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Abstract：
Lineage analysis studies in the avian embryo have identified two types of smooth muscle cells (SMCs) in the tunica media of large elastic arteries; one that originates within the cardiac neural crest and is ectoderm in origin (Ect) and another that arises from local mesenchyme of mesodermal origin (Mes). To determine if differences in primary embryonic lineage can give rise to SMCs with stable differences in growth and differentiation properties, we isolated Ect and Mes SMCs from the Day 14 chick embryo aorta. We report that despite different primary embryonic origins, Ect and Mes SMCs express nearly identical levels of seven SMC differentiation markers in vitro, consistent with their common smooth muscle developmental fates in vivo. By contrast, Ect SMCs displayed a greater capacity for growth in serum-free medium than Mes SMCs, but only under conditions permitting short-range cell-cell interactions. Most of the peptide growth factors tested that might account for serum-independent growth (PDGF-AA, PDGF-BB, basic FGF, EGF, or activin) stimulated DNA synthesis to similar extents in Ect and Mes SMCs. However, we found dramatic, lineage-dependent differences in SMC responses to transforming growth factor-beta (TGF-beta). Exposure to TGF-beta 1 (0.4 to 400 pmole/liter) consistently increased DNA synthesis in Ect SMCs, whereas in paired cultures of Mes SMCs, TGF-beta 1 was growth inhibitory. In SMC cultures transfected with p3TP-lux, a luciferase reporter controlled by the TGF-beta 1-response elements of the human PAI-1 promoter, TGF-beta 1 (120 pM) produced 12 +/- 2-fold increases in luciferase activity in Ect SMCs and only 3 +/- 1.5-fold increases in Mes SMCs. Analysis of TGF-beta receptor phenotypes by Northern blot, radioligand binding, and crosslinking assays showed that Ect and Mes SMCs expressed similar levels of types I, II, and III TGF-beta receptors. However, using a polyclonal antibody specific for the chick type II TGF-beta receptor subunit, we demonstrate that Mes SMCs produce a fully glycosylated form of this protein while Ect SMCs elaborate only an unglycosylated type II TGF-beta receptor. These results show that Ect and Mes SMCs exhibit lineage-dependent differences in growth and receptor-mediated transcriptional responses to at least one important class of SMC morphogens and growth modifiers, e.g., the TGF-betas. Our findings suggest that different SMC populations within a common vessel wall may respond in lineage-dependent ways to signals that direct formation of the tunica media in the embryo and to factors involved in the progression of vascular disease later in life.
摘要：
禽类胚胎的谱系分析研究已经确定了大弹性动脉中膜中的两种类型的平滑肌细胞（SMC）；一种起源于心脏神经c内，起源于外胚层（Ect），另一种起源于中胚层起源的局部间充质（Mes）。为了确定初级胚胎谱系的差异是否可以产生具有稳定生长和分化特性差异的SMC，我们从第14天鸡胚主动脉中分离出Ect和MesSMC。我们报告说，尽管原始胚胎起源不同，Ect和MesSMC在体外表达几乎相同水平的7个SMC分化标记，与它们在体内常见的平滑肌发育命运一致。相比之下,EctSMC在无血清培养基中表现出比MesSMC更大的生长能力，但仅在允许短距离细胞间相互作用的条件下。大多数测试的肽生长因子可能导致血清非依赖性生长（PDGF-AA，PDGF-BB,基本FGF，EGF,或激活素）刺激Ect和MesSMC中相似程度的DNA合成。然而,我们发现戏剧性，SMC对转化生长因子-β（TGF-β）反应的谱系依赖性差异。暴露于TGF-β1（0.4至400pmole/L）持续增加EctSMC中的DNA合成，而在MesSMC的配对培养中，TGF-β1具有生长抑制作用。在用p3TP-lux转染的SMC培养物中，由人PAI-1启动子的TGF-β1反应元件控制的荧光素酶报告基因，TGF-β1（120μM）在EctSMC中产生12+/-2倍的荧光素酶活性增加，而在MesSMC中仅增加3+/-1.5倍。通过Northern印迹分析TGF-β受体表型，放射性配体结合，交联实验表明，Ect和MesSMC表达相似的I型水平，II,和IIITGF-β受体。然而,使用对小鸡II型TGF-β受体亚基具有特异性的多克隆抗体，我们证明MesSMC产生该蛋白的完全糖基化形式，而EctSMC仅产生未糖基化的II型TGF-β受体。这些结果表明，Ect和MesSMC对至少一类重要的SMC形态发生素类和生长调节剂的生长和受体介导的转录反应表现出谱系依赖性差异，例如，TGF-β.我们的发现表明，共同血管壁中的不同SMC种群可能会以谱系依赖性方式对指导胚胎中膜形成的信号以及与以后血管疾病进展有关的因素做出反应。
