| BackgroundAs the starting trunk of the systemic circulation system,the aorta has a high cushioning capacity,transforming the high-pressure pulsating blood flow output by the left ventricle into a moderate-pressure stable blood flow.According to the anatomical structure,the aorta is divided into two parts,the thoracic aorta and the abdominal aorta,bounded by the diaphragm.The thoracic aorta can be further subdivided into the ascending aorta,aortic arch,and part of the descending aorta,and the abdominal aorta is divided into the suprarenal abdominal aorta and the infrarenal abdominal aorta bounded by the renal artery.From the perspective of embryonic development,the embryonic origin of aorta has regional specificity and diversity,especially vascular smooth muscle cells.The abdominal aorta arises from the somites of the paraxial mesoderm,whereas the ascending aorta arises from the cardiac neural progenitors of the neuroectoderm.In addition,there are complex and variable hemodynamic properties in the aortic lumen,including spatial gradient changes of blood flow shear stress,wall shear stress,and oscillatory shear index.These index changes directly affect the functional regulation and phenotypic transformation of endothelial cells.Moreover,turbulent flow in aortic branches and laminar flow in straight lumens are associated with susceptibility to different aortic diseases.Therefore,the unique anatomical and embryological basis of the aorta creates its unique vascular microenvironment.It has become an important evidence for revealing the heterogeneity of aortic cell composition and the corresponding pathophysiological mechanism.Previous studies have shown that different vascular diseases have different susceptibility sites,such as aneurysm and atherosclerosis.Abdominal aortic aneurysm(AAA)is a serious aortic disease with high morbidity,high risk of death,difficult detection,and lack of effective treatment drugs.The most classic and generally accepted animal model of abdominal aortic aneurysm is to implant a subcutaneous injection pump in high-fat-fed ApoE-/-mice and continuously infuse high-dose angiotensin Ⅱ.However,more than 95%of the mice only caused local aneurysm lesions in the abdominal aorta under the action of non-selective systemic pumping of angiotensin Ⅱ,and the proportion and severity of lesions were significantly higher than those in other parts of the aorta.The same risk factor that acts on the entire circulatory system leads to the generation of local lesions.According to existing studies,the reasons for the selectivity of the disease site and the mechanism of the disease cannot be explained.In terms of atherosclerosis,in the early and late stages of atherosclerotic plaque,the lesions in the aortic arch and vascular branches appeared earlier and more severely than the other parts.These phenomena indicated that there might be great differences in cell types and gene expression in different parts of aorta.It may be an important reason why the same risk factor has great differences in the pathological changes of adjacent blood vessels.In previous studies,when constructing single-cell atlases of all major organ tissues in humans or mice,the cellular composition and gene expression of aorta was often ignored.However,the existing single-cell atlas of normal aorta only provided basic transcriptome analysis and preliminary defined cell subtypes,lacking aortic heterogeneity analysis and regional division.And most studies focused on endothelial cell subtypes and ignored the major cell types such as vascular smooth muscle cells.Additionally,single-cell analysis of aortic disease often overlooked disease susceptibility in distinct locations such as the thorax and abdomen.Regarding the regional heterogeneity of the aorta,single-cell atlases of different segments of the aorta under normal or pathological conditions have not been reported.Therefore,it is of great scientific significance and clinical value to construct an aortic single-cell transcriptome atlas that simulate spatial distribution and identify cell subsets that specifically exist in different regions.ObjectivesThe purpose of this study is to define and classify aortic cell subtypes more precisely by single-cell transcriptome sequencing technology.Focus on the spatial distribution characteristics of aortic cell composition and gene expression and conduct relevant verification experiments to deeply study the relationship between aortic heterogeneity and susceptibility to different vascular diseases.Explore potential new intervention targets for the prevention and treatment of cardiovascular diseases.Methods1.Preparation of segmented single cell suspension from healthy mouse aortaThe aortas of 12-week-old C57BL/6J mice were dissected according to the anatomical structure,and the single cell suspensions were prepared by simultaneous digestion and dissociation.The quality of the single cell suspension was determined by trypan blue staining and fluorescent cell counting.The overall cell viability was more than 90%and the cell concentration was adjusted to 700-1200 cells/μl before single cell analysis.2.Single-cell sequencing and data analysisSingle-cell suspensions were loaded to 10x Chromium to capture 5000 single cells according to the manufacturer’s instructions of 10X Genomics Chromium Single-Cell 3’kit(V3).The following cDNA amplification and library construction steps were performed according to the standard protocol.Libraries were sequenced on an Illumina NovaSeq 6000 sequencing system at a minimum depth of 20,000 reads.Data quantification and identification of cell expression matrix were processed by CellRanger software.The R package Seurat was used for quality control and cell filtering,data dimensionality reduction,marker gene analysis,cell type and subtype definition.GO and KEGG differential gene enrichment analysis were performed and Monocle2 was used for pseudo-chronological analysis.WGCNA was selected for cell co-expression gene modules analysis and CellPhoneDB was used for cell interaction analysis.3.Histological staining and immunofluorescence validation experiments of mouse aortaParaffin sections of mouse aortic tissues were prepared.The structure of vascular wall and the integrity of elastic fiber in each segment of mouse aorta were detected by HE and Weigert staining.The immunofluorescence colocalization method was used to verify the specific gene expression of cell subtypes in different sections of mouse aortic tissue.Results1.Aortic single-cell transcriptome atlas that simulate spatial distribution was constructedThe aorta was divided into five segments,aortic arch(AOAR),thoracic aorta 1(TA1),thoracic aorta 2(TA2),abdominal aorta 1(AA1),and abdominal aorta 2(AA2).A total of 26,257 high-quality cells were captured,with an average of 5,250 cells per section.Eighteen cell subpopulations were obtained by clustering with unsupervised cluster analysis.Eight cell types were identified by marker gene expression,including endothelial cells,vascular smooth muscle cells,fibroblasts,and macrophages,etc.2.ECs and fibroblasts showed segmental differentially expressed genesAccording to re-clustering analysis and comparison of marker genes,endothelial cells were divided into two cell subpopulations,EC 1 and EC 2.There was no statistical difference in the number and proportion of EC 1 subtypes among the five segments,but there were abundant differentially expressed genes.The EC 2 subtype had a relatively high proportion of cells in the abdominal aorta(49.15%),but its differential gene expression was not significant.EC 1 highly expressed gene Vcaml and Scarb2 in AOAR segment specifically.Collagen-related genes,such as Colla1,were enriched in EC 1 of AA1 segment.The enrichment analysis of differentially expressed genes(DEGs)also reflected that EC 1 was mainly enriched in signaling pathways such as cell adhesion and antigen processing and presentation in the AOAR segment.The expression of Vcaml and Scarb2 in endothelial cells of different segments was verified by immunofluorescence colocalization staining,and the conclusion was consistent with the single-cell data.According to differential gene expression and functional analysis,fibroblasts were defined as two cell subtypes,Fibro 1 and Fibro 2.The distribution of Fibro 1 in the aortic arch segment was relatively high(36.92%).The distribution of Fibro 2 in each segment was relatively uniform.It was found that Fbnl was specifically highly expressed in the fibroblast subtype Fibro 1 by comparing with all the other cell subtypes.Moreover,Fbn1+fibroblasts were mainly distributed in the thoracic aorta,especially in the AOAR segment,which was verified by immunofluorescence colocalization detection.In addition,Fibro 1 also highly expressed inflammation and chemotaxis-related genes such as 116 and Cxcl12 in the AA segment.The differentially expressed genes of Fibro 2 were mainly concentrated in the AOAR segment.3.Identification of a novel constitution system of VSMC subpopulationsA more detailed classification of healthy mouse aortic smooth muscle cells was performed and six distinct cell subtypes were finally identified,including VSMC 1 to 5 and Sca1+ VSMC.The definition of VSMC 1 to 5 is determined by specific differentially expressed genes and functional characteristics.The marker gene of VSMC 1(Cluster 0,1,2,4)is Ramp1,and its functional analysis shows the characteristics of smooth muscle contraction.The marker gene of VSMC 2(Cluster 5)is Vim,which is mainly related to inflammation and blood shear response.The marker gene of VSMC 3(Cluster 6)is Camk2d,and the gene expression characteristics are mainly cell senescence and apoptosis.However,functional enrichment analysis found that VSMC 2 and VSMC 3 have some similar functional characteristics.The marker gene of VSMC 4(Cluster 8)is Malatl,and its functional characteristics are mainly related to the function of extracellular matrix.The marker gene of VSMC 5(Cluster 16)is Mrcl,and the gene expression has an immune response function similar to that of monocytes.Smooth muscle progenitor Sca1+VSMCs were present in small amounts in all segments and maintained stem cell-like properties.4.Pseudotime analysis of three VSMC subtypes characterized with gradient-distributionVSMC 1,2,3 showed gradient changes in spatial distribution across the five segments.The number of VSMC 2 decreased from the AOAR to the AA2 segment and was concentrated in the AOAR section(81.94%).In contrast,VSMC 4 showed an increasing trend and was mainly distributed in the abdominal region,particularly in the AA2 section(70.39%).The transition state VSMC 3 had a smaller amplitude of variation and gradually increased from the thoracic to the abdominal part.Pseudotime analysis of VSMC subtypes found that there were three different cell differentiation directions.And the distribution of subtypes dominates the direction of segmental cell differentiation.Among them,VSMC 2 and 4 developed into two totally different differentiation directions,marked with gene Vim and Malat1,respectively.Conclusions1.A total of 18 cell subpopulations were obtained from the five segments of the aorta,and eight cell types were identified.Aortic cell composition and gene expression showed obvious spatial distribution characteristics and heterogeneity.2.Endothelial cell subtype EC 1 specifically highly expressed Vcaml and Scarb2 at the AOAR,and DEGs were enriched in signaling pathways such as cell adhesion and antigen processing and presentation.Fbn1+fibroblasts were specifically localized to the Fibro 1 subtype and concentrated in the AOAR segment of the thoracic aorta.3.Vascular smooth muscle cells were rich in composition including VSMC 1 to 5 and Sca1+VSMC.VSMC 2,3,and 4 had significant segmental gradient distribution characteristics.BackgroundAbdominal aortic aneurysm(AAA)is a potentially fatal large vessel disease,characterized by high morbidity,high mortality,low detection rate,and low control rate.Once rupture occurs,the mortality rate is as high as 85%to 90%.So far,there is no effective drug treatment for AAA,and clinical interventions are mainly open surgery or endovascular repair.AAA is characterized by extensive degradation of the aortic wall matrix and chronic aortic wall inflammation,leading to arterial wall remodeling and eventual rupture,in which phenotypic switching of vascular smooth muscle cells(VSMCs),matrix metalloproteinases(MMPs)and inflammatory factors play a key role.Vascular smooth muscle cells are very important for maintaining vascular homeostasis.Loss of aortic tissue structure leads to weakening of vessel walls and dilation of the aorta.Although other cell types,including endothelial cells,neutrophils,macrophages,and lymphocytes,are involved in aortic aneurysm development,VSMCs dysfunction is critical for the loss of structural integrity of the aortic wall.Loss of VSMCs,phenotypic switching,secretion of inflammatory cytokines,increased activity of matrix metalloproteinases,elevated reactive oxygen species,defective autophagy,and increased aging all aggravated AAA development.It is necessary to further study the pathogenesis of aortic aneurysm and elucidate the underlying signaling pathways to identify more novel targets for the treatment of abdominal aortic aneurysm.Although studies have confirmed that VSMCs dysfunction including apoptosis and phenotypic switching are involved in the pathological process of AAA,there is a lack of research exploring the regulatory mechanism at the single-cell level.Metastasis Associated Lung Adenocarcinoma Transcript 1(MALAT1)is a widely studied cancer IncRNA associated with various signaling pathways such as MAPK/ERK,PI3K/AKT,β-catenin/Wnt,and VEGF.MALAT1 plays an important role in regulating cancer progression such as proliferation,invasion and metastasis.Cardiovascular disease(CVD)is a leading cause of human morbidity and mortality worldwide.Recent studies in the field of lncRNA MALAT1 pave the way for new therapeutic targets to prevent and even treat cardiovascular diseases.Studies have found that MALAT1 regulates various pathophysiological processes such as apoptosis,autophagy and pyroptosis under conditions of hypoxia,high glucose,cytokines and oxidative stress.In addition,MALAT1 plays an important role in inflammatory responses,macrophage dysfunction and transformation,and other immune system diseases.The segmental single-cell transcriptional atlas of normal aorta in the first part of this study found that Malat1+VSMCs were highly specific in normal abdominal aortic segments.This cell subtype may be the key to the susceptibility of AAA in abdominal aortic segment under AngⅡ-induced conditions.So far,none of the existing studies revealed the role and mechanism of Malat1 in the AngⅡ-induced abdominal aortic aneurysm model.In the second part,we will further explore the molecular mechanism of Malatl in AAA through in vivo and in vitro experiments,in order to find new targets for the prevention and treatment of abdominal aortic aneurysm.Objectives1.Verify the expression of in healthy mouse aorta and AAA tissue.2.To explore the role and pathogenesis of Malatl in AngⅡ-induced AAA formation.3.Evaluate the protective effect of Malat1 inhibitors on AAA in vivo.4.To evaluate the protective effect and mechanism of Malatl inhibitor on AAA at single cell level.Methods1.Construction of mouse aortic aneurysm model and statistics analysisThe aortic aneurysm model of AngⅡ-induced ApoE-/-mice with high-fat feeding was used,and the implanted osmotic pump was continuously pumped AngⅡ at a rate of 1000ng/kg/min for 28 days.The proportion of aortic aneurysms in each segment(aortic arch,thoracic aorta,abdominal aorta)were observed and evaluated.2.Animal experiments grouping of the inhibitorPrevention experiment:80 male ApoE-/-mice were randomly divided into 4 groups(DMSO group,inhibitor group,4-week AngⅡ+DMSO group,4-week AngⅡ +inhibitor group),20 in each group,and given high High-fat and high-cholesterol feeding.Malatl inhibitor was injected for 2 weeks before AngⅡ pumping until the end of modeling.Inhibition experiment:80 male ApoE-/-mice were randomly divided into 4 groups(DMSO group,inhibitor group,4-week AngⅡ+2-week DMSO group,4-week AngⅡ+2-week inhibitor group),20 in each group,and given high-fat and high-cholesterol feeding.During the Ang Ⅱ pumping process,the inhibitors in the inhibitor groups began to be injected in the last 2 weeks until the end of modeling.Reversal experiment:60 male ApoE-/-mice were randomly divided into 3 groups(control group,2-week AngⅡ+2-week DMSO group,2-week AngⅡ+2-week inhibitor group),20 in each group,and given High-fat and high-cholesterol feeding.After 2 weeks of AngⅡ infusion,the pump was removed to terminate the AngⅡ infusion,and the inhibitor or DMSO were injected for 2 weeks respectively.3.Construction of Malat1 knockout cell linePrepare appropriate density MOVAS cells before lentivirus infection.Set up an appropriate virus concentration gradient for lentivirus infection.Monoclonal selection was performed using puromycin after 24 h virus infection.The diluted cells were plated into 96-well plates by the gradient dilution method,and single clones were selected for gene sequencing.According to the sequencing results,the single clones knocked out of the target gene were screened and amplified,and the expanded culture was used for the next experiment.4.Bulk-RNA sequencing of Malat1 knockout cellsVascular smooth muscle cells in the Malat1 gene knockout group(KO group)and control group(WT group)were subjected to bulk-RNA sequencing at physiological levels and AngⅡ stimulation respectively,to detect the corresponding gene expression changes.5.Cellular RNA extraction,reverse transcription and real-time fluorescent quantitative PCRRNA was extracted from MOVAS cells.The TAKARA reverse transcription kit was used for mRNA reverse transcription,and RT-PCR were applied to get Ct values of targeted genes.Using β-actin as internal parameter,the Ct values were calculated by 2-△△CT formula.6.Western blot analysisAorta tissue protein of mice in each group were extracted by protein extraction kit,and protein of MOVAS cells was extracted by cell lysate.After the protein concentration was adjusted by BCA,SDS-PAGE gel electrophoresis was performed to detect the target protein contents.7.IHC,FISH and WISH staining of mouse aorta tissueParaffin sections of mouse aortic tissues were prepared.The expression difference of the target protein in mouse aortic tissue was detected by IHC staining.In situ hybridization was detected using the Malatl target probe and RNAScope 2.5 HD kit-RED,and the dapB probe was used as a negative control.Malatl expression was detected in lung tissue as a positive control.Whole-Mount fluorescence in situ hybridization is a reference method for embryonic vascular permeabilization and uses the same probes and kits as in FISH staining.8.Statistics AnalysisThe FindAllMarkers function in Seurat was used to identify the marker genes of cell subpopulations in the single-cell sequencing data,the minimum logarithmic fold change threshold was 0.25,and the p-value was calculated using the Wilcoxon rank sum test.Gene set scores and expression signatures were compared between cell populations using the Mann-Whitney U test.All statistical analyzes were performed using GraphPad Prism 8(GraphPad,San Diego,CA),each experiment was independently repeated at least three times,and all data are presented as mean ± SEM.In all statistical comparisons,p-values<0.05 were considered statistically significant.Results1.Validation of Malat1+VSMCs in mouse healthy aorta and AAA tissue The number of Malat1+VSMCs in the AA segment was much higher than that in the thoracic aorta segment by FISH detection.In the AngⅡ-induced mouse abdominal aortic aneurysm model,the expression level of Malatl in AAA tissues was also much higher than that in healthy abdominal aorta,especially in the aneurysm part.2.Analysis of human AAA tissue RNA-seq data to verify the expression of MALAT1 Through online analysis of RNA-seq data from two public databases(GSE47472,GSE57691),it was found that the relative expression level of MALAT1 in the AAA group was significantly higher than that in the healthy control group,and the expression of MALAT1 increased when the tumor size was small.3.Inhibition of Malat1 expression under AngⅡ stimulation can effectively protect vascular smooth muscle cellsThe effects of inhibiting the expression of Malat1 on the levels of MMPs and inflammatory factors in vascular smooth muscle cells were detected and verified by transcriptome sequencing,Western Blot and RT-PCR.The results showed that both the knockout of Malat1 gene and the use of inhibitors significantly reduced the levels of MMPs and inflammatory factors in VSMCs at physiological levels and under AngⅡ-stimulated conditions.4.Malat1 inhibitors can effectively prevent,inhibit and reverse the occurrence and progression of abdominal aortic aneurysmsDifferent AngⅡ and inhibitor time point strategies were used for comparison.Based on CT angiography imaging,AAA evaluation related indicators(survival curve,AAA incidence,aortic diameter,etc.)and histopathological results.It is found that Malatl inhibitor can effectively prevent,inhibit and reverse AngⅡ-induced AAA,improve the survival rate of mice,improve the remodeling of vascular structure,reduce the damage of elastic lamina,reduce the expression of MMPs and the infiltration of inflammatory factors,which is its clinical application Therapeutic value provides new evidence.5.The protective effect and target verification of Malat1 inhibitor on AAA at the single cell levelThe cell composition and gene expression changes in AAA under pathological conditions were detected from the single cell level.The protective effect of Malatl inhibitor on AAA was verified,and the subtype changes of VSMCs and the dominant subtype target were Malat1+VSMCs.Conclusions1.The expression of Malat1+ VSMCs in abdominal aorta of healthy mice,AAA tissue and human AAA tissue was significantly increased.2.Malat1 knockout in mouse smooth muscle cells and Malat1 inhibitor significantly reduced the expression of MMPs and inflammatory factors induced by AngⅡ.3.Malat1 inhibitor can effectively prevent,inhibit and reverse AngⅡ-induced AAA.4.Single-cell level detection verified the changes in cell composition and gene expression during AAA,indicating that Malat1+VSMCs played a dominant role,and verified the protective effect of Malat1 inhibitors on AAA. |
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