were angiographically confirmed preopera-
tively, and fresh tissues were acquired as part
of planned surgical resection. For scRNA-seq,
only unruptured brain AVMs were enrolled
into the study. Specimen orientation was main-
tained to ensure coverage of arteriovenous axis
with surgical clips of different sizes with aide
of intraoperative fluorescent angiography. All
tissues were acquired in close collaboration
with neurosurgeons trained in tissue isolationtechniques to minimize tissue disruption such
as avoidance of electrocautery. For bulk se-
quencing experiments, we utilized snap-frozen
ruptured and unruptured AVM specimens as
part of our biorepository. Patient demographicWinkleret al.,Science 375 , eabi7377 (2022) 4 March 2022 8 of 12
F G** **
** **nsCC3+
cellsper 103SMCsOPN - +++ +
CD44 --+ --
RGD --- + -
CD44 + RGD --- - +GPNMB+cells per mm3800040000UnrupturedRupturedRupturedGPNMBPODOXLSMADAPIDifferential Outgoing
Interaction StrengthDifferential IncomingInteraction Strength042 61-102SPP1THBSCollagenFN1
APP
CD45MIFVISFATINNOTCHMHC−IILamininAnnexin
CCLANGPTLCTRL Outgoing
BidirectionalRuptured AVMsC
Unrupturedns0125255075100- In silico
cell deconvolution
Unruptured
AVM (n =13)Ruptured AVM
(n =26)- Bulk RNAseq
differential expression
Rupture gene
module Cell proportions- Cell-specific
Gene expression
scRNAseq Ruptured genes
referenceArt1Art2Art3BCCTL1DVExVFB1FB2FbM1MGMo1Mo2Mo3Nd1NKPC
pDCPVM1PVM2SMC2SMC4SMC5SMC6SMC7SMC8TCVnNd23
2
1
0-3-2-1**
* *Change in Cell Proportion *(T-Statistic)A BVehicle OPN CD44 RGD CocktailCC3DAPIDCC3+cellsper 103
SMCsGPNMB-Mo
GPNMB+Mo+
-+ns50025*No Mo GPNMB-MoCC3DAPIGPNMB+Mo
*EColor Shape
Shared Shared
AVM IncomingFig. 5. Cell states implicated in brain AVM rupture.(A) Cellular deconvolution
and cell-specific differential expression analysis of bulk RNA-seq from
ruptured and unruptured brain AVM. (B) Bar graph of change in cell
proportiontstatistic 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), endo-
thelial cells [cyan, podocalyxin (PODOXL)], and smooth muscle cells [magenta,
aÐsmooth muscle actin (SMA)] in unruptured and ruptured AVMs. Scale bar,
100 mm. (D) Quantification of GPNMB+monocytes in unruptured (blue) and
ruptured (purple) AVMs (n= 3 donors per condition; three nonadjacent
sections per donor; 8 to 10 random images per section). Mean ± SEM,
two-tailedttest. P< 0.05. (E) (Top) Confocal microscopy analysis of cleaved
caspase-3+(green, CC3) human primary SMCs after coculture with GPNMB+
and GPNMB−monocytes isolated from ruptured AVMs. DAPI (magenta)
stains are cell nuclei. White, colocalization of CC3 and DAPI; arrow, CC3+cell.
Scale bar, 20mm. (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 AVMGPMNB+
monocytes (Mo3) relative to controls by scRNA-seq. Red, up-regulated in
AVM; blue, up-regulated in control; gray, shared between conditions; triangle,
outgoing network; square, incoming network; and 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 bySPP1) and CD44-neutralizing antibody, RGD integrin inhibitor,
or inhibitor cocktail. DAPI (magenta) stains are cell nuclei. White, colocalization
of CC3 and DAPI; arrow, CC3+cell. Scale bar, 20mm. (Bottom) Quantification of
CC3+smooth muscle cells (n= 5 to 6 independent cultures per condition).
Mo, monocytes. Mean ± SEM, ANOVA with Tukey post hoc test. **P< 0.01;
ns, not statistically significant.RESEARCH | RESEARCH ARTICLE