Role Of MiR-206-mediated Phenotypic Conversion Of Vascular Smooth Muscle Cells In The Progression Of Abdominal Aortic Aneurysm

Objectives:The aim of this study was to investigate the link between vascular smooth muscle cells(VSMCs)phenotype conversion and abdominal aortic aneurysm progression,to investigate the specific mechanism by which miR-206 regulates VSMCs phenotype conversion in abdominal aortic aneurysms,and to investigate the role of knockdown of miR-206 in promoting VSMCs conversion to a highly proliferative phenotype to reduce the risk of abdominal aortic dissection and inhibit abdominal aortic aneurysm expansion,in order to provide a new approach and idea for the treatment of abdominal aortic aneurysms.This study provides a novel approach and a new target of the treatment of abdominal aortic aneurysms.Methods:The study is divided into a total of two parts: bioinformatic analysis and in vivo experiments.(1)The aortic aneurysm single cell sequencing dataset GSE155468 was analyzed by the Seurat package,common cell types were annotated by classical cell markers.VSMCs were subsequently extracted individually for re-clustering,and all VSMCs phenotypes present in aortic aneurysms were manually annotated by the definitions of various VSMCs phenotypes and marker profiles in the published literature.Differential analysis of the aortic aneurysm miRNA sequencing dataset GSE110527 was performed by the Limma package to calculate the top 5 highly expressed miRNAs,and the miRNAs regulated by VSMCs phenotypes were identified by literature search.Cell-cell communication was analyzed using Cell Chat packets.(2)In vivo experiments: mice model of AAA was constructed by infiltration of elastase solution and oral administration of 0.2% BAPN solution for 4 weeks to form abdominal aortic aneurysms.Differences in miRNA expression levels between the AAA model group and the sham-operated group were quantified by qPCR.Immunofluorescence staining,qPCR,and western blot were used to analyze the difference in expression of VSMCs markers(ACTA2,CD68)between the AAA model group and the sham-operated group.(2)Using AAV-miR-206-sponge,miR-206 was knocked down in mice to observe its effect on AAA and VSMCs phenotypes.The inner diameter of aorta was measured by ultrasound in mice,and the outer diameter was measured in gross specimens.VSMCs markers were analyzed by immunofluorescence,qPCR and western blot.(3)Establishment of mouse VSMCs lineage tracing model Myh11-Cre ERT2-tdTomato mice by crossing Myh11-Cre ERT2-tdTomato mice and Rosa26-tdTomato mice.Results:A total of 42,611 cells were identified as CD4+ T cells,CD8+ T cells,VSMCs,monocytes,macrophages,fibroblasts,endothelial cells and B cells.VSMCs were further classified as contractile VSMCs,synthetic VSMCs,macrophage-like VSMCs,mesenchymal VSMCs,adipocyte-like VSMCs and T cell-like VSMCs.The proportion of synthetic VSMCs and macrophage-like VSMCs was significantly higher in aortic aneurysms.The top5 highly expressed miRNAs in aortic aneurysms included: hsa-miR-3148,hsa-miR-670-5p,hsamiR-206,hsa-miR-7844-5p,and hsa-miR-32-3p.A search of the literature showed that hsa-miR-206 was highly associated with VSMCs phenotype conversion.Compared with the sham surgery group,RT-PCR,western blot and immunofluorescence experiments revealed changes in the transcript levels and protein levels of VSMCs markersαSMA and CD68 in the AAA model group.Immunofluorescence showed a decrease inαSMA fluorescence intensity in the AAA model group,but an increase in the number ofαSMA+ cells,together with the presence of ACTA2+CD68+ cells.In the mouse AAA model,transfection with AAV-miR-206-sponge knocked down miR206 and reduced rupture rate,decreased diameter and enhanced VSMCs phenotypic transformation were observed in the transfected group.Lineage tracing experiments showed that the tdTomato+region in normal aorta completely overlapped with the αSMA+ region,whereas some tdTomato+ cells in the AAA model did not express αSMA,while the percentage of tdTomato+CD68+ cells increased in the AAA model.Transfection with miR-206-sponge further increased the proportion of tdTomato+αSMA-cells and tdTomato+CD68+ cells.Conclusion:VSMCs undergo phenotypic switching in abdominal aortic aneurysms,with the contractile VSMCs dedifferentiating into synthetic VSMCs and macrophage-like VSMCs.miR-206 is highly expressed in abdominal aortic aneurysms and exerts an inhibitory effect on VSMCs phenotype switching by suppressing KLF4 expression.The conversion of VSMCs to synthetic phenotype may play a protective role in the early stage of AAA formation.The high proliferation capacity of synthetic VSMCs thickens the aortic media,thus inhibiting the progression of AAA and preventing the rupture of AAA.