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.

概括 人们发现血管功能的表观遗传调控在心血管疾病中发挥着至关重要的作用。 血管内皮细胞（EC）暴露于不同的流动模式，调节血管稳态。 差异表观遗传变化，例如由不同流动模式引起的组蛋白修饰调节 EC 基因 表达谱以及因此的功能后果。组蛋白磷酸化、甲基化的偶联， 最近已发现乙酰化和乙酰化可通过不同的染色质调节基因表达 重塑复合物，这将改变相应的表型结果。然而，却有少量 研究血管细胞中组蛋白修饰的流量调节。我们假设之间的耦合 表观遗传组蛋白磷酸化、甲基化和乙酰化可能作为一种转导机制 调节不同流模式下的 EC 基因表达。我们将制定定向进化策略 对具有不同颜色的 FRET 生物传感器进行系统优化和调整，以同时监测不同的 组蛋白修饰具有高灵敏度和特异性。这些生物传感器将用于追踪多个组蛋白 在同一活细胞中同时进行修饰并揭示组蛋白不断演变的多重景观 不同流程下的修改。我们将进一步使用核酸内切酶缺陷型Cas9 (dCas9)，小指南 RNA (sgRNA) 和分割 FP 来追踪 EC 表型特定位点组蛋白修饰的动态 标记基因。然后我们的表观遗传操纵系统将用于调节这些表观遗传学 特定位点并确定它们对单个活细胞中基因表达和随后的细胞功能的影响 不同流量下。然后，确定的表观遗传图谱将在体内进行调节，随后的基因 检查表达和表型结果。提出了四个具体目标：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）解开时空耦合 组蛋白磷酸化-甲基化-乙酰化在不同流量下调节EC功能的作用，3)建立 位点特异性组蛋白修饰在流下调节 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 细胞核内组蛋白修饰与细胞增殖的时空动态 单个活细胞中的炎症将有助于阐明细胞中的时空转导机制 调节 EC 暴露后的表观遗传调节和病理生理学后果 血流动力学线索。获得的机制见解应该使我们能够识别潜在的分子靶点 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 在血管力学生物学领域，特别是与分子生物学相关的领域具有变革性影响 细胞周期和炎症的调节介导动脉粥样硬化的发展。

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