Endothelial Cell Cycle Responses to Fluid Shear Stress

项目摘要/摘要 在发育、疾病和伤口愈合过程中适当的血管网络形成和重塑 依赖于内皮细胞(EC)对输入信号(包括生理血液)的异质反应 流。一旦成熟，我们的大多数血管系统被理解为处于G0，一种静止和停滞的细胞周期状态。 然而，流如何实现静止以及如何潜在地调节它还没有很好的定义。我们的初步数据 提示p27和DYRK1a这两种细胞周期抑制蛋白都是减少细胞增殖所必需的。 在流下。P27或DYRK1a基因敲除处理的细胞没有经历细胞增殖下降的情况 在流动下，表明这些细胞没有进入静止状态。批量RNA-seq数据已在 在静态或流动条件下对细胞的实验也显示流动状态下p27的上调，突出了其 重要性。然而，其他细胞周期抑制蛋白很可能在对流体剪切的反应中发挥关键作用。 我们仍然不知道这在所有内皮细胞中看起来是一样的，还是在很大程度上 异质的。这些结果对疾病有重要意义，特别是在动脉粥样硬化方面。 和伤口愈合。我们假设层流切应力通过改变细胞周期来诱导内皮细胞内环境平衡。 抑制蛋白活性。首先，我们将通过以下方法在体外确定内皮细胞对层流的周期反应 操纵p27和静止期梦境复合通路的成员(目标1)。为了测试这一点，我们将使用 内皮细胞周期抑制物基因敲除的2D和3D微流控单元。利用手机面临的一个挑战 周期工具，如抗体或流式细胞仪，是它只允许细胞周期曲线的固定快照。vt.给出 我们想要了解随着时间的推移细胞周期是如何变化的，我们将利用PIP-Fucci，一种荧光 细胞周期报告器，这将使我们能够确定细胞周期时相在静止之前是如何在流动中变化的。 接下来，我们将使用CRISPR在体内确定是否需要细胞周期抑制蛋白来进行静止反应 PIP-Fucci斑马鱼模型中的流动敲除和操纵(目标2)。能够在以下位置执行实时成像 透明的鱼，以及通过化学抑制心脏收缩来操纵血流，使斑马鱼成为 模型生物。这些实验的成功完成将有助于深入了解流动如何调节血管 在生理性血管生成过程中的静止，并将作为提高理解的基础 动脉粥样硬化和伤口愈合。

Project Summary/Abstract Proper blood vessel network formation and remodeling during development, disease, and wound healing depend on heterogeneous responses of endothelial cells (EC) to incoming signals, including physiological blood flow. Once mature, most of our vasculature is understood to be in G0, a quiescent and arrested cell cycle state. However, how quiescence is achieved and potentially regulated by flow is not well defined. Our preliminary data suggests that both p27 and DYRK1a, cell cycle inhibitor proteins, are required for the reduction in cell proliferation under flow. Cells that are treated with p27 or DYRK1a knockdown do not experience a decline in cell proliferation under flow, suggesting that these cells are not entering a quiescent state. Bulk RNA-seq data completed in the lab on cells under static or flow conditions also show an upregulation of p27 under flow, highlighting its importance. However, it is likely that other cell cycle inhibitor proteins play a critical role in response to fluid shear stress and we still do not understand if this looks the same across all endothelial cells or if responses are largely heterogeneous. These results have important implications for disease, specifically in regard to atherosclerosis and wound healing. We hypothesize that laminar shear stress induces EC homeostasis via changes in cell cycle inhibitor protein activity. First, we will determine endothelial cell cycle responses to laminar flow in vitro by manipulating p27 and members of the quiescence DREAM complex pathway (aim 1). To test this, we will utilize 2D and 3D microfluidic units with endothelial cell cycle inhibitor knockdown. One challenge about utilizing cell cycle tools, such as antibodies or flow cytometry, is that it only allows a fixed snapshot of cell cycle profile. Given that we want to understand how cell cycle phase is changing overtime, we will utilize PIP-FUCCI, a fluorescent cell cycle reporter, that will allow us to determine how cell cycle phases change under flow prior to quiescence. Next, we will determine in vivo if cell cycle inhibitor proteins are required for quiescence response using CRISPR knockout and manipulation of flow in PIP-FUCCI zebrafish models (aim 2). The ability to perform live imaging on transparent fish as well as manipulate flow by chemical inhibition of heart contraction make zebrafish an optimal model organism. Successful completion of these experiments will provide insight on how flow regulates vessel quiescence during physiological angiogenesis and will serve as groundwork towards an improved understanding of atherosclerosis and wound healing.