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: 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
• 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

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.

内皮细胞在调节血管功能中起着重要作用。我们和其他人已经证明了 通过表观遗传和转录调控，层流脉动剪切应力（PS）诱导动脉粥样硬化， 保护基因，以维持EC稳态，而振荡剪切（OS）干扰流提高 动脉粥样硬化倾向基因导致EC功能障碍。我们进行了单细胞RNA测序（scRNA-seq） 分析表明，PS的转录组学效应与OS的转录组学效应不同。此外，我们还 表明PS引起EC稳态相关基因组蛋白活性标记（H3 K27 ac）的富集， 组蛋白抑制标记（H3 K9 me 3）在炎症相关基因。我们还证明了PS- 诱导的H3 K9 me 3依赖于核膜蛋白lamin/emerin。这些发现使我们 假设PS和OS通过核纤层蛋白/核突蛋白和染色质偶联来募集细胞， 组蛋白修饰剂，从而导致组蛋白表观遗传学和相关的基因组和 转录调控，因此相反的功能结果。lamin/emerin之间的耦合 而染色质/基因组可以将机械信号从物理空间传递到基因组空间， 和细胞命运的规定。为了验证我们的假设，我们将进行ChIP-seq以鉴定核纤层蛋白/核突蛋白。 PS和OS下的相关基因组区域（LEAGR），并确定LEAGR相关组蛋白 修改（即，表观基因组）。为了可视化不同的流动调制之间的动态相互作用， 在单个活细胞中，我们将使用内切核酸酶缺陷型Cas9（dCas 9） 用小向导RNA（sgRNA）和工程生物传感器来跟踪组蛋白谱的动态， 这些基因组位点，特别是那些与EC稳态或炎症相关的基因组位点。然后我们将确定 基因座特异性表观遗传谱在调节转录组和细胞功能中的作用， 流动。我们将在小鼠主动脉弓（OS）和胸主动脉（PS）上进行体内研究以验证我们的体外研究。 结果，并通过使用动脉粥样硬化小鼠模型评估它们对动脉粥样硬化形成的影响。具体来说，MR （磁共振）引导FUS（聚焦超声）（MRg-FUS）系统将用于远程和 非侵入性激活诱导型shRNA和CRISPR a/i（CRISPR激活或干扰）系统， 在小鼠的局部组织区域操纵核纤层蛋白/核突蛋白和基因座特异性组蛋白表观遗传学， 结扎颈动脉以检查它们在体内的功能作用。因此，提出了三个具体目标： 在不同流动下的核纤层蛋白/突蛋白和EC表观基因组/转录组的体外研究，2）基因座成像， 在单个活EC中的特异性表观遗传和染色质重塑，3）在单个活EC中的特异性表观遗传和染色质重塑的体内检查和验证， 小鼠动脉粥样硬化模型中的表观基因组/转录组调控。随着集成多组学，单- 细胞成像和非侵入性位点特异性调制，我们将能够识别和减轻关键分子 来发展血管疾病的机械医学。