A single-cell atlas of the normal and malformed human brain vasculature

Abstract

Cerebrovascular diseases are a leading cause of death and neurologic disability. Further understanding of disease mechanisms and therapeutic strategies requires a deeper knowledge of cerebrovascular cells in humans. We profiled transcriptomes of 181,388 cells to define a cell atlas of the adult human cerebrovasculature, including endothelial cell molecular signatures with arteriovenous segmentation and expanded perivascular cell diversity. By leveraging this reference, we investigated cellular and molecular perturbations in brain arteriovenous malformations, which are a leading cause of stroke in young people, and identified pathologic endothelial transformations with abnormal vascular patterning and the ontology of vascularly derived inflammation. We illustrate the interplay between vascular and immune cells that contributes to brain hemorrhage and catalog opportunities for targeting angiogenic and inflammatory programs in vascular malformations.

Fig. 1.. Cells of the human cerebrovasculature.

(A) Isolation and cell sampling from human cerebral cortex. scRNA, single cell mRNA. (B) UMAP visualization showing cell states from control adult cerebrovasculature (n=5 donors). (C) Dot plot showing expression of cell state markers. EC, endothelial cell; PC, pericyte; SMC, smooth muscle cell; FB, perivascular fibroblast; FbM, fibromyocyte; Mϕ, macrophage; TC, T cell; BC, B cell; Neu, neuron; AC, astrocyte; MG, microglia; OL, oligodendrocyte; OPC, oligodendrocyte precursor cell; RBC, erythrocyte. (D) Bar graph showing cell state proportion by donor. Number of cells sequenced by donor: control1, 6,033 cells; control2, 25,730 cells; control3, 22,816 cells; control4, 19,302 cells; control5, 654 cells. (E) UMAP visualization of endothelial cell states. Art, arterial; Cap, capillary. (F) Dot plot showing expression of endothelial cell state markers. (G) Correlation matrix of gene expression profiles between mouse and human cerebrovascular endothelial cell states. Mouse data obtained from a previously published database. Art, arterial; Cap, capillary; SS, shear stress. (H) UMAP visualization of perivascular cell states. FB, perivascular fibroblast; FbM, fibromyocyte; PC, pericyte; SMC, smooth muscle cell. (I) Dot plot showing expression of perivascular cell state markers.

Fig. 2.. Spatial RNA analysis resolves the cells of the human cerebrovasculature.

(A) UMAP visualization of spatially defined cerebrovascular cell gene expression profiles identified by multiplexed, iterative single molecule fluorescent in situ hybridization (smFISH). RNA molecules were quantified and assigned to cells via automated spot detection and nuclei segmentation. aEC, arterial endothelial cell; cEC, capillary endothelial cell; vEC, venous endothelial cell; FbM, fibromyocte; PC, pericyte; SMC, smooth muscle cell. (B) Dotplot showing expression of cell state markers. (C) Representative high-magnification microscopy images of merged smFISH and expression distribution of cell state markers projected on UMAP embeddings from (A). DAPI (blue) stains cell nuclei. Scale bar: 10 μm. (D) Representative merged smFISH images showing cellular expression of CLDN5 (magenta, endothelial cells), VEGFC (cyan, arterial endothelial cells), MFSD2A (green, capillary endothelial cells), ACKR1 (yellow, venous endothelial cells), and PDGFRB (red, mural cells). DAPI (blue) stains cell nuclei. Left panel, artery; *, endothelial cell co-expressing CLDN5 (magenta) and VEGFC (cyan). Scale bar: 20 μm. Middle panel, capillary; *, endothelial cell co-expressing CLDN5 (magenta) and MFSD2A (green); arrow, PDGFRB-expressing pericyte (red). Scale bar: 15 μm. Right panel, vein; *, endothelial cell co-expressing CLDN5 (magenta) and ACKR1 (yellow). Scale bar: 15 μm.

Fig. 3.. Cellular aberrancy in the malformed cerebrovasculature.

(A) Isolation and cell sampling from human brain arteriovenous malformations (AVMs). scRNA, single cell mRNA. (B) UMAP visualization showing cell states from AVMs (n=5 donors). (C) Bar graph showing cell state proportion by donor. Number of cells sequenced by donor: AVM1, 26,122 cells; AVM2, 28,868 cells; AVM3, 14,541 cells; AVM4, 26,660 cells; AVM5, 10,662 cells. EC, endothelial cell; SMC, smooth muscle cell; FB, perivascular fibroblast; FbM, fibromyocyte; Myl, myeloid cells; TC, T cell; BC, B cell; Neu, neuron; AC, astrocyte; MG, microglia; OL, oligodendrocyte; CP, choroid plexus. (D) Representative microscopy image of single molecule fluorescent in situ hybridization showing expression of CLDN5 (yellow, endothelial cells (EC)), TAGLN (magenta, smooth muscle cells (SMC)), COL1A2 (red, perivascular fibroblasts (FB)), and CCL19 (cyan, fibromyocytes (FbM)). DAPI (blue) stains cell nuclei. Boxes highlight representative cells. Scale bar: 50 μm. i, TAGLN⁺ smooth muscle cell. ii, CCL19⁺ and TAGLN⁺ fibromyocyte. iii, CLDN5⁺ endothelial cell. iv, COL1A2⁺ perivascular fibroblast. Inset scale bars: 5 μm. (E) Left, schematic describing computational pipeline. Endothelial cells (orange) are identified in silico by marker expression, co-embedded for downstream analytics, and iteratively clustered. An identical workflow was applied to perivascular cells (fig. S7). Middle, UMAP visualization of co-embedded endothelial cell states in control (gray) and AVMs (red). Right, UMAP visualization of iteratively clustered endothelial cell states. Art, arterial; Cap, capillary; Vn, venous; Nd, nidus. (F) Upset plot of differentially expressed genes (DEGs) (horizontal bars) by cell class. Number of DEGs exclusive to one cell class (black circles) or shared between multiple cell classes (linked black circles). Vertical bars show the number of genes per intersection. (G) Heatmap visualization of arteriovenous transcriptional identity in control (top, CTRL) and AVM (bottom) endothelial cell states. Art, arterial; Cap, capillary; Vu, venule; Vn, venous; Nd, nidus. Exp., expression, Blue, low expression; Yellow, high expression. (H) Upset plot showing intersections of DEGs in AVM endothelial cell states compared to controls. (I) UMAP visualization of AVM endothelial cell RNA velocity reveals two divergent trajectories from Nd1 (yellow). Upregulation of PLVAP and PGF occurs with endothelial Nd1-to-Nd2 transitions. Exp., expression. (J) Gene set enrichment analysis of DEGs in AVM endothelial Nd2. padj, false discovery rate adjusted P-value; NES, normalized enrichment score. (K) Dotplot showing top marker gene expression for control capillary and AVM Nd2 endothelial cells. Avg. Exp., average expression; Exp., expression (L) Violin plot of PLVAP expression showing specificity to AVM Nd2. (M) Representative-confocal microscopy analysis of PLVAP- (yellow) and ANGPT2-positivity (magenta) in PECAM1⁺ endothelial cells (cyan) in AVM nidus. Vessel shown in cross section. Colocalization of fluorescence results in white coloration. Scale bar: 50 μm.

Fig. 4.. Cerebrovascular inflammation with malformation.

(A) UMAP visualizations of co-embedded immune cells states in control cerebrovasculature and brain arteriovenous malformations (AVMs) (n=5 donors per condition). Top, colored by condition; control, gray; AVM, red; Bottom, colored by cell state. pvMϕ, perivascular macrophage; pvMϕ*, activated perivascular macrophage; Mo, monocyte; MG, microglia; ExV, ex vivo activated myeloid cells; cDC, conventional dendritic cells; pDC, plasmacytoid dendritic cells; CD8 TC, CD8⁺ T cells; CD4 TC, CD4⁺ T cells; T_reg, regulatory T cells; NK, natural killer cells; BC, B cells; Div, dividing immune cells. (B) Dot plot showing expression of immune cell markers. (C) Pie charts showing immune cell state proportions in controls (left) and AVMs (right). (D) Bar graph showing proportion of myeloid and lymphoid immune cells captured in AVMs (red) and controls (gray). n=5 donors per condition, Mean ± SEM, two-tailed t-test.*P< 0.05; ns, not significant. (E) Bar graph of relative immune cell state proportions normalized to total cells sequenced in AVMs (red) and controls (gray). (F) Representative confocal microscopy analysis of endothelial cells (cyan, podocalyxin (PODOXL)), smooth muscle cells (blue, smooth muscle α−actin (SMA)), macrophages (magenta, ionized calcium binding adaptor molecule 1 (IBA1) which is encoded by the gene AIF1 in (B)) and microglia (green, purinergic receptor P2Y12 (P2Y12)). Scale bar: 1 mm. i, layered ameboid perivascular macrophages. ii, perivascular microglial response. Inset scale bars: 50 μm. (G) Quantification of abundance (top left), cell ratio per image (top right) and perivascular distance (bottom) of IBA1⁺P2R12⁻ macrophages and IBA1⁺P2R12⁻ microglia (n=3 donors per condition; three non-adjacent sections per donor; 8–10 images per section). Mean ± SEM, two-tailed t-test.**P<0.01; ns, not significant.

Fig. 5.. Cell states implicated in brain arteriovenous malformation rupture.

(A) Cellular deconvolution and cell-specific differential expression analysis of bulk RNAseq from ruptured and unruptured brain arteriovenous malformation (AVM). (B) Bar graph of change in cell proportion t-statistic in ruptured AVMs. Purple, increased cell abundance. Blue, decreased cell abundance. *P<0.05; **P<0.01. (C) Representative confocal microscopy imaging showing GPNMB⁺ monocytes (green), endothelial cells (cyan, podocalyxin (PODOXL)), and smooth muscle cells (magenta, smooth muscle α actin (SMA)) in unruptured and ruptured AVMs. Scale bar: 100 μm. (D) Quantification of GPNMB⁺ monocytes in unruptured (blue) and ruptured (purple) AVMs (n=3 donors per condition; three non-adjacent sections per donor; 8–10 random images per section). Mean ± SEM, two-tailed t-test.*P< 0.05. (E) Top, Confocal microscopy analysis of cleaved caspase-3⁺ (green, CC3) human primary smooth muscle cells following co-culture with GPNMB⁺ and GPNMB⁻ monocytes isolated from ruptured AVMs. DAPI (magenta) stains cell nuclei. White, colocalization of CC3 and DAPI. Arrow, CC3⁺ cell. Scale bar: 20 μm. Bottom, quantification of CC3⁺ smooth muscle cells (n=3 independent cultures per condition). Mo, monocytes. Mean ± SEM, ANOVA with Tukey post-hoc test. *P<0.05; ns, not statistically significant. (F) Scatterplot of dysregulated cell communication pathways in AVM GPMNB⁺ monocytes (Mo3) relative to controls by scRNAseq. Red, upregulated in AVM; Blue, upregulated in control; Gray, shared between conditions. Triangle, outgoing network; square, incoming network; diamond, outgoing and incoming network. (G) Top, Confocal microscopy analysis of cleaved caspase-3⁺ (green, CC3) human primary smooth muscle cells treated with osteopontin (OPN, encoded by SPP1) and CD44-neutralizing antibody, RGD integrin inhibitor or inhibitor cocktail. DAPI (magenta) stains cell nuclei. White, colocalization of CC3 and DAPI. Arrow, CC3⁺ cell. Scale bar: 20 μm. Bottom, quantification of CC3⁺ smooth muscle cells (n = 5–6 independent cultures per condition). Mo, monocytes. Mean ± SEM, ANOVA with Tukey post-hoc test. **P< 0.01; ns, not statistically significant.