Extended Data Fig. 8: Phenotypic characterization of endothelial cells in healthy and cirrhotic human livers.
From: Resolving the fibrotic niche of human liver cirrhosis at single-cell level
a, Clustering and selected genes expressed in 8,020 endothelial cells from healthy (n = 4) and cirrhotic (n = 3) human livers. b, Scaled gene expression of endothelial cluster markers across endothelial cells from healthy (n = 4) and cirrhotic (n = 3) livers. c, Top, digital pixel quantification of PLVAP immunostaining of cirrhotic liver sections (n = 10) in parenchymal nodules and fibrotic septae. Bottom, ACKR1 immunostaining of cirrhotic liver sections (n = 10) in parenchymal nodules and fibrotic septae. d, Flow cytometry analysis of PLVAP, CD34 and ACKR1 in endothelial cells from healthy (n = 3, grey) or cirrhotic (n = 7, red) livers. Top, representative histograms; bottom, MFI values. e, Flow-based adhesion assay. Peripheral blood monocytes assessed for adhesion to primary human liver endothelial cells (top) and the percentage of adherent monocytes that transmigrate (bottom); endothelial cells isolated from healthy (n = 5) or cirrhotic (n = 4) livers. f, Endothelial cell gene knockdown. Cirrhotic endothelial cells were treated with siRNA against PLVAP (n = 6) or ACKR1 (n = 5) or with control siRNA (n = 6). Top, representative flow cytometry histograms for stated markers, with comparison to isotype control antibody. Bottom, flow-based adhesion assay, with PBMCs assessed for adhesion (bottom left) and the percentage of adherent cells that transmigrate (bottom right) after siRNA treatment of endothelial cells. g, Top left, self-organizing map (60 × 60 grid) of smoothed scaled metagene expression of endothelia lineage. In total, 21,237 genes, 3,600 metagenes and 45 signatures were identified. A–E denote metagene signatures overexpressed in one or more endothelial subpopulations. Bottom left, smoothed mean metagene expression profile for each endothelial subpopulation. Middle, radar plots of metagene signatures A–E showing distribution of signature expression across endothelial subpopulations, exemplar genes (middle) and Gene Ontology enrichment (right). h, Heat map of endothelial subpopulation transcription factor regulon expression (colour-coded by cluster and condition) across 8,020 endothelial cells from healthy (n = 4) and cirrhotic (n = 3) human livers. Exemplar regulons are labelled (right). Columns denote cells; rows denote regulons. i, t-SNE visualization of endothelial lineage (8,020 cells from healthy (n = 4) and cirrhotic (n = 3) livers), annotating monocle pseudotemporal dynamics (purple to yellow; grey indicates lack of inferred trajectory). RNA velocities (red arrows) visualized using Gaussian smoothing on regular grid. j, Representative immunofluorescence images (n ≥ 3) of RSPO3, PDPN, AIF1L, VWA1 or ACKR1 (red), CD34 (white), PLVAP (green) and DAPI (blue) in healthy and cirrhotic liver. Scale bars, 50 μm. k, Annotation of 8,020 endothelial cells by subpopulation and injury condition. LSEC, liver sinusoidal endothelial cells. Data are mean ± s.e.m. P values determined by two-tailed Wilcoxon test (c), two-tailed Mann–Whitney test (d, e), Kruskal–Wallis and Dunn test (f), or Fisher’s exact test (g).
