Development of Coronary Smooth Muscle

该项目的总体目标是确定控制冠状动脉平滑肌细胞(COSMc)从心前器官(PEO)的前体细胞发育和分化的分子途径。我们先前发现，在心前膜细胞上皮向间充质转化(EMT)过程中，Cosmc的分化与细胞骨架重排密切相关。细胞-细胞接触丧失后，RhoA-GTP酶激活，富含半胱氨酸的LIM结构域蛋白Crp2从局部接触移位到细胞核，以及依赖血清反应因子(SRF)的SMC靶基因转录。在对促进移植的PEO细胞内EMT的因素进行筛选时，我们发现Sonic Hedgehog(Shh)激活了Patted-1 (PTC，Shh受体)诱导EMT，刺激Cosmc分化。然后，我们检查了PTC-LacZ基因敲入小鼠，发现β-Gal染色集中在发育中的冠状动脉的内侧-外膜边界的细胞中。此外，免疫组织化学染色将Shh定位于SMC和外膜细胞之间的界面。在发育中的心脏中，PTC-LacZ的活性与PDGFbeta受体阳性的CoSMCs的出现在E15.5到P3的远端序列中是平行的。为了明确Shh信号在Cosmc发育和分化中的作用，我们提出如下建议：特异性目标1将定位HH配体、受体和修饰物基因在冠状动脉形成过程中的表达。我们将探索外膜细胞作为Shh反应信号媒介的一个新的和意想不到的角色，它协调这一独特的血管床的发育。特殊目标2将研究心外膜前细胞分化为CoSMC的分子途径。我们将集中于强大的SRF共激活因子的作用，包括Crp2和myocardin家族蛋白，作为Shh信号的靶点，以及作为Cosmc分化的中介。具体目标3将利用HH信号基因缺陷的小鼠，研究Shh信号在冠状动脉血管发育和修复中的功能作用。

The overall goal of this project is to identify molecular pathways that control development and differentiation of coronary smooth muscle cells (CoSMC) from progenitors in the proepicardial organ (PEO). We previously found that CoSMC differentiation is tightly linked to cytoskeletal rearrangements during epithelial to mesenchymal transformation (EMT) of proepicardial cells. Loss of cell-cell contacts is followed by rhoA-GTPase activation, translocation of the cysteine-rich LIM domain-containing protein Crp2 from focal contacts to the nucleus, and serum response factor (SRF)-dependent transcription of SMC target genes. In a screen for factors that promote EMT in explanted PEO cells, we found that sonic hedgehog (Shh) activated patched-1 (ptc, a Shh receptor), induced EMT and stimulated CoSMC differentiation. We then examined ptc-lacZ knock-in mice and found that beta-gal staining was concentrated in cells at the medial-adventitial border in developing coronary vessels. Moreover, immunostaining localized Shh specifically to the interface between SMC and adventitial cells. In developing hearts, ptc-lacZ activity paralleled the appearance of PDGFbeta-receptor-positive CoSMCs in a proximal to distal sequence from E15.5 to P3. To luther define the roles of Shh signaling during CoSMC development and differentiation, we propose the following: Specific Aim 1 will map the expression of hh ligand, receptor and modifier genes during formation of the coronary vessels. We will explore a novel and unsuspected role for adventitial cells as Shh-responsive signal mediators that orchestrate development of this unique vascular bed. Specific Aim 2 will examine molecular pathways by which proepicardial cells differentiate to CoSMCs. We will focus on the role of potent SRF coactivators, including Crp2 and the myocardin family of proteins, as targets of Shh signaling, and as mediators of CoSMC differentiation. Specific Aim 3 will examine the functional roles of Shh signaling during development and repair of the coronary vasculature using mice that are genetically-deficient in hh signaling.