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

血管内皮细胞(ECs)在调节血管功能中起着关键作用。我们和其他人已经证明 通过表观遗传和转录调控，层流脉动切应力(PS)诱导动脉粥样硬化。 保护基因维持EC动态平衡，而具有振荡切变(OS)的扰动流动升高 易患动脉粥样硬化的基因导致内皮细胞功能障碍。我们进行了单细胞rna测序(scrna-seq)。 分析表明，PS的转录效应与OS不同。此外，我们还有 结果表明，PS引起EC动态平衡相关基因组蛋白活性标记(H3K27ac)的丰富， 组蛋白抑制标记(H3K9me3)位于炎症相关基因。我们还演示了PS- H3K9me3的诱导依赖于核膜蛋白lamin/Emerin。这些发现导致了我们 PS和OS通过Lamin/Emerin和染色质偶联来调节EC功能的假说 组蛋白修饰物，从而导致组蛋白表观遗传学和相关的基因组和 转录调控，因此产生相反的功能结果。Lamin/Emerin之间的耦合 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进行基因 以及细胞命运规则。为了验证我们的假设，我们将进行CHIP-SEQ来鉴定lamin/Emerin PS和OS下的相关基因组区域(LEAGR)，并确定LEAGR相关的组蛋白 修饰(即，表观基因组)。可视化动态相互作用的不同流量调制 LEAGRs和Lamin/Emerin在单个活细胞中，我们将联合使用核酸内切酶缺陷Cas9(DCas9) 使用小引导RNA(SgRNA)和工程生物传感器来跟踪组蛋白图谱的动态 这些基因组位点，特别是与EC动态平衡或炎症相关的那些。然后我们将确定 不同环境下基因座特异性表观遗传学特征对转录组和细胞功能的调控作用 流动。我们将在小鼠的主动脉弓(OS)和胸主动脉(PS)上进行体内研究，以验证我们的体外实验 结果，并通过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。具体地说，MR (磁共振)引导的FUS(聚焦超声)(MRG-FUS)系统将用于远程和 非侵入性地激活可诱导的shRNA和CRISPRA/I(CRISPR激活或干扰)系统以 部分地在小鼠局部组织区域操纵层蛋白/组蛋白和位点特异性组蛋白表观遗传学 结扎颈动脉以检测其在体内的功能作用。据此，提出了三个具体目标：1) 不同流动条件下Lamin/Emerin和EC表观基因组/转录组的体外研究2)基因座成像 单个活体内皮细胞中特定的表观遗传学和染色质重塑，3)体内检测和验证 小鼠动脉粥样硬化模型中的表观基因组/转录组调控。随着集成的多组学，单一的- 细胞成像和非侵入性的位点特异性调制，我们将能够识别和缓解关键分子 开发治疗血管疾病的机械医学。

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.