Development of Coronary Smooth Muscle

该项目的总体目标是确定控制心外膜前器官（PEO）中冠状动脉平滑肌细胞（CoSMC）从祖细胞发育和分化的分子途径。我们之前发现，在心外膜前细胞上皮向间充质转化（EMT）过程中，CoSMC分化与细胞骨架重排密切相关。细胞-细胞接触丧失后，rhoA-GTPase被激活，富含半胱氨酸的LIM结构域蛋白Crp2从局灶接触转移到细胞核，以及SMC靶基因的血清反应因子（SRF）依赖性转录。在筛选外植PEO细胞中促进EMT的因子时，我们发现sonic hedgehog （Shh）激活了patched-1

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.