Mechanisms of epicardium-directed coronary vessel patterning

冠状动脉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成熟， 血管内皮细胞（EC）根据其在心脏中的位置进入动脉和静脉的命运。而 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行为和身份。心外膜是一层 心脏表面上的间皮细胞，是心血管疾病的重要群体。 祖先我们以前报道过，心外膜上皮细胞向间质细胞转化（EMT）， 需要冠状动脉EC成熟。新的初步数据揭示了深刻的EC模式， 心外膜破裂时的质量标准缺陷，最终导致不适当的 心外膜下血管生成内皮细胞的定位。来定义细胞和分子 将心外膜EMT与EC模式化和成熟联系起来的机制，我们进行了单细胞（sc） 小鼠胚胎心脏心外膜细胞和内皮细胞的RNA序列分析 在关键的发展时间点。这项研究定义了心外膜衍生的“牧羊”， “路标”细胞表达独特的血管生成趋化因子的签名。我们提供体外和 体内证据表明，一个共同的机制控制EMT和基因的表达， 编码了重要的导航信号我们还发现EC定位和动静脉命运 可以通过共同的分子机制来控制特定。基于先前 根据已发表的和初步的数据，我们假设EMT控制表达， 协调冠状动脉EC模式的心外膜衍生趋化因子的定位 和胎儿心脏中AV命运的特化。目前的研究将审问这部小说 心外膜导向的冠状动脉EC模式（定位和分支）的范例， 成熟（动静脉规范）。在这里，我们将使用转基因小鼠， 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 生物学方法：1）定义一个共同的机制，调节EMT和表达 编码EC指导线索的基因;和2）询问协调 心外膜定向EC模式和AV命运特化。我们希望这些研究能提供 对控制血管模式的机制的重要见解。这项研究也可能 促进我们对冠状动脉疾病发展起源的理解，并导致 刺激缺血心脏组织的血管再生和修复的治疗策略。

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.