening started in the first decade and could reach 3-5 times thicker than the media by the fourth decades. The phenotypic modulation of smooth muscles in human coronary arteries was disappearance of SM2 and SM1 in the intima-media border regions, followed by accumulation of macrophages, thus indicating that human coronary atherosclerosis develops in a completely different fashion from those found in animal models. However, in the restenotic lesions of coronary arteries, we found smooth muscle cells with the embryonic phenotype vigorously proliferated. These results indicate that immunohistochemistry using anti-smooth muscle MHC isoforms is highly useful to study the process of athero- and arteriosclerosis.The promoter regions of SMemb and SM1/2 were also characterized in this study. The 5'-promoter region of the SMemb gene does not have a TATA box. However, a novel cis-element at around -100 bp seemed to play a key role in the activation of this gene. SM1 gene, on the other hand, has a TATA box as well as several cis-elements involved in muscle development and differentiation.Finally we tried to induce smooth muscle cell differentiation in vitro using a P19 mouse embryonic carcinoma cell line. We obtained evidence showing that P19 could be induced to smooth muscle cell with retinoic acid because SM1 MHC were positive under this regimen. Less