Endothelial Cell Cycle Responses to Fluid Shear Stress

内皮细胞周期对流体剪切应力的反应

• 批准号: 10624370
• 负责人: Natalie Theresa Tanke
• 金额: $ 3.88万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10624370
• 关键词: 3-Dimensional; Address; Adult; Affect; Antibodies; Aorta; Atherosclerosis; Biology; Blood Circulation; Blood Vessels; Blood flow; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Cycle Regulation; Cell Proliferation; Cell division; Cells; Chemicals; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complex; Data; Detection; Development; Disease; Dorsal; Endothelial Cells; Event; Exposure to; Fertilization; Fishes; Flow Cytometry; Goals; Homeostasis; Hour; Image; Imaging Techniques; In Vitro; Knock-out; Label; Liquid substance; Measures; Microfluidics; Microscopy; Modeling; Molecular; Morphogenesis; Myocardial Contraction; Outcome Assessment; Pathway interactions; Phase; Physiologic Neovascularization; Physiological; Play; Proliferation Marker; Proteins; Reporter; Repression; Role; S phase; Signal Transduction; Testing; Time; Training; Up-Regulation; Zebrafish; angiogenesis; blood vessel development; cell motility; design; experience; experimental study; improved; in vivo; inhibitor; innovation; insight; knock-down; live cell imaging; member; model organism; mutant; phase change; prevent; programs; protein function; response; shear stress; three dimensional cell culture; tool; transcriptome sequencing; wound healing

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.

项目总结/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认为是在G0，一个静止和停滞的细胞周期状态。 然而，静止是如何实现的，并可能通过流量进行调节，还没有很好的定义。我们的初步数据 表明细胞周期抑制蛋白p27和DYRK1a都是细胞增殖减少所必需的 下流。用p27或DYRK1a敲低处理的细胞不经历细胞增殖的下降 在流动下，表明这些细胞没有进入静止状态。批量RNA测序数据已在 在静态或流动条件下对细胞进行的实验也显示了在流动下p27的上调，突出了其 重要性然而，很可能其他细胞周期抑制蛋白在响应流体剪切中起关键作用 我们仍然不知道这是否在所有内皮细胞中看起来都是一样的，或者反应在很大程度上是 异质的这些结果对疾病，特别是动脉粥样硬化有重要意义 和伤口愈合。我们假设层流切应力通过改变细胞周期诱导EC稳态 抑制蛋白活性。首先，我们将确定内皮细胞周期的反应，层流在体外， 操纵p27和静止期DREAM复合物通路的成员（目的1）。为了验证这一点，我们将使用 具有内皮细胞周期抑制剂敲低的2D和3D微流体单元。利用细胞的一个挑战是 细胞周期工具（如抗体或流式细胞术）的缺点在于它仅允许细胞周期概况的固定快照。给定 我们想了解细胞周期阶段是如何随着时间的推移而变化的，我们将利用PIP-FUCCI，一种荧光 细胞周期报告基因，这将使我们能够确定细胞周期阶段如何在静止前的流动下变化。 接下来，我们将使用CRISPR在体内确定细胞周期抑制剂蛋白是否是静止反应所必需的。 敲除和操纵PIP-FUCCI斑马鱼模型中的流动（目的2）。能够对 透明鱼以及通过化学抑制心脏收缩来操纵血流使斑马鱼成为最佳的 模式生物这些实验的成功完成将提供关于流量如何调节血管的见解 在生理血管生成过程中的静止，并将作为基础，以提高理解 动脉粥样硬化和伤口愈合。