Science - USA (2022-03-04)

Winkleret al.,Science 375 , eabi7377 (2022) 4 March 2022 5 of 12

A

Vascular

Neurons/glia

Immune

Endothelial

Smooth muscle

Fibroblast

Fibromyocyte

Myeloid

T cell

B cell

Astrocytes

Oligodendrocytes

Other

Choroid plexus

Neurons

B

J

UMAP

D

UMAP

Condition

AVM

Control

Endothelial

Art1 Art3 Nd1

Art2 Vn Nd2

UMAP

Exp.

Latent time

Exp.

Latent time

PLVAP PGF

PLVAP

ANGPT2

PECAM1

Merged

PLVAP

3

Art1

Art2

Art3

Art4

Nd1

Nd2

Nd2

AV

M

Control

SLC3A2

MFSD2A

SLC16A1

SLC38A5

CCL14

PGF

ANGPT2

PLVAP

STC1

20

40

60

Avg. Exp.

1.5

0

% Exp.

Gene Set Enrichment

Complement

Hypoxia

Inflammatory

response

EMT

Angiogenesis

MTORC1

Signaling

padj

0.06

0.02

0

021

NES

Expression

M

E

C

I

KL

Cell Proportion (%)

ACCPBCECFBFbMMylNeuOL

SMC

TC

0

50

100

AVM 3 AVM 5

AVM 4

AVM 1

AVM 2

Donor

AVM 1

AVM 2

Donor

i.

CLDN5

TAGLN

COL1A2

CCL19

DAPI

i. SMCii. FbMiii. ECiv. FB

ii.

iii.

iv.

AVM

Control

UMAP

Endothelial Cells Endothelial

Art1 Art3 Nd1

Art2 Vn Nd2

SMC

FbM

FB

EC

PC

400

200

0

750 0

Intersection Size

F

Art1Art3Nd1

Nd2

Art2

Vn

250

125

0

10005000

DEGs

Intersection Size

H

DEGs

Exp.

0.5

0.1

CTRL

AVM

G

Art

Art1 Art2Art3

Art1Art2Art3

Vn

Vn

Vn

Vu

Vu

Cap

Art

Vn

Vu

Cap

Cap

Nd1Nd2

Cap

Fig. 3. Cellular aberrancy in the malformed cerebrovasculature.(A) Isolation
and cell sampling from human brain 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 are AVM1,
26,122 cells; AVM2, 28,868 cells; AVM3, 14,541 cells; AVM4, 26,660 cells; and
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; and CP, choroid plexus.
(D) Representative microscopy image of single-molecule fluorescent in situ
hybridization showing expression ofCLDN5[yellow, endothelial cells (EC)],TAGLN

[magenta, smooth muscle cells (SMC)],COL1A2[red, perivascular fibroblasts

(FB)], andCCL19[cyan, fibromyocytes (FbM)]. DAPI (blue) stains are cell nuclei.

Boxes highlight representative cells. Scale bar, 50mm. (i)TAGLN+smooth

muscle cell. (ii)CCL19+andTAGLN+fibromyocyte. (iii)CLDN5+endothelial cell.

(iv)COL1A2+perivascular fibroblast. Scale bars, insets, 5mm. (E) (Left) Schematic

describing computational pipeline. Endothelial cells (orange) are identified in silico

by marker expression, coembedded for downstream analytics, and iteratively

clustered. An identical workflow was applied to perivascular cells (fig. S7). (Middle)

UMAP visualization of coembedded endothelial cell states in control (gray) and

AVMs (red). (Right) UMAP visualization of iteratively clustered endothelial cell

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