Supplemental Material

Comprehensive Transcriptome Analysis of Cerebral Cavernous Malformation Across Multiple Species and Genotypes

Janne Koskimäki *,1; Romuald Girard*,1; Yan Li2; Laleh Saadat1; Hussein A. Zeineddine1; Rhonda Lightle1; Thomas Moore1; Seán Lyne1; Kenneth Avner1; Robert Shenkar1; Ying Cao1; Changbin Shi1; Sean P. Polster1; Dongdong Zhang1; Julián Carrión-Penagos1; Sharbel Romanos1; Gregory Fonseca3; Miguel A. Lopez-Ramirez4; Eric M. Chapman5; Evelyn Popiel5; Alan T. Tang6; Amy Akers7; Pieter Faber8; Jorge Andrade2; Mark Ginsberg4; W. Brent Derry5,9; Mark L. Kahn6; Douglas A. Marchuk10; Issam A. Awad1

From: 1Neurovascular Surgery Program, Section of Neurosurgery, The University of Chicago Medicine and Biological Sciences, Chicago, IL 2Center for Research Informatics, The University of Chicago, Chicago, IL 3Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 4Department of Medicine, University of California San Diego, La Jolla, CA 5Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada 6Department of Medicine and Cardiovascular Institute, University of Pennsylvania, Philadelphia, PA 7Angioma Alliance Norfolk, Virginia 8University of Chicago Genomics Facility, The University of Chicago, Chicago, IL 9Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, ON, Canada. 10The Molecular Genetics and Microbiology Department, Duke University Medical Center, Durham, NC *Equal Contribution Correspondence to: Issam A. Awad, MD, MSc, FACS, MA (hon), Section of Neurosurgery, University of Chicago Medicine, 5841 S. Maryland, MC3026/Neurosurgery J341, Chicago, IL 60637. Telephone +1 773-702-2123; Fax +1 773-702-3518. E-mail [email protected]

1 Supplemental Material and Methods

Patient demographics

The descriptive data of the subjects for whom 5 human cerebral cavernous malformation (CCM) lesions were surgically resected are presented in Table S1. Neurovascular unit (NVU) controls were collected from the brains of autopsy subjects free of neurological diseases.

Laser capture microdissection

NVUs from CCMs and normal capillaries were collected using laser capture microdissection at 40x magnification, and cut NVUs were subsequently stored at -80°C (Leica LMD 6500, Leica Biosystems Inc.) (1).

Bioinformatics analyses

The quality of raw sequencing reads was assessed by FastQC (v0.11.2; http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The post-alignment quality control was evaluated using RSeQC(2), and Picard tools (v1.117; http://broadinstitute.github.io/picard/). Reads were mapped to University of California Santa Cruz (UCSC) human genome model (hg19), GENCODE mouse model (GRCm38), Ensembl Caenorhabditis elegans genome model (WBcel235) separately with respect to different species with STAR v2.5.3a (3). Gene transcripts were assembled and quantified using featureCounts (4) with respect to corresponding reference genome gene annotations. Differentially expressed genes (DEGs) analyses were conducted using DESeq2(5), with an additive model for batch effect correction if necessary. The human homologous genes of DEGs from mouse and C. elegans animal models were annotated based on the National Center for Biotechnology Information (NCBI) HomoloGene database. Out of 18,038 corresponding human genes in the database, 16,200 (around 90%) genes from mouse and 3013 (~17%) genes from C. elegans have corresponding human homologous genes. Because the homologous genes limitation from C. elegans, we implemented different threshold to DEGs from C. elegans (p≤0.25, false discovery rate (FDR) corrected; and |fold change (FC)|≥1.2) compared to human lesional ECs and the 2 murine models (p≤0.05, FDR corrected; and |FC|≥1.2) when comparing all the models together. Flow chart of the study is presented in Figure S1. We implemented 2 approaches to study the transcriptome of CCM models using sub-network analysis and gene ontology (GO) enrichment analyses. In the first, DEGs of engineered animal models were compared with the DEGs from human lesional NVUs to identify common DEGs across species (Ccm1/Krit1 or Ccm3/Pdcd10). In the second approach, genotype specific associated DEGs from mouse and C. elegans were compared separately.

2 Both approaches were followed with GO enrichment analyses, network essential genes identification, network clustering analysis, and where sub-network analysis, network essential genes identification, and network clustering analysis. GO enrichment analysis was conducted with R bioconductor package clusterProfiler. Network analyses were performed with ReactomeFIViz (6) in Cytoscape (http://www.cytoscape.org/) based on a highly reliable Reactome function interaction network (7) (version 2016). The entire FI network was constructed by merging interactions extracted from human curated pathways with interactions predicted using a machine learning approach. The individual CCM gene expression profiles for each human patient have been generated following trimmed mean of M-values (TMM) normalization implemented into the raw count reads after low expressed gene features removal. The TMM normalized expression values can be compared directly across different patients and genes.

Supplemental Results

RNA quality

For each sample of laser-captured NVUs from human surgically resected CCM lesion and normal capillaries the concentration of RNA was between 145 and 3360 pg/µl (RNA integrity numbers = [1.8 - 5.1]). For each sample of BMEC models and their respective controls, the concentration of RNA was between 322 and 89 ng/µl (RNA integrity numbers = [6.0 - 8.7]). Similarly, for C. elegans models and their respective controls the concentration of RNA was between 55 and 309 ng/µl (RNA integrity numbers = [9.8 - 10]).

Definition of neurovascular unit and restrictions of laser capture microdissection in humans

NVUs in capillaries are composed of endothelial cells, pericytes, astrocytes and neurons (8). As we acknowledge the technical limitations of laser capture microdissection to isolate pure capillary endothelial cells (Figure S7, video), we determined the proportion of DEGs that may be attributed to other cell types than endothelial cells. We first defined the top 500 human homologous DEGs derived for each of the following analyses: neurons versus endothelial cell (ECs), astrocytes versus ECs, new oligodendrocytes versus ECs, myelinating oligodendrocytes versus ECs and microglia versus ECs (https://web.stanford.edu/group/barres_lab/brain_rnaseq.html) (9). We then cross-compared each top 500 DEGs to the 915 DEGs from human lesional NVUs. The results showed that 655 of the 915 DEGs (72%) in the human lesional NVUs represented EC DEGs versus other non-EC cell types. Among the 260 non-EC DEGs, 108 were common DEGs with neurons, 50 with astrocytes, 70 with new oligodendrocytes, 72 with myelinating and 108 with microglia.

Supplementary References

1. Espina V, Milia J, Wu G, Cowherd S, Liotta LA. Laser capture microdissection. Methods Mol Biol. 2006;319:213-29. 2. Wang L, Wang S, Li W. RSeQC: quality control of RNA-seq experiments. Bioinformatics. 2012;28(16):2184-5. 3. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29(1):15-21. 4. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30(7):923-30. 5. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. 6. Wu G, Dawson E, Duong A, Haw R, Stein L. ReactomeFIViz: a Cytoscape app for pathway and network-based data analysis. F1000Res. 2014;3:146. 7. Wu G, Feng X, Stein L. A human functional protein interaction network and its application to cancer data analysis. Genome Biol. 2010;11(5):R53. 8. Iadecola C. The Neurovascular Unit Coming of Age: A Journey through Neurovascular Coupling in Health and Disease. Neuron. 2017;96(1):17-42. 9. Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O'Keeffe S, et al. An RNA- sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014;34(36):11929-47. 10. Berman JR, Kenyon C. Germ-cell loss extends C. elegans life span through regulation of DAF-16 by kri-1 and lipophilic-hormone signaling. Cell. 2006;124(5):1055-68. 11. Ito S, Greiss S, Gartner A, Derry WB. Cell-nonautonomous regulation of C. elegans germ cell death by kri-1. Curr Biol. 2010;20(4):333-8.

Supplementary Figures and Tables

Figure S1. Flowchart of the study. 1. We analyzed differentially expressed genes (DEGs) from human lesional neurovascular units, mouse brain microvascular endothelial cell Ccm1/Krit1ECKO and Ccm3/Pdcd10 ECKO, as well as C. elegans ccm1/kri-1 and ccm3/pdcd10 compared with the appropriate controls for each model. 2. Genes from the animal models were annotated based on the National Center for Biotechnology Information (NCBI) HomoloGene database to allow direct comparison with human genes. 3. We compared DEGs across species and genotypes. 4. We finally performed gene set ontology enrichment, clustering, and network analyses.

Human microdissected lesional NVUs A 371 DEGs 544 DEGs 10.0 ● ● ● ● ● ●

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● ● ● ● 7.5 ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●

(FDR) ● ● ● ● ● ● ● Up ● ● ● ● ● ● ● ● ● ● ● ● 5.0 ● ● ● ● ● ●● ● ● ●● ● 10 ● ● ● ● ● ●● ● Down ● ● ●● ● ● ●●●● ●●● ● ● ● ●● ● ● ● ●●● ● ● ● ● ●●●● ● ● ● ● ● ● ● g ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ●●● ● ● ● ● ● ● ●●● ● ● ●●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● o ● ● ● ● ●● ● ● ● ●●●●● ● ● ● Non− DEGs ●● ●● ●● ● ● ● ● ● ● ●● ●●● ●● ● ●●●● ● ● ● ●● ● ● ●● ●● ● l ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ●●● ● ● ● ●● ●● ● ●●● ● ● ● ● ● ● ● ●● ●● ● ● ●● ● ● ● ●●● ● ● ●● ● ●● ● ● ●● ●● ●●● ● ●● ● ●● ●● ● ● ● ● ● ● ● ●● ●●●●● ● ●● ● ●● ● ● ●●● ●● ● ● ● ●●●●● ●●●● ●●●● ● ●●●● ● 2.5 ● ● ● ● ●● ●● ● ● ●●●● ●●●●●●● ● ● ● ● ● ● ● ●● ● − ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ●● ● ● ●● ● ●● ● ● ● ● ●●●● ●●●●● ●● ● ● ● ● ● ●●●●● ● ● ● ●●●●●●●● ● ●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ● ● ● ● ● ●● ●●●●●● ● ●●● ●●● ● ● ●● ● ● ●●● ●● ●● ● ●●●●●● ●●●●●●●●●●●●●●●●●●●●● ● ● ● ●● ● ●●● ● ●● ●●●●● ●● ● ●● ●●●● ●●●●●●●● ●● ● ●●●● ● ● ●●●●● ●● ●● ● ●●●●●●● ● ●●●●●●●●●●●●● ●● ● ● ●●● ● ● ●●●● ● ● ● ● ●● ●●●●●●●●●●● ●●●●●● ●●●●●●● ● ●● ● ●●● ● ●● ● ● ●● ●●●●●●● ●●●●●● ●●● ●●●●●●●●● ●●●● ●●● ● ● ●●● ● ● ●●●●● ● ●●● ●●●●●●● ●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●● ●● ● ●●●● ● ●●● ● ●●●●● ● ● ● ● ●●● ●●●●●●●●●●●●● ●●●●●●●●● ●●●●●●●●●●●●● ●● ● ●●● ● ●●●●●● ● ● ●●●● ●●●● ●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●● ●●●● ●●●●●● ● ●● ● ●● ●●●●● ●●● ●●●●● ●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ●●● ●●● ● ● ●● ●● ● ●● ● ●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●● ● ●●● ●●●●●●● ● ●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●● ●●●● ● ● ● ● ● ●● ●●● ●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●● ●●●●●●●●●● ● ● ● ●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ●●●●●●●●●●● ●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●● 0.0 ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● − 10.0 − 5.0 0.0 5.0 10.0 log 2 (FC) ECKO C. elegans ccm1/kri-1 B Mouse BMEC Ccm1/Krit1 C 840 DEGs 1092 DEGs 229 DEGs 1414 DEGs ● ●●● ●● ●●●● ●●●●●●● ●●●●●●●●● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●● ●●● ● ●●● ●● ● ● ● ● ● ●●● ●● ●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●● ●●● ● ●● ● ● ●● ● ●● ● ●●● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● 10.0 ● ● ● 10.0 ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ●●● ● ● ● ● ●● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ●●● ● ● ● ● 7.5 ● ● ● ● 7.5 ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ●●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ●●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ●●●● ● ●● ● ● ● ● ● ● ●●●● ● ●● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● (FDR) ● (FDR) ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ●● ● ● ● ● ● ● ●

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● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 7.5 7.5 ● ● ● ● ● ● ● ● ● ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● (FDR) (FDR) ● ● ●● ● ● ● ●●● ● ● ● ●

● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ● ●● ● ● ●● ● ● ● ● ● ●● ● ● ●● ●●● ● ● ● ● ● ● ● ● ● ● 5.0 ● ● ● ● ● 5.0 ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ●●●●● ● ● 10 10 ● ● ● ● ●●●● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ●● ●● ● ● ●● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● g ● g ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ●● ● ● ● ● ●●●●● ● ●●●● ●● ●● ● ● ● ● ● ●● ●●● ● ●● ●●●● ●● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●● ● ●●● ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● ●● ●●●● ●● ● ● ● o ● ● ● ● ● ● o ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ●●●●● ●● ● ● ● ● ● ● ● ●● ●● ● ● ●● ●●● ●●●●●●●●●● ●● ● ● l ● ● ● ● ●● l ● ● ● ● ●●● ●●●●● ● ● ● ● ●●● ● ● ● ● ●● ● ● ●●●●● ● ● ● ●●●● ● ● ● ●●● ●●●●●●●● ● ●●● ●● ● ● ● ● ● ●● ●●● ●● ● ● ● ●● ● ● ● ● ●●●● ●●● ●● ● ●● ●● ● ● ● ● ●●●●●●●●●● ● ●● ● ●● ●●●●●● ●●● ● ● ●●●●●●●● ●●● ●●● ●●● ●●●● ●● ● ● ● ● ● ● ● ● ● ●●● ●●●●●●●●●●● ● ● ● ● ● ● ● ●● ● ● ●●● ●●●● ●●●●● ● ● ● ● ● ● ●● ●●●● ●●●● ● ●●●● ●●●●● ●● ● ● ● ● ●●●●● ● ● ●● ●● ● ● ● ● ●● ● ● ●●●● ● ●●●●●●●●● ● ● − − ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ●●●●● ● ●●●●●●●●●●●● ● ● ● ●●●●● ●● ● ● ● ● ● ●● ● ●● ● ●● ●●●●●● ●●●●●●●●●●●●●●●●●● ● ● ● 2.5 ● ●● ● ● ●● ● ● 2.5 ● ●● ●● ● ●●● ●●●●● ●●●●●● ●●●●●●●●●●●● ● ●● ●●● ● ●● ●●●● ●●●●●●●●● ● ● ● ● ● ●●●●●●● ●●●●●●●●● ● ● ● ● ● ●●● ● ● ● ●●●●●●●●●●●●● ● ●●●●●●●●●●●●●● ● ● ● ●● ●●●●● ●●● ●●● ●●● ● ● ● ●● ● ● ●● ●●●●●●●●●● ●●●●●●●●●●●●●●●●● ● ● ● ● ●● ●● ●●●●●● ● ● ● ●●● ●● ●●●●● ●●●●●●●●●●●●●●● ●●● ● ● ● ●●● ●●●● ●●●●● ● ● ● ● ● ●● ●●●●● ●●●●●●●●●●●●●●● ● ● ●●●●●● ● ●● ● ●● ● ● ●● ●● ● ● ● ●●●●●● ● ●●●●●●●●●●● ● ●● ● ●● ●● ●● ● ●● ● ●● ●●●● ●●●●●●●●●●● ●●●●●●●●●●●●●●●●● ● ●● ● ●●●● ●● ●●● ●●●● ● ● ● ●●● ● ●● ●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●● ● ● ● ●● ●●●● ●●●●●●●●●● ● ● ● ●● ●● ● ●●●● ● ●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ●●●●●●●●●● ●●●●●●●●●●●●● ● ● ●● ● ● ●●●●● ●●●● ●● ●●●●●●●●●●●●●●● ●● ● ●● ●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●● ●●● ● ● ●● ●●●●● ●●●● ●●● ●●● ●●●●●●●●●●●●●●●●●●●●●●● ● ● ●●●●●●●●●●●●●●● ●●●●●●● ●● ●●●●●● ●● ● ●● ●● ●●●●● ●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●● ●● ●●●●●●●●●●●●●●● ●●●●●●●●●● ●●● ●● ● ● ● ●●● ●● ● ●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●● ●● ● ●● ●●●●●●●●●●● ●●●●●●●●●●● ●● ●● ● ● ● ● ● ●●●●●●● ● ●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●● ●● ● ● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●● ●● ● ●● ● ● ●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●● ●●● ●● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●● ●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●● ●●● ●●● ● ● ● ● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●● ●●● ●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●● ● ● ● ● ● ●●● ●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ● ●● ●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●● ●● ● ● ●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●● ● ●●● ● ●●●●● ●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●● ●● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ●● ● ●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●● ●●●●● ●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●● ● ●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●● ● ●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●● ●●●● ● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●● ●● ● ●● ●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●● ●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● 0.0 ● ●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● 0.0 ●●●●●●●●●●●●●●●●●●●●●●●●●●● − 3.0 0.0 3.0 − 3.0 0.0 3.0 log (FC) log (FC) 2 2 Figure S2. Volcano plots of differentially expressed genes (DEGs) in human lesional neurovascular units (NVUs), mouse brain microvascular endothelial cell (BMEC) Ccm1/Krit1 and Ccm3/Pdcd10, as well as C. elegans ccm1/kri-1 and ccm3/pdcd10 models. The transcriptome analyses identified (A) 915 DEGs in human lesional NVUs, (B) 1932 DEGs in BMEC Ccm1/Krit1ECKO, (C) 1643 DEGs in C. elegans ccm1/kri-1 (D) 524 DEGs in BMEC Ccm3/Pdcd10ECKO, and (E) 1581 DEGs in C. elegans ccm3/pdcd10. All DEGs were considered at a fold change (FC)≥1.2; significance level p<0.05, false discovery rate (FDR) corrected. Each dot represents an individual DEG with respect to the control. Red dots represent up-regulated DEGs and blue dots are down-regulated DEGs. Results are presented as x-axis log2 FC and y-axis log10 FDR corrected p-value.

−1.5 −0.5 0.5 1.5 Scaled log2 (CPM)

CCM1 CCM2 CCM3

1 3 4 5

Patient Patient 2 Patient Patient Patient Control 1 Control 2Control 3

Figure S3. Individual gene expression profiles for CCM1, CCM2 and CCM3 of each patients with cerebral cavernous malformation (CCM) and autopsy controls. The results suggest that at least one of the CCM genes was down-regulated in the human microdissected lesional neurovascular units (NVUs). Particularly, patient 4 has been clinically genotyped and identified with a familial CCM1 mutation. Her gene expression profile supports this genotype by showing a down-regulation of CCM1 (darkest blue box). CPM = counts per million.

establishment of organelle localization ● ● ● ● ● vesicle localization ● ● ● ● establishment of vesicle localization ● ● ● ● organelle localization ● ● ● ● granulocyte activation ● ● ● neutrophil mediated immunity ● ● ● neutrophil activation involved in immune response ● ● ● ● neutrophil degranulation ● ● ● ● neutrophil activation ● ● ● ● cell−substrate junction ● ● ● ● focal adhesion ● ● ● ● cell−substrate adherens junction ● ● ● ● response to drug ● ● ● ● active ion transmembrane transporter activity ● ● ● vacuolar lumen ● ● ● ATPase activity, coupled to movement of substances ● ● ● ● organic anion transport ● ● ● ● ATPase activity, coupled to transmembrane movement of substances ● ● ● hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances ● ● ● secretory granule lumen ● ● ● cytoplasmic vesicle lumen ● ● ● vesicle lumen ● ● ● phosphoric ester hydrolase activity ● ● ● ● primary active transmembrane transporter activity ● ● ● P−P−bond−hydrolysis−driven transmembrane transporter activity ● ● ● glycoprotein metabolic process ● ● ● Golgi subcompartment ● ● ● glycoprotein biosynthetic process ● ● ● carbohydrate catabolic process ● ● ● cadherin binding ● ● ● ● response to oxidative stress ● ● ● ● establishment of protein localization to plasma membrane ● ● ● ● response to reactive oxygen species ● ● ● ● response to hydrogen peroxide ● ● ● ● extracellular matrix organization ● ● ● extracellular structure organization ● ● ● axon ● ● ● T cell activation ● ● ● leukocyte cell−cell adhesion ● ● ● leukocyte differentiation ● ● ● blood vessel morphogenesis ● ● ● regulation of cell−cell adhesion ● ● ● regulation of leukocyte cell−cell adhesion ● ● ● regulation of lymphocyte proliferation ● ● ● integrin−mediated signaling pathway ● ● ● regulation of mononuclear cell proliferation ● ● ● lymphocyte differentiation ● ● ● positive regulation of cellular component movement ● ● ● regulation of leukocyte differentiation ● ● ● T cell differentiation ● ● ● leukocyte migration ● ● ● leukocyte proliferation ● ● ● positive regulation of cell migration ● ● ● regulation of T cell activation ● ● ● positive regulation of locomotion ● ● ● positive regulation of cell motility ● ● ● regulation of lymphocyte activation ● ● ● protein secretion ● ● ● cellular metal ion homeostasis ● ● ● skeletal system development ● ● ● regulation of actin filament−based process ● ● ● negative regulation of cell adhesion ● ● ● regulation of actin cytoskeleton organization ● ● ● cell−matrix adhesion ● ● ● adherens junction ● ● ● ● small GTPase mediated signal transduction ● ● ● ● regulation of supramolecular fiber organization ● ● ● side of membrane ● ● ● leukocyte chemotaxis ● ● ● cell adhesion molecule binding ● ● ● ● positive regulation of cell projection organization ● ● ● positive regulation of cell development ● ● ● blood coagulation ● ● ● regulation of cytoskeleton organization ● ● ● coagulation ● ● ● hemostasis ● ● ● cell leading edge ● ● ● ● growth cone ● ● ● regulation of peptide secretion ● ● ● regulation of protein secretion ● ● ● positive regulation of neuron differentiation ● ● ● response to mechanical stimulus ● ● ● regulation of protein serine/threonine kinase activity ● ● ● positive regulation of protein kinase activity ● ● ● homotypic cell−cell adhesion ● ● ● actin filament organization ● ● ● positive regulation of neurogenesis ● ● ● tubulin binding ● ● ● response to molecule of bacterial origin ● ● ● regulation of cellular component size ● ● ● negative regulation of intracellular signal transduction ● ● ● cartilage development ● ● ● regulation of protein complex assembly ● ● ● membrane raft ● ● ● protein polymerization ● ● ● membrane microdomain ● ● ● kinase regulator activity ● ● ● regulation of actin polymerization or depolymerization ● ● ● heart development ● ● ● regulation of actin filament length ● ● ● regulation of protein polymerization ● ● ● regulation of long−term neuronal synaptic plasticity ● ● ● cell−cell junction ● ● ● connective tissue development ● ● ● membrane region ● ● ● negative regulation of protein phosphorylation ● ● ● positive regulation of protein serine/threonine kinase activity ● ● ● response to lipopolysaccharide ● ● ● actin filament bundle assembly ● ● ● positive regulation of cellular component biogenesis ● ● ● regulation of B cell proliferation ● ● ● microtubule binding ● ● ● actin filament bundle organization ● ● ● cell cortex part ● ● ● response to metal ion ● ● ● ● negative regulation of cell−cell adhesion ● ● ● negative regulation of phosphorylation ● ● ● regulation of apoptotic signaling pathway ● ● ● positive regulation of MAPK cascade ● ● ● tube morphogenesis ● ● ● cell projection membrane ● ● ● morphogenesis of a branching epithelium ● ● ● dendritic spine ● ● ● neuron spine ● ● ● regulation of extrinsic apoptotic signaling pathway ● ● ● negative regulation of leukocyte differentiation ● ● ● response to inorganic substance ● ● ● ● bone remodeling ● ● ● regulation of developmental growth ● ● ● peptidyl−tyrosine modification ● ● ● muscle cell migration ● ● ● response to oxygen levels ● ● ● positive regulation of protein transport ● ● ● morphogenesis of a branching structure ● ● ● regulation of MAP kinase activity ● ● ● epithelial cell proliferation ● ● ● cellular response to iron ion ● ● ● regulation of insulin secretion ● ● ● cell junction organization ● ● ● platelet dense tubular network ● ● ● microtubule ● ● ● cell−cell junction organization ● ● ● regulation of cell shape ● ● ● negative regulation of protein kinase activity ● ● ● extrinsic component of membrane ● ● ● regulation of integrin−mediated signaling pathway ● ● ● morphogenesis of embryonic epithelium ● ● ● negative regulation of MAPK cascade ● ● ● regulation of homotypic cell−cell adhesion ● ● ●

ECKO ECKO Human ccm1/kri-1 ccm3/pdcd10 Ccm1/Krit1 human ccm 915 Ccm3/Pdcd10 C. elegans worm ccm1 homo worm ccm3 homo mouse ccm1 homo mouse ccm3 homo C. elegans

Mouse BMEC adjusted p−value Mouse BMEC 0.025 0.050 0.075 Gene Count ● 30 ● 60 ● 90

Figure S4. Common gene ontology (GO) terms across species. One GO term (GO:0051656 establishment of organelle localization) was common across the 5 models studied. In addition, 24 enriched GO terms were present in 4 of the 5 models; and 149 GOs were commonly identified in at least 3 (p<0.1, false discovery rate corrected for all comparisons). These identified GO terms were related to cell-cell adhesion, vesicle transportation, cell organelle localization and movement, immunity and inflammation, ion transmembrane transporter activity, MAPK signaling and responses to oxidative stress. A GO term was defined as common between species if it was identified in both genotypes of the same species in order to exclude GOs that may be specific for either genotype.

A B

736 837

137 34 45 32 8 1

1579 128 934 420 40 1011

Ccm1/Krit1ECKO ccm1/kri-1 Ccm3/Pdcd10ECKO ccm3/pdcd10

Figure S5. Venn diagrams of human homologous differentially expressed genes (DEGs) in Ccm1/Krit1 and Ccm3/Pdcd10 genotype models. A total number of 915 DEGs were identified in human lesional neurovascular units, 1852 and 506 in respectively brain microvascular endothelial cell (BMEC) Ccm1/Krit ECKO and Ccm3/Pdcd10 ECKO, as well as 1104 and 1084 in C. elegans ccm1/kri-1 and ccm3/pdcd10 respectively (for human and mouse BMEC FC≥1.2; p<0.05 false discovery rate (FDR) corrected, for C. elegans FC>1.0; p<0.25 FDR corrected). (A) Eight DEGs were common across species in Ccm1/Krit1 models: SPARCL1, PDGRFA, FAXC, UNC13A, FAT1, PLXDC2, PLCD3, and GNAO1. (B) One DEG was common across species in Ccm3/Pdcd10 models: FAT1.

760

109 10 36

1432 275 185

Ccm1/Krit1ECKO Ccm3/Pdcd10 ECKO

Figure S6. Venn diagram of human homologous differentially expressed genes (DEGs) between human micro-dissected lesional neurovascular units (NVUs) and mouse brain microvascular endothelial cell (BMEC) Ccm1/Krit1ECKO and Ccm3/Pdcd10ECKO models. The transcriptomes analyses identified 36 common DEGs between the human microdissected lesional neurovascular units (NVUs), mouse BMECs Ccm1/Krit1ECKO and Ccm3/Pdcd10ECKO models. In addition, further analyses showed that 109 and 10 DEGs were common between human NVUs and respectively Ccm1/Krit1ECKO and Ccm3/Pdcd10ECKO (FC≥1.2, p<0.05; FDR corrected).

Figure. S7. Laser capture microdissection of a cerebral cavernous malformation in vivo (video).

Table S1: Characteristics of CCM subjects used in neurovascular unit RNAseq.

Patient Age Sex Sporadic/Familial Indication of resection

1 13 Female Sporadic Hemorrhagic expansion

2 31 Male Sporadic Seizures

3 34 Female Sporadic Size of the lesion 4 4 Female Familial (CCM1) Hemorrhagic expansion

5 40 Female Sporadic Hemorrhagic expansion/Seizures

Table S2. List of identified differentially expressed genes (DEGs) in all models. (For human and mouse BMEC FC≥1.2; p<0.05 false discovery rate corrected (FDR), for C. elegans FC>1.0; p<0.25 FDR corrected).

Table S3. List of identified GO terms in all models (p<0.10, false discovery rate corrected).

Table S4. List of 95 enriched gene ontology terms using the 16 genes with more than 20 edges in the analysis (p<0.01, false discovery rate corrected).

Table S5. Identified differentially expressed genes between human microdissected lesional neurovascular units and mouse BMEC Ccm1/Krit1ECKO and Ccm3/Pdcd10ECKO models.

Table S6. List of 71 differently expressed genes (DEGs) common across mouse brain microvascular endothelial cells (BMECs) and C. elegans that were identified only in the Ccm1/Krit1 mutant models regardless of the direction of the fold change. Mouse BMEC DEGs were considered at a fold change (FC)≥1.2; significance level p<0.05, false discovery rate (FDR) corrected. C. elegans DEGs were considered at a FC≥1.2; significance level p<0.25, FDR corrected.

Table S7. List of 11 differently expressed genes (DEGs) common across mouse brain microvascular endothelial cells (BMECs) and C. elegans that were identified only in the Ccm3/Pdcd10 mutant models regardless of the direction of the fold change. Mouse BMEC DEGs were considered at a fold change (FC)≥1.2; significance level p<0.05, false discovery rate (FDR) corrected. C. elegans DEGs were considered at a FC≥1.2; significance level p<0.25, FDR corrected.

Table S8. List of genes with a documented role in CCM disease for each model. We identified a list of 54 genes with a documented role in CCM disease using DisGeNet (disease ID C2919945, Cavernous Hemangioma of Brain). Among the 54 documented genes, 42 were found in the human lesional neurovascular units transcriptome, 32 human homologues in mouse BMEC Ccm1/Krit1ECKO, 32 in mouse BMEC Ccm3/Pdcd10ECKO, 6 in C. elegans ccm1/kri1 and 6 in C. elegans ccm3/pdcd10. C. elegans ccm1/kri1 mRNA levels are upregulated but the transcripts have been shown to be non-functional(10, 11).

Table S9. Full list of enriched gene ontology terms present only within the Ccm1/Krit1 genotype models (p<0.1, false discovery rate (FDR) corrected).

Table S10. Full list of enriched gene ontology terms present only within the Ccm3/Pdcd10 genotype models (p<0.1, false discovery rate (FDR) corrected).