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，如冠状动脉成形术后发生的动脉粥样硬化或再狭窄。血管平滑肌在增殖过程中从收缩状态(成年型)转变为合成状态(胚胎型)。在这个项目中，我们从收缩蛋白基因表达的角度研究了在动物模型和人类中，平滑肌肉的表型改变何时发生以及如何发生，并参与了血管病变的形成。我们首先分离了三种类型的平滑肌肌球蛋白重链(MHC)亚型(SM1、SM2和SMemb)的cDNA克隆，并建立了敏感的免疫组织学。在球囊损伤的兔主动脉中，损伤后2~3周可见新生内膜细胞由具有胚胎型表型的平滑肌组成，但在4周内仍能向成体表型分化。在人的冠状动脉中，Inti…更多的基质增厚开始于第一个十年，到第四个十年可能达到介质厚度的3-5倍。人冠状动脉内膜-中层交界区SM2和SM1的表型改变是SM2和SM1的消失，随后是巨噬细胞的聚集，这表明人类冠状动脉粥样硬化的发展方式与动物模型完全不同。然而，在冠状动脉再狭窄的病变中，我们发现具有胚胎表型的平滑肌细胞增殖旺盛。这些结果表明，用抗平滑肌MHC亚型的免疫组织化学方法研究动脉粥样硬化和动脉硬化的过程是非常有用的。本研究还对SMemb和SM1/2启动子区域进行了研究。SMemb基因的5‘-启动子区域没有TATA盒。然而，-100bp左右的一个新的顺式元件似乎在该基因的激活中发挥了关键作用。另一方面，SM1基因具有一个TATA盒，以及几个参与肌肉发育和分化的顺式元件。最后，我们尝试用小鼠胚胎癌细胞系P19在体外诱导平滑肌细胞分化。我们获得的证据表明，P19可以被维甲酸诱导为平滑肌细胞，因为SM1 MHC在这种方案下是阳性的。较少

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