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）从心外膜前器官（PEO）祖细胞发育和分化的分子途径。我们以前发现，CoSMC分化与前心外膜细胞上皮间质转化（EMT）过程中的细胞骨架重排密切相关。细胞-细胞接触的丧失之后是rhoA-GT3活化，富含半胱氨酸的LIM结构域蛋白Crp 2从病灶接触易位到细胞核，以及SMC靶基因的血清反应因子（SRF）依赖性转录。在筛选促进凋亡的PEO细胞EMT的因素时，我们发现音刺猬（Shh）激活了patched-1， (ptc Shh受体）诱导EMT并刺激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血管的内外膜边缘的细胞中。此外，免疫组化定位Shh特别是SMC和外膜细胞之间的界面。在发育中的心脏中，ptc-lacZ活性在E15.5至P3的近端至远端序列中抑制PDGF β受体阳性CoSMC的出现。为了进一步确定Shh信号在CoSMC发育和分化过程中的作用，我们提出以下建议：特异性Aim 1将绘制冠状动脉血管形成过程中hh配体、受体和修饰基因的表达。我们将探索一种新的和未知的作用外膜细胞作为Shh反应的信号介质，协调发展这一独特的血管床。具体目标2将检查心外膜细胞分化为CoSMC的分子途径。我们将集中在有效的SRF辅激活剂，包括Crp 2和myocardin家族的蛋白质，作为目标的Shh信号，并作为调解员的CoSMC分化的作用。具体目标3将使用hh信号传导遗传缺陷的小鼠检查Shh信号传导在冠状血管系统发育和修复期间的功能作用。

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.