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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：
10543036
负责人：
Natalie Theresa Tanke
金额：
$ 3.78万
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/10543036
关键词：
3-Dimensional Address Adult Affect Animal Model 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 Pathway interactions Phase Physiologic Neovascularization Physiological Play Proliferating Proliferation Marker Proteins Reporter 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 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.

项目总结/文摘