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

• 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
• 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

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.

总结

项目成果

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
• 作者: Qian,Tongcheng;Lu,Shaoying;Ma,Hongwei;Fang,Jing;Zhong,Wenxuan;Wang,Yingxiao
• 通讯作者: Wang,Yingxiao

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

• DOI: 10.1038/s41551-021-00779-w
• 发表时间: 2021-11
• 期刊: Nature biomedical engineering
• 影响因子: 28.1

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

跟踪活癌细胞中H3K27的动态组蛋白甲基化。

• 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

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

电穿孔的荧光蛋白生物传感器，用于探测无基因编码的细胞中的分子活性。

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

Visualizing Spatiotemporal Dynamics of Intercellular Mechanotransmission upon Wounding.

• DOI: 10.1021/acsphotonics.8b00383
• 发表时间: 2018-08
• 期刊: ACS photonics
• 影响因子: 7
• 作者: Pengzhi Wang;Jing Liang;Linda Z. Shi;Yi Wang;Ping Zhang;Mingxing Ouyang;D. Preece;Qin Peng;Lunan Shao;Jason Fan;Jie Sun;Shawn S. Li;M. Berns;Huimin Zhao;Yingxiao Wang