Endothelial Cell Cycle State and Cell Fate

建立功能性血管网络是胚胎发育的限速步骤， 受伤的组织和组织替换工程。尽管我们在识别 促进内皮细胞增殖和发芽的因素，我们缺乏对如何正确控制 血管重塑过程中内皮细胞生长和表型特化，这已经产生了显着的 临床治疗、组织工程和再生医学的障碍。虽然多个信令 这些通路与动脉-静脉网络形成的调节有关，包括流动诱导的 机械传导和Notch信号传导，这些信号协调调节的机制 内皮细胞生长抑制和身份尚不清楚。我们最近的研究表明， 血管丛经受全身血液循环，并且不同大小的剪切应力促进 差异生长反应和基因表达。也就是说，动脉/小动脉剪切应力水平促进Notch 信号传导和下游p27诱导的晚期G1期阻滞，使动脉基因表达成为可能（Fang 2017）。 相反，静脉/小静脉的典型流量大小诱导早期G1停滞，并使静脉血中的G1和G2浓度上调。 基因.有趣的是，不同的内皮细胞周期状态似乎在出生后动脉与静脉中维持。 我们对细胞周期控制在内皮细胞命运决定中的作用或差异信号传导知之甚少， 血管特定流量大小诱导的途径，以及它们如何协调诱导和维持 内皮细胞周期状态和身份。我们研究的科学前提是内皮细胞周期 需要控制适当的动脉和静脉规格，使得当内皮细胞处于不同的 在细胞周期状态下，它们表现出动脉与静脉基因表达的不同倾向。支持这个想法 来自于胚胎干细胞的研究，该研究表明，处于G1期早期与晚期的细胞有一种倾向， 中胚层/内胚层与外胚层的命运（Paulkin 2014）。因此，我们假设， 动脉和静脉中的流动力诱导不同的细胞内信号传导途径，促进不同的细胞内信号传导途径 内皮细胞周期状态，创造不同的机会窗口调节动脉与 静脉基因表达为了确保科学的严谨性，我们将在动脉模型中测试这一假设- 静脉网的形成和修复，并在体外的人内皮细胞培养系统， 操纵我们将定义血管特异性血流幅度调节内皮细胞的机制， 周期状态，确定不同的内皮细胞周期状态如何使差异表型特化 （动脉与静脉），并确定内皮细胞周期状态的操纵是否可以预防或纠正 动脉-静脉畸形和增强损伤后血管修复。对这一假设的评估将产生 对血管形成和再生的新的基本见解，可用于创造人类 离体微脉管系统和治疗血管病变。

Establishing a functional vascular network is a rate-limiting step in embryonic development, the repair of injured tissues, and the engineering of tissue replacements. Although we have made progress in identifying factors that promote endothelial cell proliferation and sprouting, we lack understanding of how to properly control endothelial cell growth and phenotypic specialization during vascular remodeling, which has created a significant roadblock for clinical therapies, tissue engineering and regenerative medicine. Although multiple signaling pathways have been implicated in the regulation of arterial-venous network formation, including flow-induced mechanotransduction and Notch signaling, the mechanisms by which these signals coordinately regulate endothelial cell growth suppression and identity were unclear. Our recent studies revealed that remodeling vascular plexi are subject to systemic blood circulation, and that shear stress of different magnitudes promotes differential growth responses and gene expression. That is, arterial/arteriolar shear stress levels promote Notch signaling, and downstream p27-induced late G1 phase arrest that enables arterial gene expression (Fang 2017). Conversely, flow magnitudes typical of veins/venules induce early G1 arrest, and enables upregulation of venous genes. Interestingly, distinct endothelial cell cycle states appear to be maintained in arteries vs. veins postnatally. We know very little about the role of cell cycle control in endothelial cell fate decisions, or the differential signaling pathways induced by vessel-specific flow magnitudes, and how they may coordinately induce and maintain endothelial cell cycle state and identity. The scientific premise of our research is that endothelial cell cycle control is required for proper arterial and venous specification, such that when endothelial cells are in different cell cycle states, they exhibit different propensity for arterial vs. venous gene expression. Support for this idea comes from studies in embryonic stem cells that show cells in early vs. late G1 phase have a propensity for mesoderm/endoderm vs. ectoderm fate, respectively (Paulkin 2014). Thus, our hypothesis is that differential flow forces in arteries and veins induce different intracellular signaling pathways that promote distinct endothelial cell cycle states, creating distinct windows of opportunity for the regulation of arterial vs. venous gene expression. To ensure scientific rigor, we will test this hypothesis in vivo in models of arterial- venous network formation and repair, and in vitro in human endothelial cell culture systems that allow flow manipulation. We will define mechanisms by which vessel-specific flow magnitudes modulate endothelial cell cycle state, determine how distinct endothelial cell cycle states enable differential phenotypic specialization (artery vs. vein), and determine whether manipulation of endothelial cell cycle state can prevent or correct arterial-venous malformations and enhance post-injury vascular repair. Evaluation of this hypothesis will yield novel fundamental insights into blood vessel formation and regeneration that can be used to create human microvasculature ex vivo and treat vascular pathologies.