Integration of single-cell imaging and multi-omics sequencing to study EC mechano-pathophysiology

整合单细胞成像和多组学测序来研究 EC 机械病理生理学

基本信息

批准号：
10825307
负责人：
SHU CHIEN
金额：
$ 62.38万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10825307
关键词：
Acceleration Acetylation Animals Aorta Atherosclerosis Biosensor Blood Vessels Cardiovascular Diseases Cause of Death Cell Cycle Cell Cycle Regulation Cell Nucleus Cell Proliferation Cell physiology Cells Chromatin Chromatin Remodeling Factor Color Coupling Cues DNA Methylation Developed Countries Development Directed Molecular Evolution Disease Progression Endothelial Cells Endothelium Epigenetic Process Exposure to Fluorescence Fluorescence Resonance Energy Transfer Functional disorder Gene Expression Gene Expression Profile Gene Expression Regulation Generations Genes Genetic Guide RNA Histone Acetylation Histone Code Histones Homeostasis Inflammation Inflammatory Intervention Lesion Ligation Maps Masks Mediating Methylation Modeling Modification Molecular Molecular Target Monitor Nucleic Acid Regulatory Sequences Outcome Pathologic Processes Pattern Pharmacologic Substance Phenotype Phosphorylation Play Regulation Reporting Role Sensitivity and Specificity Signal Transduction System Vascular Endothelial Cell Visualization aortic arch atherogenesis atheroprotective cellular imaging design endonuclease epigenetic regulation genomic locus hemodynamics histone methylation histone modification in vivo inhibitor insight microscopic imaging multiple omics phenotypic biomarker recruit response spatiotemporal

项目摘要

Summary Epigenetic regulation of vascular functions has been found to play crucial roles in cardiovascular diseases. Vascular endothelial cells (ECs), which are exposed to different flow patterns, regulate vascular homeostasis. Differential epigenetic changes, e.g. histone modifications, caused by different flow patterns regulate EC gene expression profile and hence functional consequences. The coupling of histone phosphorylation, methylation, and acetylation have recently been identified to regulate gene expressions through the distinct chromatin remodeling complexes, which would alter the consequential phenotypic outcome. However, there is a paucity of study in the flow-regulation of histone modifications in vascular cells. We hypothesize that the coupling among epigenetic histone phosphorylation, methylation, and acetylation may serve as a transducing mechanism to regulate EC gene expressions under different patterns of flows. We will develop a directed evolution strategy for the systematic optimization and tuning of FRET biosensors with distinct colors to simultaneously monitor different histone modifications with high sensitivity and specificity. These biosensors will be used to track multiple histone modifications simultaneously in the same live cell and unravel the evolving multiplex landscape of histone modifications under different flows. We will further employ the endonuclease-deficient Cas9 (dCas9), small guide RNAs (sgRNAs) and split FPs to track the dynamics of histone modifications at the specific loci of EC phenotype marker genes. Our epigenetic manipulation system will then be employed to modulate epigenetics at these specific loci and determine their effects on gene expressions and consequent cellular functions in single live cells under different flows. The identified epigenetic profiles will then be modulated in vivo, and the consequent gene expression and phenotypic outcome examined. Four specific aims are proposed: 1) Develop and optimize FRET biosensors to visualize the dynamic histone modifications in single cells, 2) Unravel the spatiotemporal coupling of histone phosphorylation-methylation-acetylation in regulating EC functions under different flows, 3) Establish the roles of locus-specific histone modifications in regulating EC gene expression under flows, 4) Elucidate the effect of histone modifications on gene expression and lesion formation in vivo. The simultaneous tracking of the spatiotemporal dynamics of histone modifications in the nucleus in conjunction with cell proliferation and inflammation in a single live cell will allow the elucidation of the spatiotemporal transducing mechanism in regulating epigenetic modulations and pathophysiological consequences upon the exposure of ECs to hemodynamic cues. The mechanistic insights obtained should allow us to identify the potential molecular targets and facilitate the design of pharmaceutical interventions for pathologic processes. As such, the project should have transformative impact in the field of vascular mechanobiology, particularly related to the molecular regulations of cell cycle and inflammation in mediating the development of atherosclerosis.

概括已经发现血管功能的表观遗传调节在心血管疾病中起着至关重要的作用。暴露于不同流动模式的血管内皮细胞（EC）调节血管稳态。差异表观遗传学变化，例如由不同流动模式引起的组蛋白修饰调节EC基因表达曲线及功能后果。组蛋白磷酸化，甲基化，偶联最近已经鉴定出乙酰化来通过不同的染色质调节基因表达重塑配合物，这将改变结果表型结果。但是，很少研究血管细胞中组蛋白修饰的流动调节。我们假设耦合表观遗传组蛋白磷酸化，甲基化和乙酰化可能是转导机制在不同的流量模式下调节EC基因表达。我们将制定一个定向的进化策略具有不同颜色的FRET生物传感器的系统优化和调整，可以同时监视不同的不同具有高灵敏度和特异性的组蛋白修饰。这些生物传感器将用于跟踪多个组蛋白同时在同一活细胞中进行修改，并揭示组蛋白不断发展的多重景观在不同的流下进行修改。我们将进一步采用核酸内切酶的CAS9（DCAS9），小指南 RNA（SGRNA）和拆分FPS以跟踪EC表型特定基因座的组蛋白修饰的动力学标记基因。然后，我们的表观遗传操纵系统将被用来调节这些表观遗传学特定基因座并确定它们对基因表达的影响，并确定单个活细胞中的细胞功能在不同的流下。然后将在体内调节所鉴定的表观遗传谱，然后将其基因调节。表达和表型结果检查了。提出了四个具体目的：1）开发和优化FRET 生物传感器可视化单细胞中的动态组蛋白修饰，2）揭开时空耦合在不同流动下调节EC功能中的组蛋白磷酸化 - 甲基化 - 乙酰化，3）建立基因座特异性组蛋白修饰在调节流动下的EC基因表达中的作用，4）阐明组蛋白修饰对体内基因表达和病变形成的影响。同时跟踪核中组蛋白修饰的时空动力学与细胞增殖和单个活细胞中的炎症将允许阐明时空转导机制调节EC暴露于EC暴露于血液动力学提示。获得的机械见解应使我们能够识别潜在的分子靶标并促进针对病理过程的药物干预措施的设计。因此，该项目应该在血管机械生物学领域具有变革性影响，特别是与分子有关细胞周期和炎症的法规介导动脉粥样硬化的发展。

项目成果

期刊论文数量（65）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

FRET imaging of calcium signaling in live cells in the microenvironment.

微环境中活细胞中钙信号传导的 FRET 成像。

DOI：
10.1039/c2ib20264f
发表时间：
2013
期刊：
Integrative biology : quantitative biosciences from nano to macro
影响因子：
0
作者：
Qian,Tongcheng;Lu,Shaoying;Ma,Hongwei;Fang,Jing;Zhong,Wenxuan;Wang,Yingxiao
通讯作者：
Wang,Yingxiao

Electroporation-delivered fluorescent protein biosensors for probing molecular activities in cells without genetic encoding.

DOI：
10.1039/c4cc04730c
发表时间：
2014-10-09
期刊：
Chemical communications (Cambridge, England)
影响因子：
0
作者：
Sun C;Ouyang M;Cao Z;Ma S;Alqublan H;Sriranganathan N;Wang Y;Lu C
通讯作者：
Lu C

Monocytes engineered with iSNAP inhibit human B-lymphoma progression.

DOI：
10.1002/btm2.10285
发表时间：
2022-05
期刊：
Bioengineering & translational medicine
影响因子：
7.4
作者：
通讯作者：

Tracking the Dynamic Histone Methylation of H3K27 in Live Cancer Cells.

DOI：
10.1021/acssensors.1c01670
发表时间：
2021-12-24
期刊：
ACS SENSORS
影响因子：
8.9
作者：
Gong, Ya;Wei, Chujun;Cheng, Leonardo;Ma, Fengyi;Lu, Shaoying;Peng, Qin;Liu, Longwei;Wang, Yingxiao
通讯作者：
Wang, Yingxiao

Control of the activity of CAR-T cells within tumours via focused ultrasound.