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取决于内皮细胞（EC）对传入信号的异质反应，包括生理血液流动。一旦成熟，我们的大多数脉管系统就被认为是G0，是一种静止和被捕的细胞周期状态。但是，如何实现静止和可能受流量调节的定义。我们的初步数据表明p27和dyRK1a（细胞周期抑制剂蛋白）都是细胞增殖所必需的下流。用p27或dyrk1a敲低处理的细胞不会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-seq数据在在静态或流动条件下的细胞上的实验室也显示了流动下p27的上调，突出显示其重要性。但是，其他细胞周期抑制剂蛋白可能在响应液体剪切方面起关键作用压力，我们仍然不明白在所有内皮细胞中看起来都一样，还是反应在很大程度上是异质。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。我们假设层状剪切应力通过细胞周期变化诱导EC稳态抑制剂蛋白活性。首先，我们将确定内皮细胞周期对层流对层流的反应，并通过操纵p27和静止不动的梦complecter道途径的成员（AIM 1）。为了测试这一点，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周期工具，例如抗体或流式细胞术，它仅允许固定的细胞周期轮廓。给出我们想了解细胞周期阶段的加时性变化，我们将利用Pip-fucci，一种荧光细胞周期记者，这将使我们能够确定细胞周期阶段在静止之前如何在流动下发生变化。接下来，我们将在体内确定是否需要使用CRISPR进行静止反应的细胞周期抑制剂蛋白敲除斑线斑马鱼模型中流动的敲除和操纵（AIM 2）。执行实时成像的能力透明鱼类以及通过化学抑制心脏收缩而操纵流动使斑马鱼成为最佳模型生物。这些实验的成功完成将提供有关流动如何调节血管的见解生理血管生成期间的静止，将作为改善理解的基础动脉粥样硬化和伤口愈合。