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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：
10443151
负责人：
SHU CHIEN
金额：
$ 79万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-12-20 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10443151
关键词：
Area Atherosclerosis Biosensor Blood Vessels Blood flow CRISPR interference CRISPR-mediated transcriptional activation Carotid Arteries Cell Fate Control Cell physiology Cells ChIP-seq Chemicals Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Coupling Endothelial Cells Engineering Epigenetic Process Fluorescence Resonance Energy Transfer Focused Ultrasound Functional disorder Gene Targeting Genes Genetic Genome Genomic Segment Genomics Guide RNA Histones Homeostasis Image In Vitro Individual Inflammation Intravenous Investigation Knowledge Laboratories Lamins Ligation Link Location Magnetic Resonance Modeling Monitor Mus Nature Nuclear Nuclear Envelope Pattern Play Prevention Proteins Regulation Role Series Site System Testing Therapeutic Thoracic aorta Time Tissues Transcriptional Regulation Validation Vascular Diseases Vascular Endothelial Cell Viral aortic arch atherogenesis atheroprotective base cellular imaging chromatin remodeling emerin endonuclease epigenetic regulation epigenome experimental study functional outcomes genomic locus genomic profiles histone modification in vivo inhibitor loss of function mechanical signal mouse model multiple omics novel recruit shear stress single-cell RNA sequencing small hairpin RNA tool transcriptome transcriptomics

项目摘要

Endothelial cells (ECs) play a critical role in regulating vascular functions. We and others have demonstrated that, through epigenetic and transcriptional regulations, laminar pulsatile shear stress (PS) induces athero- protective genes to maintain EC homeostasis, whereas disturbed flow with oscillatory shear (OS) elevates athero-prone genes to cause EC dysfunctions. We have performed single-cell RNA sequencing (scRNA-seq) analyses to demonstrate that the transcriptomic effects of PS are distinct from those of OS. In addition, we have shown that PS caused enrichments of histone active mark (H3K27ac) at genes related to EC homeostasis and histone repressing mark (H3K9me3) at genes related to inflammation. We also demonstrated that the PS- induced H3K9me3 is dependent on the nuclear envelop proteins lamin/emerin. These findings have led to our hypothesis that PS and OS modulate EC functions through the coupling of lamin/emerin and chromatin to recruit histone modifiers, thus leading to differential changes in histone epigenetics and the associated genomic and transcriptomic regulations, and hence the opposite functional outcomes. The couplings between lamin/emerin and chromatin/genome can transduce the mechanical signals from physical space into genome space for gene and cell fate regulations. In order to test our hypothesis, we will conduct ChIP-seq to identify the lamin/emerin associated genome regions (LEAGRs) under PS and OS, and determine the LEAGR-associated histone modifications (i.e., epigenome). To visualize the differential flow-modulations of the dynamic interaction between LEAGRs and lamin/emerin in single live cells, we will employ endonuclease-deficient Cas9 (dCas9) together with small guide RNAs (sgRNAs) and engineered biosensors to track the dynamics of the histone profiles of these genomic loci, particularly those related to EC homeostasis or inflammation. We will then determine the roles of the locus-specific epigenetic profiles in regulating the transcriptome and cellular functions under different flows. We will conduct studies in vivo on aorta arch (OS) and thoracic aorta (PS) in mice to validate our in vitro results, and assess their impacts on atherogenesis by using atherosclerotic mouse models. Specifically, the MR (magnetic resonance)-guided FUS (focused ultrasound) (MRg-FUS) system will be used to remotely and noninvasively activate the inducible shRNA and CRISPRa/i (CRISPR activation or interference) systems to manipulate lamin/emerin and locus-specific histone epigenetics at local tissue areas of mouse with partially ligated carotid arteries to examine their functional roles in vivo. Accordingly, three specific aims are proposed: 1) In vitro investigation of lamin/emerin and EC epigenome/transcriptome under different flows, 2) Imaging of locus- specific epigenetic and chromatin remodeling in single live ECs, 3) In vivo examination and validation of the epigenome/transcriptome regulation in mouse atherosclerosis models. With the integrated multi-omics, single- cell imaging, and noninvasive locus-specific modulation, we will be able to identify and mitigate the key molecules to develop mechanomedicine for vascular diseases.

内皮细胞（ECs）在调节血管功能中起着至关重要的作用。我们和其他人已经证明了这一点