图1 动脉粥样硬化小鼠肝脏中PGAM5表达升高

Fig.1 Increased hepatic PGAM5 expression in atherosclerotic mice. A: Immunofluorescence staining with DAPI (blue) and PGAM5 (red) of mouse liver tissues (Original magnification: ×100; inset magnification: ×600). B: Quantitative analysis of relative mean fluorescence intensity (MFI) of PGAM5 in mouse liver tissues (n=4). **P<0.01 vs NC group.

图2 PGAM5缺失改善动脉粥样硬化表型

Fig.2 PGAM5 deficiency attenuates atherosclerosis in mice. A: Oil Red O staining of mouse whole aorta. B: Oil Red O, HE, and Masson staining of mouse aortic root sections (×60). C: Quantification of aortic root lipid deposition with the lesion area presented as percentage of total vessel area (NC, n=3; ND, n=4; WD, n=6; ATOR, n=5; PGAM5 KO, n=3). D: Quantification of aortic root lesion area in HE staining (NC, n=5; ND, n=4; WD, n=6; ATOR, n=5; PGAM5 KO, n=5). ***P<0.001, ****P<0.0001 vs NC group; ####P<0.0001 vs WD group.

图3 PGAM5缺失改善动脉粥样硬化小鼠的肝脏脂质沉积和血浆脂质异常

Fig.3 PGAM5 deficiency alleviates hepatic lipid deposition and plasma lipid abnormalities in atherosclerotic mice. A: Oil Red O staining and HE staining of mouse liver tissues (×20). B: Quantitative analysis of hepatic lipid accumulation area (expressed as the percentage of lipid-positive area relative to total tissue area) (NC, n=7; ND, n=3; WD, n=7; ATOR, n=3; PGAM5 KO, n=5). C: Plasma levels of LDL-C (n=6), TC (n=6), TG (n=6), and HDL-C (NC, n=7; ND, n=6; WD, n=6; ATOR, n=6; PGAM5 KO, n=6) in mice of each group. *P<0.05, ***P<0.001, ****P<0.0001 vs NC group; ##P<0.01, ###P<0.001, ####P<0.0001 vs WD group.

图4 两组肝脏差异蛋白KEGG通路富集分析

Fig.4 Enrichment analysis of differential liver proteins between the two groups using KEGG pathways.

图5 小鼠肝脏单细胞RNA测序分析

Fig.5 Single-cell RNA sequencing analysis of mouse liver tissues. A: UMAP visualization of major hepatic cell clusters in WD and PGAM5 KO groups. B: Marker gene expression used for cell-type annotation. C: Proportional distribution of major cell populations between groups (n=4).

图6 内皮细胞与肝脏其他细胞差异基因的GO与KEGG富集分析

Fig.6 GO and KEGG enrichment analysis of differential genes between endothelial cells and other hepatic cell populations. A-C: Gene Ontology (GO) enrichment analysis of differentially expressed genes in ECs, including biological process (A), cellular component (B), and molecular function (C). D: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes in ECs.

图7 肝脏内皮细胞亚群的转录特征

Fig.7 Transcriptional characteristics of hepatic endothelial subpopulations. A: UMAP dimensionality reduction plot of liver endothelial cells subpopulations. B: Proportional distribution of different endothelial cells subpopulations across samples. C: Heatmap of differentially expressed genes among endothelial cells subpopulations.

图8 肝脏内皮细胞亚群标志基因表达图谱

Fig.8 Expression of marker genes in hepatic endothelial subpopulations. A: UMAP visualization and violin plots of Lyve1, Fcgr2b, Flt4, and Stab2 expression across clusters. B: UMAP visualization and violin plots of Vwf and Pecam1 expression across clusters.

图9 中心静脉内皮细胞标志基因表达图谱

Fig.9 Expression of central vein endothelial cells marker genes. A: UMAP visualization and violin plots of Rspo3 and Wnt9b expression across clusters. B: UMAP visualization and violin plots of Wnt2, Kit, Cdh13, Thbd, Fabp4, Plpp1, Gas6, and Lhx6 expression across clusters.

图10 动脉样内皮细胞标志基因表达图谱

Fig.10 Expression profile of marker genes in arterial-like endothelial cells. A: UMAP visualization and violin plots of Dll4 and Efnb2 expression across clusters. B: UMAP visualization and violin plots of Msr1, Ltbp4, Ntn4, Adam23, Ldb2, Adgrg6, and Aass expression across clusters.

图11 肝窦内皮细胞差异基因的GO与KEGG富集分析

Fig.11 GO/KEGG enrichment analysis of differential genes in LSECs. A-C: Gene Ontology enrichment analysis of differentially expressed genes in LSECs, including biological process (A), cellular component (B), and molecular function (C). D: KEGG pathway enrichment analysis of differentially expressed genes in LSECs.

图12 HLSECs中PGAM5敲降效果验证

Fig.12 Validation of PGAM5 knockdown in HLSECs. A: Representative fluorescence images (×100) showing viral transduction efficiency in HLSECs. B: Western blotting of PGAM5 protein and quantitative analysis (n=3). C: Relative PGAM5 mRNA expression levels (n=3). ****P<0.0001 vs NC group.

图13 oxLDL处理后HLSECs的Bodipy染色结果

Fig.13 Bodipy staining of HLSECs after oxLDL treatment (×200).

图14 PGAM5缺失显著改变AS小鼠血浆代谢物谱

Fig.14 PGAM5 deficiency reshapes plasma metabolite profile in AS mice. A: Relative abundance of L-palmitoylcarnitine. B: Relative abundance of arachidonic acid. C: Pathway enrichment analysis of top differential metabolites (n=5). ##P<0.01, ###P<0.001 vs WD group.

图15 HLSECs对Dil-oxLDL摄取的荧光检测结果

Fig.15 Dil-oxLDL uptake assay in HLSECs (×600).

图16 PGAM5缺失显著改变AS小鼠血浆代谢物谱

Fig.16 PGAM5 deficiency significantly alters the plasma metabolite profile in AS mice. A: Heatmap of the representative differential lipids. B: Distribution of differential lipid classes. C: Distribution of differential lipid subclasses.