| Objective:We aimed to generate a gene expression and cell atlas of Stanford Type A aortic dissection aorta based purely on the unbiased identification of spatially defining features by employing spatial transcriptomics.The unsupervised classification reveals different position of the aorta(ascending aorta,brachiocephalic artery,left subclavian artery,and left common carotid artery)and different severity of ascending aorta,which of ligand-receptor pairs can be used to infer intercellular communication from cell pairs.Methods:Fresh aorta tissue was washed in cold PBS and embedded in OCT sectioning by submersion in isopentane pre-cooled to-80℃ in dry ice for spatial transcriptomics.All raw sequencing data was used Spaceranger software from 10×Genomics to process,align and summarize UMI counts against hg38 human reference genome for each spot on the Visium spatial transcriptomics array.Subsequent cell type annotation results also showed that the effect obtained by PCA dims 30 combined with UMAP dimensionality reduction cluster analysis was more consistent with the actual cell type distribution.Cell type prediction probabilities were calculated for each spot using factor analysis via Find All Markers in Seurat.For all marker gene expression,we used R packages Seurat,WGCNA,and GSEA enrichment for statistical testing.Gene Ontology and pathway enrichment analyses were performed using the ‘cluster Profiler’ R package.Trajectories on compartment embeddings were calculated using Monocle 3 algorithm.To define intercellular communication networks within the aortic cellulome,we used a dataset of human ligandreceptor pairs to develop a list of mouse orthologs comprising 2,009ligand-receptor pairs.Opal multi-colour immunohistochemistry staining with anti-CD31,-HLA-DR,-α-SMA,-CD163 and-CDM antibodies to validate the existence of cell types.Result:(1)According to the standard in the diagnosis and artificial vascular replacement of patients with Stanford Type A aortic dissection,develop into the exclusion criteria aorta tissue 15 screening of 8 cases of sample collection,among them 6 samples of the ascending aorta(1sample with both the ascending aorta and left subclavian artery and left common carotid artery,1 sample with both the tear part and untear part),2 cases of brachiocephalic artery samples,total 19 sections for spatial transcription sequencing.In the universal analysis process of normalization,component analysis,and dimensionality reduction analysis,different algorithms were compared to establish a suitable pipeline for human aortic tissues.We identified 19,879 genes and 7 major cell populations and divided them based on their positions in the aortic tissue.This approach provides a spatially resolved transcriptome-and tissue-wide perspective of the adult human aorta and will allow the application of human fibrous aortic tissues.(2)We applied ST to spatially profile gene expression in different stages of ascending aorta and different parts of the aorta(ascending aorta,left subclavian artery,left common carotid artery and brachiocephalic artery).We analyzed 19 section tissues using spatial transcriptomics.A total of 33,201 spots,an average of 19202 genes per Spot,428,338,634 reads per Spot and 1370 Median Genes per Spot were detected,and the sequencing saturation was more than 90%.We present a molecular approach that reveals a comprehensive transcriptional landscape of aortic dissection cell types with varying degrees of severity and maps cell-type-specific gene expression to specific anatomical domains.Spatial transcriptomics identified unique gene profiles corresponding to different anatomical regions at distinct sevirity of development.(3)Through the analysis of cellular communication in different stages of ascending aorta,the occurrence of Stanford type A aortic dissection is closely related to the location of cells,and there are different degrees of interaction between cells and cells.With the increase of the widest diameter of ascending aorta,different cells in the aorta play different roles and functional states are also different.In the mild Stanford type A aortic dissection,the cells were mainly fibroblasts.Participate in the maintenance of vascular morphology and vascular development.In the moderate Stanford type A aortic dissection,the cells were mainly stromal cells,which regulate the movement of cell components,promote cell migration and other biological activities;in the severe Stanford type A aortic dissection,the cells are mainly macrophages,which participate in the immune response and make an immune response to inflammation and injury of the aorta.(4)The cell distribution characteristics of different types of cells were quantitatively analyzed by Opal multi-colour immunohistochemistry staining,and the cell statistical methods in spatial transcriptomics were verified at the single-cell resolution.Conclusion:Space transcriptome study techniques can be applied to low cell density,high fiber of the multilayer structure of aorta tissue,grope for the pipeline preliminary biological information analysis is suitable for the sample analysis,this method is preliminarily describes comprehensive gene expression and cell atlas of Stanford type A aortic dissection were established under the pathological state,and cell types at different positions of aorta and distinct sverity of ascending aorta were analyzed in detail,and the roles,functions and functional states of various cells were elaborated under the pathological state.The occurrence and development of Stanford Type A aortic dissection was closely related to the location of cells and the active degree of cell communication.In distinct sverity of ascending aorta,cell emitters and receivers were different,which participate in different biological activities and play different roles in the body.Our findings will pave the way toward both a better understanding of Stanford type A aortic dissection pathogenesis and heterogeneity and the implementation of more effective personalized therapeutic approaches. |
PDF Full Text Request