Single-Cell RNA Sequencing And Assay For Transposase-Accessible Chromatin Using Sequencing Reveals Cellular And Molecular Dynamics Of Aortic Aging In Mice

Background and Aim: Age-related vascular aging,a major risk factor for cardiovascular diseases,results in decreased vascular function,which damaging the function of tissues and organs.Although the influence of vascular aging on cardiovascular diseases has been extensively studied,little is known about the cellular and molecular mechanisms underlying age-related vascular aging at the single-cell level.Therefore,this study aims to investigate the changes in gene expression and chromatin accessibility during aortic aging at the single-cell level,in order to provide an important theoretical basis for the prevention and treatment of age-related cardiovascular diseases.Methods: Firstly,the systolic blood pressure,diastolic blood pressure,pulse pressure,heart rate,and pulse wave velocity in C57/BL6 J mice at different ages(4,26 and 86 weeks old)were evaluated using a non-invasive blood pressure tester and a non-invasive doppler blood flow monitor,respectively.The structure and expression of inflammatory cytokines in aortas of mice were tested using hematoxylin-eosin staining,verhoeff’s van gieson staining,and immunohistochemical staining.Secondly,transcriptome profile from the aortas of 4-,26-,and 86-week-old C57/BL6 J mice was analyzed using single-cell RNA sequencing(sc RNA-seq).Cell type-specific marker genes,transcriptional regulons,biological function were identified based on sc RNA-seq data.The composition of cell types,cell-cell communication,inflammation level,and transcriptional noise during aortic aging in mice were analyzed.Multiplex immunohistochemistry staining,flow cytometry,and wire myography were used to verify the results from single-cell analysis.Then,transposase-accessible chromatin profile from the aortas of mice at different age was analyzed using single-cell assay for transposase-accessible chromatin sequencing(sc ATAC-seq).The chromatin accessibility status of cell type-specific transcriptional factors was identified based on sc ATAC-seq data.The distribution characteristics of chromatin open regions in mice at different age was analyzed.Senescence-associated differential chromatin accessibility regions were identified and annotated.Finally,multi-omics integration analysis was performed to explore the key regulators of endothelial cells(EC)and fibroblasts during aortic aging.Results: We founded that systolic and diastolic blood pressure increased with age in mice.Vascular remodeling related to aging,increased artery stiffness,and increased expression of TNF-α,IL-1β,IL-6,and IL-10 were observed in mice with age.Based on single-cell RNA sequencing analysis,16 cell types,including two EC subtypes(EC1and EC2),were identified and verified using multiplex immunohistochemistry staining and flow cytometry.EC1 had higher gene set activity associated with cellular senescence and aging than do EC2,while EC2 highly expressed genes involved in vascular tone regulation,such as Nos3,Ace,and Edn1,and had higher gene set activity associated with blood pressure regulation.EC-dependent vasodilation decreased with age,as did the EC2 ratio in aorta.By integrating the transcriptome and chromatin accessibility data,we identified cell type-specific transcription factor regulatory networks and open chromatin states.Furthermore,we observed that aortic aging caused alteration of cell communication,increased level of inflammation,alteration of cell type composition,and increased transcriptional noise.Results from sc ATAC-seq analysis revealed that senescence mainly affected the degree of chromatin accessibility in promoters and distant intergenic regions of mouse aortic cells,rather than altering the distribution of chromatin open regions.Senescence trajectories analysis revealed that EC1 and fibroblast senescence are linked to different m RNA expression patterns and open chromatin states of transcription factors.Conclusions: In this study,the transcriptome and chromatin accessibility profiles during aortic aging in mice were constructed by sc RNA-seq and sc ATAC-seq analysis.Aortic EC was found to be heterogeneous,including two functionally distinct subtypes,EC1 and EC2.Aortic cell type-specific transcriptional regulons and chromatin accessibility status were determined.Aortic aging caused an increased EC1 to EC2 ratio,increased level of inflammation,increased transcriptional noise,and alteration of cell communication and chromatin accessibility.Senescence-associated regulators Tead1,Gata2,Mafk in EC1 and Stat3,Nfil3,Hoxa5 in fibroblast were identified by integrating the dual-omics data.