Mechanisms of epicardium-directed coronary vessel patterning

冠状血管系统为心脏提供氧气和营养，并清除 代谢废物。这种连续网络的组织需要血管的成熟 血管内皮细胞(EC)根据它们在心脏中的位置决定动脉和静脉的命运。而当 许多控制血管构型的指导因素已经被定义，但尚不清楚 空间信息如何控制细胞的行为和身份。心外膜是一层单层 心脏表面的间皮细胞，它容纳了重要的心血管疾病人群 祖先。我们以前报道过心外膜上皮向间充质转化(EMT)是 冠状动脉内皮细胞成熟所必需的。新的初步数据揭示了深刻的EC模式和 心外膜破裂时的规格缺陷，最终导致不适当的 新生血管内皮细胞在心外膜下的定位。为了定义细胞和分子 心外膜EMT与EC构型和成熟的联系我们进行了单细胞(Sc)实验 小鼠胚胎心脏心外膜来源细胞和内皮细胞的RNA测序 在关键的发展时间点。这项研究定义了心外膜起源的“牧羊人”和 表达独特的血管生成趋化因子信号的“路标”细胞。我们提供体外培养和 体内证据表明控制EMT和基因表达的共同机制 对重要的指导线索进行编码。我们还发现EC的定位和动静脉命运 规格可能由一个共同的分子机制控制。基于之前的 已发表的和初步的数据，我们假设EMT控制表达和 协调冠状动脉内皮细胞构型的心外膜来源趋化因子的定位 以及胎儿心脏中的房室命运规范。目前的研究将审问这部小说 心外膜引导的冠状动脉内皮细胞模式(定位和分支)和 成熟(动静脉规范)。在这里，我们将使用转基因小鼠，延时 活胚胎多光子成像、scRNA-seq和空间转录组学，以及细胞和分子 生物学方法：1)定义一种共同的机制，调节EMT和 编码EC引导信号的基因；以及2)询问协调机制 心外膜指导的EC图案化和AV命运规范。我们预计这些研究将提供 对控制血管图案化的机制的重要见解。这项研究还可能 促进我们对冠状动脉疾病的发育起源的理解，并导致 刺激缺血心脏组织血运重建和修复的治疗策略。

The coronary blood vasculature provides the heart with oxygen and nutrients, and removes metabolic waste. Organization of this contiguous network requires the maturation of vascular endothelial cells (EC) into arterial and venous fates based upon their location in the heart. While many of the guidance factors that control vascular patterning have been defined, it is not clear how spatial information controls cell behavior and identity. The epicardium is a single layer of mesothelial cells on the surface of the heart that harbors an important population of cardiovascular progenitors. We previously reported that epicardial epithelial-to-mesenchymal transition (EMT) is required for coronary EC maturation. New preliminary data reveals profound EC patterning and specification defects upon disruption of the epicardium, culminating with the inappropriate localization of angiogenic ECs in the sub-epicardium. To define the cellular and molecular mechanisms linking epicardial EMT to EC patterning and maturation we performed single cell (sc) RNA-sequencing of epicardium-derived cells and ECs isolated from the embryonic mouse heart at key developmental timepoints. This study defined epicardium-derived “shepherding” and “guidepost” cells that express unique angiogenic chemokine signatures. We provide in vitro and in vivo evidence that suggest a common mechanism controls EMT and the expression of genes that encode important guidance cues. We also find that EC localization and arteriovenous fate specification may be controlled by a common molecular mechanism. Based on previously published and preliminary data, we hypothesize that EMT controls the expression and localization of epicardium-derived chemokines that coordinate coronary EC patterning and AV fate specification in the fetal heart. The current study will interrogate this novel paradigm of epicardium-directed coronary EC patterning (localization and branching) and maturation (arteriovenous specification). Here, we will use genetically modified mice, time-lapse live embryo multi-photon imaging, scRNA-seq and spatial transcriptomics, and cell and molecular biology approaches to: 1) Define a common mechanism regulating EMT and the expression of genes that encode EC guidance cues; and 2) Interrogate the mechanisms coordinating epicardium-directed EC patterning and AV fate specification. We expect these studies will provide important insights into the mechanisms that control vascular patterning. This study may also advance our understanding of the developmental origins of coronary artery disease, and lead to therapeutic strategies that stimulate revascularization and repair of ischemic heart tissue.