Nakajima Y, Krug E L, Markwald R R
Department of Cellular Biology and Anatomy, Medical College of Wisconsin, Milwaukee 53226.
Dev Biol. 1994 Oct;165(2):615-26. doi: 10.1006/dbio.1994.1280.
We have demonstrated previously that the epithelial-mesenchymal transformation of cardiac endothelium in early chick heart development is induced by EDTA-soluble (ES) extracellular molecules synthesized by the myocardium of specific regions, i.e., the outflow tract (OT) and atrioventricular (AV) canal. Polyclonal antibodies (ES3) prepared against these molecules recognized two major bands, 28 and 46 kDa, in immunoblots and blocked the transformation of OT endothelial cells into mesenchyme in a three-dimensional collagen gel culture system. The studies of Potts et al. (Proc. Natl. Acad. Sci. USA 88, 1516-1520 (1991)) and Potts and Runyan (Dev. Biol. 134, 392-401 (1989)) indicate that transforming growth factor (TGF)-beta expression is necessary for the formation of mesenchyme from cardiac endothelium. In this study, we used ES3 antibodies to test the hypothesis that TGF-beta expression by transforming endothelial cells is regulated by ES antigens. OT and AV endothelial cells treated with embryonic cardiocyte conditioned medium (CCM), which elicits epithelial-mesenchymal transformation, were shown by immunohistochemistry to increase expression of TGF-beta 1-like protein immediately prior to and during their transformation in culture. Endothelium from a nontransforming region of the heart (i.e., ventricle) did not express detectable levels of TGF-beta under similar conditions. The staining pattern for TGF-beta 1-like protein was characterized by a distinct particulate or granular distribution within the Golgi and cytoplasm and at cell surfaces. However, when endothelial transformation was blocked by immunoadsorption of ES proteins from CCM, increased staining for TGF-beta was not observed. These findings suggest an inductive relationship between myocardially derived ES proteins and TGF-beta expression by chick heart endothelial cells which is requisite for their transformation into cushion mesenchyme.
我们之前已经证明，在鸡胚心脏发育早期，心脏内皮细胞的上皮-间质转化是由特定区域（即流出道（OT）和房室（AV）管）心肌合成的EDTA可溶性（ES）细胞外分子诱导的。针对这些分子制备的多克隆抗体（ES3）在免疫印迹中识别出两条主要条带，分别为28 kDa和46 kDa，并在三维胶原凝胶培养系统中阻断了OT内皮细胞向间充质的转化。Potts等人（《美国国家科学院院刊》88, 1516 - 1520 (1991)）以及Potts和Runyan（《发育生物学》134, 392 - 401 (1989)）的研究表明，转化生长因子（TGF）-β的表达对于心脏内皮细胞形成间充质是必需的。在本研究中，我们使用ES3抗体来检验以下假设：转化内皮细胞中TGF-β的表达受ES抗原调节。用胚胎心肌细胞条件培养基（CCM）处理可引发上皮-间质转化的OT和AV内皮细胞，免疫组织化学显示，在培养过程中，它们在转化之前及转化过程中立即增加了TGF-β1样蛋白的表达。来自心脏非转化区域（即心室）的内皮细胞在类似条件下未表达可检测水平的TGF-β。TGF-β1样蛋白的染色模式表现为在高尔基体、细胞质以及细胞表面有明显的颗粒状或点状分布。然而，当通过免疫吸附CCM中的ES蛋白来阻断内皮细胞转化时，未观察到TGF-β染色增加。这些发现表明，心肌来源的ES蛋白与鸡胚心脏内皮细胞中TGF-β的表达之间存在诱导关系，这对于它们转化为垫状间充质是必需的。
