GPRASP1 loss-of-function links to arteriovenous malformations by endothelial activating GPR4 signals.

Arteriovenous malformations (AVMs) are fast-flow vascular malformations and refer to important causes of intracerebral haemorrhage in young adults. Getting deep insight into the genetic pathogenesis of AVMs is necessary. Herein, we identified two vital missense variants of G protein-coupled receptor (GPCR) associated sorting protein 1 (GPRASP1) in AVM patients for the first time and congruously determined to be loss-of-function variants in endothelial cells. GPRASP1 loss-of-function caused endothelial dysfunction in vitro and in vivo. Endothelial Gprasp1 knockout mice suffered a high probability of cerebral haemorrhage, AVMs and exhibited vascular anomalies in multiple organs. GPR4 was identified to be an effective GPCR binding with GPRASP1 to develop endothelial disorders. GPRASP1 deletion activated GPR4/cAMP/MAPK signalling to disturb endothelial functions, thus contributing to vascular anomalies. Mechanistically, GPRASP1 promoted GPR4 degradation. GPRASP1 enabled GPR4 K63-linked ubiquitination, enhancing the binding of GPR4 and RABGEF1 to activate RAB5 for conversions from endocytic vesicles to endosomes, and subsequently increasing the interactions of GPR4 and ESCRT members to package GPR4 into multivesicular bodies or late endosomes for lysosome degradation. Notably, the GPR4 antagonist NE 52-QQ57 and JNK inhibitor SP600125 effectively rescued the vascular phenotype caused by endothelial Gprasp1 deletion. Our findings provided novel insights into the roles of GPRASP1 in AVMs and hinted at new therapeutic strategies.

Somatic BrafV600E mutation in the cerebral endothelium induces brain arteriovenous malformations.

Current treatments of brain arteriovenous malformation (BAVM) are associated with considerable risks and at times incomplete efficacy. Therefore, a clinically consistent animal model of BAVM is urgently needed to investigate its underlying biological mechanisms and develop innovative treatment strategies. Notably, existing mouse models have limited utility due to heterogenous and untypical phenotypes of AVM lesions. Here we developed a novel mouse model of sporadic BAVM that is consistent with clinical manifestations in humans. Mice with BrafV600E mutations in brain ECs developed BAVM closely resembled that of human lesions. This strategy successfully induced BAVMs in mice across different age groups and within various brain regions. Pathological features of BAVM were primarily dilated blood vessels with reduced vascular wall stability, accompanied by spontaneous hemorrhage and neuroinflammation. Single-cell sequencing revealed differentially expressed genes that were related to the cytoskeleton, cell motility, and intercellular junctions. Early administration of Dabrafenib was found to be effective in slowing the progression of BAVMs; however, its efficacy in treating established BAVM lesions remained uncertain. Taken together, our proposed approach successfully induced BAVM that closely resembled human BAVM lesions in mice, rendering the model suitable for investigating the pathogenesis of BAVM and assessing potential therapeutic strategies.

Somatic PIK3CA Mutations in Sporadic Cerebral Cavernous Malformations.

BACKGROUND: Cerebral cavernous malformations (CCMs) are common sporadic and inherited vascular malformations of the central nervous system. Although familial CCMs are linked to loss-of-function mutations in KRIT1 (CCM1), CCM2, or PDCD10 (CCM3), the genetic cause of sporadic CCMs, representing 80% of cases, remains incompletely understood. METHODS: We developed two mouse models harboring mutations identified in human meningiomas with the use of the prostaglandin D2 synthase (PGDS) promoter. We performed targeted DNA sequencing of surgically resected CCMs from patients and confirmed our findings by droplet digital polymerase-chain-reaction analysis. RESULTS: We found that in mice expressing one of two common genetic drivers of meningioma - Pik3ca H1047R or AKT1 E17K - in PGDS-positive cells, a spectrum of typical CCMs develops (in 22% and 11% of the mice, respectively) instead of meningiomas, which prompted us to analyze tissue samples from sporadic CCMs from 88 patients. We detected somatic activating PIK3CA and AKT1 mutations in 39% and 1%, respectively, of lesion tissue from the patients. Only 10% of lesions harbored mutations in the CCM genes. We analyzed lesions induced by the activating mutations Pik3ca H1074R and AKT1 E17K in mice and identified the PGDS-expressing pericyte as the probable cell of origin. CONCLUSIONS: In tissue samples from sporadic CCMs, mutations in PIK3CA were represented to a greater extent than mutations in any other gene. The contribution of somatic mutations in the genes that cause familial CCMs was comparatively small. (Funded by the Fondation ARC pour la Recherche contre le Cancer and others.).

Comment on, "Increased expression of ephrin A1 in brain arteriovenous malformation: DNA microarray analysis".

This study by Sasahara et al. explores the role of ephrin A1 in brain arteriovenous malformations (AVM) using DNA microarray analysis, quantitative real-time RT-PCR, and immunohistochemistry. The research identifies significant upregulation of ephrin A1 in AVM, suggesting its potential involvement in the abnormal vascular architecture characteristic of this condition. The study's innovative methodology and thorough exploration of gene expression patterns contribute valuable insights into AVM pathogenesis, highlighting ephrin A1 as a potential therapeutic target. However, the study's limitations include clinical variability among patient samples and the use of draining veins as controls, which may affect the robustness of the findings. Future research should address these limitations by using more homogeneous samples and expanding the investigation to include other ephrin family members. This could provide a broader understanding of ephrin signaling in AVM and guide the development of targeted therapies.

Pathologic features of brain hemorrhage after radiation treatment: case series with somatic mutation analysis.

BACKGROUND: Radiation treatment for diseases of the brain can result in hemorrhagic adverse radiation effects. The underlying pathologic substrate of brain bleeding after irradiation has not been elucidated, nor potential associations with induced somatic mutations. METHODS: We retrospectively reviewed our department's pathology database over 5 years and identified 5 biopsy specimens (4 patients) for hemorrhagic lesions after brain irradiation. Tissues with active malignancy were excluded. Samples were characterized using H&E, Perl's Prussian Blue, and Masson's Trichrome; immunostaining for B-cells (anti-CD20), T-cells (anti-CD3), endothelium (anti-CD31), macrophages (anti-CD163), α-smooth muscle actin, and TUNEL. DNA analysis was done by two panels of next-generation sequencing for somatic mutations associated with known cerebrovascular anomalies. RESULTS: One lesion involved hemorrhagic expansion among multifocal microbleeds that had developed after craniospinal irradiation for distant medulloblastoma treatment. Three bleeds arose in the bed of focally irradiated arteriovenous malformations (AVM) after confirmed obliteration. A fifth specimen involved the radiation field distinct from an irradiated AVM bed. From these, 2 patterns of hemorrhagic vascular pathology were identified: encapsulated hematomas and cavernous-like malformations. All lesions included telangiectasias with dysmorphic endothelium, consistent with primordial cavernous malformations with an associated inflammatory response. DNA analysis demonstrated genetic variants in PIK3CA and/or PTEN genes but excluded mutations in CCM genes. CONCLUSIONS: Despite pathologic heterogeneity, brain bleeding after irradiation is uniformly associated with primordial cavernous-like telangiectasias and disruption of genes implicated in dysangiogenesis but not genes implicated as causative of cerebral cavernous malformations. This may implicate a novel signaling axis as an area for future study.

Understanding the pathogenesis of brain arteriovenous malformation: genetic variations, epigenetics, signaling pathways, and immune inflammation.

Brain arteriovenous malformation (BAVM) is a rare but serious cerebrovascular disease whose pathogenesis has not been fully elucidated. Studies have found that epigenetic regulation, genetic variation and their signaling pathways, immune inflammation, may be the cause of BAVM the main reason. This review comprehensively analyzes the key pathways and inflammatory factors related to BAVMs, and explores their interplay with epigenetic regulation and genetics. Studies have found that epigenetic regulation such as DNA methylation, non-coding RNAs and m6A RNA modification can regulate endothelial cell proliferation, apoptosis, migration and damage repair of vascular malformations through different target gene pathways. Gene defects such as KRAS, ACVRL1 and EPHB4 lead to a disordered vascular environment, which may promote abnormal proliferation of blood vessels through ERK, NOTCH, mTOR, Wnt and other pathways. PDGF-B and PDGFR-ß were responsible for the recruitment of vascular adventitial cells and smooth muscle cells in the extracellular matrix environment of blood vessels, and played an important role in the pathological process of BAVM. Recent single-cell sequencing data revealed the diversity of various cell types within BAVM, as well as the heterogeneous expression of vascular-associated antigens, while neutrophils, macrophages and cytokines such as IL-6, IL-1, TNF-α, and IL-17A in BAVM tissue were significantly increased. Currently, there are no specific drugs targeting BAVMs, and biomarkers for BAVM formation, bleeding, and recurrence are lacking clinically. Therefore, further studies on molecular biological mechanisms will help to gain insight into the pathogenesis of BAVM and develop potential therapeutic strategies.

Key biomarkers in cerebral arteriovenous malformations: Updated review.

The formation of vascular networks consisting of arteries, capillaries, and veins is vital in embryogenesis. It is also crucial in adulthood for the formation of a functional vasculature. Cerebral arteriovenous malformations (CAVMs) are linked with a remarkable risk of intracerebral hemorrhage because arterial blood is directly shunted into the veins before the arterial blood pressure is dissipated. The underlying mechanisms responsible for arteriovenous malformation (AVM) growth, progression, and rupture are not fully known, yet the critical role of inflammation in AVM pathogenesis has been noted. The proinflammatory cytokines are upregulated in CAVM, which stimulates overexpression of cell adhesion molecules in endothelial cells (ECs), leading to improved leukocyte recruitment. It is well-known that metalloproteinase-9 secretion by leukocytes disrupts CAVM walls resulting in rupture. Moreover, inflammation alters the angioarchitecture of CAVMs by upregulating angiogenic factors impacting the apoptosis, migration, and proliferation of ECs. A better understanding of the molecular signature of CAVM might allow us to identify biomarkers predicting this complication, acting as a goal for further investigations that may be potentially targeted in gene therapy. The present review is focused on the numerous studies conducted on the molecular signature of CAVM and the associated hemorrhage. The association of numerous molecular signatures with a higher risk of CAVM rupture is shown through inducing proinflammatory mediators, as well as growth factors signaling, Ras-mitogen-activated protein kinase-extracellular signal-regulated kinase, and NOTCH pathways, which are accompanied by cellular level inflammation and endothelial alterations resulting in vascular wall instability. According to the studies, it is assumed that matrix metalloproteinase, interleukin-6, and vascular endothelial growth factor are the biomarkers most associated with CAVM and the rate of hemorrhage, as well as diagnostic methods, with respect to enhancing the patient-specific risk estimation and improving treatment choices.

Evolution of clinical and translational advances in the management of pediatric arteriovenous malformations.

Arteriovenous malformations (AVMs) represent one of the most challenging diagnoses in pediatric neurosurgery. Until recently, the majority of AVMs was only identified after hemorrhage and primarily treated with surgery. However, recent advances in a wide range of fields-imaging, surgery, interventional radiology, radiation therapy, and molecular biology-have profoundly advanced the understanding and therapy of these complex lesions. Here we review the progress made in pediatric AVMs with a specific focus on innovations relevant to clinical care.

Selective Endothelial Hyperactivation of Oncogenic KRAS Induces Brain Arteriovenous Malformations in Mice.

OBJECTIVE: Brain arteriovenous malformations (bAVMs) are a leading cause of hemorrhagic stroke and neurological deficits in children and young adults, however, no pharmacological intervention is available to treat these patients. Although more than 95% of bAVMs are sporadic without family history, the pathogenesis of sporadic bAVMs is largely unknown, which may account for the lack of therapeutic options. KRAS mutations are frequently observed in cancer, and a recent unprecedented finding of these mutations in human sporadic bAVMs offers a new direction in the bAVM research. Using a novel adeno-associated virus targeting brain endothelium (AAV-BR1), the current study tested if endothelial KRASG12V mutation induces sporadic bAVMs in mice. METHODS: Five-week-old mice were systemically injected with either AAV-BR1-GFP or -KRASG12V . At 8 weeks after the AAV injection, bAVM formation and characteristics were addressed by histological and molecular analyses. The effect of MEK/ERK inhibition on KRASG12V -induced bAVMs was determined by treatment of trametinib, a US Food and Drug Administration (FDA)-approved MEK/ERK inhibitor. RESULTS: The viral-mediated KRASG12V overexpression induced bAVMs, which were composed of a tangled nidus mirroring the distinctive morphology of human bAVMs. The bAVMs were accompanied by focal angiogenesis, intracerebral hemorrhages, altered vascular constituents, neuroinflammation, and impaired sensory/cognitive/motor functions. Finally, we confirmed that bAVM growth was inhibited by trametinib treatment. INTERPRETATION: Our innovative approach using AAV-BR1 confirms that KRAS mutations promote bAVM development via the MEK/ERK pathway, and provides a novel preclinical mouse model of bAVMs which will be useful to develop a therapeutic strategy for patients with bAVM. ANN NEUROL 2021;89:926-941.

miR-18a Inhibits BMP4 and HIF-1α Normalizing Brain Arteriovenous Malformations.

RATIONALE: Brain arteriovenous malformations (AVMs) are abnormal tangles of vessels where arteries and veins directly connect without intervening capillary nets, increasing the risk of intracerebral hemorrhage and stroke. Current treatments are highly invasive and often not feasible. Thus, effective noninvasive treatments are needed. We previously showed that AVM-brain endothelial cells (BECs) secreted higher VEGF (vascular endothelial growth factor) and lower TSP-1 (thrombospondin-1) levels than control BEC; and that microRNA-18a (miR-18a) normalized AVM-BEC function and phenotype, although its mechanism remained unclear. OBJECTIVE: To elucidate the mechanism of action and potential clinical application of miR-18a as an effective noninvasive treatment to selectively restore the phenotype and functionality of AVM vasculature. METHODS AND RESULTS: The molecular pathways affected by miR-18a in patient-derived BECs and AVM-BECs were determined by Western blot, RT-qPCR (quantitative reverse transcription polymerase chain reaction), ELISA, co-IP, immunostaining, knockdown and overexpression studies, flow cytometry, and luciferase reporter assays. miR-18a was shown to increase TSP-1 and decrease VEGF by reducing PAI-1 (plasminogen activator inhibitor-1/SERPINE1) levels. Furthermore, miR-18a decreased the expression of BMP4 (bone morphogenetic protein 4) and HIF-1α (hypoxia-inducible factor 1α), blocking the BMP4/ALK (activin-like kinase) 2/ALK1/ALK5 and Notch signaling pathways. As determined by Boyden chamber assays, miR-18a also reduced the abnormal AVM-BEC invasiveness, which correlated with a decrease in MMP2 (matrix metalloproteinase 2), MMP9, and ADAM10 (ADAM metallopeptidase domain 10) levels. In vivo pharmacokinetic studies showed that miR-18a reaches the brain following intravenous and intranasal administration. Intranasal co-delivery of miR-18a and NEO100, a good manufacturing practices-quality form of perillyl alcohol, improved the pharmacokinetic profile of miR-18a in the brain without affecting its pharmacological properties. Ultra-high-resolution computed tomography angiography and immunostaining studies in an Mgp-/- AVM mouse model showed that miR-18a decreased abnormal cerebral vasculature and restored the functionality of the bone marrow, lungs, spleen, and liver. CONCLUSIONS: miR-18a may have significant clinical value in preventing, reducing, and potentially reversing AVM.

Somatic Gain of KRAS Function in the Endothelium Is Sufficient to Cause Vascular Malformations That Require MEK but Not PI3K Signaling.

RATIONALE: We previously identified somatic activating mutations in the KRAS (Kirsten rat sarcoma viral oncogene homologue) gene in the endothelium of the majority of human sporadic brain arteriovenous malformations; a disorder characterized by direct connections between arteries and veins. However, whether this genetic abnormality alone is sufficient for lesion formation, as well as how active KRAS signaling contributes to arteriovenous malformations, remains unknown. OBJECTIVE: To establish the first in vivo models of somatic KRAS gain of function in the endothelium in both mice and zebrafish to directly observe the phenotypic consequences of constitutive KRAS activity at a cellular level in vivo, and to test potential therapeutic interventions for arteriovenous malformations. METHODS AND RESULTS: Using both postnatal and adult mice, as well as embryonic zebrafish, we demonstrate that endothelial-specific gain of function mutations in Kras (G12D or G12V) are sufficient to induce brain arteriovenous malformations. Active KRAS signaling leads to altered endothelial cell morphogenesis and increased cell size, ectopic sprouting, expanded vessel lumen diameter, and direct connections between arteries and veins. Furthermore, we show that these lesions are not associated with altered endothelial growth dynamics or a lack of proper arteriovenous identity but instead seem to feature exuberant angiogenic signaling. Finally, we demonstrate that KRAS-dependent arteriovenous malformations in zebrafish are refractory to inhibition of the downstream effector PI3K but instead require active MEK (mitogen-activated protein kinase kinase 1) signaling. CONCLUSIONS: We demonstrate that active KRAS expression in the endothelium is sufficient for brain arteriovenous malformations, even in the setting of uninjured adult vasculature. Furthermore, the finding that KRAS-dependent lesions are reversible in zebrafish suggests that MEK inhibition may represent a promising therapeutic treatment for arteriovenous malformation patients. Graphical Abstract: A graphical abstract is available for this article.

Molecular and genetic mechanisms in brain arteriovenous malformations: new insights and future perspectives.

Brain arteriovenous malformations (bAVMs) are rare vascular lesions made of shunts between cerebral arteries and veins without the interposition of a capillary bed. The majority of bAVMs are asymptomatic, but some may be revealed by seizures and potentially life-threatening brain hemorrhage. The management of unruptured bAVMs remains a matter of debate. Significant progress in the understanding of their pathogenesis has been made during the last decade, particularly using genome sequencing and biomolecular analysis. Herein, we comprehensively review the recent molecular and genetic advances in the study of bAVMs that not only allow a better understanding of the genesis and growth of bAVMs, but also open new insights in medical treatment perspectives.

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

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.

Hypoxia Promotes Angiogenic Effect in Extracranial Arteriovenous Malformation Endothelial Cells.

Arteriovenous malformation (AVM) is characterized by high-flow blood vessels connecting arteries and veins without capillaries. This disease shows increased angiogenesis and a pathophysiological hypoxic environment in proximal tissues. Here, we analyzed the effects of hypoxia on angiogenesis in the endothelial cells (ECs) of AVM and normal tissues. ECs from human normal and AVM tissues were evaluated using immunocytochemistry with CD31. In vitro tube formation under hypoxia was tested in both ECs using Matrigel. The relative expression of angiogenesis-related genes was measured using real-time PCR. Under normoxia, CD31 was significantly higher in AVM ECs (79.23 ± 0.65%) than in normal ECs (74.15 ± 0.70%). Similar results were observed under hypoxia in AVM ECs (63.85 ± 1.84%) and normal ECs (60.52 ± 0.51%). In the tube formation test under normoxic and hypoxic conditions, the junction count and total vessel length were significantly greater in AVM ECs than normal ECs. Under both normoxia and hypoxia, the angiogenesis-related gene FSTL1 showed a significantly higher expression in AVM ECs than in normal ECs. Under hypoxia, CSPG4 expression was significantly lower in AVM ECs than in normal ECs. Accordingly, the angiogenic effect was increased in AVM ECs compared with that in normal ECs. These results provide a basic knowledge for an AVM treatment strategy.

Somatic Activating KRAS Mutations in Arteriovenous Malformations of the Brain.

BACKGROUND: Sporadic arteriovenous malformations of the brain, which are morphologically abnormal connections between arteries and veins in the brain vasculature, are a leading cause of hemorrhagic stroke in young adults and children. The genetic cause of this rare focal disorder is unknown. METHODS: We analyzed tissue and blood samples from patients with arteriovenous malformations of the brain to detect somatic mutations. We performed exome DNA sequencing of tissue samples of arteriovenous malformations of the brain from 26 patients in the main study group and of paired blood samples from 17 of those patients. To confirm our findings, we performed droplet digital polymerase-chain-reaction (PCR) analysis of tissue samples from 39 patients in the main study group (21 with matching blood samples) and from 33 patients in an independent validation group. We interrogated the downstream signaling pathways, changes in gene expression, and cellular phenotype that were induced by activating KRAS mutations, which we had discovered in tissue samples. RESULTS: We detected somatic activating KRAS mutations in tissue samples from 45 of the 72 patients and in none of the 21 paired blood samples. In endothelial cell-enriched cultures derived from arteriovenous malformations of the brain, we detected KRAS mutations and observed that expression of mutant KRAS (KRASG12V) in endothelial cells in vitro induced increased ERK (extracellular signal-regulated kinase) activity, increased expression of genes related to angiogenesis and Notch signaling, and enhanced migratory behavior. These processes were reversed by inhibition of MAPK (mitogen-activated protein kinase)-ERK signaling. CONCLUSIONS: We identified activating KRAS mutations in the majority of tissue samples of arteriovenous malformations of the brain that we analyzed. We propose that these malformations develop as a result of KRAS-induced activation of the MAPK-ERK signaling pathway in brain endothelial cells. (Funded by the Swiss Cancer League and others.).

Somatic Mosaicism in the Pathogenesis of de novo Cerebral Arteriovenous Malformations: A Paradigm Shift Implicating the RAS-MAPK Signaling Cascade.

Cerebral arteriovenous malformations (AVMs) are leading causes of lesional hemorrhagic stroke in both the pediatric and young adult population, with sporadic AVMs accounting for the majority of cases. Recent evidence has identified somatic mosaicism in key proximal components of the RAS-MAPK signaling cascade within endothelial cells collected from human sporadic cerebral AVMs, with early preclinical models supporting a potential causal role for these mutations in the pathogenesis of these malformations. Germline mutations that predispose to deregulation of the RAS-MAPK signaling axis have also been identified in hereditary vascular malformation syndromes, highlighting the key role of this signaling axis in global AVM development. Herein, we review the most recent genomic and preclinical evidence implicating somatic mosaicism in the RAS-MAPK signaling pathway in the pathogenesis of sporadic cerebral AVMs. Also, we review evidence for RAS-MAPK dysregulation in hereditary vascular malformation syndromes and present a hypothesis suggesting that this pathway is central for the development of both sporadic and syndrome-associated AVMs. Finally, we examine the clinical implications of these recent discoveries and highlight potential therapeutic targets within this signaling pathway.

RNA-Sequencing Highlights Inflammation and Impaired Integrity of the Vascular Wall in Brain Arteriovenous Malformations.

Background and Purpose- Interventional treatment of unruptured brain arteriovenous malformations (BAVMs) has become increasingly controversial. Because medical therapy is still lacking, we aimed to obtain insight into the disease mechanisms implicated in BAVMs and to identify potential targets for medical treatment to prevent rupture of a BAVM. Methods- We used next-generation RNA sequencing to identify differential expression on a transcriptome-wide level comparing tissue samples of 12 BAVMs to 16 intracranial control arteries. We identified differentially expressed genes by negative binominal generalized log-linear regression (false discovery rate corrected P<0.05). We selected 10 genes for validation using droplet digital polymerase chain reaction. We performed functional pathway analysis accounting for potential gene-length bias, to establish enhancement of biological pathways involved in BAVMs. We further assessed which Gene Ontology terms were enriched. Results- We found 736 upregulated genes in BAVMs including genes implicated in the cytoskeletal machinery and cell-migration and genes encoding for inflammatory cytokines and secretory products of neutrophils and macrophages. Furthermore, we found 498 genes downregulated including genes implicated in extracellular matrix composition, the binary angiopoietin-TIE system, and TGF (transforming growth factor)-ß signaling. We confirmed the differential expression of top 10 ranked genes. Functional pathway analysis showed enrichment of the protein digestion and absorption pathway (false discovery rate-adjusted P=1.70×10-2). We identified 47 enriched Gene Ontology terms (false discovery rate-adjusted P<0.05) implicated in cytoskeleton network, cell-migration, endoplasmic reticulum, transmembrane transport, and extracellular matrix composition. Conclusions- Our genome-wide RNA-sequencing study points to involvement of inflammatory mediators, loss of cerebrovascular quiescence, and impaired integrity of the vascular wall in the pathophysiology of BAVMs. Our study may lend support to potential receptivity of BAVMs to medical therapeutics, including those promoting vessel maturation, and anti-inflammatory and immune-modifying drugs.

Defective adgra2 (gpr124) splicing and function in zebrafish ouchless mutants.

A hitherto unidentified N-ethyl-N-nitrosourea (ENU)-induced mutation affects dorsal root ganglia (DRG) formation in ouchless mutant zebrafish larvae. In contrast to previous findings assigning the ouchless phenotypes to downregulated sorbs3 transcript levels, this work re-attributes the phenotypes to an essential splice site mutation affecting adgra2 (gpr124) splicing and function. Accordingly, ouchless mutants fail to complement previously characterized adgra2 mutants and exhibit highly penetrant cerebrovascular defects. The aberrantly spliced adgra2 transcript found in ouchless mutants encodes a receptor lacking a single leucine-rich repeat (LRR) within its N-terminus.

N6-methyladenosine methyltransferase METTL3 affects the phenotype of cerebral arteriovenous malformation via modulating Notch signaling pathway.

BACKGROUND: Cerebral arteriovenous malformation (AVM) is a serious life-threatening congenital cerebrovascular disease. Specific anatomical features, such as nidus size, location, and venous drainage, have been validated to affect treatment outcomes. Until recently, molecular biomarkers and corresponding molecular mechanism related to anatomical features and treatment outcomes remain unknown. METHODS: RNA N6-methyladenosine (m6A) Methyltransferase METTL3 was identified as a differentially expressed gene in groups with different lesion sizes by analyzing the transcriptome sequencing (RNA-seq) data. Tube formation and wound healing assays were performed to investigate the effect of METTL3 on angiogenesis. In addition, Methylated RNA Immunoprecipitation Sequencing technology (MeRIP-seq) was performed to screen downstream targets of METTL3 in endothelial cells and to fully clarify the specific underlying molecular mechanisms affecting the phenotype of cerebral AVM. RESULTS: In the current study, we found that the expression level of METTL3 was reduced in the larger pathological tissues of cerebral AVMs. Moreover, knockdown of METTL3 significantly affected angiogenesis of the human endothelial cells. Mechanistically, down-regulation of METTL3 reduced the level of heterodimeric Notch E3 ubiquitin ligase formed by DTX1 and DTX3L, thereby continuously activating the Notch signaling pathway. Ultimately, the up-regulated downstream genes of Notch signaling pathway dramatically affected the angiogenesis of endothelial cells. In addition, we demonstrated that blocking Notch pathway with DAPT could restore the phenotype of METTL3 deficient endothelial cells. CONCLUSIONS: Our findings revealed the mechanism by which m6A modification regulated the angiogenesis and might provide potential biomarkers to predict the outcome of treatment, as well as provide suitable pharmacological targets for preventing the formation and progression of cerebral AVM.

High-Flow Vascular Malformations in Children.

Children can have a variety of intracranial vascular anomalies ranging from small and incidental with no clinical consequences to complex lesions that can cause substantial neurologic deficits, heart failure, or profoundly affect development. In contrast to high-flow lesions with direct arterial-to-venous shunts, low-flow lesions such as cavernous malformations are associated with a lower likelihood of substantial hemorrhage, and a more benign course. Management of vascular anomalies in children has to incorporate an understanding of how treatment strategies may affect the normal development of the central nervous system. In this review, we discuss the etiologies, epidemiology, natural history, and genetic risk factors of three high-flow vascular malformations seen in children: brain arteriovenous malformations, intracranial dural arteriovenous fistulas, and vein of Galen malformations.