Endothelial Cell Cycle Responses to Fluid Shear Stress

内皮细胞周期对流体剪切应力的反应

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要在发育、疾病和伤口愈合过程中正确的血管网络形成和重塑取决于内皮细胞 (EC) 对传入信号（包括生理血液）的异质反应流动。一旦成熟，我们的大部分脉管系统就处于 G0，一种静止和停滞的细胞周期状态。然而，如何实现静止以及如何通过流动进行潜在调节尚不清楚。我们的初步数据表明 p27 和 DYRK1a（细胞周期抑制剂蛋白）对于减少细胞增殖是必需的流动下。用 p27 或 DYRK1a 敲低处理的细胞不会出现细胞增殖下降流动下，表明这些细胞没有进入静止状态。批量 RNA-seq 数据完成于静态或流动条件下细胞的实验室也显示 p27 在流动下的上调，突出了其重要性。然而，其他细胞周期抑制剂蛋白可能在响应流体剪切中发挥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相同，或者反应是否在很大程度上异质。这些结果对疾病具有重要意义，特别是在动脉粥样硬化方面和伤口愈合。我们假设层流剪切应力通过细胞周期的变化诱导 EC 稳态抑制剂蛋白活性。首先，我们将通过体外确定内皮细胞周期对层流的反应操纵 p27 和静止 DREAM 复合体通路的成员（目标 1）。为了测试这一点，我们将利用具有内皮细胞周期抑制剂敲低的 2D 和 3D 微流体单元。利用细胞的一项挑战细胞周期工具（例如抗体或流式细胞术）的一个缺点是它只允许细胞周期概况的固定快照。给定我们想要了解细胞周期阶段如何随时间变化，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在静止之前在流动下如何变化。接下来，我们将使用 CRISPR 在体内确定静止反应是否需要细胞周期抑制剂蛋白 PIP-FUCCI 斑马鱼模型中的基因敲除和血流操纵（目标 2）。执行实时成像的能力透明的鱼以及通过化学抑制心脏收缩来控制流量使斑马鱼成为最佳选择模式生物。成功完成这些实验将有助于了解流量如何调节容器生理性血管生成过程中的静止状态，并将作为加深理解的基础动脉粥样硬化和伤口愈合。