Single-cell Sequencing Analyzes Human Aortic Valves To Explore The Main Pathogenic Factor Of Calcific Aortic Valve Disease

Part 1: Single-cell sequencing to determine the cell composition of human aortic valvesObject: Isolated valve tissue cells from human aortic valve tissues for performing single-cell sequencing to determine the cell types in the valve tissue.Methods: Approved by the Ethics Committee of Tongji Medical College of Huazhong University of Science and Technology,this study collected aortic valves of recipients undergoing heart transplantation due to non-valvular diseases and patients undergoing active valve replacement surgery due to CAVD.The samples were divided into the control young group,the control elderly group,and the calcified lesion group after ultrasonic examination,inspection by the surgeon,and pathological examination.After physically cutting and dispersing digestive enzymes into a single cell suspension,the process from sending the samples to the machine to collecting cell transcriptome information was completed within 6hours after the samples were obtained.After collecting the data,UMAP clustering and cell typing were performed,also immunofluorescence and immunohistochemical identification on aortic tissues and primary cells were performed.Results: 94,277 cells were obtained from 9 valve tissue cells,and 11 cell subgroups were identified.CD68 and CCL3L1 were used as marker genes to identify valve macrophage cluster 6;and CD3 D and CD3 E were used as marker genes to identify valve lymphocyte cluster 9;The valve endothelial cell cluster 8 was identified with ECSCR and TEK as marker genes.In VICs: LUM and MFAP4 were identified as cluster 0 markers in interstitial cells;FOS and PTN were cluster 2 markers;SPARC and COL1A2 were cluster 3 markers;C7 was cluster 4 marker;SPOCK3 was cluster 7 marker;MT1A and CDH19 were cluster 10 markers.Cluster 1 and cluster 5,the main cells of calcification lesions were identified,and CCL20,RPL17,CMSS1,AKR1C1 and S100A11 were used as their marker genes,respectively.In the control elderly group,the proportion of cluster 0 cells was significantly increased to34.5%.The proportions of cluster 1 and cluster 5 in the calcified lesion group were 47% and30%.Conclusion: There are 11 groups of cells in the valve tissue,respectively,valve lymphocytes,valve macrophages,valve endothelial cells and valve interstitial cells.The valve interstitial cells are divided into 8 clusters:cluster 0,cluster 2,cluster 3,cluster 4,cluster 7,cluster 10,cluster 1,cluster 5.The interstitial cells of the diseased valve tissue are mainly cluster 1 and cluster 5.The proportion of cluster 0 in the interstitial cells of the control elderly aortic valve tissue was significantly increased,which may be the protagonist of the disease.Part 2:Based on single-cell sequencing analysis reveals that inflammation is the key initiating factor for CAVDObjective: To analyze the characteristics of valve interstitial cell subsets and explore the transcription changes of different valve cells under different pathophysiological conditions,recognize the pathophysiological changes of the valve from the single-cell level,and explore the key pathogenic factor.Method: A reasonable and orderly screening of massive single cell data and perform GO and KEGG analysis for each cell types,PAGA differentiation trajectory analysis and Monocle pseudo-time sequence analysis were used to detect the internal connections between the different cell subgroups,and verification at the tissue level,try to isolate the cells and perform verification in vitro.Results: The study found that valve interstitial cells have a universal extracellular matrix remodeling function.In cluster 0,bone formation is prominent,cluster 2 is prominent in Wnt signaling pathway,cluster 3 is prominent in cartilage differentiation,cluster 4 is prominent in glial cell differentiation,and cluster 7 is prominent in differentiation of myoblasts,cluster 10 has the potential for multi-directional differentiation.Cluster 1 in the main cells of calcification disease is the result of abnormal activation of multiple signal pathways,and cluster 5 has significant metabolic-related signal pathways and autophagy regulation characteristics.Analysis of gene changes in each cell subset showed that immune cells release inflammatory factors and promote the occurrence of aortic valve inflammation,while valve endothelial cells transform into mesenchymal cells to promote inflammation regulation and interstitial remodeling.Up-regulation of Cluster 0 ratio promoted TNF signaling pathway activation and extracellular matrix remodeling.This study verified the differentiation of valve endothelial cells into mesenchyme at the histological level,and isolated valve endothelial cells for in vitro experiments to verify the characteristics of endothelial to mesenchymal transition.Conclusion: The increase in age promotes the decline of valve immune cells and valve endothelial cells,promotes the release of inflammatory factors in the aortic valve,and promotes the occurrence of inflammatory reactions.The transformation of valve endothelial cells to mesenchymal cells makes the protective effect of the aortic valve endothelial layer disappear and accelerates aortic valve disease occur.The 6 populations of valve interstitial cells have universal extracellular matrix remodeling functions but have their own characteristics.Among them,cluster 0 has significant bone formation characteristics.The increase in the proportion of Cluster 0 cell subpopulation promotes the activation of TNF signaling pathway and extracellular matrix remodeling in the aortic valve interstitium.The main cells of calcification lesions are divided into two types: abnormal activation of inflammatory signaling pathways and abnormal activation of metabolism-related signaling pathways.Part 3: Research on the mechanism of andrographolide inhibiting inflammatory response to delay the progression of CAVDObjective: To explore the mechanism of the Andrographolide(AGP)inhibiting the osteogenic differentiation of valve interstitial cells in vitro.Methods: In an cellular pathological model,AGP was given to valve interstitial cells.Alizarin red staining was used to detect the formation of calcium nodules in the cells,and gene detection and protein detection were used to detect the expression of key osteogenic proteins.Use transcriptome sequencing to analyze the main target signal pathways of AGP,and detect the phosphorylation of key proteins in related signal pathways.Results: In the in vitro model,AGP significantly inhibited the formation of calcium nodules.PCR,Western Bloting,and immunofluorescence detection all showed that AGP inhibited the expression of key osteogenic proteins RUNX2 and ALP.Further transcriptome sequencing analysis showed that AGP inhibited the TNF,PI3K-AKT and ERK signaling pathways during the process of osteogenic induction.Protein detection revealed that the phosphorylation of ERK,I?B? and AKT molecules was inhibited,and the activation of the signaling pathway was blocked,which inhibited osteogenesis differentiation of valve interstitial cells.Conclusion: The Andrographolide can inhibit the osteogenic differentiation of valve interstitial cells in vitro,and regulate PI3K-AKT,ERK1/2,NF-kappa B,TNF,etc.Signal pathways by inhibiting the phosphorylation of ERK,I?B? and AKT molecules.