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

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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.