Emerging pathogenic mechanisms in human brain arteriovenous malformations: a contemporary review in the multiomics era

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  • 1 Department of Neurological Surgery, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, Phoenix; and
  • | 2 Barrow Aneurysm and AVM Research Center, Department of Translational Neuroscience, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, Phoenix, Arizona
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A variety of pathogenic mechanisms have been described in the formation, maturation, and rupture of brain arteriovenous malformations (bAVMs). While the understanding of bAVMs has largely been formulated based on animal models of rare hereditary diseases in which AVMs form, a new era of “omics” has permitted large-scale examinations of contributory genetic variations in human sporadic bAVMs. New findings regarding the pathogenesis of bAVMs implicate changes to endothelial and mural cells that result in increased angiogenesis, proinflammatory recruitment, and breakdown of vascular barrier properties that may result in hemorrhage; a greater diversity of cell populations that compose the bAVM microenvironment may also be implicated and complicate traditional models. Genomic sequencing of human bAVMs has uncovered inherited, de novo, and somatic activating mutations, such as KRAS, which contribute to the pathogenesis of bAVMs. New droplet-based, single-cell sequencing technologies have generated atlases of cell-specific molecular derangements. Herein, the authors review emerging genomic and transcriptomic findings underlying pathologic cell transformations in bAVMs derived from human tissues. The application of multiple sequencing modalities to bAVM tissues is a natural next step for researchers, although the potential therapeutic benefits or clinical applications remain unknown.

ABBREVIATIONS

bAVM = brain arteriovenous malformation; ddPCR = digital droplet polymerase chain reaction; DNM = de novo germline mutation; EndMT = endothelial-to-mesenchymal transition; HHT = hereditary hemorrhagic telangiectasia; HUVEC = human umbilical vascular endothelial cell; RNA-seq = RNA sequencing; scRNA-seq = single-cell mRNA sequencing.

A variety of pathogenic mechanisms have been described in the formation, maturation, and rupture of brain arteriovenous malformations (bAVMs). While the understanding of bAVMs has largely been formulated based on animal models of rare hereditary diseases in which AVMs form, a new era of “omics” has permitted large-scale examinations of contributory genetic variations in human sporadic bAVMs. New findings regarding the pathogenesis of bAVMs implicate changes to endothelial and mural cells that result in increased angiogenesis, proinflammatory recruitment, and breakdown of vascular barrier properties that may result in hemorrhage; a greater diversity of cell populations that compose the bAVM microenvironment may also be implicated and complicate traditional models. Genomic sequencing of human bAVMs has uncovered inherited, de novo, and somatic activating mutations, such as KRAS, which contribute to the pathogenesis of bAVMs. New droplet-based, single-cell sequencing technologies have generated atlases of cell-specific molecular derangements. Herein, the authors review emerging genomic and transcriptomic findings underlying pathologic cell transformations in bAVMs derived from human tissues. The application of multiple sequencing modalities to bAVM tissues is a natural next step for researchers, although the potential therapeutic benefits or clinical applications remain unknown.

Brain arteriovenous malformations (bAVMs) are rupture-prone dysplastic vascular tangles that are a potentially treatable cause of hemorrhagic stroke.1 Dynamic cellular interactions within the affected vasculature are responsible for the formation, progression, and ultimate bleeding of a bAVM. However, to date, mechanistic insights into these phenomena have largely come from rodent models of genetic syndromes of which bAVMs form only a small part, such as hereditary hemorrhagic telangiectasia (HHT).2,3 While such insights are of tremendous value and have been reviewed elsewhere,4 the molecular events catalyzing the cellular transformations in more common sporadic bAVMs have remained enigmatic. As a result, medical therapies have remained elusive.5

With completion of the Human Genome Project, emerging technological development has lent rise to the scientific era of “omics” and has revolutionized the study of human diseases, most notably cancer. Differing omics technologies permit large-scale surveys of genes (genomics), RNA expression (transcriptomics), proteins (proteomics), and metabolites (metabolomics).6 These emerging applications of omics have begun to uncover the acquired genetic mutations, alterations in gene expression, and cell transformations responsible for sporadic bAVMs (Fig. 1). As this new molecular era of vascular malformations begins, many questions remain unanswered and yet highly relevant to the neurosurgical community. Herein, we review the latest insights derived from sequencing applications of human bAVM tissue and discuss future challenges and opportunities for the discovery and eventual translation into personalized therapy.

FIG. 1.
FIG. 1.

Sequencing technologies to decipher molecular mechanisms in human bAVMs. Nucleic acids are extracted from either tissue homogenates or single cells following resection. Utilization of different high-throughput sequencing modalities allows genomic or transcriptomic profiling to deduce pathologic aberrations in bAVMs. Whole-exome sequencing sequences protein encoding gene regions in a genome and analyzes to discover pathologic DNA variants, such as DNMs or somatic activating mutations, or alterations in copy number. Microarray or next-generation RNA-seq detects and quantifies differences in gene expression. Functional enrichment analyses applied to sets of differentially expressed genes may be used to deduce comparative alterations in biological pathways or processes. Single-cell RNA-seq deduces gene expression profiles from individual cells. Clustering algorithms are employed to systematically determine cell composition and variation within the studied tissue. Differential gene expression analyses, functional enrichment analyses, or ligand-receptor interactions may be analyzed to infer alterations in gene expression, biological pathways or processes, or cell-to-cell communications, respectively, for individual cell classes. Illustrative findings highlight molecular or cellular aberrancies uncovered with each sequencing modality. Created with Biorender.com.

Cellular Aberrancy in Human bAVMs

The cerebral vasculature is a complex, multicellular conduit through which blood circulates.7,8 At the cellular level, the cerebral vessels are classically thought to be composed of endothelial cells, pericytes, vascular smooth muscle cells, and perivascular immune cells, such as perivascular macrophages.7,9,10 Research into cellular alterations in bAVMs has largely focused on these classic cell populations. However, emerging research has uncovered a greater level of cellular diversity, including cells such as perivascular fibroblasts and fibromyocytes, although their role in the formation and maturation of bAVMs remains unexplored.11,12 In this section, we describe works highlighting the cellular abnormalities in the traditional cell populations that compose human bAVMs.

Endothelial Cells

In the normal cerebral vasculature, endothelial cells form a single-cell thick inner lining of the vessel lumen.8,13 Outside of the circumventricular organs, the endothelial cell membrane is continuous. Adjacent endothelial cells are connected by tight junction and adherens junction protein complexes, and each cell displays low rates of nonspecific vesicular transport.8 Together, this forms the basis of the blood-brain barrier, which restricts the unregulated influx of circulating cells and macromolecules into the brain.8,14

Endothelial cells have been a focal point for bAVM research in humans. In bAVMs, endothelial cells become abnormal and morphologically assume an immature, hyperactive phenotype with filopodia, cytoplasmic vesicles, and vacuolization.15 Heightened endothelial turnover and proliferation have been observed and contribute to endothelial hyperplasia.16–18 In culture, bAVM endothelial cells proliferate and migrate more rapidly and express higher levels of proangiogenic factors, such as vascular endothelial growth factor A.19,20 Endothelial proliferation and blood-brain barrier properties are also spatially varied in situ. Endothelial hypoactivity and microvascular collapse or hyperplasia may be observed even within the same vessel segment.8 Within the nidus, some endothelial cell tight junction proteins are intact, whereas others become discontinuous with both disruptions in tight junctions and the presence of fenestrae (small pores) in the endothelial cell membranes.8,21,22 Functionally, this results in discrete areas of blood-brain barrier breakdown or microhemorrhage in some bAVMs.8 This, in tandem with endothelial upregulation of proinflammatory adhesion molecules (such as intercellular adhesion molecule-1) or cytokine (such as interleukin-8), results in increased infiltration of immune cells into the perivascular microenvironment.19,23–25

Mural Cells

Mural cells, a colloquial term to refer to both pericytes and smooth muscle cells, are contractile perivascular cells that stabilize the cerebral vasculature.7,9 Mural cell composition varies along the arteriovenous axis. For example, pericytes are found in capillaries, venules, and some arterioles and contribute to the regulation of the blood-brain barrier, angiogenesis, and cerebral blood flow.7,9 Smooth muscle cells are found in larger vessels, such as arteries and veins; they regulate cerebral blood flow, maintain vascular integrity, and phagocytose extracellular macromolecules.26–28

Although less well characterized compared with endothelial cells, reductions in both pericytes and smooth muscle cells have been reported in bAVMs.26–30 Pericyte reductions are greatest in ruptured AVMs and correlate with the severity of blood-brain barrier breakdown or microhemorrhage.31 Residual pericytes also have a number of histological abnormalities, including a greater abundance of pinocytotic vesicles, vacuoles, and cytoskeletal filaments.15 Restoration of pericyte populations has been shown to decrease vascular dysplasia and hemorrhage in animal models, supporting a contributing role of pericyte loss to destabilization.26–28 Smooth muscle cells have shown alterations in cytoskeletal and contractile proteins in human bAVMs and have displayed impaired migration and survival in vitro.22,26–28,32,33 While this evidence supports a contributory role of mural cells via either cell loss or phenotypic alterations in bAVMs, further characterization is needed.

Molecular Mechanisms Underlying Cellular Transformation in bAVMs

Like in many diseases, the cellular transformations responsible for bAVM formation are believed to result from acquired or inherited molecular aberrancies, such as genetic mutations or alterations in gene expression. These are thought to manifest in vascular malformations through environmental contexts that stimulate angiogenesis, such as tissue injury or inflammation. Recent large-scale molecular profiling of bAVMs has permitted deeper understanding of genetic underpinnings facilitating pathogenic cell transformations necessary for the formation of bAVMs.

Inherited Germline Mutations

Less than 5% of bAVMs occur in autosomal dominant inherited genetic syndromes such as HHT or capillary malformation–arteriovenous malformation syndrome.34 These syndromes have been shown to arise from mutations in TGF-β and Ras vascular signaling pathways, including endoglin, ACVRL1, RASA1, and EPHB4B.35–37 Knockout of HHT genes in endothelial cells results in bAVMs in rodents.2,3 Heritable genetic mutations may therefore contribute to development of bAVMs.

In humans without genetic syndromes, targeted genomic analyses of blood or saliva have suggested that polymorphisms in ACVRL1 or ENG may be associated with heightened risk of bAVMs.38,39 Whole-exome sequencing, a genomics technology that sequences protein encoding gene regions in a genome, has been used to identify previously undescribed germline loss-of-function mutations in SMAD9, a modulator of bone morphogenetic protein signaling and member of the TGF-β superfamily of proteins, in a nonsyndromic bAVM.40 Knockdown of smad9 led to abnormal arteriovenous connections in the cranial vasculature of zebrafish.40 This has yet to be replicated in other patients with nonsyndromic bAVMs, and unbiased genome-wide association studies failed to identify single-nucleotide polymorphisms associated with increased risk for AVMs.41 Thus, most AVMs are thought not to arise from heritable germline genetic mutations.

De Novo Germline Mutations

For most AVMs deemed sporadic or of no clear heritable cause, active investigations into noninherited mutagenesis have begun to shed light on additional genetic contributions to bAVM pathogenesis. Such genetic contributions may be due to de novo germline mutations (DNMs), which occur within one generation due to mutagenesis in parental gametes or early on in embryonic development.42,43 DNMs are therefore present in most cells of an individual but not in the majority of their parents’ cells. Using whole-exome sequencing in 60 case-unaffected-parental trios, investigators identified and prioritized 16 DNMs associated with sporadic bAVMs, including ENG, EPAS1 (also known as hypoxia inducible factor 2A), DACT1, EMILIN1, GAS6, JUP, COL1A1, and EXPH5.44 As initial proof of concept, knockdown of epas1 resulted in normal trunk vasculature but significant brain vascular malformations in zebrafish.44 Others have reported de novo cases of HHT with AVMs in other organs, such as the lung.45 Future studies are needed to better characterize contributions of DNMs to AVMs, especially in younger patients.

Somatic Activating Mutations

Somatic mutations occur in normal cells through the course of life as a result of environmental and/or endogenous mutagens.46,47 These mutations are passed on to the progeny of the mutated cell with each cell division and result in mosaicism. Most accumulate passively and do not influence cell behavior, whereas others lead to a pathogenic gain or loss of cellular function and result in clonal expansion.48 In a landmark study, investigators employed whole-exome sequencing in blood-tissue pairs and identified somatic gain-of-function or activating mutations in the proto-oncogene KRAS in bAVMs, but not circulating leukocytes.49 Using whole-exome sequencing in tissues or targeted digital droplet polymerase chain reaction (ddPCR), the investigators uncovered that somatic activating KRAS mutations occur in a majority of sporadic bAVMs, specifically the G12V, G12D, and Q61H variants.49 These findings have been confirmed using targeted gene panel–based deep sequencing approaches in tandem with ddPCR. In a recent panel of 422 solid tumor–related genes, investigators identified additional mutations in KRAS and the V600E activating mutation in BRAF.50 In their cohort, activating KRAS and BRAF mutations occurred in 76.2% and 4.8% of sporadic bAVMs, respectively.

KRAS is an effector molecule downstream of receptor tyrosine kinases that can activate multiple signal transduction cascades (summarized in Fig. 2). One of the downstream pathways is the MAPK-ERK signaling pathway.51 Other activating mutations in the MAPK signaling pathway are associated with extracranial vascular malformations.52–55 Targeted analysis of the MAPK signaling pathway identified 24 prospective candidate bAVM-associated somatic variants in 11 MAPK pathway genes. However, mutations in KRAS were the only recurrent mutations in multiple individuals.56 Many independent groups have focused on these KRAS and related BRAF mutations with targeted analyses such as ddPCR; a recent metanalysis summarizing these works found the prevalence of KRAS and BRAF mutations in bAVMs to be 55% and 7.5%, respectively.57

FIG. 2.
FIG. 2.

Ras-MAPK signaling pathway in arteriovenous malformations. Ligand binding and activation of receptor tyrosine kinases, such as VEGFR2, leads to activation of KRAS and the MAPK ERK signaling pathway, resulting in endothelial migration and angiogenesis. Molecular abnormalities in the Ras-MAPK signaling pathway have been documented in human bAVM tissues, genetically engineered HUVECs, and preclinical mouse or zebrafish animal models. The experimental source for each molecular abnormality is described. Created with BioRender.com.

How mutations in KRAS influence the clinical presentation or natural history of bAVMs is less clear. A single report has found that KRAS-mutated bAVMs are more likely to present with hemorrhage at first presentation, whereas others have found no association with bAVM size or location, age at diagnosis, presenting symptom, or cell proliferation on histology.44,56,58,59 To shed light on these questions and therapeutic implications, groups have recently generated mice and zebrafish that express KRASG12V and KRASG12D and confirmed that this is sufficient to induce bAVMs via excessive angiogenic sprouting of endothelial cells.60,61 Importantly, works in these model systems have supported that mutant KRAS results in bAVMs via MAPK signaling and that targeted inhibition of the downstream effector, MEK, may reverse KRAS-dependent AVMs.60,61 These works have therefore suggested that acquired molecular aberrancies may have therapeutic relevance in preclinical models; whether they may be effectively translated into humans, however, remains to be seen.

Differential Gene Expression

In addition to studying genetic polymorphisms or mutations within individual genes, others have studied patterns of aberrant gene expression in bAVMs, so-called transcriptomics, to deduce genes or pathways that may be implicated in pathologic changes. Early insights into differential gene expression came from groups using microarray, an early omics technology that profiles gene expression. Microarray studies have demonstrated upregulation of several genes implicated in angiogenesis within bAVMs, including EFNA1.62 Upregulation of other angiogenic factors, such as VEGFA, as well as cell surface adhesion receptors important to cell-matrix and cell-cell interactions, such as ITGAV, has also been demonstrated.63

Due to computational limitations, many early microarray-based studies focused on expression patterns of individual genes. Newer, high-throughput next-generation RNA-sequencing (RNA-seq) technologies are now employed with systems-biology approaches that employ functional enrichment analyses (such as pathway or ontological analyses) to detect relevant groups of differentially regulated genes and aid interpretation with respect to biological function of certain genes.64 For example, more recent RNA-seq–based gene expression profiling in bAVMs has identified 736 upregulated genes, including genes encoding cytoskeletal machinery, cell migration, and inflammatory and secretory products of neutrophils and macrophages. In addition, 498 downregulated genes were identified, including genes encoding extracellular matrix components and the stabilizing angiopoietin-TIE pathway. Gene ontological analysis of these differentially expressed genes implicated the cytoskeleton network, cell migration, and extracellular matrix composition in the pathogenesis of bAVMs.65 RNA-seq and gene-set enrichment analysis have also been used to discover reductions in mature brain endothelial marker genes within bAVMs.44 Such analysis has also been applied to profile transcriptomes of bAVMs with variable phenotypes (such as those with lower or higher blood flow). Differential gene expression was found in 368 genes along with enrichment of Wnt signaling in low-flow bAVMs.66

The sources of these variations in gene expression or molecular variations within bAVMs have yet to be fully characterized. One hypothesis is that dysregulated gene expression may be the downstream by-product of somatic mutations, such as KRAS. By expressing in human umbilical vascular endothelial cells (HUVECs), groups have profiled aberrancies in gene expression with RNA-seq and found upregulation of gene sets in bAVM causative pathways, such as Notch signaling, angiogenesis, and endothelial-to-mesenchymal transition (EndMT; a process through which endothelial cells undergo a series of molecular transformations that lead to a change in phenotype toward a mesenchymal cell).49 Employing a similar approach, others have shown that expression of KRASG12V in HUVECs leads to upregulation of gene sets implicated in angiogenesis, cell cycle, and regulation of MAPK signaling, while increasing endothelial cell permeability.60 RNA-seq profiling with gene-set enrichment analysis in HUVECs following knockdown of newly discovered DNMs, such as JUP or ENG, has further implicated EndMT.44 Whether acquired genetic mutations account for all observed transcriptomic alterations is unlikely, and other mechanisms such as flow-induced modulation of gene expression or other epigenetic mechanisms of transcriptional regulation have yet to be explored.

Single-Cell Transcriptomics

Traditionally, sequencing technologies are used on tissue homogenates in which the nucleic acids from millions of cells are pooled together, a process known as bulk sequencing. In pooling cells, however, relevant transcriptional changes occurring in only a minority of cells may be hidden. Bulk sequencing precludes the deduction of the cell of origin of detected DNA mutations or gene expression changes. These are typically either inferred or validated via orthogonal techniques. To overcome these limitations, emerging droplet-based sequencing technologies have been developed that utilize molecular barcoding tags such that sequencing information retains its cell of origin. To employ technologies such as single-cell mRNA sequencing (scRNA-seq), organs or tissues are deconstructed into single-cell suspensions. Large-scale profiling of single-cell gene expression profiles is then used to assemble comprehensive cell censes, known as cell atlases, to more comprehensively deduce cell composition of an organ such as the brain; they may also be especially powerful in defining rare cell types or variations.67,68

Initial scRNA-seq applications to the mouse cerebrovasculature demonstrated the utility of this approach and identified previously unrecognized cell variation, such as the presence of perivascular fibroblasts and a molecular basis for phenotypic changes with arteriovenous transitions known as zonations.9,11,69 In the human cerebrovasculature, emerging reports have confirmed conservation of endothelial arteriovenous zonations at a molecular level.12,70,71 Unlike mice, however, there is expansion in transcriptional variation and diversity of perivascular cells in humans, including the presence of smooth muscle–like cells called fibromyocytes and previously unrecognized variations in pericytes, smooth muscle cells, and perivascular fibroblasts.12,70,71 By creating these cellular atlases or blueprints of the normal cerebrovasculature, each cell type or subtype may be compared with analogous cells in diseased tissues to systematically define cell-specific abnormalities in human cerebrovascular diseases.

Use of this technology to decipher cell-specific molecular aberrancy in bAVMs has only recently been reported (Fig. 3). Employing scRNA-seq, one group profiled 13,328 cells and discovered a distinct mesenchymal population in bAVMs that coexpresses endothelial and mesenchymal markers suggestive of EndMT.44 We recently profiled 106,853 single cells and systematically reported cell-specific patterns of aberrant gene expression within AVMs. Molecular aberration was found in each cerebrovascular cell population, including endothelial cells, pericytes, smooth muscle cells, fibromyocytes, and fibroblasts (Fig. 3A).12 Within endothelial cells, we found loss of normal arteriovenous zonations. We identified nidal endothelial cells with unique signatures, such as inappropriate expression of PLVAP and PGF, and expression of gene sets to support heightened angiogenic potential and abnormal immune cell crosstalk.12 While immune infiltration has been previously described in bAVMs, prior studies have resorted to only studying a handful a markers at a time.24 By employing scRNA-seq, we identified 17 molecularly distinct immune cells within bAVMs (Fig. 3B). With bAVM rupture, we observed enrichment of specific immune cells, such as a specialized type of monocyte identified by expression of GPNMB, which destabilizes the vasculature by depleting smooth muscle cells (Fig. 3B).12 As with other atlases, we created a freely searchable cell viewer to allow neurosurgeons and other investigators to map gene expression in the human cerebrovasculature and bAVMs.12,72

FIG. 3.
FIG. 3.

Single-cell sequencing reveals cell-resolution alterations in AVMs. A: Single-cell mRNAseq of unruptured human bAVMs has uncovered further diversity in vascular cell composition, including unrecognized transcriptional variation in endothelial cells (purple), smooth muscle cells (red), fibromyocytes (brown), and perivascular fibroblasts (yellow). Illustrative examples are provided of cell-resolution gene expression changes for each vascular cell type, such as a unique molecular signature of endothelial cells in the bAVM nidus. B: Heightened vascular immune cell crosstalk results in immune cell infiltration and gives rise to a diverse and spatially heterogeneous cerebrovascular inflammatory response in bAVMs. The center panels show the 17 distinct immune cells of myeloid or lymphoid origin that have been identified. Enrichment of a specialized monocyte subtype, known as GPNMB+ monocytes (light blue), occurs with bAVM rupture and destabilizes the vasculature by depleting smooth muscle cells (red) via secretion of osteopontin (yellow, encoded by the gene SPP1). Created with Biorender.com.

Future Directions

Emerging applications of sequencing-based technologies have now granted unparalleled insights into the molecular genetics and cellular diversity of human bAVMs. A majority of solitary lesions hitherto deemed sporadic because of an absence of familial genetic components now appear to be the result of acquired genetic events.44,49,50 Multiple cell-signaling pathways have been implicated, including TGF-β, NOTCH, and Ras-MAPK. How these signaling pathways converge to result in similar phenotypes or whether this implies molecular heterogeneity and distinct subtypes, however, remains to be seen. Many of the published studies have only applied a single sequencing modality to examine genetic mutations or gene expression changes in relative isolation. To allow more comprehensive molecular classification, studies that integrate multiple omics platforms, so-called multiomics, are needed to understand relationships between genetics, epigenetics, and transcriptomics in human bAVMs.

Conclusions

Molecular classification of tumors has revolutionized the field of neuro-oncology and medicine. Neurosurgeons will play a vital role in determining whether the same may be true for vascular malformations. An isolated case report has suggested that genotype-targeted molecular inhibition may have a role in obliterating an AVM.73 However, most molecular profiling has been performed on tissues excised in the operating room, as stereotactic biopsy would be far too dangerous in a high-flow vascular lesion, such as bAVM. Because of the much lower recurrence rates of bAVMs when compared with cancer, molecular information obtained at the time of definitive treatment (surgery) is unlikely to inform patient care. Efforts to develop technologies to permit molecular stratification in living patients, such as endovascular or liquid biopsy, are ongoing.74,75 Despite these persistent barriers and shortcomings, the dawn of a molecular era in vascular malformations is upon us, and its clinical utility will be decided in the decades to come.

Disclosures

The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

Author Contributions

Conception and design: Lawton, Winkler, Catapano. Drafting the article: Winkler, Pacult. Critically revising the article: Lawton, Pacult, Catapano, Scherschinski, Srinivasan, Graffeo, Oh. Reviewed submitted version of manuscript: all authors. Study supervision: Lawton.

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    Gallione CJ, Richards JA, Letteboer TG, et al. SMAD4 mutations found in unselected HHT patients. J Med Genet. 2006;43(10):793797.

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    Revencu N, Boon LM, Mendola A, et al. RASA1 mutations and associated phenotypes in 68 families with capillary malformation-arteriovenous malformation. Hum Mutat. 2013;34(12):16321641.

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    Simon M, Franke D, Ludwig M, et al. Association of a polymorphism of the ACVRL1 gene with sporadic arteriovenous malformations of the central nervous system. J Neurosurg. 2006;104(6):945949.

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    Pawlikowska L, Tran MN, Achrol AS, et al. Polymorphisms in transforming growth factor-beta-related genes ALK1 and ENG are associated with sporadic brain arteriovenous malformations. Stroke. 2005;36(10):22782280.

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    Walcott BP, Winkler EA, Zhou S, et al. Identification of a rare BMP pathway mutation in a non-syndromic human brain arteriovenous malformation via exome sequencing. Hum Genome Var. 2018;5:18001.

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    Weinsheimer S, Bendjilali N, Nelson J, et al. Genome-wide association study of sporadic brain arteriovenous malformations. J Neurol Neurosurg Psychiatry. 2016;87(9):916923.

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    Goldmann JM, Veltman JA, Gilissen C. De novo mutations reflect development and aging of the human germline. Trends Genet. 2019;35(11):828839.

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    Kessler MD, Loesch DP, Perry JA, et al. De novo mutations across 1,465 diverse genomes reveal mutational insights and reductions in the Amish founder population. Proc Natl Acad Sci U S A. 2020;117(5):25602569.

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    Li H, Nam Y, Huo R, et al. De novo germline and somatic variants convergently promote endothelial-to-mesenchymal transition in simplex brain arteriovenous malformation. Circ Res. 2021;129(9):825839.

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    Cheng C, Zhu X, Yang F, Zhao S, Chen X. Identification of a novel de novo mutation of ENG gene in a fetus with pulmonary arteriovenous malformation. Clin Genet. 2020;98(6):626627.

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    Martincorena I, Fowler JC, Wabik A, et al. Somatic mutant clones colonize the human esophagus with age. Science. 2018;362(6417):911917.

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    Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, Kinzler KW. Cancer genome landscapes. Science. 2013;339(6127):15461558.

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    Nikolaev SI, Vetiska S, Bonilla X, et al. Somatic activating KRAS mutations in arteriovenous malformations of the brain. N Engl J Med. 2018;378(3):250261.

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    Hong T, Yan Y, Li J, et al. High prevalence of KRAS/BRAF somatic mutations in brain and spinal cord arteriovenous malformations. Brain. 2019;142(1):2334.

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    Gimple RC, Wang X. RAS: Striking at the core of the oncogenic circuitry. Front Oncol. 2019;9:965.

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    Couto JA, Huang AY, Konczyk DJ, et al. Somatic MAP2K1 mutations are associated with extracranial arteriovenous malformation. Am J Hum Genet. 2017;100(3):546554.

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    Couto JA, Vivero MP, Kozakewich HP, et al. A somatic MAP3K3 mutation is associated with verrucous venous malformation. Am J Hum Genet. 2015;96(3):480486.

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    Limaye N, Kangas J, Mendola A, et al. Somatic activating PIK3CA mutations cause venous malformation. Am J Hum Genet. 2015;97(6):914921.

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    Al-Olabi L, Polubothu S, Dowsett K, et al. Mosaic RAS/MAPK variants cause sporadic vascular malformations which respond to targeted therapy. J Clin Invest. 2018;128(4):14961508.

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    Gao S, Nelson J, Weinsheimer S, et al. Somatic mosaicism in the MAPK pathway in sporadic brain arteriovenous malformation and association with phenotype. J Neurosurg. 2021;136(1):148155.

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    Bameri O, Salarzaei M, Parooie F. KRAS/BRAF mutations in brain arteriovenous malformations: a systematic review and meta-analysis. Interv Neuroradiol. 2021;27(4):539546.

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    Goss JA, Huang AY, Smith E, et al. Somatic mutations in intracranial arteriovenous malformations. PLoS One. 2019;14(12):e0226852.

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    Priemer DS, Vortmeyer AO, Zhang S, Chang HY, Curless KL, Cheng L. Activating KRAS mutations in arteriovenous malformations of the brain: frequency and clinicopathologic correlation. Hum Pathol. 2019;89:3339.

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    Fish JE, Flores Suarez CP, Boudreau E, et al. Somatic gain of KRAS function in the endothelium is sufficient to cause vascular malformations that require MEK but not PI3K signaling. Circ Res. 2020;127(6):727743.

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    Hashimoto T, Lawton MT, Wen G, et al. Gene microarray analysis of human brain arteriovenous malformations. Neurosurgery. 2004;54(2):410425.

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    García-Campos MA, Espinal-Enríquez J, Hernández-Lemus E. Pathway analysis: state of the art. Front Physiol. 2015;6:383.

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    Hauer AJ, Kleinloog R, Giuliani F, et al. RNA-sequencing highlights inflammation and impaired integrity of the vascular wall in brain arteriovenous malformations. Stroke. 2020;51(1):268274.

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    Huo R, Fu W, Li H, et al. RNA sequencing reveals the activation of Wnt signaling in low flow rate brain arteriovenous malformations. J Am Heart Assoc. 2019;8(12):e012746.

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Illustration from Agosti et al. (E5). Used with permission of Mayo Foundation for Medical Education and Research. All rights reserved.

  • View in gallery

    Sequencing technologies to decipher molecular mechanisms in human bAVMs. Nucleic acids are extracted from either tissue homogenates or single cells following resection. Utilization of different high-throughput sequencing modalities allows genomic or transcriptomic profiling to deduce pathologic aberrations in bAVMs. Whole-exome sequencing sequences protein encoding gene regions in a genome and analyzes to discover pathologic DNA variants, such as DNMs or somatic activating mutations, or alterations in copy number. Microarray or next-generation RNA-seq detects and quantifies differences in gene expression. Functional enrichment analyses applied to sets of differentially expressed genes may be used to deduce comparative alterations in biological pathways or processes. Single-cell RNA-seq deduces gene expression profiles from individual cells. Clustering algorithms are employed to systematically determine cell composition and variation within the studied tissue. Differential gene expression analyses, functional enrichment analyses, or ligand-receptor interactions may be analyzed to infer alterations in gene expression, biological pathways or processes, or cell-to-cell communications, respectively, for individual cell classes. Illustrative findings highlight molecular or cellular aberrancies uncovered with each sequencing modality. Created with Biorender.com.

  • View in gallery

    Ras-MAPK signaling pathway in arteriovenous malformations. Ligand binding and activation of receptor tyrosine kinases, such as VEGFR2, leads to activation of KRAS and the MAPK ERK signaling pathway, resulting in endothelial migration and angiogenesis. Molecular abnormalities in the Ras-MAPK signaling pathway have been documented in human bAVM tissues, genetically engineered HUVECs, and preclinical mouse or zebrafish animal models. The experimental source for each molecular abnormality is described. Created with BioRender.com.

  • View in gallery

    Single-cell sequencing reveals cell-resolution alterations in AVMs. A: Single-cell mRNAseq of unruptured human bAVMs has uncovered further diversity in vascular cell composition, including unrecognized transcriptional variation in endothelial cells (purple), smooth muscle cells (red), fibromyocytes (brown), and perivascular fibroblasts (yellow). Illustrative examples are provided of cell-resolution gene expression changes for each vascular cell type, such as a unique molecular signature of endothelial cells in the bAVM nidus. B: Heightened vascular immune cell crosstalk results in immune cell infiltration and gives rise to a diverse and spatially heterogeneous cerebrovascular inflammatory response in bAVMs. The center panels show the 17 distinct immune cells of myeloid or lymphoid origin that have been identified. Enrichment of a specialized monocyte subtype, known as GPNMB+ monocytes (light blue), occurs with bAVM rupture and destabilizes the vasculature by depleting smooth muscle cells (red) via secretion of osteopontin (yellow, encoded by the gene SPP1). Created with Biorender.com.

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    Simon M, Franke D, Ludwig M, et al. Association of a polymorphism of the ACVRL1 gene with sporadic arteriovenous malformations of the central nervous system. J Neurosurg. 2006;104(6):945949.

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    Pawlikowska L, Tran MN, Achrol AS, et al. Polymorphisms in transforming growth factor-beta-related genes ALK1 and ENG are associated with sporadic brain arteriovenous malformations. Stroke. 2005;36(10):22782280.

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    Walcott BP, Winkler EA, Zhou S, et al. Identification of a rare BMP pathway mutation in a non-syndromic human brain arteriovenous malformation via exome sequencing. Hum Genome Var. 2018;5:18001.

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    • Export Citation
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    Weinsheimer S, Bendjilali N, Nelson J, et al. Genome-wide association study of sporadic brain arteriovenous malformations. J Neurol Neurosurg Psychiatry. 2016;87(9):916923.

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    Goldmann JM, Veltman JA, Gilissen C. De novo mutations reflect development and aging of the human germline. Trends Genet. 2019;35(11):828839.

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    Kessler MD, Loesch DP, Perry JA, et al. De novo mutations across 1,465 diverse genomes reveal mutational insights and reductions in the Amish founder population. Proc Natl Acad Sci U S A. 2020;117(5):25602569.

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    Li H, Nam Y, Huo R, et al. De novo germline and somatic variants convergently promote endothelial-to-mesenchymal transition in simplex brain arteriovenous malformation. Circ Res. 2021;129(9):825839.

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    • Export Citation
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    Cheng C, Zhu X, Yang F, Zhao S, Chen X. Identification of a novel de novo mutation of ENG gene in a fetus with pulmonary arteriovenous malformation. Clin Genet. 2020;98(6):626627.

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    • Export Citation
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    Martincorena I, Fowler JC, Wabik A, et al. Somatic mutant clones colonize the human esophagus with age. Science. 2018;362(6417):911917.

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    Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, Kinzler KW. Cancer genome landscapes. Science. 2013;339(6127):15461558.

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    • Crossref
    • Search Google Scholar
    • Export Citation
  • 50

    Hong T, Yan Y, Li J, et al. High prevalence of KRAS/BRAF somatic mutations in brain and spinal cord arteriovenous malformations. Brain. 2019;142(1):2334.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51

    Gimple RC, Wang X. RAS: Striking at the core of the oncogenic circuitry. Front Oncol. 2019;9:965.

  • 52

    Couto JA, Huang AY, Konczyk DJ, et al. Somatic MAP2K1 mutations are associated with extracranial arteriovenous malformation. Am J Hum Genet. 2017;100(3):546554.

    • Crossref
    • Search Google Scholar
    • Export Citation
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    Couto JA, Vivero MP, Kozakewich HP, et al. A somatic MAP3K3 mutation is associated with verrucous venous malformation. Am J Hum Genet. 2015;96(3):480486.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 54

    Limaye N, Kangas J, Mendola A, et al. Somatic activating PIK3CA mutations cause venous malformation. Am J Hum Genet. 2015;97(6):914921.

  • 55

    Al-Olabi L, Polubothu S, Dowsett K, et al. Mosaic RAS/MAPK variants cause sporadic vascular malformations which respond to targeted therapy. J Clin Invest. 2018;128(4):14961508.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 56

    Gao S, Nelson J, Weinsheimer S, et al. Somatic mosaicism in the MAPK pathway in sporadic brain arteriovenous malformation and association with phenotype. J Neurosurg. 2021;136(1):148155.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 57

    Bameri O, Salarzaei M, Parooie F. KRAS/BRAF mutations in brain arteriovenous malformations: a systematic review and meta-analysis. Interv Neuroradiol. 2021;27(4):539546.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 58

    Goss JA, Huang AY, Smith E, et al. Somatic mutations in intracranial arteriovenous malformations. PLoS One. 2019;14(12):e0226852.

  • 59

    Priemer DS, Vortmeyer AO, Zhang S, Chang HY, Curless KL, Cheng L. Activating KRAS mutations in arteriovenous malformations of the brain: frequency and clinicopathologic correlation. Hum Pathol. 2019;89:3339.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 60

    Fish JE, Flores Suarez CP, Boudreau E, et al. Somatic gain of KRAS function in the endothelium is sufficient to cause vascular malformations that require MEK but not PI3K signaling. Circ Res. 2020;127(6):727743.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 61

    Park ES, Kim S, Huang S, et al. Selective endothelial hyperactivation of oncogenic KRAS induces brain arteriovenous malformations in mice. Ann Neurol. 2021;89(5):926941.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 62

    Sasahara A, Kasuya H, Akagawa H, et al. Increased expression of ephrin A1 in brain arteriovenous malformation: DNA microarray analysis. Neurosurg Rev. 2007;30(4):299305.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 63

    Hashimoto T, Lawton MT, Wen G, et al. Gene microarray analysis of human brain arteriovenous malformations. Neurosurgery. 2004;54(2):410425.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 64

    García-Campos MA, Espinal-Enríquez J, Hernández-Lemus E. Pathway analysis: state of the art. Front Physiol. 2015;6:383.

  • 65

    Hauer AJ, Kleinloog R, Giuliani F, et al. RNA-sequencing highlights inflammation and impaired integrity of the vascular wall in brain arteriovenous malformations. Stroke. 2020;51(1):268274.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 66

    Huo R, Fu W, Li H, et al. RNA sequencing reveals the activation of Wnt signaling in low flow rate brain arteriovenous malformations. J Am Heart Assoc. 2019;8(12):e012746.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 67

    Hodge RD, Bakken TE, Miller JA, et al. Conserved cell types with divergent features in human versus mouse cortex. Nature. 2019;573(7772):6168.

  • 68

    Nowakowski TJ, Bhaduri A, Pollen AA, et al. Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex. Science. 2017;358(6368):13181323.

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