| Background:Lymphedema commonly occurs following lymph node resection for cancer treatment,placing a significant physical and psychological burden on cancer survivors and severely affecting their quality of life.Lymphedema is characterized by progressive swelling,chronic inflammation,excessive fibrosis,and deposition of adipose in the affected limb.Previous studies have shown that the stromal vascular fraction(SVF)in lymphedema undergoes significant changes in cell types,proliferation,and differentiation,but most studies have only focused on known marker genes.An in-depth elucidation of SVF in lymphedema is essential for medical development.Therefore,this study used the subcutaneous adipose tissue of patients with lymphedema as a key point to understand the pathogenesis of lymphedema,combined with single-cell RNA sequencing techniques.This study attempts to investigate cellular heterogeneity,lineage-specific regulatory changes,and cell-cell communication alterations in SVF of subcutaneous adipose tissue from lymphedema,in order to identify the pathogenesis and find novel medical targets or therapies for the treatment of lymphedema.Methods:In this study,single-cell RNA sequencing of SVF from subcutaneous adipose tissue was performed from five patients with lymphedema and four healthy donors.This study identified the cellular heterogeneity through differential expression and functional enrichment analysis,and revealed significantly expanded or contracted cell lineages and subpopulations by differential proportional analysis.This study explored differential gene expression through differential regulatory network analysis and gene set enrichment analysis,and uncovered changes in ligand-receptor interactions through cell-cell communication analysis.This study verified the change in proportion of the key subpopulations of adipose-derived stromal cells(ASCs)in lymphedema using flow cytometry,and showed the subpopulations of ASCs using immunofluorescence staining.Transcriptome analysis was performed to detect the differential gene expression in freshly isolated ASCs from three lymphedema patients and three healthy donors.In order to detect the influence of key genes in ASCs on the fibrosis of adipose tissue during the pathogenesis of lymphedema,gene silencing experiments were performed.In this study,a mouse model of tail lyphedema was developed and targeted drugs were injected to explore the effect of inhibiting key subpopulations of macrophages on lymphedema progression.Results:Single-cell RNA sequencing yielded high-quality data.Unbiased clustering of 70,209 cells revealed 21 cell clusters,which could be assigned to 10 cell lineages.This study identified the heterogeneity of ASCs and macrophages as well as key genes and cell subpopulations in SVF.This study uncovered a perivascular ligand-receptor interaction module and communication changes for ASCs.There were four subpopulations of ASCs,and the PRG4+/CLEC3B+ASCs was extremely expanded in lymphedema.The knockdown of CLEC3B in vitro could significantly attenuate the fibrogenesis of ASCs from patients.Three subpopulations of macrophages were identified,and LYVE1+anti-inflammatory macrophage was significantly contracted in lymphedema.The ratio of anti-inflammatory macrophages and pro-inflammatory macrophages greatly reduced in lymphedema,indicating a pro-inflammatory microenvironment in subcutaneous adipose tissue.Pharmacological blockage of Treml+pro-inflammatory macrophages using mLR12 could significantly alleviate lymphedema in a mouse tail model.Conclusions:This study provided a comprehensive and in-depth elucidation of cellular heterogeneity,lineage-specific regulatory changes,and cell-cell communication alterations of SVF in lymphedema at single-cell resolution for the first time,which also provided valuable single-cell RNA data for further exploration of the pathological mechanisms of lymphedema.This research revealed lymphedema-associated cell subpopulations.This study demonstrated that the knockdown of CLEC3B could significantly attenuate the fibrogenesis of ASCs from patients,and thus CLEC3B could serve as a potential target for alleviating adipose tissue fibrosis in lymphedema.In vivo experiments in mice suggested that pharmacologically blocking TREM1 with LR12 could serve as a promising medical therapy for treating lymphedema.This study had significant implications for the clinical development of lymphedema treatments based on molecular targets.Background: The incidence of coronary artery disease(CAD)is increasing year by year in our country.Its risk factors mainly include obesity,diabetes,hypertension,hyperlipidemia and unhealthy lifestyles,placing a significant financial burden on society and severely affecting the quality of life of patients.A growing nximber of studies have reported the association of pericoronary epicardial adipose tissue(PEAT)with CAD,but none have explored the pathological mechanisms of PEAT in CAD at single-cell resolution.The aim of this study is to explore cellular heterogeneity,differential expression of genes and pathways,and cell-cell communication alterations of PEAT in CAD,in order to identify pathogenesis and find key subpopulations and genes for the treatment of CAD.Methods: In this study,single-nucleus RNA sequencing of PEAT was performed from eight patients of CAD,eight patients of CAD with Type 2 Diabetes Mellitus(T2DM),and eight patients of valvular heart disease.This study explored the cellular heterogeneity and CAD-associated cell subpopulations despite plenty of bioinformatics analysis,such as differential proportion analysis,differential expression analysis,hdWGCNA analysis,and biological theme comparison analysis.Differential expression and functional enrichment analysis identified genes that play an essential role in the progression of CAD.This study uncovered changes in ligand-receptor interactions and pathways across different cell types of CAD through cell-cell communication analysis.Results: Single-nucleus RNA sequencing yielded high-quality data.This study analyzed the cellular heterogeneity of PEAT,which could be assigned to 16 cell lineages.This study identified heterogeneity in adipocytes,macrophages,and adipose-derived stromal cells.Macrophages were included in six subpopulations and NAMPT+/CD83+ macrophages were significantly expanded in CAD.There were five subpopulations of adipocytes,and the ANXAl+ adipocytes were extremely expanded in CAD.CD83 and ANXAl which had previously been strongly expressed in these two subgroups,had effects of inflammation resolution.In this study,it was found that the Circadian Clock pathway is up-regulated in adipocytes in CAD,with the key genes like PER2 and PERI,suggesting that circadian clock genes might play an important role for CAD treatment.In this study,enhanced VISFATIN,ANNEXIN and SEMA3 pathways were found in CAD.Conclusions: This study provided the first elucidation of cellular heterogeneity,differential expression of genes and pathways,and cell-cell communication alterations in PEAT from CAD at single-cell resolution,as well as valuable single-cell RNA data for further exploration of the pathological mechanisms of CAD.The research revealed CADassociated cell subpopulations,such as NAMPT+/CD83+ macrophages and ANXAl+adipocytes.This study prompted the key circadian clock genes such as PER2 and PER1 and key pathways such as VISFATIN,ANNEXIN and SEMA3 in the pathogenesis of CAD.This study played an important role in exploring the key cell subpopulations and genes for the treatment of CAD. |
