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Endoluminal Biopsy for Vein of Galen Malformation. BACKGROUND AND OBJECTIVES: Vein of Galen malformation (VOGM), the result of arteriovenous shunting between choroidal and/or subependymal arteries and the embryologic prosencephalic vein, is among the most severe cerebrovascular disorders of childhood. We hypothesized that in situ analysis of the VOGM lesion using endoluminal tissue sampling (ETS) is feasible and may advance our understanding of VOGM genetics, pathogenesis, and maintenance. METHODS: We collected germline DNA (cheek swab) from patients and their families for genetic analysis. In situ VOGM "endothelial" cells (ECs), defined as CD31 + and CD45 - , were obtained from coils through ETS during routine endovascular treatment. Autologous peripheral femoral ECs were also collected from the access sheath. Single-cell RNA sequencing of both VOGM and peripheral ECs was performed to demonstrate feasibility to define the transcriptional architecture. Comparison was also made with a published normative cerebrovascular transcriptome atlas. A subset of VOGM ECs was reserved for future DNA sequencing to assess for somatic and second-hit mutations. RESULTS: Our cohort contains 6 patients who underwent 10 ETS procedures from arterial and/or venous access during routine VOGM treatment (aged 12 days to ∼6 years). No periprocedural complications attributable to ETS occurred. Six unique coil types were used. ETS captured 98 ± 88 (mean ± SD; range 17-256) experimental ECs (CD31 + and CD45 - ). There was no discernible correlation between cell yield and coil type or route of access. Single-cell RNA sequencing demonstrated hierarchical clustering and unique cell populations within the VOGM EC compartment compared with peripheral EC controls when annotated using a publicly available cerebrovascular cell atlas. CONCLUSION: ETS may supplement investigations aimed at development of a molecular-genetic taxonomic classification scheme for VOGM. Moreover, results may eventually inform the selection of personalized pharmacologic or genetic therapies for VOGM and cerebrovascular disorders more broadly.

Institutional review board approval was granted by the author's institution for this prospective observational study. Parents provided informed consent to either (1) deploy and remove coils specifically for research purposes (ie, only to collect cells for ETS) or (2) only if the coil had to be removed otherwise for clinical purposes. Six pediatric (range: 12 days to ∼6 years) VOGM patients undergoing standard-of-care embolization were recruited between 2022 and 2023. Diagnosis was confirmed by catheter angiography. Clinical presentation included neonatal congestive heart failure and hydrovenous disorder. All patients were seemed suitable for clinical intervention based on Bicetre score.1 During each endovascular procedure, ETS was performed if deemed safe by the primary operator (J.G.J.). Staged embolization patients underwent ETS on more than one occasion. Information regarding the sampling location, coil type and other data were recorded on a templated form and entered into a secured Redcap database. Germline DNA was collected from all subjects and first-degree relatives who were available for consent through cheek swab. Pedigrees were assembled to eventually compare germline DNA against EC's retrieved by ETS to identify somatic and second-hit mutations.

a templated form and entered into a secured Redcap database. Germline DNA was collected from all subjects and first-degree relatives who were available for consent through cheek swab. Pedigrees were assembled to eventually compare germline DNA against EC's retrieved by ETS to identify somatic and second-hit mutations. ETS protocols have been established previously for other cerebrovascular lesions.17,22-24 However, VOGM poses novel challenges because of the shearing effect of high flowing blood and small patient size, which precludes many common endovascular devices. Clinical factors alone determined vessel selection, catheter selection, and access route without consideration for anticipated ETS cell yield. Thus, ETS is considered US Food and Drug Administration off-label use. Transarterial embolization more often characterized earlier stages of treatment. Flow-directed Magic (Balt, Montmorency) microcatheters are not amenable to ETS. Thus, the sample selection was limited to treatments using a Marathon microcatheter (EV3) which accommodates smaller (upto 6 mm) Blockade coils. Achieving a stable coil nest in such high flow pedicles is challenging and best done at a curve or narrowing in the vessel. Transvenous embolization often allowed standard 0.017” microcatheters (and larger coils) given the less tortuous and delicate access route, except in cases of retrograde pedicle catheterization when an Apollo (EV3) was used in conjunction with Onyx. Like Marathon, Apollo accommodates smaller Blockade coils for ETS. Coils were sized to approximate the vessel's diameter and conform to its walls. After 2 minutes of endoluminal dwell time, the coil was resheathed through the microcatheter, protecting it from contact with other vessel segments. At procedure's end, the vascular access sheath(s) were collected, and ECs were isolated.

S. Coils were sized to approximate the vessel's diameter and conform to its walls. After 2 minutes of endoluminal dwell time, the coil was resheathed through the microcatheter, protecting it from contact with other vessel segments. At procedure's end, the vascular access sheath(s) were collected, and ECs were isolated. The explanted coil is immediately placed into a conical tube of Dulbecco's Modified Eagle Medium (DMEM) on ice. The coil is then transported to the flow cytometry core within 30 minutes. DMEM is aspirated and the coil rinsed in Dulbecco's phosphate-buffered saline (DPBS) (without Ca or Mg), incubated in 0.25% Trypsin-EDTA at room temperature for 5 minutes before neutralization with 1 mL of fetal bovine serum. The coil is then washed with a trypsin–fetal bovine serum solution to ensure complete dissociation. The dissociate is then spun down (∼500 g, 5 minutes), the supernatant removed, and the pellet resuspended in ammonium-chloride-potassium lysis buffer on ice, for 3∼4 minutes to lyse red blood cell contaminants. The ammonium-chloride-potassium lysis buffer is then neutralized using 1× PBS or DMEM. Cells are pelleted again and resuspended in PBS 100 µl with Alexa Fluor 647–conjugated monoclonal anti-human CD31 antibody (BD Biosciences; 1:200 dilution) and anti-human CD45 antibody (BD Biosciences; 1:200 dilution). The cell solution containing CD31 and CD45 antibodies are incubated on ice for 20 minutes before addition of propidium iodide (to exclude dead cells, 120 µg/mL, 1:100 dilution in DPBS without Ca or Mg) for an additional 10-minute incubation on ice. Finally, 500 µl DPBS (without Ca or Mg) is added, centrifuged for 5 minutes at 500 g, the supernatant is removed, the cell pellet is resuspended in 300 µl DPBS, and individual cells are sorted into a 96-well plate. Cells obtained from the shealth are processed in an analogous fashion.

ional 10-minute incubation on ice. Finally, 500 µl DPBS (without Ca or Mg) is added, centrifuged for 5 minutes at 500 g, the supernatant is removed, the cell pellet is resuspended in 300 µl DPBS, and individual cells are sorted into a 96-well plate. Cells obtained from the shealth are processed in an analogous fashion. Cells are subsequently fluorescently sorted on a BD FACSAria II Flow Cytometer (BD Biosciences). Nonviable cells were excluded based on propidium iodide positivity, monocyte populations were excluded based on CD45 positivity, and endothelial enrichment was performed through positive selection of CD31 cells. Viable cells enriched for endothelium were therefore considered to be CD31-positive, CD45-negative, and PI-negative cells. Cells are then sorted into single cells in a 96-well plate (already containing 4 µl of DPBS as indicated above) for either single-cell or bulk analysis. The plate is then sealed and frozen at −80°C for downstream human genetic and functional genomic analyses. Pooled experimental cells lysed for DNA purification using standard methodologies25 can then be used for downstream WES experiments where a sufficiently large amount of DNA is required.

ngle-cell or bulk analysis. The plate is then sealed and frozen at −80°C for downstream human genetic and functional genomic analyses. Pooled experimental cells lysed for DNA purification using standard methodologies25 can then be used for downstream WES experiments where a sufficiently large amount of DNA is required. Raw sequencing data were processed with the Cell Ranger pipeline software (v.3.0.2; 10× Genomics). The Cell Ranger count pipeline was used to perform quality control, sample demultiplexing, barcode processing, alignment, and single-cell 5ʹ gene counting. Cell ranger “count” was used to align raw reads against the hg38 genome (refdata-gex-GRCh38-2020-A) using CellRanger software (v.4.0.0) (10× Genomics). Subsequently, cell barcodes and unique molecular identifiers underwent filtering and correction using default parameters in Cell Ranger. Reads with the retained barcodes were quantified and used to build the gene expression matrix.

8 genome (refdata-gex-GRCh38-2020-A) using CellRanger software (v.4.0.0) (10× Genomics). Subsequently, cell barcodes and unique molecular identifiers underwent filtering and correction using default parameters in Cell Ranger. Reads with the retained barcodes were quantified and used to build the gene expression matrix. Seurat (v.3.0.0), implemented using the R package, was applied to exclude low-quality cells.26 Cells that expressed fewer than 200 and larger than 5000 genes were filtered out. The processed data were normalized using Seurat's “NormalizeData” function, which used a global scaling normalization method, LogNormalize, to normalize the gene expression measurements for each cell to the total gene expression. Highly variable genes were then identified using the function “FindVariableGenes” in Seurat. The anchors were identified using the “FindIntegrationAnchors” function, and thus, the matrices from different samples were integrated with the “IntegrateData” function. The variation arising from library size and percentage of mitochondrial genes was regressed out using the function “ScaleData” in Seurat. Principal component analysis was performed using the Seurat function “RunPCA”, and K-nearest neighbor graph was constructed using “FindNeighbors” function in Seurat with the number of significant PCs identified from Principle Components Analysis. Clusters were identified using “FindClusters” function with resolution of 0.4. The clusters were visualized in 2 dimensions with Uniform Manifold Approximation and Projection for Dimension Reduction. The normalization, integration, and clustering were performed under standard Seurat workflow.

ETS protocols have been established previously for other cerebrovascular lesions.17,22-24 However, VOGM poses novel challenges because of the shearing effect of high flowing blood and small patient size, which precludes many common endovascular devices. Clinical factors alone determined vessel selection, catheter selection, and access route without consideration for anticipated ETS cell yield. Thus, ETS is considered US Food and Drug Administration off-label use. Transarterial embolization more often characterized earlier stages of treatment. Flow-directed Magic (Balt, Montmorency) microcatheters are not amenable to ETS. Thus, the sample selection was limited to treatments using a Marathon microcatheter (EV3) which accommodates smaller (upto 6 mm) Blockade coils. Achieving a stable coil nest in such high flow pedicles is challenging and best done at a curve or narrowing in the vessel. Transvenous embolization often allowed standard 0.017” microcatheters (and larger coils) given the less tortuous and delicate access route, except in cases of retrograde pedicle catheterization when an Apollo (EV3) was used in conjunction with Onyx. Like Marathon, Apollo accommodates smaller Blockade coils for ETS. Coils were sized to approximate the vessel's diameter and conform to its walls. After 2 minutes of endoluminal dwell time, the coil was resheathed through the microcatheter, protecting it from contact with other vessel segments. At procedure's end, the vascular access sheath(s) were collected, and ECs were isolated.

The explanted coil is immediately placed into a conical tube of Dulbecco's Modified Eagle Medium (DMEM) on ice. The coil is then transported to the flow cytometry core within 30 minutes. DMEM is aspirated and the coil rinsed in Dulbecco's phosphate-buffered saline (DPBS) (without Ca or Mg), incubated in 0.25% Trypsin-EDTA at room temperature for 5 minutes before neutralization with 1 mL of fetal bovine serum. The coil is then washed with a trypsin–fetal bovine serum solution to ensure complete dissociation. The dissociate is then spun down (∼500 g, 5 minutes), the supernatant removed, and the pellet resuspended in ammonium-chloride-potassium lysis buffer on ice, for 3∼4 minutes to lyse red blood cell contaminants. The ammonium-chloride-potassium lysis buffer is then neutralized using 1× PBS or DMEM. Cells are pelleted again and resuspended in PBS 100 µl with Alexa Fluor 647–conjugated monoclonal anti-human CD31 antibody (BD Biosciences; 1:200 dilution) and anti-human CD45 antibody (BD Biosciences; 1:200 dilution). The cell solution containing CD31 and CD45 antibodies are incubated on ice for 20 minutes before addition of propidium iodide (to exclude dead cells, 120 µg/mL, 1:100 dilution in DPBS without Ca or Mg) for an additional 10-minute incubation on ice. Finally, 500 µl DPBS (without Ca or Mg) is added, centrifuged for 5 minutes at 500 g, the supernatant is removed, the cell pellet is resuspended in 300 µl DPBS, and individual cells are sorted into a 96-well plate. Cells obtained from the shealth are processed in an analogous fashion.

Cells are subsequently fluorescently sorted on a BD FACSAria II Flow Cytometer (BD Biosciences). Nonviable cells were excluded based on propidium iodide positivity, monocyte populations were excluded based on CD45 positivity, and endothelial enrichment was performed through positive selection of CD31 cells. Viable cells enriched for endothelium were therefore considered to be CD31-positive, CD45-negative, and PI-negative cells. Cells are then sorted into single cells in a 96-well plate (already containing 4 µl of DPBS as indicated above) for either single-cell or bulk analysis. The plate is then sealed and frozen at −80°C for downstream human genetic and functional genomic analyses. Pooled experimental cells lysed for DNA purification using standard methodologies25 can then be used for downstream WES experiments where a sufficiently large amount of DNA is required.

Raw sequencing data were processed with the Cell Ranger pipeline software (v.3.0.2; 10× Genomics). The Cell Ranger count pipeline was used to perform quality control, sample demultiplexing, barcode processing, alignment, and single-cell 5ʹ gene counting. Cell ranger “count” was used to align raw reads against the hg38 genome (refdata-gex-GRCh38-2020-A) using CellRanger software (v.4.0.0) (10× Genomics). Subsequently, cell barcodes and unique molecular identifiers underwent filtering and correction using default parameters in Cell Ranger. Reads with the retained barcodes were quantified and used to build the gene expression matrix.

All 6 families that were solicited to participate in the study agreed and provided informed consent. ETS was performed a total of 10 times (owing to staged procedures involving the same subject), in 3 arteries and 7 veins. All coils yielded CD31+, CD45−experimental cells, although the number varied widely (98 ± 88 mean ± SD; range 17-256) (Table). Yield tended to improve later in the course of treatment (for subjects who underwent >1 ETS), suggesting flow reduction may shear fewer cells off the coil. Multiple coil types were used to capture cells through ETS, suggesting that this technique is not limited to 1 coil manufacturer or design. No periprocedural complications related to ETS were encountered, and no subjects were lost to follow-up. Overview of ETS Results From Patients With VOGM Indicating Participant ID, Age at ETS, Ethnicity, Route of Access for Routine Treatment and ETS, Vessel Sampling, Coil Type Used for ETS, and Experimental Cell (ie, CD31+, CD45−) Yield ETS, endoluminal tissue sampling; PVM, prosencephalic vein of Markowski; VOGM, vein of Galen malformation. No patient identifiers were available to anyone outside of the research group. Age ranges were determined as follows: neonates (younger than 1 mo), infant (1 mo and younger than 2 y), and children (older than 2 y).

Overview of ETS Results From Patients With VOGM Indicating Participant ID, Age at ETS, Ethnicity, Route of Access for Routine Treatment and ETS, Vessel Sampling, Coil Type Used for ETS, and Experimental Cell (ie, CD31+, CD45−) Yield ETS, endoluminal tissue sampling; PVM, prosencephalic vein of Markowski; VOGM, vein of Galen malformation. No patient identifiers were available to anyone outside of the research group. Age ranges were determined as follows: neonates (younger than 1 mo), infant (1 mo and younger than 2 y), and children (older than 2 y). This report focuses on technical aspects of ETS in VOGM. Complete trios were assembled in 2/6 families, mother and child only in 3/6, and father and child only in 1/6 (Figure 1). Cheek swab DNA from all individuals listed in Figure 1 was collected. An overview of our approach can be found in Figure 2. ETS is performed (see Methods for full details), and specimens are immediately transported for flow cytometry sorting. After positive selection with CD31 and negative selection of CD45, experimental “endothelial” cells are obtained (Figure 3). This finding demonstrates ETS’ potential to enable identification of coding mutations, copy number variants, deletions, insertions, genomic rearrangements, and the single cell/nucleus RNA transcriptional landscape unique to VOGM tissue in situ. Finally, scRNA seq was performed to demonstrate feasibility of this approach on VOGM samples obtained through ETS and femoral access control (Figure 4). Using hierarchical clustering, we identify 12 unique cell populations VOGM in situ and femoral access samples (Figure 4A), where the proportion of cells within each population differs between VOGM lesion and control (Figure 4B). Collectively, these data illustrate the feasibility of ETS for VOGM and we offer an integrative strategy for advancing our molecular genetic understanding of VOGM pathogenesis and maintenance.

samples (Figure 4A), where the proportion of cells within each population differs between VOGM lesion and control (Figure 4B). Collectively, these data illustrate the feasibility of ETS for VOGM and we offer an integrative strategy for advancing our molecular genetic understanding of VOGM pathogenesis and maintenance. Pedigrees of VOGM families where the VOGM patient (proband) underwent endothelial sampling (red). Male patients are indicated with squares and female patients indicated with circles. No patient identifiers were available to anyone outside of the research group. VOGM, vein of Galen malformation. Overview of our approach to elucidate the genetic basis of VOGM. Figure was designed using Biorender. FACS, fluorescence-activated cell sorting; VOGM, vein of Galen malformation. Representative flow cytometry plot demonstrating experimental “endothelial” cell population (blue; CD31+, CD45−) obtained during endoluminal tissue sampling of vein of Galen malformation lesion. The yellow cells represent the excluded fraction which are likely monocyte and red blood cell in origin.

Overview of our approach to elucidate the genetic basis of VOGM. Figure was designed using Biorender. FACS, fluorescence-activated cell sorting; VOGM, vein of Galen malformation. Representative flow cytometry plot demonstrating experimental “endothelial” cell population (blue; CD31+, CD45−) obtained during endoluminal tissue sampling of vein of Galen malformation lesion. The yellow cells represent the excluded fraction which are likely monocyte and red blood cell in origin. Single-cell RNA sequencing of VOGM in situ identifies distinct cell types. A, Pink represents all cells obtained from the femoral access sheath, and blue represents in situ VOGM cells obtained by endoluminal tissue sampling. Both cell populations did not undergo antibody selection. B, Hierarchical clustering identifies 12 unique cell populations across control and VOGM “endothelial” cells using a published cerebrovascular transcriptome atlas for annotation.27 UMAP, Uniform Manifold Approximation and Projection for Dimension Reduction; VOGM, vein of Galen malformation.

While VOGM represents a severe cerebrovascular anomaly with profound systemic and neurologic consequences,27 it is also rare and until now, pathologic studies were limited to postmortem specimens.5 For these reasons, investigative studies into VOGM embryologic development, vascular maintenance, postnatal growth through angiogenesis, and cellular response to treatment have been hindered. Here we demonstrate feasibility of obtaining cells from the VOGM lesion directly with the goal of identifying in situ genomic alterations that are potentially disease altering. Our results suggest that as flow is reduced through the lesion, ETS cell yield improves. This is perhaps not surprising, but highlights the challenges in obtaining cellular information from cerebrovascular lesions. Selection of coils was determined only with clinical efficacy in mind and without bias toward utility for cell yield. We suspect that development of coil technology designed for biopsy (ie, antibody-coating, biomaterials with vessel wall adhesive properties, etc.) may overcome these limitations. Overall, we demonstrate feasibility for ETS in VOGM and discuss our approach to using this technique to identify disease-modifying genomic alterations with potential pharmacologic solutions.

signed for biopsy (ie, antibody-coating, biomaterials with vessel wall adhesive properties, etc.) may overcome these limitations. Overall, we demonstrate feasibility for ETS in VOGM and discuss our approach to using this technique to identify disease-modifying genomic alterations with potential pharmacologic solutions. Recent WES studies have identified putatively causative de novo and inherited gene mutations in ∼30% of sporadic VOGM cases.8,28 These studies identified a genome-wide significant burden of rare, damaging mutations in Ephrin B4 (EphB4).28,29 Interestingly, deletion of EphB4 in zebrafish recapitulates anomalies in the dorsal cranial vessels representing the phylogenic homologue of the vein of Galen.29 EphB4's role as a critical regulator of AV specification28,29 suggests VOGM may share a common pathogenetic mechanism with other congenital AV shunts such as AVM. Specifically, incomplete AV differentiation and zonation are hallmarks of AVM, driven in part by EphB430 mutations as well as several other genes regulating the developmental cerebrovascular niche.8,31 ETS, for the first time, enables in situ analysis to delineate these pathogenic mechanisms and pathways responsible for VOGM and cerebrovascular development more broadly.

tion are hallmarks of AVM, driven in part by EphB430 mutations as well as several other genes regulating the developmental cerebrovascular niche.8,31 ETS, for the first time, enables in situ analysis to delineate these pathogenic mechanisms and pathways responsible for VOGM and cerebrovascular development more broadly. The genetics underlying VOGM, and their relative contribution (compared with environmental factors) to disease, however, remain poorly understood.27 ETS represents a novel method for investigating these unanswered questions by screening lesional VOGM EC's for de novo, somatic, and second hit mutations.27,32 Complementary in situ scRNA seq may elucidate vascular maintenance and progrowth molecular programs that are not strictly genetic or heritable, but triggered in response to environmental factors. The PI3K/AKT/MTOR33 and RAS/RAF/MEK/ERK34 signaling programs, for example, influence angiogenesis, cell growth, apoptosis, and differentiation. Both are dysregulated in AVM, but at various frequency and intensity. Upstream EphB4 mutations are responsible in some instances yet absent in others. Additional mutations and transcriptional aberrations have been described in other vascular malformations, the vast majority being mosaic and restricted to pathologic tissue. This highlights the potential role for ETS and other evolving approaches such as liquid biopsy as tools to understand VOGM biology and to guide treatment.35-38

tations and transcriptional aberrations have been described in other vascular malformations, the vast majority being mosaic and restricted to pathologic tissue. This highlights the potential role for ETS and other evolving approaches such as liquid biopsy as tools to understand VOGM biology and to guide treatment.35-38 Future studies of ETS for genomic analysis may inform personalized therapies for VOGM and cerebrovascular diseases more broadly. While several existing compounds modulate RAS/RAF/MAPK and PI3K/AKT/mTOR39-42 signaling, these treatments may be misguided without patient-specific data. Somatic WES and scRNA seq have the potential to identify disease-causing mutations and disease-propagating transcriptional aberrations. Both represent potential targets for gene therapy, novel or repurposed pharmacotherapies. However, protocols for reliably sequencing picogram DNA quantities and interpreting the “big data” inherent to scRNA seq remain a focus of ongoing study. VOGM is a rare disease and treatment limited to few comprehensive pediatric hospitals with the requisite expertise. However, even at tertiary referral centers, only 2 to 3 new VOGM cases may be seen annually. The small sample size reported here reflects this limitation, which reduces statistical power and challenges the generalizability of our results. We have formed the VOGM Genetics Research Consortium (VOGM-GRC, www.vogm-genetics.com), a multi-institutional consortium of leading pediatric centers across the world to address this shortcoming in future studies.

VOGM is a rare disease and treatment limited to few comprehensive pediatric hospitals with the requisite expertise. However, even at tertiary referral centers, only 2 to 3 new VOGM cases may be seen annually. The small sample size reported here reflects this limitation, which reduces statistical power and challenges the generalizability of our results. We have formed the VOGM Genetics Research Consortium (VOGM-GRC, www.vogm-genetics.com), a multi-institutional consortium of leading pediatric centers across the world to address this shortcoming in future studies.

We describe here a method for successfully performing ETS in VOGM that addresses unique properties of this formidable arteriovenous shunt. The potential benefits of ETS in VOGM are numerous and include (1) acquisition of lesional tissue to identify somatic mutations, (2) ability to study molecular alterations in the shunt over time as they remodel, and (3) enabling precision medicine–based approaches to pharmacologically targetable mutations. Nevertheless, ETS devices, sequencing platforms, and genomic informatics all demonstrate substantial opportunities to improve. Our understanding of VOGM pathophysiology and vascular maintenance will grow in concert with such advances, potentially informing novel treatments to better outcomes across this specific population and cerebrovascular disease more broadly.