Inflammation and neutrophil extracellular traps in cerebral cavernous malformation.
Cellular and molecular life sciences : CMLS
Cerebral Cavernous Malformation (CCM) is a brain vascular disease with various neurological symptoms. In this study, we describe the inflammatory profile in CCM and show for the first time the formation of neutrophil extracellular traps (NETs) in rodents and humans with CCM. Through RNA-seq analysis of cerebellum endothelial cells from wild-type mice and mice with an endothelial cell-specific ablation of the Ccm3 gene (Ccm3), we show that endothelial cells from Ccm3 mice have an increased expression of inflammation-related genes. These genes encode proinflammatory cytokines and chemokines, as well as adhesion molecules, which promote recruitment of inflammatory and immune cells. Similarly, immunoassays showed elevated levels of these cytokines and chemokines in the cerebellum of the Ccm3 mice. Consistently, both flow cytometry and immunofluorescence analysis showed infiltration of different subsets of leukocytes into the CCM lesions. Neutrophils, which are known to fight against infection through different strategies, including the formation of NETs, represented the leukocyte subset within the most pronounced increase in CCM. Here, we detected elevated levels of NETs in the blood and the deposition of NETs in the cerebral cavernomas of Ccm3 mice. Degradation of NETs by DNase I treatment improved the vascular barrier. The deposition of NETs in the cavernomas of patients with CCM confirms the clinical relevance of NETs in CCM.
Overview of cerebral cavernous malformations: comparison of treatment approaches.
Journal of neurology, neurosurgery, and psychiatry
OBJECTIVES:The comparison of treatment efficacy for cerebral cavernous malformations (CCMs) has not yet been well researched. DESIGN:PubMed, Cochrane Library, Science Direct, ISI Web of Science, Embase and additional sources were searched to identify cohort studies about the treatment of CCMs published between 1990 and 2020. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed; the Newcastle-Ottawa Scale was used to assess the risk of bias and to evaluate limitations based on selection/outcome biases. The cumulative incidences with 95% CIs were calculated using the random effects model. The models of Poisson distribution were applied to evaluate risk factors of poorer treatment outcome by calculating rate ratios within 100 person-years with 95% CIs. RESULTS:A total of 100 cohorts yielding 8994 patients treated for CCMs within 41 098 person-years of follow-up were analysed. The efficacy of ensuring the prevention of haemorrhage was 97% in surgical, 86% in radiosurgical and 77% in the conservative treatment. The lowest mortality (1%) was after radiosurgery, and the highest persistent morbidity (22%) was in natural history series. Deep-seated and brainstem CCMs were associated with higher bleeding rates. Lobar localisation was a protective factor in all analyses. Patients with history of previous haemorrhage were exposed to higher risk of rebleeding. Male gender was a protective factor associated with lower risk of post-treatment haemorrhage. CONCLUSIONS:Surgical resection of CCM is effective in ensuring the prevention of haemorrhage with acceptable morbidity and mortality, but conservative and radiosurgical management is a justified treatment alternative. Brainstem and deep-seated CCMs are predominantly associated with higher haemorrhage rates.
New Insights Into Cerebrovascular Pathophysiology and Hypertension.
Despite advances in acute management and prevention of cerebrovascular disease, stroke and vascular cognitive impairment together remain the world's leading cause of death and neurological disability. Hypertension and its consequences are associated with over 50% of ischemic and 70% of hemorrhagic strokes but despite good control of blood pressure (BP), there remains a 10% risk of recurrent cerebrovascular events, and there is no proven strategy to prevent vascular cognitive impairment. Hypertension evolves over the lifespan, from predominant sympathetically driven hypertension with elevated mean BP in early and mid-life to a late-life phenotype of increasing systolic and falling diastolic pressures, associated with increased arterial stiffness and aortic pulsatility. This pattern may partially explain both the increasing incidence of stroke in younger adults as well as late-onset, chronic cerebrovascular injury associated with concurrent systolic hypertension and historic mid-life diastolic hypertension. With increasing arterial stiffness and autonomic dysfunction, BP variability increases, independently predicting the risk of ischemic and intracerebral hemorrhage, and is potentially modifiable beyond control of mean BP. However, the interaction between hypertension and control of cerebral blood flow remains poorly understood. Cerebral small vessel disease is associated with increased pulsatility in large cerebral vessels and reduced reactivity to carbon dioxide, both of which are being targeted in early phase clinical trials. Cerebral arterial pulsatility is mainly dependent upon increased transmission of aortic pulsatility via stiff vessels to the brain, while cerebrovascular reactivity reflects endothelial dysfunction. In contrast, although cerebral autoregulation is critical to adapt cerebral tone to BP fluctuations to maintain cerebral blood flow, its role as a modifiable risk factor for cerebrovascular disease is uncertain. New insights into hypertension-associated cerebrovascular pathophysiology may provide key targets to prevent chronic cerebrovascular disease, acute events, and vascular cognitive impairment.
Deep Learning in the Management of Intracranial Aneurysms and Cerebrovascular Diseases: A Review of the Current Literature.
Intracranial aneurysms are a common asymptomatic vascular pathology, the rupture of which is a devastating event with a significant risk of morbidity and mortality. Aneurysm detection and risk stratification before rupture events are, therefore, imperative to guide prophylactic measures. Artificial intelligence has shown great promise in the management pathway of aneurysms, through automated detection, the prediction of rupture risk, and outcome prediction after treatment. The complementary use of these programs, in addition to clinical practice, has demonstrated high diagnostic and prognostic accuracy, with the potential to improve patient outcomes. In the present review, we explored the role and limitations of deep learning, a subfield of artificial intelligence, in the aneurysm patient journey. We have also briefly summarized the application of deep learning models in automated detection and prediction in cerebral arteriovenous malformations and Moyamoya disease.
Brain arteriovenous malformations.
Lawton Michael T,Rutledge W Caleb,Kim Helen,Stapf Christian,Whitehead Kevin J,Li Dean Y,Krings Timo,terBrugge Karel,Kondziolka Douglas,Morgan Michael K,Moon Karam,Spetzler Robert F
Nature reviews. Disease primers
An arteriovenous malformation is a tangle of dysplastic vessels (nidus) fed by arteries and drained by veins without intervening capillaries, forming a high-flow, low-resistance shunt between the arterial and venous systems. Arteriovenous malformations in the brain have a low estimated prevalence but are an important cause of intracerebral haemorrhage in young adults. For previously unruptured malformations, bleeding rates are approximately 1% per year. Once ruptured, the subsequent risk increases fivefold, depending on associated aneurysms, deep locations, deep drainage and increasing age. Recent findings from novel animal models and genetic studies suggest that arteriovenous malformations, which were long considered congenital, arise from aberrant vasculogenesis, genetic mutations and/or angiogenesis after injury. The phenotypical characteristics of arteriovenous malformations differ among age groups, with fistulous lesions in children and nidal lesions in adults. Diagnosis mainly involves imaging techniques, including CT, MRI and angiography. Management includes observation, microsurgical resection, endovascular embolization and stereotactic radiosurgery, alone or in any combination. There is little consensus on how to manage patients with unruptured malformations; recent studies have shown that patients managed medically fared better than those with intervention at short-term follow-up. By contrast, interventional treatment is preferred following a ruptured malformation to prevent rehaemorrhage. Management continues to evolve as new mechanistic discoveries and reliable animal models raise the possibility of developing drugs that might prevent the formation of arteriovenous malformations, induce obliteration and/or stabilize vessels to reduce rupture risk. For an illustrated summary of this Primer, visit: http://go.nature.com/TMoAdn.
A Transvenous Endovascular Approach in Straight Sinus Has Minor Impacts on Chordae Willisii.
Ye Yuanliang,Ding Jiuyang,Liu Shoutang,Lan Tiancai,Chen Long,Wang Yingxue,Xia Bing,Yang Jianqing
Frontiers in neurology
Cerebral dural sinuses contain different types of chordae willisii (CW). The transvenous endovascular approach, which has become an optimal method for the treatment of cerebrovascular diseases, such as malformation, fistula, and chronic intracranial hypertension, due to sinus thromboses, frequently uses retrograde navigation through dural sinuses. Whether or how much the endoscopic procedure damages the chordae willisii is often not well-assessed. In our study, an overall number of 38 cadaveric heads were analyzed for the distribution and features of the chordae willisii in the straight sinus. We used an endoscope on these samples mimicking a mechanical thrombectomy procedure performed in the straight sinus. Both endoscopic gross observation and light microscopic histological examination were used to assess the damages to the chordae willisii by the procedure. We found that the valve-like lamellae and longitudinal lamellae structures were mainly found in the posterior part of straight sinus whereas trabeculae were present in both anterior and posterior portions. We treated a group of samples with a stent and another with a balloon. The stent-treated group had a significantly higher rate of Grade 1 damage comparing with the balloon-treated group ( = 0.02). The incidence of damage to the surface of chordae willisii was also higher in the stent-treated group ( = 0.00). Neither the use of stent nor of balloon increased the rate of damage to chordae willisii during repeated experiments. These findings indicated that stent or balloon navigation through the straight sinus can cause minor damages to the chordae willisii and frequent uses of retrograde navigation through the straight sinus do not appear to increase the rates of damage to chordae willisii.
A single-cell atlas of the normal and malformed human brain vasculature.
Science (New York, N.Y.)
Cerebrovascular diseases are a leading cause of death and neurologic disability. Further understanding of disease mechanisms and therapeutic strategies requires a deeper knowledge of cerebrovascular cells in humans. We profiled transcriptomes of 181,388 cells to define a cell atlas of the adult human cerebrovasculature, including endothelial cell molecular signatures with arteriovenous segmentation and expanded perivascular cell diversity. By leveraging this reference, we investigated cellular and molecular perturbations in brain arteriovenous malformations, which are a leading cause of stroke in young people, and identified pathologic endothelial transformations with abnormal vascular patterning and the ontology of vascularly derived inflammation. We illustrate the interplay between vascular and immune cells that contributes to brain hemorrhage and catalog opportunities for targeting angiogenic and inflammatory programs in vascular malformations.
Molecular feature of arterial remodeling in the brain arteriovenous malformation revealed by arteriovenous shunt rat model and RNA sequencing.
PURPOSE:Morphological research suggested the feeding artery of brain arteriovenous malformation (bAVM) had vascular remodeling under the high blood flow; however, the underlying molecular mechanisms were unclear. METHODS:We constructed 32 simplified AVM rat models in four groups: the control group (n = 6), 1-week high-blood-flow group (n = 9), 3-week high-blood-flow group (n = 7) and 6-week high-blood-flow group (n = 10). The circumference, blood velocity, blood flow, pressure, and wall shear of the feeding artery were measured or calculated. The arterial wall change was observed by Masson staining. RNA sequencing (RNA-seq) of feeding arteries was performed, followed by bioinformatics analysis to detect the potential molecular mechanism for bAVM artery remodeling under the high blood flow. RESULTS:We observed hemodynamic injury and vascular remodeling on the feeding artery under the high blood flow. RNA-seq showed immune/inflammation infiltration and vascular smooth muscle cell (VSMC) phenotype transformation during remodeling. Weighted gene co-expression network analysis (WGCNA) and time series analysis further identified 27 key genes and pathways involved in remodeling. Upstream miRNA and molecular drugs were predicted targeting these key genes. CONCLUSIONS:We depicted molecular change of bAVM arterial remodeling via RNA-seq in high-blood-flow rat models. Twenty-seven key genes may regulate immune/inflammation infiltration and VSMC phenotype transform in bAVM arterial remodeling.
The Transcriptional Landscapes and Key Genes in Brain Arteriovenous Malformation Progression in a Venous Hypertension Rat Model Revealed by RNA Sequencing.
Journal of inflammation research
BACKGROUND:Brain arteriovenous malformations (bAVM) are abnormal vascular lesions characterized by direct connections between arteries and veins without an intervening capillary bed. The primary goal for brain AVM treatment is to prevent rupture and hemorrhage; however, the underlying molecular mechanisms are still unknown. METHODS:We constructed venous hypertension (VH) rat model with end-to-end anastomosis of the proximal left common carotid artery and the left distal external jugular vein. Thirty-eight adult rats were randomly assigned to four groups: the 0-week (n=5), the 1-week VH group (n=12), the 3-week VH group (n=9), and the 6-week VH group (n=12). We measured the hemodynamics and diameter of the arterialized veins. An RNA sequencing of arterialized veins was conducted, followed by comprehensive bioinformatics analysis to identify key genes and biological pathways involved in VH progression. The candidate genes from RNA-Seq were validated by RT-qPCR and immunostaining in human tissues. RESULTS:We observed high-flow and low resistance characteristics in VH models. A total of 317 upregulated and 258 downregulated common genes were consistently differentially expressed during VH progression. Thirteen co-expression modules were obtained by WGCNA analysis, and 4 key modules were identified. Thirteen genes: Adamts8, Adamtsl3, Spon2, Adamtsl2, Chad, Itga7, Comp, Itga8, Bmp6, Fst, Smad6, Smad7, Grem1, and Nog with differential expressions were identified using the density of maximum neighborhood component (DMNC) algorithm in Cytohubba. The expression of five potential genes (Adamts8, Adamtsl3, Spon2, Adamtsl2, Itga8) were increased in RT-qPCR, while in human bAVM tissue, the protein levels of Adamtsl2 and Itga8 were significant elevated and Spon2 and Adamtsl3 were decreased. CONCLUSION:The identified gene networks of Adamtsl3, Spon2, Adamtsl2, and Itga8 provided key genes for further intervention.
Molecular Signature of Brain Arteriovenous Malformation Hemorrhage: A Systematic Review.
Germans Menno R,Sun Wenhua,Sebök Martina,Keller Annika,Regli Luca
BACKGROUND:The mechanisms of brain arteriovenous malformation (bAVM) development, formation, and progress are still poorly understood. By gaining more knowledge about the molecular signature of bAVM in relation to hemorrhage, we might be able to find biomarkers associated with this serious complication, which can function as a goal for further research and can be a potential target for gene therapy. AIMS:To provide a comprehensive overview of the molecular signature of bAVM-related hemorrhage We conducted a systematic review, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, of articles published in Embase, Medline, Cochrane central, Scopus, and Chinese databases (CNKI, Wanfang). SUMMARY OF REVIEW:Our search identified 3944 articles, of which 3108 remained after removal of duplicates. After title, abstract, and full-text screening, 31 articles were included for analysis. The results show an overview of molecular characteristics. Several genetic polymorphisms are identified that increase the risk of bAVM rupture by increasing the expression of certain inflammatory cytokines (interleukin [IL]-6, IL-17A, IL-1β, and tumor necrosis factor-α), NOTCH pathways, matrix metalloproteinase-9, and vascular endothelial growth factor-α. CONCLUSIONS:Several molecular factors are associated with the risk of bAVM-related hemorrhage. These factors are associated with increased inflammation on the cellular level and changes in the endothelium leading to instability of the vessel wall. Further investigation of these biomarkers regarding hemorrhage rates, together with their relationship with noninvasive diagnostic methods, should be a goal of future studies to improve the patient specific risk estimation and future treatment options.
Homocysteine Level and Risk of Hemorrhage in Brain Arteriovenous Malformations.
Zeng Chaofan,Lin Fa,Ge Peicong,Zhang Dong,Wang Shuo,Zhao Jizong
Objective:We aimed to investigate the risk factors associated with hemorrhage and clarify the relation of homocysteine (Hcy) with brain arteriovenous malformations (bAVMs). Method:We retrospectively reviewed bAVM patients from Beijing Tiantan Hospital between January 2019 and December 2019. Clinical and laboratory variables were analyzed in enrolled patients with bAVMs. Potential predictors associated with hemorrhage were evaluated by logistic regression analysis. Results:A total of 143 bAVM patients were identified in the study, including 69 unruptured and 74 ruptured cases. Patients with hemorrhage were less likely to have hyperhomocysteinemia ( = 0.023). Logistic regression analysis showed that increased maximum diameter of bAVM lesions (odds ratio (OR) 0.634, 95% confidence intervals (CI) 0.479-0.839; = 0.001) and serum Hcy level (OR 0.956, 95% CI 0.920-0.993; = 0.021) were associated with lower risk of hemorrhage in bAVMs. Conclusion:The present study provided evidence regarding the association between serum Hcy and hemorrhage in patients with bAVMs. Higher Hcy level was correlated with a lower risk of rupture. Detection of factors for subsequent hemorrhage is necessary to develop therapeutic strategies for bAVMs preferably.
Association of Variants in FCGR2A, PTPN2, and GM-CSF with Cerebral Cavernous Malformation: Potential Biomarkers for a Symptomatic Disease.
da Fontoura Galváo Gustavo,Fontes-Dantas Fabrícia Lima,da Silva Elielson Veloso,Alves-Leon Soniza Vieira,de Souza Jorge Marcondes
Current neurovascular research
BACKGROUND:Cerebral Cavernous Malformations (CCM) predispose patients to a lifetime risk of seizures and symptomatic hemorrhage. Only a small percentage of people affected will develop clinical symptoms and the molecular mechanisms underlying lesional activity remain unclear. We analyzed a panel of Single Nucleotide Polymorphisms (SNPs) in CCM patients. We looked for plasmatic inflammatory cytokines, checking for a pattern of plasma expression heterogeneity and any correlation with genetic variations identified with different CCM clinical phenotypes. METHODS:This was a case-control study from a long-term follow-up cohort including 23 CCM patients, of which 16 were symptomatic, and 7 were asymptomatic. A 200-SNP panel was considered through next-generation sequencing and 18 different plasma molecules were assessed through a suspension array system. RESULTS:Fcγ receptor IIa rs1801274 (FCGR2A) and protein tyrosine phosphatase non-receptor type 2 rs72872125 PTPN2 were statistically different between groups. Patients who had a combination of the presence of FCGR2A and the absence of PTPN2 also had symptoms earlier in life. The combination of genetic polymorphisms and serum level of GM-CSF showed the best diagnostic biomarker to distinguish symptomatic patients as formulated: [0.296*(FCGR2A)] + [-0.788*(PTPN2)] + [-0.107*(GM-CSF)]. CONCLUSION:We have shown that SNPs in inflammation genes might be related to a symptomatic phenotype in CCM. We also demonstrated that a formula based on two of these polymorphisms (FCGR2A+ and PTPN2+) is possibly capable of predicting a symptomatic phenotype during a patient's lifetime.
Prognostic Significance of Homocysteine Level on Neurological Outcome in Brain Arteriovenous Malformations.
Lin Fa,Zeng Chaofan,Ge Peicong,Zhang Dong,Wang Shuo,Zhao Jizong
Objective:We aimed to investigate the serum homocysteine (Hcy) level in patients with brain arteriovenous malformation (bAVM) and their impact on neurological outcome during hospitalization. Method:We retrospectively reviewed patients diagnosed with bAVMs in Beijing Tiantan Hospital from January 2019 to August 2020. Patients were divided into two groups according to the mRS (modified Rankin Scale) score at discharge. Clinical and laboratory characteristics were compared. Logistic regression analyses were performed to identify the potential risk factors for short-term neurological outcome. Results:A total of 175 bAVM patients were enrolled in the study, including 139 patients with favorable outcome (mRS ≤ 2) and 36 patients with unfavorable outcome (mRS > 2). Hyperhomocysteinemia was identified in 32.6% of cases ( = 57). Serum Hcy level was related to seizure manifestation ( = 0.034) and short-term neurological outcome ( = 0.027). Logistic regression analysis showed that serum glucose (OR 1.897, 95% CI 1.115-3.229; = 0.018) and Hcy level (OR 0.838, 95% CI 0.720-0.976; = 0.023) were significantly associated with short-term disability. Conclusion:Our results indicated that the lower serum Hcy level is strongly associated with in-hospital unfavorable outcome. Further prospective studies of Hcy natural history and managements in bAVMs are required, which would be valuable for evaluating the disease-modifying efficacy of oral nutritional supplements in bAVM patients.
Secondary S100B Protein Increase Following Brain Arteriovenous Malformation Rupture is Associated with Cerebral Infarction.
Garzelli Lorenzo,Jacquens Alice,Amouyal Caroline,Premat Kevin,Sourour Nader,Cortese Jonathan,Haffaf Idriss,Mathon Bertrand,Lenck Stéphanie,Clarençon Frédéric,Degos Vincent,Shotar Eimad
Molecules (Basel, Switzerland)
Early S100B protein serum elevation is associated with poor prognosis in patients with ruptured brain arteriovenous malformations (BAVM). The purpose of this study is to determine whether a secondary elevation of S100B is associated with early complications or poor outcome in this population. This is a retrospective study of patients admitted for BAVM rupture. A secondary increase of S100B was defined as an absolute increase by 0.1 μg/L within 30 days of admission. Fisher's and unpaired t tests followed by multivariate analysis were performed to identify markers associated with this increase. Two hundred and twenty-one ruptures met inclusion criteria. Secondary S100B protein serum elevation was found in 17.1% of ruptures and was associated with secondary infarction ( < 0.001), vasospasm-related infarction ( < 0.001), intensive care ( = 0.009), and hospital length of stay ( = 0.005), but not with early rebleeding ( = 0.07) or in-hospital mortality ( = 0.99). Secondary infarction was the only independent predictor of secondary increase of S100B (OR 9.9; 95% CI (3-35); < 0.001). Secondary elevation of S100B protein serum levels is associated with secondary infarction in ruptured brain arteriovenous malformations.
Cell-free DNA as a diagnostic analyte for molecular diagnosis of vascular malformations.
Zenner Kaitlyn,Jensen Dana M,Cook Tori T,Dmyterko Victoria,Bly Randall A,Ganti Sheila,Mirzaa Ghayda M,Dobyns William B,Perkins Jonathan A,Bennett James T
Genetics in medicine : official journal of the American College of Medical Genetics
PURPOSE:Vascular malformations (VM) are primarily caused by somatic activating pathogenic variants in oncogenes. Targeted pharmacotherapies are emerging but require molecular diagnosis. Since variants are currently only detected in malformation tissue, patients may be ineligible for clinical trials prior to surgery. We hypothesized that cell-free DNA (cfDNA) could provide molecular diagnoses for patients with isolated VM. METHODS:cfDNA was isolated from plasma or cyst fluid from patients with arteriovenous malformations (AVM), venous malformations (VeM), or lymphatic malformations (LM), and assayed for known pathogenic variants using droplet digital polymerase chain reaction (ddPCR). Cyst fluid cfDNA from an independent cohort of LM patients was prospectively screened for variants using a multiplex ddPCR assay. RESULTS:Variants were detected in plasma cfDNA in patients with AVM (2/8) and VeM (1/3). Variants were detected in cyst fluid cfDNA (7/7) but not plasma (0/26) in LM patients. Prospective testing of cyst fluid cfDNA with multiplex ddPCR identified variants in LM patients who had never undergone surgery (4/5). CONCLUSION:Variants were detected in plasma from AVM and VeM patients, and in cyst fluid from patients with LM. These data support investigation of cfDNA-based molecular diagnostics for VM patients, which may provide opportunities to initiate targeted pharmacotherapies without prior surgery.
Plasma Biomarkers of Inflammation and Angiogenesis Predict Cerebral Cavernous Malformation Symptomatic Hemorrhage or Lesional Growth.
Girard Romuald,Zeineddine Hussein A,Koskimäki Janne,Fam Maged D,Cao Ying,Shi Changbin,Moore Thomas,Lightle Rhonda,Stadnik Agnieszka,Chaudagar Kiranj,Polster Sean,Shenkar Robert,Duggan Ryan,Leclerc David,Whitehead Kevin J,Li Dean Y,Awad Issam A
RATIONALE:The clinical course of cerebral cavernous malformations is highly unpredictable, with few cross-sectional studies correlating proinflammatory genotypes and plasma biomarkers with prior disease severity. OBJECTIVE:We hypothesize that a panel of 24 candidate plasma biomarkers, with a reported role in the physiopathology of cerebral cavernous malformations, may predict subsequent clinically relevant disease activity. METHODS AND RESULTS:Plasma biomarkers were assessed in nonfasting peripheral venous blood collected from consecutive cerebral cavernous malformation subjects followed for 1 year after initial sample collection. A first cohort (N=49) was used to define the best model of biomarker level combinations to predict a subsequent symptomatic lesional hemorrhagic expansion within a year after the blood sample. We generated the receiver operating characteristic curves and area under the curve for each biomarker individually and each weighted linear combination of relevant biomarkers. The best model to predict lesional activity was selected as that minimizing the Akaike information criterion. In this cohort, 11 subjects experienced symptomatic lesional hemorrhagic expansion (5 bleeds and 10 lesional growths) within a year after the blood draw. Subjects had lower soluble CD14 (cluster of differentiation 14; =0.05), IL (interleukin)-6 (=0.04), and VEGF (vascular endothelial growth factor; =0.0003) levels along with higher plasma levels of IL-1β (=0.008) and soluble ROBO4 (roundabout guidance receptor 4; =0.03). Among the 31 weighted linear combinations of these 5 biomarkers, the best model (with the lowest Akaike information criterion value, 25.3) was the weighted linear combination including soluble CD14, IL-1β, VEGF, and soluble ROBO4, predicting a symptomatic hemorrhagic expansion with a sensitivity of 86% and specificity of 88% (area under the curve, 0.90; <0.0001). We then validated our best model in the second sequential independent cohort (N=28). CONCLUSIONS:This is the first study reporting a predictive association between plasma biomarkers and subsequent cerebral cavernous malformation disease clinical activity. This may be applied in clinical prognostication and stratification of cases in clinical trials.
Deep Sequencing of Small RNAs in Blood of Patients with Brain Arteriovenous Malformations.
Chen Yong,Li Zhili,Shi Yi,Huang Guangfu,Chen Longyi,Tan Haibin,Wang Zhenyu,Yin Cheng,Hu Junting
BACKGROUND:Deregulation of circulating microRNAs (miRNAs) is always associated with development and progression of human diseases. We aimed to assess whether patients with brain arteriovenous malformations (BAVMs) possess a distinct miRNA signature compared with healthy subjects. METHODS:Three patients with unruptured BAVMs and 3 normal control subjects were recruited as case and control groups. Peripheral blood was collected, and miRNA signature was obtained by next-generation sequencing, followed by comparative, functional, and network analyses. Quantitative reverse transcription polymerase chain reaction was performed to validate expression of specific miRNAs. RESULTS:Deep sequencing detected 246 differentially expressed miRNAs in blood samples of patients with BAVMs compared with normal control subjects. For the top 5 miRNAs, 946 target genes were predicted, and a BAVM-specific miRNA-target gene regulatory network was constructed. Functional annotation suggested that 15 of the predicted miRNA-targeted genes were involved in vascular endothelial growth factor signaling, in which 3 critical miRNAs were involved: miR-7-5p, miR-199a-5p, and miR-200b-3p. CONCLUSIONS:We explored the miRNA expression signature of BAVMs, which will provide an important foundation for future studies on the regulation of miRNAs involved in BAVMs.
Values of vanillylmandelic acid and homovanillic acid in the urine as potential prognostic biomarkers in ischaemic stroke patients.
Bonifačić David,Aralica Merica,Sotošek Tokmadžić Vlatka,Rački Valentino,Tuškan-Mohar Lidija,Kučić Natalia
Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals
BACKGROUND:Suitable biomarkers that have prognostic values are one of the key points of interest in ischaemic stroke. Increased sympathetic nervous system activity in ischaemic stroke causes multiple local and systemic effects that can be detrimental to the outcome. The mechanism of action is increased secretion and activity of catecholamines, whose end metabolic products are vanillylmandelic acid and homovanilic acid. Aim of our study was to determine whether these compounds can be used as potential prognostic biomarkers in ischaemic stroke, as a unique insight into the activity of the sympathetic nervous system. METHODS:Urine samples of 96 patients with ischaemic stroke and transitory ischaemic attacks were analysed. Values of vanillylmandelic and homovanillic acids in urine were tested using liquid chromatography on the first and third day post-stroke. Severity of stroke was determined using the NIHSS scale, while functional outcome was determined using the Modified Rankin Scale. RESULTS:Values of vanillylmandelic and homovanillic acids positively correlated with functional outcome of ischaemic stroke. Favorable outcomes correlated with decreased values, on contrary to increased values, which were associated with unfavourable outcomes. CONCLUSION:Determining the values of these compounds in the urine is an easily available prognostic tool for the ischaemic stroke outcome, while also influencing potential therapeutic changes.
Brain arteriovenous malformations: implications of CEACAM1-positive inflammatory cells and sex on hemorrhage.
Neyazi Belal,Herz Angelika,Stein Klaus-Peter,Gawish Islam,Hartmann Christian,Wilkens Ludwig,Erguen Sueleyman,Dumitru Claudia A,Sandalcioglu I Erol
Brain arteriovenous malformations (bAVMs) are severe conditions which, upon rupture, cause debilitating neurological deficits and even death. The exact cellular and molecular mechanisms associated with bAVM rupture are currently unclear. The objective of this study was to explore the potential role of CEA-related cell adhesion molecule-1 (CEACAM1) in bAVM pathophysiology. Expression and localization of CEACAM1 were assessed immunohistochemically in tissue microarrays from bAVM patients (n = 60). The association of CEACAM1 with clinical parameters was analyzed with Spearman's rank correlation coefficient and chi-square test. The predictive value of CEACAM1 was tested using logistic regression analysis. CEACAM1 was highly expressed in tissue-infiltrating neutrophil granulocytes. High levels of CEACAM1-positive cells were associated with bAVM rupture (hemorrhage), but not with arteriovenous malformation (AVM) size, preoperative embolization, or seizure. This association was significant (p = 0.029, chi-square) in male but not in female patients, and high CEACAM1-positive immune infiltration showed predictive significance for hemorrhage in male bAVM patients only (OR = 6.50, 95 % CI 1.09-38.63, p = 0.040). Within the ruptured bAVM group, patients with a short hemorrhage to surgery (HTS) time interval had higher levels of CEACAM1 immune infiltration than patients with long HTS. This decrease in the levels of CEACAM1 immune infiltration between the HTS short and HTS long groups was, however, significant only in female patients (p = 0.022, chi-square). Our findings substantiate the role of inflammation in the pathophysiology of bAVM and suggest the presence of sexual dimorphism in this disease.
Polymorphisms in ACVRL1 and endoglin genes are not associated with sporadic and HHT-related brain AVMs in Dutch patients.
Boshuisen Kim,Brundel Manon,de Kovel Carolien G F,Letteboer Tom G,Rinkel Gabriel J E,Westermann Cornelis J J,Kim Helen,Pawlikowska Ludmila,Koeleman Bobby P C,Klijn Catharina J M
Translational stroke research
We aimed to replicate the association of the IVS3-35A>G polymorphism in the activin receptor-like kinase (ACVRL) 1 gene and the 207G>A polymorphism in the endoglin (ENG) gene with sporadic brain arteriovenous malformations (BAVM) in Dutch BAVM patients. In addition, we assessed whether these polymorphisms contribute to the risk of BAVM in patients with hereditary haemorrhagic telangiectasia type 1 (HHT1). We genotyped 143 Dutch sporadic BAVM patients and 360 healthy volunteers for four variants in the ACVRL1 gene including IVS3-35A>G and two variants in the ENG gene including 207G>A. Differences in allele and genotype frequencies between sporadic BAVM patients and controls and their combined effect were analysed with a likelihood ratio test. Furthermore, we compared the allele and genotype frequencies between 24 HHT1 patients with a BAVM with those of a relative with HHT1 without a BAVM in a matched pair analysis using Wilcoxon signed rank test. No significant differences in allele frequency were found between sporadic BAVM cases and controls or between HHT1 patients with and without BAVM for any of the polymorphisms or the combination of ACVRL1 and ENG polymorphisms. Meta-analysis of the current and the two previous studies for the ACVRL1 IVS3-35A polymorphism showed a persisting association between the ACVRL1 IVS3-35A polymorphism and risk of sporadic BAVM (odds ratio, 1.86; 95% CI: 1.32-2.61, p<0.001). We did not replicate the previously found association between a polymorphism in ACVRL1 IVS3-35A>G and BAVM in Dutch patients. However, meta-analysis did not rule out a possible effect.
Ferumoxytol-enhanced MRI to Image Inflammation within Human Brain Arteriovenous Malformations: A Pilot Investigation.
Hasan David M,Amans Matthew,Tihan Tarik,Hess Christopher,Guo Yi,Cha Soonmee,Su Hua,Martin Alastair J,Lawton Michael T,Neuwelt Edward A,Saloner David A,Young William L
Translational stroke research
Inflammation cell infiltration and cytokine expression are seen in the vascular walls and intervening stroma of resected brain arteriovenous malformation (bAVM) specimens, even in unruptured and previously untreated lesions. Macrophages may play a critical role in bAVM progression to rupture, and could serve as a marker for rupture risk. We assessed feasibility of imaging macrophages within the bAVM nidus using ferumoxytol-enhanced MRI in four patients with already diagnosed bAVMs using iron-sensitive imaging (ISI; T2*-GE-MRI sequence). Patients were imaged at baseline and at either 1 day (n=2) or 5 days (n=2) after infusion of 5mg/kg of ferumoxytol. Residual intravascular ferumoxytol obscured evaluation for uptake in bAVM vascular walls and stroma at the 1-day time point. The two cases imaged at 5 days showed less intravascular tracer but had signal loss in the nidal region consistent with ferumoxytol localization. One case underwent surgical resection; there was prominent vascular wall CD68 staining. Ferumoxytol-enhanced-MRI for assessing bAVM inflammatory cell burden appears feasible and has the potential to be developed as a biomarker to study lesional inflammatory events.
Silent intralesional microhemorrhage as a risk factor for brain arteriovenous malformation rupture.
Guo Yi,Saunders Tara,Su Hua,Kim Helen,Akkoc Deniz,Saloner David A,Hetts Steven W,Hess Christopher,Lawton Michael T,Bollen Andrew W,Pourmohamad Tony,McCulloch Charles E,Tihan Tarik,Young William L,
BACKGROUND AND PURPOSE:We investigated whether brain arteriovenous malformation silent intralesional microhemorrhage, that is, asymptomatic bleeding in the nidal compartment, might serve as a marker for increased risk of symptomatic intracranial hemorrhage (ICH). We evaluated 2 markers to assess the occurrence of silent intralesional microhemorrhage: neuroradiological assessment of evidence of old hemorrhage-imaging evidence of bleeding before the outcome events-and hemosiderin positivity in hematoxylin and eosin-stained paraffin block sections. METHODS:We identified cases from our brain arteriovenous malformation database with recorded neuroradiological data or available surgical paraffin blocks. Using 2 end points, index ICH or new ICH after diagnosis (censored at treatment, loss to follow-up, or death), we performed logistic or Cox regression to assess evidence of old hemorrhage and hemosiderin positivity adjusting for age, sex, deep-only venous drainage, maximal brain arteriovenous malformation size, deep location, and associated arterial aneurysms. RESULTS:Evidence of old hemorrhage was present in 6.5% (n=975) of patients and highly predictive of index ICH (P<0.001; OR, 3.97; 95% CI, 2.1-7.5) adjusting for other risk factors. In a multivariable model (n=643), evidence of old hemorrhage was an independent predictor of new ICH (hazard ratio, 3.53; 95% CI, 1.35-9.23; P=0.010). Hemosiderin positivity was found in 36.2% (29.6% in unruptured; 47.8% in ruptured; P=0.04) and associated with index ICH in univariate (OR, 2.18; 95% CI, 1.03-4.61; P=0.042; n=127) and multivariable models (OR, 3.64; 95% CI, 1.11-12.00; P=0.034; n=79). CONCLUSIONS:The prevalence of silent intralesional microhemorrhage is high and there is evidence for an association with both index and subsequent ICH. Further development of means to detect silent intralesional microhemorrhage during brain arteriovenous malformation evaluation may present an opportunity to improve risk stratification, especially for unruptured brain arteriovenous malformations.
Gene expression profiling of blood in brain arteriovenous malformation patients.
Weinsheimer Shantel M,Xu Huichun,Achrol Achal S,Stamova Boryana,McCulloch Charles E,Pawlikowska Ludmila,Tian Yingfang,Ko Nerissa U,Lawton Michael T,Steinberg Gary K,Chang Steven D,Jickling Glen,Ander Bradley P,Kim Helen,Sharp Frank R,Young William L
Translational stroke research
Brain arteriovenous malformations (BAVMs) are an important cause of intracranial hemorrhage (ICH) in young adults. Gene expression profiling of blood has led to the identification of stroke biomarkers, and may help identify BAVM biomarkers and illuminate BAVM pathogenesis. It is unknown whether blood gene expression profiles differ between 1) BAVM patients and healthy controls, or 2) unruptured and ruptured BAVM patients at presentation. We characterized blood transcriptional profiles in 60 subjects (20 unruptured BAVM, 20 ruptured BAVM, and 20 healthy controls) using Affymetrix whole genome expression arrays. Expression differences between groups were tested by ANOVA, adjusting for potential confounders. Genes with absolute fold change ≥ 1.2 (false discovery rate corrected p ≤ 0.1) were selected as differentially expressed and evaluated for over-representation in KEGG biological pathways (p ≤ 0.05). Twenty-nine genes were differentially expressed between unruptured BAVM patients and controls, including 13 which may be predictive of BAVM. Patients with ruptured BAVM compared to unruptured BAVM differed in expression of 1490 genes, with over-representation of genes in 8 pathways including MAPK, VEGF, Wnt signaling and several inflammatory pathways. These results suggest clues to the pathogenesis of BAVM and/or BAVM rupture and point to potential biomarkers or new treatment targets.
Pathogenesis and radiobiology of brain arteriovenous malformations: implications for risk stratification in natural history and posttreatment course.
Achrol Achal S,Guzman Raphael,Varga Monika,Adler John R,Steinberg Gary K,Chang Steven D
Brain arteriovenous malformations (BAVMs) are an important cause of intracerebral hemorrhage (ICH) in young adults. Biological predictors of future ICH risk are lacking, and controversy exists over previous studies of natural history risk among predominantly ruptured BAVM cohorts. Recent studies have suggested that the majority of BAVMs are now diagnosed as unruptured lesions, and that the risk according to natural history among these lesions may be less than previously assumed. In the first part of this review, the authors discuss available data on the natural history of BAVMs and highlight the need for future studies that aim to develop surrogate biomarkers of disease progression that accurately predict future risk of ICH in BAVMs. The etiology of BAVM remains unknown. Recent studies have suggested a role for genetic factors in the pathogenesis of sporadic BAVM, which is further supported by reports of familial occurrence of BAVM and association with known systemic genetic disorders (such as Osler-Weber-Rendu disease, Sturge-Weber disease, and Wyburn-Mason syndrome). Molecular characterization of BAVM tissue demonstrates a highly angiogenic milieu with evidence of increased endothelial cell turnover. Taken together with a number of reports of de novo BAVM formation, radiographic growth after initial BAVM diagnosis, and regrowth after successful treatment of BAVM, these findings challenge the long-held assumption that BAVMs are static lesions of congenital origin. In the second part of this review, the authors discuss available data on the origins of BAVM and offer insights into future investigations into genetics and endothelial progenitor cell involvement in the pathogenesis of BAVM. Current treatment options for BAVM focus on removal or obliteration of the lesion in an attempt to protect against future ICH risk, including microsurgical resection, endovascular embolization, and stereotactic radiosurgery (SRS). In the third part of this review, the authors discuss available data on SRS in BAVMs and highlight the need for future studies on the radiobiology of BAVMs, especially in regard to biomarker detection for tracking SRS response during the latency period. Insights from future investigations in BAVM may not only prove important for the development of novel therapies and relevant biomarkers for BAVM, but could also potentially benefit a variety of other disorders involving new vessel formation in the CNS, including stroke, tumors, moyamoya disease, and other cerebrovascular malformations.
MMP-9 expression is associated with leukocytic but not endothelial markers in brain arteriovenous malformations.
Chen Yongmei,Fan Yongfeng,Poon K Y Trudy,Achrol Achal S,Lawton Michael T,Zhu Yiqian,McCulloch Charles E,Hashimoto Tomoki,Lee Chanhung,Barbaro Nicholas M,Bollen Andrew W,Yang Guo-Yuan,Young William L
Frontiers in bioscience : a journal and virtual library
Brain arteriovenous malformations (BAVM) have high matrix metalloproteinase-9 (MMP-9) expression, the source of which is unclear. We hypothesized MMP-9 production might be due to inflammation in BAVM. Compared to control brain tissues (n = 5), BAVM tissue (n = 139) had a higher expression (by ELISA) of myeloperoxidase (MPO) (193 +/- 189 vs. 6 +/- 3, ng/mg, P < .001), MMP-9 (28 +/- 32 vs. 0.7 +/- 0.6, ng/mg, P < .001), and IL-6 (102 +/- 218 vs. 0.1 +/- 0.1, pg/mg, P < .001), but not eNOS (114 +/- 87 vs. 65 +/- 9, pg/mg, P = .09). MMP-9 expression in BAVM highly correlated with myeloperoxidase (R2 = .76, P < .001), as well as with IL-6 (R2 = .32, P < .001). In contrast, MMP-9 in BAVM poorly correlated with the endothelial marker, eNOS (R2 = .03, P = .05), and CD31 (R2 = .004, P = .57). Compared to non-embolized patients (n = 46), patients with pre-operative embolization (n = 93) had higher levels of myeloperoxidase (236 +/- 205 vs. 106 +/- 108, ng/mg, P < .001) and MMP-9 (33 +/- 35 vs. 16 +/- 20, ng/mg, P < .001), however the correlation between MMP-9 and myeloperoxidase was equally strong for both groups (R2 = .69, n = 93, P < .001, for both). MMP-9 expression correlated with the lipocalin-MMP-9 complex, suggesting neutrophils as the MMP-9 source. MPO co-localized with majority of MMP-9 signal by immunohistochemistry. Our data suggest that inflammation is a prominent feature of BAVM lesional phenotype, and neutrophils appear to be a major source of MMP-9 in these lesions.
Polymorphisms in transforming growth factor-beta-related genes ALK1 and ENG are associated with sporadic brain arteriovenous malformations.
Pawlikowska Ludmila,Tran Mary N,Achrol Achal S,Ha Connie,Burchard Esteban,Choudhry Shweta,Zaroff Jonathan,Lawton Michael T,Castro Richard,McCulloch Charles E,Marchuk Douglas,Kwok Pui-Yan,Young William L,
BACKGROUND AND PURPOSE:Mutations in endoglin (ENG) and activin-like kinase (ALK1) cause hereditary hemorrhagic telangiectasias, disorders characterized by pulmonary and brain arteriovenous malformations (BAVMs). We investigated whether polymorphisms in these genes are also associated with sporadic BAVM. METHODS:A total of 177 sporadic BAVM patients and 129 controls (all subjects white) were genotyped for 2 variants in ALK1 and 7 variants in ENG. RESULTS:The ALK1 IVS3-35A>G polymorphism was associated with BAVM: (AnyA [AA+AG] genotype: odds ratio, 2.47; 95% CI, 1.38 to 4.44; P=0.002). Two ENG polymorphisms, ENG -1742A>G and ENG 207G>A, showed a trend toward association with BAVM that did not reach statistical significance. CONCLUSIONS:A common polymorphism in ALK1 is associated with sporadic BAVM, suggesting that genetic variation in genes mutated in familial BAVM syndromes may play a role in sporadic BAVMs.
Abnormal expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in brain arteriovenous malformations.
Hashimoto Tomoki,Wen Gen,Lawton Michael T,Boudreau Nancy J,Bollen Andrew W,Yang Guo-Yuan,Barbaro Nicholas M,Higashida Randall T,Dowd Christopher F,Halbach Van V,Young William L,
BACKGROUND AND PURPOSE:Excessive degradation of the vascular matrix by matrix metalloproteinases (MMPs) can lead to structural instability of vessels. In this study we examined the expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in brain arteriovenous malformations (BAVMs). METHODS:We performed gelatin zymography for MMPs and Western blot for MMP-9, MMP-2, TIMP-1, TIMP-2, TIMP-3, and TIMP-4. MMP-9 expression was localized by immunohistochemistry. RESULTS:We analyzed 37 BAVM specimens and 9 control brain specimens from epilepsy surgery. Thirty-two BAVM patients had embolization treatment before resection. Eighteen BAVM patients had a history of hemorrhage from BAVMs. Neither MMP-2 nor TIMP-2 was detected in BAVMs or control brain specimens. Compared with control brain samples, BAVM samples had higher levels of total MMP-9, active MMP-9, pro-MMP-9, TIMP-1, and TIMP-3. TIMP-4 levels were higher in the control brain than in BAVM specimens. MMP-9 was localized to the endothelial cell/peri-endothelial cell layer and infiltrating neutrophils of BAVMs. BAVMs with venous stenosis >or=50% had higher expression of MMP-9 than BAVMs with venous stenosis <50%. There was no apparent association between total MMP-9, pro-MMP-9, or active MMP-9 levels and (1) feeding artery pressure, (2) pattern of draining vein (exclusively deep venous drainage versus any superficial drainage), and (3) BAVM size. CONCLUSIONS:We found increased levels of MMP-9 and TIMPs in BAVMs. Abnormal balance of MMP-9 and TIMPs may contribute to vascular instability of BAVMs.
Genetics and Emerging Therapies for Brain Arteriovenous Malformations.
Scherschinski Lea,Rahmani Redi,Srinivasan Visish M,Catapano Joshua S,Oh S Paul,Lawton Michael T
Brain arteriovenous malformations (AVMs) are characterized by a high-pressure, low-resistance vascular nidus created by direct shunting of blood from feeding arteries into arterialized veins, bypassing intervening capillaries. AVMs pose a risk of spontaneous rupture because the vessel walls are continuously exposed to increased shear stress and abnormal flow phenomena, which lead to vessel wall inflammation and distinct morphologic changes. The annual rupture rate is estimated at 2%, and once an AVM ruptures, the risk of rerupture increases 5-fold. The ability of AVMs to grow, regress, recur, and undergo remodeling shows their dynamic nature. Identifying the underlying cellular and molecular pathways of AVMs not only helps us understand their natural physiology but also allows us to directly block vital pathways, thus preventing AVM development and progression. Management of AVMs is challenging and often necessitates a multidisciplinary approach, including neurosurgical, endovascular, and radiosurgical expertise. Because many of these procedures are invasive, carry a risk of inciting hemorrhage, or are controversial, the demand for pharmacologic treatment options is increasing. In this review, we introduce novel findings of cellular and molecular AVM physiology and highlight key signaling mediators that are potential targets for AVM treatment. Furthermore, we give an overview of syndromes associated with hereditary and nonhereditary AVM formation and discuss causative genetic alterations.
State of the Art and Future Direction in Diagnosis, Molecular Biology, Genetics, and Treatment of Brain Arteriovenous Malformations.
Pérez-Alfayate Rebeca,Grasso Giovanni
Brain arteriovenous malformations (bAVMs) are uncommon and represent a heterogeneous group of lesions. Although these 2 facts have delayed research on this topic, knowledge about the pathophysiology, diagnosis, and treatment of bAVMs has evolved in recent years. We conducted a review of the literature to update the knowledge about diagnosis, molecular biology, genetic, pathology, and treatment by searching for the following terms: "Epidemiology AND Natural History," "risk of hemorrhage," "intracranial hemorrhage," "diagnosis," "angiogenesis," "molecular genetics," "VEGF," "KRAS," "radiosurgery," "endovascular," "microsurgery," or "surgical resection." Our understanding of bAVMs has significantly evolved in recent years. The latest investigations have helped in defining some molecular pathways involved in the pathology of bAVM. Although there is still more to learn and discover, describing these pathways will allow the creation of targeted treatments that could improve the prognosis of patients with bAVMs.
Exome-wide Analysis of De Novo and Rare Genetic Variants in Patients With Brain Arteriovenous Malformation.
BACKGROUND AND OBJECTIVES:Brain arteriovenous malformation (bAVM) is a congenital disorder and a leading cause of hemorrhagic stroke. Germline genetic variants play an essential role in the pathogenesis of bAVM. However, the biological relevance of disease-associated genes identified in previous studies is elusive. In this study, we aim to systematically investigate the contribution of germline variants to bAVM and explore the critical molecular pathways underlying the pathogenesis of bAVM. METHODS:Probands with sporadic bAVM were consecutively recruited into this study from November 2015 to November 2018 and underwent exome sequencing. The controls were aggregated from individuals who were not known to have vascular malformation and underwent exome sequencing for clinical or research purposes. The retained control dataset included 4,609 individuals, including 251 individuals with parental samples sequenced. We first analyzed de novo variants in cases and controls and performed a pathway enrichment analysis. A gene-based rare variant association analysis was then performed to identify genes whose variants were significantly enriched in cases. RESULTS:We collected an exome-sequenced bAVM cohort consisting of 152 trios and 40 singletons. By first focusing on de novo variants, we observed a significant mutational burden of likely gene-disrupting variants in cases vs controls. By performing a pathway enrichment analysis of all nonsynonymous de novo variants identified in cases, we found the angiopoietin-like protein 8 (ANGPTL8) regulatory pathway to be significantly enriched in patients with bAVM. Through an exome-wide rare variant association analysis utilizing 4,394 in-house exome data as controls, we identified as a disease-associated gene for bAVM. In addition, we found that the variants in cases are preferably located at the N' side of the SLC19A3 protein. These findings implicate a phenotypic expansion of -related disorders with a domain-specific effect. DISCUSSION:This study provides insights into the biological basis of bAVM by identifying novel molecular pathways and candidate genes.
Mutational spectrum of syndromic genes in sporadic brain arteriovenous malformation.
Wang Kun,Zhang Mingqi,Zhao Sen,Xie Zhixin,Zhang Yisen,Liu Jian,Zhang Ying,Yang Xinjian,Wu Nan
Chinese neurosurgical journal
BACKGROUND:Brain arteriovenous malformations (BAVMs) are abnormal vessels that are apt to rupture, causing life-threatening intracranial hemorrhage (ICH). The estimated prevalence of BAVMs is 0.05% among otherwise healthy individuals. In this study, we aim to investigate the mutational spectrum of syndromic genes in sporadic BAVM. METHODS:We recruited a cohort of 150 patients with BAVM and performed whole-exome sequencing on their peripheral blood DNA. To explore the mutational spectrum of syndromic genes in sporadic brain arteriovenous malformation, we selected six genes according to the Online Mendelian Inheritance in Man (OMIM) and literature. All variants in the six candidate genes were extracted and underwent filtering for qualifying variants. RESULTS:There are a total of four patients with rare variants in hereditary hemorrhagic telangiectasia-related genes. In addition, we identified two patients have the variant of RASA1 gene in our database, which are also rare mutations that are absent from population databases. However, we did not find any patients with GNAQ mutations in our database. CONCLUSIONS:In conclusion, we demonstrated that variants in syndromic vascular malformations play important roles in the etiology of sporadic BAVM.
Endoluminal Biopsy for Molecular Profiling of Human Brain Vascular Malformations.
BACKGROUND AND OBJECTIVES:Ras-mitogen-activated protein kinase (MAPK) signaling abnormalities occur in most brain arteriovenous malformations (bAVMs). No means exist to molecularly profile bAVMs without open surgery, limiting precision medicine approaches to treatment. Here, we report use of endoluminal biopsy of the vessel lumen of bAVMs to characterize gene expression and blood flow-mediated transcriptional changes in living patients. METHODS:Endoluminal biopsy and computational fluid dynamic modeling (CFD) were performed in adults with unruptured AVMs with cerebral angiography. Each patient underwent surgical resection and cell sampling from a contiguous arterial segment. Fluorescence-assisted cell sorting enriched endothelial cells, which were sequenced on an Illumina HiSeq 4000 sequencer. Gene expression was quantified with RNA sequencing (RNAseq). Differential gene expression, ontology, and correlative analyses were performed. Results were validated with quantitative reverse transcription PCR (RT-qPCR). RESULTS:Endoluminal biopsy was successful in 4 patients without complication. Endoluminal biopsy yielded 269.0 ± 79.9 cells per biopsy (control 309.2 ± 86.6 cells, bAVM 228.8 ± 133.4 cells). RNAseq identified 106 differentially expressed genes (DEGs) in bAVMs (false discovery rate ≤0.05). DEGs were enriched for bAVM pathogenic cascades, including Ras-MAPK signaling ( < 0.05), and confirmed with RT-qPCR and a panel predictive of MAPK/extracellular signal-regulated kinase inhibitor response. Compared to patient-matched surgically excised tissues, endoluminal biopsy detected 83.3% of genes, and genome-wide expression strongly correlated (Pearson = 0.77). Wall shear stress measured by CFD correlated with inflammatory pathway upregulation. Comparison of pre-embolization and postembolization samples confirmed flow-mediated gene expression changes. DISCUSSION:Endoluminal biopsy allows molecular profiling of bAVMs in living patients. Gene expression profiles are similar to those of tissues acquired with open surgery and identify potentially targetable Ras-MAPK signaling abnormalities in bAVMs. Integration with CFD allows determination of flow-mediated transcriptomic alterations. Endoluminal biopsy may help facilitate trials of precision medicine approaches to bAVMs in humans.
Soluble Endoglin Stimulates Inflammatory and Angiogenic Responses in Microglia That Are Associated with Endothelial Dysfunction.
Park Eun S,Kim Sehee,Yao Derek C,Savarraj Jude P J,Choi Huimahn Alex,Chen Peng Roc,Kim Eunhee
International journal of molecular sciences
Increased soluble endoglin (sENG) has been observed in human brain arteriovenous malformations (bAVMs). In addition, the overexpression of sENG in concurrence with vascular endothelial growth factor (VEGF)-A has been shown to induce dysplastic vessel formation in mouse brains. However, the underlying mechanism of sENG-induced vascular malformations is not clear. The evidence suggests the role of sENG as a pro-inflammatory modulator, and increased microglial accumulation and inflammation have been observed in bAVMs. Therefore, we hypothesized that microglia mediate sENG-induced inflammation and endothelial cell (EC) dysfunction in bAVMs. In this study, we confirmed that the presence of sENG along with VEGF-A overexpression induced dysplastic vessel formation. Remarkably, we observed increased microglial activation around dysplastic vessels with the expression of NLRP3, an inflammasome marker. We found that sENG increased the gene expression of VEGF-A, pro-inflammatory cytokines/inflammasome mediators (TNF-α, IL-6, NLRP3, ASC, Caspase-1, and IL-1β), and proteolytic enzyme (MMP-9) in BV2 microglia. The conditioned media from sENG-treated BV2 (BV2-sENG-CM) significantly increased levels of angiogenic factors (Notch-1 and TGFβ) and pERK1/2 in ECs but it decreased the level of IL-17RD, an anti-angiogenic mediator. Finally, the BV2-sENG-CM significantly increased EC migration and tube formation. Together, our study demonstrates that sENG provokes microglia to express angiogenic/inflammatory molecules which may be involved in EC dysfunction. Our study corroborates the contribution of microglia to the pathology of sENG-associated vascular malformations.
No shinkei geka. Neurological surgery
Recently, a KRAS mutation located in the endothelial cells of the nidus in arteriovenous malformations(AVMs)was reported. The findings indicate the possibility of medical therapy against cerebral AVMs; the blockade of RAS-MAPK signaling cascade may stabilize AVM nidus. As for surgical treatment for AVMs, achievement of low morbidity is the most important objective. To achieve this, assessment of blood flow, usage of non-stick bipolar forceps, and 3D simulation prior to operation are important. Assessment of individual cases for surgical treatment leads to low morbidity in surgeries.
Pilot investigation of circulating angiogenic and inflammatory biomarkers associated with vascular malformations.
Wetzel-Strong Sarah E,Weinsheimer Shantel,Nelson Jeffrey,Pawlikowska Ludmila,Clark Dewi,Starr Mark D,Liu Yingmiao,Kim Helen,Faughnan Marie E,Nixon Andrew B,Marchuk Douglas A
Orphanet journal of rare diseases
BACKGROUND:Vascular malformations in the central nervous system are difficult to monitor and treat due to their inaccessible location. Angiogenic and inflammatory proteins are secreted into the bloodstream and may serve as useful biomarkers for identifying patients at risk for complications or with certain disease phenotypes. METHODS:A validated multiplex protein array consisting of 26 angiogenic and inflammatory biomarkers (Angiome) was assessed in plasma isolated from healthy controls and patients with either sporadic brain arteriovenous malformation (BAVM), familial cerebral cavernous malformation (CCM), or hereditary hemorrhagic telangiectasia (HHT). These samples were obtained from archives of ongoing research studies at the University of California San Francisco and through prospective collection at the Toronto HHT Centre at St. Michael's Hospital. RESULTS:We compared circulating biomarker levels from each patient group to healthy controls and analyzed each pairwise combination of patient groups for differences in biomarker levels. Additionally, we analyzed the HHT samples to determine the association between biomarker levels and the following HHT-specific phenotypes, BAVM, pulmonary arteriovenous malformation (PAVM), liver vascular malformation (LVM), and gastrointestinal (GI) bleeding. Compared to controls, levels of SDF1 were significantly elevated in HHT patients (Proportional Increase [PI] = 1.87, p < 0.001, q = 0.011). Levels of sENG were significantly reduced in HHT patients compared to controls (PI = 0.56, p < 0.001, q < 0.001), reflecting the prevalence of HHT1 patients in this cohort. Levels of IL6 (PI = 3.22, p < 0.001, q < 0.001) and sTGFβR3 (PI = 0.70, p = 0.001, q < 0.029) differed significantly in CCM patients compared to controls. Compared to controls, ten of the biomarkers were significantly different in sporadic BAVM patients (q-values < 0.05). Among the pairwise combinations of patient groups, a significant elevation was observed in TGFβ1 in CCM patients compared to sporadic BAVM patients (PI = 2.30, p < 0.001, q = 0.034). When examining the association of circulating biomarker levels with HHT-specific phenotypes, four markers were significantly lower in HHT patients with BAVM (q-values < 0.05), and four markers were significantly higher in patients with LVM (q-values < 0.05). CONCLUSIONS:This pilot study suggests that the profile of circulating angiogenic and inflammatory biomarkers may be unique to each type of vascular malformation. Furthermore, this study indicates that circulating biomarkers may be useful for assessing phenotypic traits of vascular malformations.
Somatic mosaicism in the MAPK pathway in sporadic brain arteriovenous malformation and association with phenotype.
Gao Sen,Nelson Jeffrey,Weinsheimer Shantel,Winkler Ethan A,Rutledge Caleb,Abla Adib A,Gupta Nalin,Shieh Joseph T,Cooke Daniel L,Hetts Steven W,Tihan Tarik,Hess Christopher P,Ko Nerissa,Walcott Brian P,McCulloch Charles E,Lawton Michael T,Su Hua,Pawlikowska Ludmila,Kim Helen
Journal of neurosurgery
OBJECTIVE:Sporadic brain arteriovenous malformation (BAVM) is a tangled vascular lesion characterized by direct artery-to-vein connections that can cause life-threatening intracerebral hemorrhage (ICH). Recently, somatic mutations in KRAS have been reported in sporadic BAVM, and mutations in other mitogen-activated protein kinase (MAPK) signaling pathway genes have been identified in other vascular malformations. The objectives of this study were to systematically evaluate somatic mutations in MAPK pathway genes in patients with sporadic BAVM lesions and to evaluate the association of somatic mutations with phenotypes of sporadic BAVM severity. METHODS:The authors performed whole-exome sequencing on paired lesion and blood DNA samples from 14 patients with sporadic BAVM, and 295 genes in the MAPK signaling pathway were evaluated to identify genes with somatic mutations in multiple patients with BAVM. Digital droplet polymerase chain reaction was used to validate KRAS G12V and G12D mutations and to assay an additional 56 BAVM samples. RESULTS:The authors identified a total of 24 candidate BAVM-associated somatic variants in 11 MAPK pathway genes. The previously identified KRAS G12V and G12D mutations were the only recurrent mutations. Overall, somatic KRAS G12V was present in 14.5% of BAVM lesions and G12D was present in 31.9%. The authors did not detect a significant association between the presence or allelic burden of KRAS mutation and three BAVM phenotypes: lesion size (maximum diameter), age at diagnosis, and age at ICH. CONCLUSIONS:The authors confirmed the high prevalence of somatic KRAS mutations in sporadic BAVM lesions and identified several candidate somatic variants in other MAPK pathway genes. These somatic variants may contribute to understanding of the etiology of sporadic BAVM and the clinical characteristics of patients with this condition.
Effects of serum starvation and vascular endothelial growth factor stimulation on the expression of Notch signalling pathway components.
Brain arteriovenous malformation (BAVM) is an abnormality in the cerebral vascular system. Although the upregulation of the Notch signalling pathway is a deterministic factor in BAVM, the mechanism by which this pathway is upregulated in patients with BAVM is uncertain. The effects of serum starvation and vascular endothelial growth factor (VEGF) stimulation on the Notch signalling pathway in brain microvascular endothelial cells (MECs) and mouse embryonic stem (mES)/embryoid body (EB)-derived endothelial cells were investigated in this study. The duration of serum starvation and VEGF concentration were changed, cell viability was measured, and reasonable time and concentration gradients were selected for subsequent studies. Protein and mRNA expression levels of Notch signalling pathway components in both MECs and mES/EB-derived endothelial cells were detected using western blotting and real-time PCR, respectively. Expression levels of the Notch1, Notch4, Jagged1, delta-like ligand 4 (Dll4) and Hes1 proteins and mRNAs were upregulated by lower VEGF concentrations and shorter-term serum starvation but inhibited by higher VEGF concentrations and longer-term serum starvation. This study revealed effects of changes in the duration of serum starvation and VEGF concentration on the expression of Notch signalling pathway components in both MECs and mES/EB-derived endothelial cells, potentially contributing to BAVM formation.
Cyclo-oxygenase 2, a putative mediator of vessel remodeling, is expressed in the brain AVM vessels and associates with inflammation.
Keränen Sara,Suutarinen Santeri,Mallick Rahul,Laakkonen Johanna P,Guo Diana,Pawlikowska Ludmila,Jahromi Behnam Rezai,Rauramaa Tuomas,Ylä-Herttuala Seppo,Marchuk Doug,Krings Timo,Koivisto Timo,Lawton Michael,Radovanovic Ivan,Kim Helen,Faughnan Marie E,Frösen Juhana
BACKGROUND:Brain arteriovenous malformations (bAVM) may rupture causing disability or death. BAVM vessels are characterized by abnormally high flow that in general triggers expansive vessel remodeling mediated by cyclo-oxygenase-2 (COX2), the target of non-steroidal anti-inflammatory drugs. We investigated whether COX2 is expressed in bAVMs and whether it associates with inflammation and haemorrhage in these lesions. METHODS:Tissue was obtained from surgery of 139 bAVMs and 21 normal Circle of Willis samples. The samples were studied with immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR). Clinical data was collected from patient records. RESULTS:COX2 expression was found in 78% (109/139) of the bAVMs and localized to the vessels' lumen or medial layer in 70% (95/135) of the bAVMs. Receptors for prostaglandin E2, a COX2-derived mediator of vascular remodeling, were found in the endothelial and smooth muscle cells and perivascular inflammatory cells of bAVMs. COX2 was expressed by infiltrating inflammatory cells and correlated with the extent of inflammation (r = .231, p = .007, Spearman rank correlation). COX2 expression did not associate with haemorrhage. CONCLUSION:COX2 is induced in bAVMs, and possibly participates in the regulation of vessel wall remodelling and ongoing inflammation. Role of COX2 signalling in the pathobiology and clinical course of bAVMs merits further studies.
Review of treatment and therapeutic targets in brain arteriovenous malformation.
Pan Peipei,Weinsheimer Shantel,Cooke Daniel,Winkler Ethan,Abla Adib,Kim Helen,Su Hua
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism
Brain arteriovenous malformations (bAVM) are an important cause of intracranial hemorrhage (ICH), especially in younger patients. The pathogenesis of bAVM are largely unknown. Current understanding of bAVM etiology is based on studying genetic syndromes, animal models, and surgically resected specimens from patients. The identification of activating somatic mutations in the Kirsten rat sarcoma viral oncogene homologue gene and other mitogen-activated protein kinase () pathway genes has opened up new avenues for bAVM study, leading to a paradigm shift to search for somatic, mutations in sporadic bAVMs instead of focusing on inherited genetic mutations. Through the development of new models and understanding of pathways involved in maintaining normal vascular structure and functions, promising therapeutic targets have been identified and safety and efficacy studies are underway in animal models and in patients. The goal of this paper is to provide a thorough review or current diagnostic and treatment tools, known genes and key pathways involved in bAVM pathogenesis to summarize current treatment options and potential therapeutic targets uncovered by recent discoveries.
An Insight into the microRNAs Associated with Arteriovenous and Cavernous Malformations of the Brain.
Florian Ioan Alexandru,Buruiana Andrei,Timis Teodora Larisa,Susman Sergiu,Florian Ioan Stefan,Balasa Adrian,Berindan-Neagoe Ioana
: Brain arteriovenous malformations (BAVMs) and cerebral cavernous malformations (CCMs) are rare developmental anomalies of the intracranial vasculature, with an irregular tendency to rupture, and as of yet incompletely deciphered pathophysiology. Because of their variety in location, morphology, and size, as well as unpredictable natural history, they represent a management challenge. MicroRNAs (miRNAs) are strands of non-coding RNA of around 20 nucleotides that are able to modulate the expression of target genes by binding completely or partially to their respective complementary sequences. Recent breakthroughs have been made on elucidating their contribution to BAVM and CCM occurrence, growth, and evolution; however, there are still countless gaps in our understanding of the mechanisms involved. : We have searched the Medline (PubMed; PubMed Central) database for pertinent articles on miRNAs and their putative implications in BAVMs and CCMs. To this purpose, we employed various permutations of the terms and idioms: 'arteriovenous malformation', 'AVM', and 'BAVM', or 'cavernous malformation', 'cavernoma', and 'cavernous angioma' on the one hand; and 'microRNA', 'miRNA', and 'miR' on the other. Using cross-reference search; we then investigated additional articles concerning the individual miRNAs identified in other cerebral diseases. : Seven miRNAs were discovered to play a role in BAVMs, three of which were downregulated (miR-18a, miR-137, and miR-195*) and four upregulated (miR-7-5p, miR-199a-5p, miR-200b-3p, and let-7b-3p). Similarly, eight miRNAs were identified in CCM in humans and experimental animal models, two being upregulated (miR-27a and mmu-miR-3472a), and six downregulated (miR-125a, miR-361-5p, miR-370-3p, miR-181a-2-3p, miR-95-3p, and let-7b-3p). : The following literature review endeavored to address the recent discoveries related to the various implications of miRNAs in the formation and growth of BAVMs and CCMs. Additionally, by presenting other cerebral pathologies correlated with these miRNAs, it aimed to emphasize the potential directions of upcoming research and biological therapies.
Selective Endothelial Hyperactivation of Oncogenic KRAS Induces Brain Arteriovenous Malformations in Mice.
Park Eun S,Kim Sehee,Huang Shuning,Yoo Ji Young,Körbelin Jakob,Lee Tae Jin,Kaur Balveen,Dash Pramod K,Chen Peng R,Kim Eunhee
Annals of neurology
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 KRAS mutation induces sporadic bAVMs in mice. METHODS:Five-week-old mice were systemically injected with either AAV-BR1-GFP or -KRAS . 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 KRAS -induced bAVMs was determined by treatment of trametinib, a US Food and Drug Administration (FDA)-approved MEK/ERK inhibitor. RESULTS:The viral-mediated KRAS 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.
Maternal and Fetal Outcomes in Women with Brain Arteriovenous Malformation Rupture during Pregnancy.
Yan Kimberly L,Ko Nerissa U,Hetts Steven W,Weinsheimer Shantel,Abla Adib A,Lawton Michael T,Kim Helen
Cerebrovascular diseases (Basel, Switzerland)
BACKGROUND:Sporadic brain arteriovenous malformations (BAVM) are a major cause of hemorrhagic stroke in younger persons. Prior studies have reported contradictory results regarding the risk of hemorrhage during pregnancy, and there are no standard guidelines for the management of pregnant women who present with BAVM rupture. The purpose of this study is to describe maternal and fetal outcomes and treatment strategies in patients with BAVM hemorrhage during pregnancy. METHODS:We performed a retrospective review of the University of California, San Francisco Brain AVM Project database for female patients who were pregnant at the time of BAVM hemorrhage between 2000 and 2017. Clinical and angiographic characteristics at presentation, BAVM treatment, and maternal outcomes using modified Rankin scale (mRS) score at presentation and 2-year follow-up were recorded. Fetal outcomes were abstracted from medical records and maternal reports. RESULTS:Sixteen patients presented with BAVM hemorrhage during pregnancy, 81% (n = 13) of whom were in their second or third trimester. Three patients (19%) who were in their first trimester terminated or miscarried pregnancy prior to BAVM intervention. Of the remaining 13 patients, 77% (n = 10) received emergent BAVM treatment at time of hemorrhage prior to delivery, and 85% of patients achieved BAVM obliteration and good maternal outcomes (mRS 0-2) at 2-year follow-up. All patients had uncomplicated deliveries (69% cesarean and 23% vaginal) with no reports of postnatal cognitive or developmental delays in infants at 2-year follow-up. CONCLUSIONS:Our study shows good long-term maternal and fetal outcomes in ruptured BAVM patients presenting during pregnancy, the majority who received BAVM interventional treatment prior to delivery.
The CTSC-RAB38 Fusion Transcript Is Associated With the Risk of Hemorrhage in Brain Arteriovenous Malformations.
Yan Zihan,Fan Guangming,Li Hao,Jiao Yuming,Fu Weilun,Weng Jiancong,Huo Ran,Wang Jie,Xu Hongyuan,Wang Shuo,Cao Yong,Zhao Jizong
Journal of neuropathology and experimental neurology
Brain arteriovenous malformations (bAVMs) are congenital anomalies of blood vessels that cause intracranial hemorrhage in children and young adults. Chromosomal rearrangements and fusion genes play an important role in tumor pathogenesis, though the role of fusion genes in bAVM pathophysiological processes is unclear. The aim of this study was to identify fusion transcripts in bAVMs and analyze their effects. To identify fusion transcripts associated with bAVM, RNA sequencing was performed on 73 samples, including 66 bAVM and 7 normal cerebrovascular samples, followed by STAR-Fusion analysis. Reverse transcription polymerase chain reaction and Sanger sequencing were applied to verify fusion transcripts. Functional pathway analysis was performed to identify potential effects of different fusion types. A total of 21 fusion transcripts were detected. Cathepsin C (CTSC)-Ras-Related Protein Rab-38 (RAB38) was the most common fusion and was detected in 10 of 66 (15%) bAVM samples. In CTSC-RAB38 fusion-positive samples, CTSC and RAB38 expression was significantly increased and activated immune/inflammatory signaling. Clinically, CTSC-RAB38 fusion bAVM cases had a higher hemorrhage rate than non-CTSC-RAB38 bAVM cases (p < 0.05). Our study identified recurrent CTSC-RAB38 fusion transcripts in bAVMs, which may be associated with bAVM hemorrhage by promoting immune/inflammatory signaling.
Neurovascular inflammation in the pathogenesis of brain arteriovenous malformations.
Krithika S,Sumi S
Journal of cellular physiology
Brain arteriovenous malformations (bAVM) arise as congenital or sporadic focal lesions with a significant risk for intracerebral hemorrhage (ICH). A wide range of interindividual differences is present in the onset, progression, and severity of bAVM. A growing body of gene expression and polymorphism-based research studies support the involvement of localized inflammation in bAVM disease progression and rupture. In this review article, we analyze the altered responses of neural, vascular, and immune cell types that contribute to the inflammatory process, which exacerbates the pathophysiological progression of vascular dysmorphogenesis in bAVM lesions. The cumulative effect of inflammation in bAVM development is orchestrated by various genetic moderators and inflammatory mediators. We also discuss the potential therapies for the treatment of brain AVM by targeting the inflammatory processes and mediators. Elucidating the precise role of inflammation in the bAVM growth and hemorrhage would open novel avenues for noninvasive and effectual causal therapy that may complement the current therapeutic strategies.
Metabolic Disorder of Extracellular Matrix Mediated by Decorin Upregulation Is Associated With Brain Arteriovenous Malformation Diffuseness.
Li Maogui,Liu Qingyuan,Yang Junhua,Jiang Pengjun,Yang Yi,Zhang Yanan,Cao Yong,Wu Jun,Wang Shuo
Frontiers in aging neuroscience
Background and Objective:Diffuse brain arteriovenous malformations (BAVMs) are mixed up with normal brain parenchyma and therefore increase the difficulty of surgical resection, leading to poor surgical prognosis. Since the mechanism underlying BAVM diffuseness remains unknown, a quantitative proteomic analysis was performed to investigate the altered expression of proteins in diffuse BAVMs compared to compact ones. Methods:We performed proteomic analysis on five diffuse BAVMs and five compact BAVMs. Bioinformatics analysis was conducted to identify potential signals related to BAVM diffuseness. Candidate proteins were then investigated in BAVM specimens using immunofluorescence and Western blot analysis. Tube formation assays were used to investigate the effects of candidate proteins on the angiogenesis of human umbilical endothelial cells (HUVECs). Finally, Masson, Sirius red staining, and immunofluorescence were used to evaluate the characteristics of extracellular matrix (ECM) in BAVM tissues. Results:A total of 58 proteins were found to be differentially expressed between diffuse and compact BAVMs via proteomic analysis. TGF-β (transforming growth factor-beta) signaling pathway, ECM-receptor pathway, relaxin signaling pathway, and several other pathways were associated with BAVM diffuseness. The TGF-β signaling pathway is associated with angiogenesis; the role of this pathway in the formation of diffuse BAVMs was investigated, and the decorin (DCN) upregulation played an important role in this process. Immunofluorescence showed that DCN was significantly upregulated within and around the malformed vessels of diffuse BAVMs. Functional assays showed that exogenous DCN could promote the tube formation ability of HUVECs through inhibiting the TGF-β signaling pathway and overproducing ECM. Histological staining demonstrated the overproduction of ECM in diffuse BAVMs. Conclusion:TGF-β signaling pathway inhibited by DCN in vascular endothelial cells is related to BAVM diffuseness. The metabolic disorder of ECM caused by DCN upregulation may significantly contribute to the formation of diffuse BAVMs.
Exome sequencing of 112 trios identifies recessive genetic variants in brain arteriovenous malformations.
Zhang Mingqi,Ding Xinghuan,Zhang Qianqian,Liu Jian,Zhang Yisen,Zhang Ying,Tian Zhongbin,Li Wenqiang,Zhu Wei,Kang Huibin,Wang Zhongxiao,Wu Xinzhi,Wang Chao,Yang Xinjian,Wang Kun
Journal of neurointerventional surgery
BACKGROUND:Brain arteriovenous malformation (BAVM) is a main cause of cerebral hemorrhage and hemorrhagic stroke in adolescents. Morphologically, a BAVM is an abnormal connection between cerebrovascular arteries and veins. The genetic etiology of BAVMs has not been fully elucidated. In this study, we aim to investigate potential recessive genetic variants in BAVMs by interrogation of rare compound heterozygous variants. METHODS:We performed whole exome sequencing (WES) on 112 BAVM trios and analyzed the data for rare and deleterious compound heterozygous mutations associated with the disease. RESULTS:We identified 16 genes with compound heterozygous variants that were recurrent in more than one trio. Two genes () were recurrently mutated in three trios. has been previously associated with BAVM pathogenesis. Fourteen genes (, , , , , , , , , , , , , ) were recurrently mutated in two trios, and five of these genes (, , , , ) have been reported to play a role in angiogenesis or vascular diseases. Additionally, abnormal expression of the MYLK protein is related to spinal arteriovenous malformations. CONCLUSION:Our study indicates that rare recessive compound heterozygous variants may underlie cases of BAVM. These findings improve our understanding of BAVM pathology and indicate genes for functional validation.
Somatic Gain of KRAS Function in the Endothelium Is Sufficient to Cause Vascular Malformations That Require MEK but Not PI3K Signaling.
Fish Jason E,Flores Suarez Carlos Perfecto,Boudreau Emilie,Herman Alexander M,Gutierrez Manuel Cantu,Gustafson Dakota,DiStefano Peter V,Cui Meng,Chen Zhiqi,De Ruiz Karen Berman,Schexnayder Taylor S,Ward Christopher S,Radovanovic Ivan,Wythe Joshua D
RATIONALE:We previously identified somatic activating mutations in the () 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 (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.
Germline Mutation Enrichment in Pathways Controlling Endothelial Cell Homeostasis in Patients with Brain Arteriovenous Malformation: Implication for Molecular Diagnosis.
Scimone Concetta,Granata Francesca,Longo Marcello,Mormina Enricomaria,Turiaco Cristina,Caragliano Antonio A,Donato Luigi,Sidoti Antonina,D'Angelo Rosalia
International journal of molecular sciences
Brain arteriovenous malformation (bAVM) is a congenital defect affecting brain microvasculature, characterized by a direct shunt from arterioles to venules. Germline mutations in several genes related to transforming growth factor beta (TGF-β)/BMP signaling are linked to both sporadic and hereditary phenotypes. However, the low incidence of inherited cases makes the genetic bases of the disease unclear. To increase this knowledge, we performed a whole exome sequencing on five patients, on DNA purified by peripheral blood. Variants were filtered based on frequency and functional class. Those selected were validated by Sanger sequencing. Genes carrying selected variants were prioritized to relate these genes with those already known to be linked to bAVM development. Most of the prioritized genes showed a correlation with the TGF-βNotch signaling and vessel morphogenesis. However, two novel pathways related to cilia morphogenesis and ion homeostasis were enriched in mutated genes. These results suggest novel insights on sporadic bAVM onset and confirm its genetic heterogeneity. The high frequency of germline variants in genes related to TGF-β signaling allows us to hypothesize bAVM as a complex trait resulting from the co-existence of low-penetrance loci. Deeper knowledge on bAVM genetics can improve personalized diagnosis and can be helpful with genotype-phenotype correlations.
Mesenchymal Behavior of the Endothelium Promoted by SMAD6 Downregulation Is Associated With Brain Arteriovenous Malformation Microhemorrhage.
Fu Weilun,Huo Ran,Yan Zihan,Xu Hongyuan,Li Hao,Jiao Yuming,Wang Linjian,Weng Jiancong,Wang Jie,Wang Shuo,Cao Yong,Zhao Jizong
BACKGROUND AND PURPOSE:In unruptured brain arteriovenous malformations (bAVMs), microhemorrhage portends a higher risk of future rupture and may represent a transitional state along the continuum of destabilization. Exploration of the molecular and cellular mechanisms of microhemorrhage will provide a possible target for medical treatment to prevent bAVM bleeding. METHODS:We performed RNA sequencing analysis on 34 unruptured bAVM surgical samples. Functional pathway analysis was performed to identify potential signals associated with the microhemorrhagic phenotype. Candidate gene was then investigated in bAVM specimens by immunohistochemical staining. Several functional assays were used to investigate the effects of candidate genes on the phenotypic properties of cultured human umbilical vein endothelial cells. Then, Masson trichrome staining and immunofluorescence staining were used to evaluate the phenotypic and molecular changes in bAVM tissue. RESULTS:Via RNA sequencing, we identified differential gene expression between 18 microhemorrhagic bAVMs and 16 nonmicrohemorrhagic bAVMs. TGFβ (transforming growth factor-beta)/BMP (bone morphogenetic protein) signaling was associated with the bAVM microhemorrhage group when SMAD6 (SMAD family member 6) was downregulated. Immunohistochemical staining showed that the vascular endothelium of microhemorrhagic bAVMs exhibited decreased SMAD6 expression. Functional assays revealed that SMAD6 downregulation promoted the formation of endothelial cell tubes with deficient cell-cell junctions and facilitated the acquisition of mesenchymal behavior by endothelial cells. Masson trichrome and immunofluorescence staining demonstrated that mesenchymal phenotype of endothelial cells is promoted in microhemorrhagic bAVMs. CONCLUSIONS:TGFβ/BMP signaling mediated by SMAD6 in vascular endothelial cells is associated with microhemorrhagic bAVMs, and mesenchymal behavior of endothelial cells induced by SMAD6 downregulation is related with bAVM microhemorrhage.
Histopathology of brain AVMs part I: microhemorrhages and changes in the nidal vessels.
Järvelin Patrik,Wright Roosa,Pekonen Henri,Keränen Sara,Rauramaa Tuomas,Frösen Juhana
BACKGROUND:Arteriovenous malformations of the brain (bAVM) may rupture from aneurysms or ectasias of the feeding, draining, or nidal vessels. Moreover, they may rupture from the immature, fragile nidal vessels that are characteristic to bAVMs. How the histopathological changes of the nidal vessels associate with clinical presentation and hemorrhage of the lesion is not well known. MATERIALS AND METHODS:We investigated tissue samples from surgically treated bAVMs (n = 85) using standard histological and immunohistochemical stainings. Histological features were compared with the clinical presentation of the patient. RESULTS:Microhemorrhages from nidal vessels were found both in bAVMs with a history of clinically evident rupture and in bAVMs considered unruptured. These microhemorrhages were associated with presence of immature, pathological nidal vessels (p = 0.010) and perivascular inflammation of these vessels (p = 0.001), especially with adhesion of neutrophils (p < 0.001). In multivariate analysis, perivascular inflammation (OR = 19, 95% CI 1.6 to 230), neutrophil infiltration of the vessel wall (OR = 13, 95% CI 1.9 to 94), and rupture status (OR = 0.13, 95% CI 0.017 to 0.92) were significantly associated with microhemorrhages. CONCLUSIONS:Clinically silent microhemorrhages from nidal vessels seem to be very common in bAVMs, and associate with perivascular inflammation and neutrophil infiltration. Further studies on the role of perivascular inflammation in the clinical course of bAVMs are indicated.
Histopathology of brain AVMs part II: inflammation in arteriovenous malformation of the brain.
Wright Roosa,Järvelin Patrik,Pekonen Henri,Keränen Sara,Rauramaa Tuomas,Frösen Juhana
BACKGROUND:Hemorrhage from an arteriovenous malformation of the brain (bAVM) has been associated with focal inflammation of the bAVM. Intrigued by the possibility of anti-inflammatory drug therapy to stabilize bAVMs and prevent hemorrhage, we investigated the association of bAVM inflammation with other histological features and clinical presentation. MATERIALS AND METHODS:Tissue samples from 85 surgically treated bAVMs were studied with histology and CD45 immunostainings. The histological data was compared with the clinical history of the patient. Univariate analysis and logistic regression were performed. RESULTS:Inflammation was found in all studied bAVMs and did not associate with rupture (p = 0.442). While multiple types of inflammatory cells were present, macrophages were clearly the dominant inflammatory cell type, especially in samples with strong inflammation (87% of the samples). Of those bAVMs that had strong inflammation, only 56% had presented with clinically evident rupture. However, hemosiderin which is a sign of prior hemorrhage was detected in 78.4% (58/74) of samples with strong inflammation and was associated with it (p = 0.003). Inflammation in the nidus and parenchyma was associated with perivascular inflammation (p < 0.001). Multivariate analysis did not reveal any independent histological or clinical risk factor for inflammation. CONCLUSIONS:Since strong inflammation is present in both unruptured and ruptured bAVMs, it is not just a reaction to rupture. Our observations suggest that inflammation of the bAVM may indeed predispose to fragility and hemorrhage of the nidal vessels. Further studies in the role of inflammation in the untreated clinical course of bAVMs are indicated.
Methylation of Phospholipase A2 Group VII Gene Is Associated with Brain Arteriovenous Malformations in Han Chinese Populations.
Liu Yuchun,Wu Xizheng,Nie Sheng,Zhou Shengjun,Xiao Shuyuan,Gao Xiang,Lin Zhiqing,Sun Jie,Huang Yi
Journal of molecular neuroscience : MN
The purpose of this study was to evaluate the contribution of DNA methylation at the phospholipase A2 group VII (PLA2G7) gene, to the risk of developing brain arteriovenous malformations (BAVMs) and intracranial aneurysms (IAs) in a Han Chinese population. Seventy patients with BAVMs or IAs and 26 control subjects were recruited to evaluate PLA2G7 methylation by bisulfite pyrosequencing. CpG3 methylation at the PLA2G7 was significantly higher in BAVM patients than in the control group (Chr6,46735560, p = 0.042). Gender subgroup analysis showed that PLA2G7 CpG4 (Chr6,46735574, p = 0.033) and mean methylation (p = 0.037) levels were significantly associated with BAVM in males. However, in females, PLA2G7 methylations were much lower in IAs than in controls [CpG2 (Chr6,46735558), p = 0.030] and BAVMs [CpG2, p = 0.001; CpG4, p = 0.007; CpG6 (Chr6, 46735579), p = 0.024; mean, p = 0.013]. In addition, mean methylation of the PLA2G7 significantly correlated with apoB levels in all individuals (r = 0.288, p = 0.006), and with apoE in BAVM patients (r = 0.259, p = 0.016). The receiver operating characteristic (ROC) curve showed that PLA2G7 CpG3 methylation might be a predictor of BAVM risk in Han Chinese (area under curve (AUC) = 0.76, p = 0.008). PLA2G7 DNA methylation was significantly associated with the risk of developing BAVMs in males or IAs in females. Future studies on the PLA2G7 mechanisms should be performed on the pathogenesis of these cerebrovascular disorders.
High-Throughput Sequencing to Detect Novel Likely Gene-Disrupting Variants in Pathogenesis of Sporadic Brain Arteriovenous Malformations.
Scimone Concetta,Donato Luigi,Alafaci Concetta,Granata Francesca,Rinaldi Carmela,Longo Marcello,D'Angelo Rosalia,Sidoti Antonina
Frontiers in genetics
Molecular signaling that leads to brain arteriovenous malformation (bAVM) is to date elusive and this is firstly due to the low frequency of familial cases. Conversely, sporadic bAVM is the most diffuse condition and represents the main source to characterize the genetic basis of the disease. Several studies were conducted in order to detect both germ-line and somatic mutations linked to bAVM development and, in this context, next generation sequencing technologies offer a pivotal resource for the amount of outputted information. We performed whole exome sequencing on a young boy affected by sporadic bAVM. Paired-end sequencing was conducted on an Illumina platform and filtered variants were validated by Sanger sequencing. We detected 20 likely gene-disrupting variants affecting as many loci. Of these variants, 11 are inherited novel variants and one is a nonsense variant, affecting gene. Moreover, we also considered rare known variants affecting loci involved in vascular differentiation. In order to explain their possible involvement in bAVM pathogenesis, we analyzed molecular networks at Cytoscape platform. In this study we focus on some genetic point variations detected in a child affected by bAVM. Therefore, we suggest these novel affected loci as prioritized for further investigation on pathogenesis of bAVM lesions.
RNA-Sequencing Highlights Inflammation and Impaired Integrity of the Vascular Wall in Brain Arteriovenous Malformations.
Hauer Allard J,Kleinloog Rachel,Giuliani Fabrizio,Rinkel Gabriël J E,de Kort Gerard A,Berkelbach van der Sprenkel Jan Willem,van der Zwan Albert,Gosselaar Peter H,van Rijen Peter C,de Boer-Bergsma Jelkje J,Deelen Patrick,Swertz Morris A,De Muynck Louis,Van Damme Philip,Veldink Jan H,Ruigrok Ynte M,Klijn Catharina J M
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 <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)-β 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 =1.70×10). We identified 47 enriched Gene Ontology terms (false discovery rate-adjusted <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.
Age-dependent changes of collagen alpha-2(IV) expression in the extracellular matrix of brain arteriovenous malformations.
Neyazi Belal,Stein Klaus-Peter,Wilkens Ludwig,Maslehaty Homajoun,Dumitru Claudia A,Sandalcioglu I Erol
Clinical neurology and neurosurgery
OBJECTIVES:Brain arteriovenous malformations (bAVM) are rare vascular lesions. Recent observations challenge the congenital nature of these lesions. The underlying cellular and molecular mechanisms associated with dynamic changes of bAVM still remain unclear. The objective of this study was to explore the potential role of COL4A2 (Collagen alpha-2(IV)) in the pathophysiology of bAVM. PATIENTS AND METHODS:Expression and localization of COL4A2 were analyzed on tissue microarrays from bAVM patients (n = 60) by immunohistochemistry. Correlations between COL4A2 levels and clinical parameters were examined with Pearson's test for normally- distibuted or Spearman's Rho for not normally distributed data. Comparison between different clinical parameters was performed using t-test, non-parametric Mann-Whitney U test or Kruskal- Wallis test. Fisher's exact test was used for categorical data. RESULTS:COL4A2 was mainly expressed beneath the endothelium of vessels in the tunica media of bAVM. This pattern of expression indicates the basement membrane of the vessel walls. High levels of COL4A2 expression correlated with the age at surgery of patients (p = 0.005; R = 0.393; Spearman's Rho). The age at surgery in young (17-25 years) and old patients (55-76 years) showed a linear correlation; a greater variance of COL4A2 expression was observed in the age group between 26-54 years. CONCLUSION:This study reports for the first time the expression of COL4A2 in bAVM and suggests a potential role of COL4A2 in bAVM pathophysiology. These findings contribute to a better understanding of the microenvironment of bAVM and may foster the development of improved therapeutic strategies for this disease.
Methylation of the CDKN2A Gene Increases the Risk of Brain Arteriovenous Malformations.
Chen Xiaosheng,Liu Yuchun,Zhou Shengjun,Nie Sheng,Lin Zhiqin,Zhou Chenhui,Sun Jie,Gao Xiang,Huang Yi
Journal of molecular neuroscience : MN
Brain arteriovenous malformations (BAVMs) and intracranial aneurysms (IAs) are the results of a combination of genetic and environmental factors. Epigenetic factors also play an important role in the pathogenesis of these disorders. The aim of this study was to determine the effect of DNA methylation at the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene on the risk of BAVMs and IAs. A total of 70 intracranial vascular malformation patients (22 patients with BAVMs and 48 patients with IAs) and 26 patients with cerebral trauma (used as controls) were included in this study. DNA methylation levels of eight cytosine-phosphate-guanine (CpG) dinucleotides present in the CDKN2A gene were measured using bisulfite pyrosequencing technology. Significant differences in methylation at CpG1 (p = 0.005), CpG5 (p = 0.011), and CpG8 (p = 0.017) were observed between BAVM patients and controls. CDKN2A methylation levels in BAVM patients were much higher than those in IA patients (CpG5: p = 0.004; CpG8: p = 0.010). Significant differences were observed between female IA patients and female BAVM patients (CpG5: p = 0.006; CpG8: p = 0.005; mean: p = 0.015). Receiver operating characteristic (ROC) curve analysis showed that the CDKN2A gene methylation trended toward a diagnostic indicator in BAVM patients (area under curve = 0.711, p = 0.013). In conclusion, our study demonstrated that the CDKN2A gene methylation levels are significantly correlated with the occurrence of BAVMs, and thus, have potential for use in the early diagnosis of BAVMs. Future research on the pathogenesis of BAVMs should focus on the role of genetic factors in aberrant venous development. The association of the CDKN2A gene with venous development also deserves further study.
Flow-induced, inflammation-mediated arterial wall remodeling in the formation and progression of intracranial aneurysms.
Frösen Juhana,Cebral Juan,Robertson Anne M,Aoki Tomohiro
OBJECTIVE:Unruptured intracranial aneurysms (UIAs) are relatively common lesions that may cause devastating intracranial hemorrhage, thus producing considerable suffering and anxiety in those affected by the disease or an increased likelihood of developing it. Advances in the knowledge of the pathobiology behind intracranial aneurysm (IA) formation, progression, and rupture have led to preclinical testing of drug therapies that would prevent IA formation or progression. In parallel, novel biologically based diagnostic tools to estimate rupture risk are approaching clinical use. Arterial wall remodeling, triggered by flow and intramural stresses and mediated by inflammation, is relevant to both. METHODS:This review discusses the basis of flow-driven vessel remodeling and translates that knowledge to the observations made on the mechanisms of IA initiation and progression on studies using animal models of induced IA formation, study of human IA tissue samples, and study of patient-derived computational fluid dynamics models. RESULTS:Blood flow conditions leading to high wall shear stress (WSS) activate proinflammatory signaling in endothelial cells that recruits macrophages to the site exposed to high WSS, especially through macrophage chemoattractant protein 1 (MCP1). This macrophage infiltration leads to protease expression, which disrupts the internal elastic lamina and collagen matrix, leading to focal outward bulging of the wall and IA initiation. For the IA to grow, collagen remodeling and smooth muscle cell (SMC) proliferation are essential, because the fact that collagen does not distend much prevents the passive dilation of a focal weakness to a sizable IA. Chronic macrophage infiltration of the IA wall promotes this SMC-mediated growth and is a potential target for drug therapy. Once the IA wall grows, it is subjected to changes in wall tension and flow conditions as a result of the change in geometry and has to remodel accordingly to avoid rupture. Flow affects this remodeling process. CONCLUSIONS:Flow triggers an inflammatory reaction that predisposes the arterial wall to IA initiation and growth and affects the associated remodeling of the UIA wall. This chronic inflammation is a putative target for drug therapy that would stabilize UIAs or prevent UIA formation. Moreover, once this coupling between IA wall remodeling and flow is understood, data from patient-specific flow models can be gathered as part of the diagnostic workup and utilized to improve risk assessment for UIA initiation, progression, and eventual rupture.
Expression of osteopontin, matrix metalloproteinase-2 and -9 proteins in vascular instability in brain arteriovenous malformation.
BACKGROUND:Matrix metalloproteinase (MMP)-2 and -9 are Osteopontin (OPN) dependent molecules implicated in the destabilization of blood vessels. OPN and MMPs have been studied in brain arteriovenous malformation (BAVM) patients' tissues and blood samples before intervention. In this study, we compared the serum level of these markers before and after treatment, as well as assessed their protein expressions in BAVM tissues to evaluate their roles in this disease. METHODOLOGY:Serum samples from six BAVM patients and three control subjects were analyzed using enzyme-linked immunoabsorbent assay (ELISA) for OPN. A total of 10 BAVM patients and five control subjects were analyzed using Multiplex ELISA for MMPs. A total of 16 BAVM tissue samples and two normal brain tissue samples were analyzed using immunohistochemistry. RESULT:MMP-2 and -9 were significantly higher in the serum of BAVM patients before and after treatment than in control patients. There were no significant differences of OPN and MMP-9 serum level in BAVM patients before and after treatment. MMP-2 showed a significant elevation after the treatment. Expression of OPN, MMP-2 and -9 proteins were seen in endothelial cells, perivascular cells and brain parenchyma of BAVM tissues. CONCLUSION:Findings revealed that the level of MMP-2 and -9 in the serum correlated well with the expression in BAVM tissues in several cases. Knockdown studies will be required to determine the relationships and mechanisms of action of these markers in the near future. In addition, studies will be required to investigate the expression of these markers' potential applications as primary medical therapy targets for BAVM patients.
RNA Sequencing Reveals the Activation of Wnt Signaling in Low Flow Rate Brain Arteriovenous Malformations.
Huo Ran,Fu Weilun,Li Hao,Jiao Yuming,Yan Zihan,Wang Linjian,Wang Jie,Wang Shuo,Cao Yong,Zhao Jizong
Journal of the American Heart Association
Background The blood flow rate of brain arteriovenous malformations ( bAVM s) is an important clinical characteristic closely associated with the hemorrhage risk and radiosurgery obliteration rate of bAVM s. However, the underlying molecular properties remain unclear. To identify potential key molecules, signaling pathways, and vascular cell types involved, we compared gene expression profiles between bAVM s with high flow rates and low flow rates ( LFR ) and validated the functions of selected key molecules in vitro. Methods and Results We performed RNA -sequencing analysis on 51 samples, including 14 high flow rate bAVM s and 37 LFR bAVM s. Functional pathway analysis was performed to identify potential signals influencing the flow rate phenotype of bAVM s. Candidate genes were investigated in bAVM specimens by immunohistochemical staining. Migration, tube formation, and proliferation assays were used to test the effects of candidate genes on the phenotypic properties of cultured human umbilical vein endothelial cells and human brain vascular smooth muscle cells. We identified 250 upregulated and 118 downregulated genes in LFR bAVM s compared with high flow rate bAVM s. Wnt signaling was activated in the LFR group via upregulation of FZD 10 and MYOC . Immunohistochemical staining showed that vascular endothelial and smooth muscle cells of LFR bAVM s exhibited increased FZD 10 and MYOC expression. Experimentally elevating these genes promoted human umbilical vein endothelial cells and migration and tube formation by activating canonical Wnt signaling in vitro. Conclusions Our results suggest that canonical Wnt signaling mediated by FZD 10 and MYOC is activated in vascular endothelial and smooth muscle cells in LFR bAVM s.
Laminar Wall Shear Stress in Brain Arteriovenous Malformations: Systematic Review of Literature.
Lo Presti Anna,Rogers Jeffrey M,Stoodley Marcus A,Assaad Nazih N A,Simons Mary,Sen Itsu,Morgan Michael Kerin
BACKGROUND:Laminar wall sheer stress (LWSS) modulates inflammatory activity of the endothelium and may be a contributing factor in many cerebrovascular pathologies. There is a lack of consensus whether significant differences in LWSS exist between feeding vessels in brain arteriovenous malformation (bAVM) and healthy vessels. A systematic review of LWSS research in bAVM was undertaken, including the methods used and the assumptions made in determining LWSS. METHODS:Ovid MEDLINE, EMBASE, and Scopus electronic databases were systematically searched from inception for articles calculating LWSS in bAVM cases. LWSS values were extracted for comparison between ipsilateral bAVM feeding arteries and healthy contralateral vessels or healthy normative data. RESULTS:Three retrospective cohort studies were identified, reporting on 42 adult and pediatric bAVM cases. Mean LWSS (mLWSS) in healthy vessels (contralateral vessels or normative controls) typically ranged from 1.2-2.7 Pa, while mLWSS values in untreated bAVM feeding arteries typically ranged from 1.6-3.6 Pa. All studies had mixed cohorts of ruptured and unruptured cases, obscuring the relationship between LWSS and bAVM history. CONCLUSIONS:mLWSS values in healthy arteries and bAVM feeding vessels tend to be low and overlapping. Further research of high scientific and methodologic quality is necessary to improve understanding of how LWSS hemodynamics relate to bAVM formation, rupture, and treatment.
Effect of elevation of vascular endothelial growth factor level on exacerbation of hemorrhage in mouse brain arteriovenous malformation.
Cheng Philip,Ma Li,Shaligram Sonali,Walker Espen J,Yang Shun-Tai,Tang Chaoliang,Zhu Wan,Zhan Lei,Li Qiang,Zhu Xiaonan,Lawton Michael T,Su Hua
Journal of neurosurgery
OBJECTIVE:A high level of vascular endothelial growth factor (VEGF) has been implicated in brain arteriovenous malformation (bAVM) bleeding and rupture. However, direct evidence is missing. In this study the authors used a mouse bAVM model to test the hypothesis that elevation of focal VEGF levels in bAVMs exacerbates the severity of bAVM hemorrhage. METHODS:Brain AVMs were induced in adult mice in which activin receptor-like kinase 1 (Alk1, a gene that causes AVM) gene exons 4-6 were floxed by intrabasal ganglia injection of an adenoviral vector expressing Cre recombinase to induce Alk1 mutation and an adeno-associated viral vector expressing human VEGF (AAV-VEGF) to induce angiogenesis. Two doses of AAV-VEGF (5 × 109 [high] or 2 × 109 [low]) viral genomes were used. In addition, the common carotid artery and external jugular vein were anastomosed in a group of mice treated with low-dose AAV-VEGF 6 weeks after the model induction to induce cerebral venous hypertension (VH), because VH increases the VEGF level in the brain. Brain samples were collected 8 weeks after the model induction. Hemorrhages in the bAVM lesions were quantified on brain sections stained with Prussian blue, which detects iron deposition. VEGF levels were quantified in bAVM tissue by enzyme-linked immunosorbent assay. RESULTS:Compared to mice injected with a low dose of AAV-VEGF, the mice injected with a high dose had higher levels of VEGF (p = 0.003) and larger Prussian blue-positive areas in the bAVM lesion at 8 or 9 weeks after model induction (p = 0.002). VH increased bAVM hemorrhage in the low-dose AAV-VEGF group. The overall mortality in the high-dose AAV-VEGF group was 26.7%, whereas no mouse died in the low-dose AAV-VEGF group without VH. In contrast, VH caused a mortality of 50% in the low-dose AAV-VEGF group. CONCLUSIONS:Using mouse bAVM models, the authors provided direct evidence that elevation of the VEGF level increases bAVM hemorrhage and mouse mortality.
KRAS G12D or G12V Mutation in Human Brain Arteriovenous Malformations.
Oka Mieko,Kushamae Mika,Aoki Tomohiro,Yamaguchi Tadashi,Kitazato Keiko,Abekura Yu,Kawamata Takakazu,Mizutani Tohru,Miyamoto Susumu,Takagi Yasushi
BACKGROUND:Brain arteriovenous malformations (BAVMs) are vascular malformations composed of tangles of abnormally developed vasculature without capillaries. Abnormal shunting of arteries and veins is formed, resulting in high-pressure vascular channels, which potentially lead to rupture. BAVMs are generally considered a congenital disorder. But clinical evidence regarding involution, regrowth, and de novo formation argue against the static condition of this disease. Recently, the presence of the somatic activating KRAS mutations in more than half of BAVM cases was reported, suggesting the role of KRAS function in the pathogenesis. METHODS:KRAS mutation in codon35 (G→A, G12D; G→T, G12V) was examined by a digital polymerase chain reaction analysis using genome purified from paraffin-embedded slides of human BAVMs. We also examined protein expression of KRAS G12D in lesions to corroborate results from digital polymerase chain reaction analysis. RESULTS:We detected codon35 G→A mutation in 15 (39.5%) among 38 samples and codon35 G→T mutation in 10 (27.0%) among 37 samples we could assess mutations. There were no samples positive for both codon35 G→A and G→T mutation. The ratio of codon35 G→A mutation ranged from 0.60% to 12.28% and that of G→T was from 1.20% to 8.99%. We next examined protein expression of KRAS G12D in BAVM lesions in immunohistochemistry. A KRAS G12D mutant was detected mainly in endothelial cells of dilated vessels in lesions. CONCLUSIONS:KRAS mutations in codon35 were detected in about two thirds of specimens examined. KRAS function may actively contribute to the pathobiology of BAVM and can become a therapeutic target.
High prevalence of KRAS/BRAF somatic mutations in brain and spinal cord arteriovenous malformations.
Hong Tao,Yan Yupeng,Li Jingwei,Radovanovic Ivan,Ma Xiangyuan,Shao Yang W,Yu Jiaxing,Ma Yongjie,Zhang Peng,Ling Feng,Huang Shuchen,Zhang Hongqi,Wang Yibo
Brain : a journal of neurology
Brain and spinal arteriovenous malformations are congenital lesions causing intracranial haemorrhage or permanent disability especially in young people. We investigated whether the vast majority or all brain and spinal arteriovenous malformations are associated with detectable tumour-related somatic mutations. In a cohort of 31 patients (21 with brain and 10 with spinal arteriovenous malformations), tissue and paired blood samples were analysed with ultradeep next generation sequencing of a panel of 422 common tumour genes to identify the somatic mutations. We used droplet digital polymerase chain reaction to confirm the panel sequenced mutations and identify the additional low variant frequency mutations. The association of mutation variant frequencies and clinical features were analysed. The average sequencing depth was 1077 ± 298×. High prevalence (87.1%) of KRAS/BRAF somatic mutations was found in brain and spinal arteriovenous malformations with no other replicated tumour-related mutations. The prevalence of KRAS/BRAF mutation was 81.0% (17 of 21) in brain and 100% (10 of 10) in spinal arteriovenous malformations. We detected activating BRAF mutations and two novel mutations in KRAS (p.G12A and p.S65_A66insDS) in CNS arteriovenous malformations for the first time. The mutation variant frequencies were negatively correlated with nidus volumes of brain (P = 0.038) and spinal (P = 0.028) arteriovenous malformations but not ages. Our findings support a causative role of somatic tumour-related mutations of KRAS/BRAF in the overwhelming majority of brain and spinal arteriovenous malformations. This pathway homogeneity and high prevalence implies the development of targeted therapies with RAS/RAF pathway inhibitors without the necessity of tissue genetic diagnosis.10.1093/brain/awy307_video1awy307media15978667388001.
Reductions in brain pericytes are associated with arteriovenous malformation vascular instability.
Winkler Ethan A,Birk Harjus,Burkhardt Jan-Karl,Chen Xiaolin,Yue John K,Guo Diana,Rutledge W Caleb,Lasker George F,Partow Carlene,Tihan Tarik,Chang Edward F,Su Hua,Kim Helen,Walcott Brian P,Lawton Michael T
Journal of neurosurgery
OBJECTIVEBrain arteriovenous malformations (bAVMs) are rupture-prone tangles of blood vessels with direct shunting of blood flow between arterial and venous circulations. The molecular and/or cellular mechanisms contributing to bAVM pathogenesis and/or destabilization in sporadic lesions have remained elusive. Initial insights into AVM formation have been gained through models of genetic AVM syndromes. And while many studies have focused on endothelial cells, the contributions of other vascular cell types have yet to be systematically studied. Pericytes are multifunctional mural cells that regulate brain angiogenesis, blood-brain barrier integrity, and vascular stability. Here, the authors analyze the abundance of brain pericytes and their association with vascular changes in sporadic human AVMs.METHODSTissues from bAVMs and from temporal lobe specimens from patients with medically intractable epilepsy (nonvascular lesion controls [NVLCs]) were resected. Immunofluorescent staining with confocal microscopy was performed to quantify pericytes (platelet-derived growth factor receptor-beta [PDGFRβ] and aminopeptidase N [CD13]) and extravascular hemoglobin. Iron-positive hemosiderin deposits were quantified with Prussian blue staining. Syngo iFlow post-image processing was used to measure nidal blood flow on preintervention angiograms.RESULTSQuantitative immunofluorescent analysis demonstrated a 68% reduction in the vascular pericyte number in bAVMs compared with the number in NVLCs (p < 0.01). Additional analysis demonstrated 52% and 50% reductions in the vascular surface area covered by CD13- and PDGFRβ-positive pericyte cell processes, respectively, in bAVMs (p < 0.01). Reductions in pericyte coverage were statistically significantly greater in bAVMs with prior rupture (p < 0.05). Unruptured bAVMs had increased microhemorrhage, as evidenced by a 15.5-fold increase in extravascular hemoglobin compared with levels in NVLCs (p < 0.01). Within unruptured bAVM specimens, extravascular hemoglobin correlated negatively with pericyte coverage (CD13: r = -0.93, p < 0.01; PDGFRβ: r = -0.87, p < 0.01). A similar negative correlation was observed with pericyte coverage and Prussian blue-positive hemosiderin deposits (CD13: r = -0.90, p < 0.01; PDGFRβ: r = -0.86, p < 0.01). Pericyte coverage positively correlated with the mean transit time of blood flow or the time that circulating blood spends within the bAVM nidus (CD13: r = 0.60, p < 0.05; PDGFRβ: r = 0.63, p < 0.05). A greater reduction in pericyte coverage is therefore associated with a reduced mean transit time or faster rate of blood flow through the bAVM nidus. No correlations were observed with time to peak flow within feeding arteries or draining veins.CONCLUSIONSBrain pericyte number and coverage are reduced in sporadic bAVMs and are lowest in cases with prior rupture. In unruptured bAVMs, pericyte reductions correlate with the severity of microhemorrhage. A loss of pericytes also correlates with a faster rate of blood flow through the bAVM nidus. This suggests that pericytes are associated with and may contribute to vascular fragility and hemodynamic changes in bAVMs. Future studies in animal models are needed to better characterize the role of pericytes in AVM pathogenesis.
The Role of Matrix Metalloproteinase-3 in the Doxycycline Attenuation of Intracranial Venous Hypertension-Induced Angiogenesis.
Zou Xiang,Wu Zehan,Huang Jun,Liu Peixi,Qin Xuanfeng,Chen Liang,Zhu Wei,Zhao Yao,Li Peiliang,Song Jianping,Yang Guo-Yuan,Mao Ying
BACKGROUND:The molecular mechanism of brain arteriovenous malformation (BAVM) is largely unknown. Intracranial venous hypertension (VH) may enhance focal angiogenesis and promote BAVM development and progression. A rat VH model effectively simulates the hemodynamic microenvironment of this disease. OBJECTIVE:To explore the effect of doxycycline in VH-related angiogenesis, as well as the role of matrix metalloproteinase-3 (MMP-3) and other molecular factors. METHODS:A rat VH model was generated by common carotid artery and distal external jugular vein anastomosis. Microvessel density (MVD) in the perisinus area and expression of MMP-3/2/9, VEGF, TIMP-1, TGF-β, and HIF-1α were examined, with and without daily doxycycline treatment for 4 wk. The effects of doxycycline were verified in Vitro using human brain microvascular endothelial cells (HBMECs). MMP-3 overexpression or knockdown in HBMECs was used to confirm the role of MMP-3 in cell functions. RESULTS:MVD in the perisinus cortex was greatly increased after VH. Doxycycline decreased MVD, suppressed MMP-3 overexpression, and reduced VEGF, TGF-β, and TIMP-1 levels compared with the controls (P < .05). In Vitro, doxycycline decreased HBMEC migration, tube formation, and the mRNA, protein, and enzymatic activity levels of MMP-3. MMP-3 overexpression in HBMECs promoted migration, while knockdown of MMP-3 significantly attenuated proliferation, migration, and tube formation (P < .05). CONCLUSION:Our findings indicate that MMP-3 plays an important role in VH-related angiogenesis and the promotion of vascular remodeling. Suppression of MMP-3 overexpression by doxycycline may provide a potential strategy for inhibiting BAVM development.
Perturbations of BMP/TGF-β and VEGF/VEGFR signalling pathways in non-syndromic sporadic brain arteriovenous malformations (BAVM).
Wang Kun,Zhao Sen,Liu Bowen,Zhang Qianqian,Li Yaqi,Liu Jiaqi,Shen Yan,Ding Xinghuan,Lin Jiachen,Wu Yong,Yan Zihui,Chen Jia,Li Xiaoxin,Song Xiaofei,Niu Yuchen,Liu Jian,Chen Weisheng,Ming Yue,Du Renqian,Chen Cong,Long Bo,Zhang Yisen,Tong Xiangjun,Zhang Shuyang,Posey Jennifer E,Zhang Bo,Wu Zhihong,Wythe Joshua D,Liu Pengfei,Lupski James R,Yang Xinjian,Wu Nan
Journal of medical genetics
BACKGROUND:Brain arteriovenous malformations (BAVM) represent a congenital anomaly of the cerebral vessels with a prevalence of 10-18/100 000. BAVM is the leading aetiology of intracranial haemorrhage in children. Our objective was to identify gene variants potentially contributing to disease and to better define the molecular aetiology underlying non-syndromic sporadic BAVM. METHODS:We performed whole-exome trio sequencing of 100 unrelated families with a clinically uniform BAVM phenotype. Pathogenic variants were then studied in vivo using a transgenic zebrafish model. RESULTS:We identified four pathogenic heterozygous variants in four patients, including one in the established BAVM-related gene, , and three damaging variants in novel candidate genes: , and , which we then functionally validated in zebrafish. In addition, eight likely pathogenic heterozygous variants (, and ) were identified in eight patients, and 16 patients carried one or more variants of uncertain significance. Potential oligogenic inheritance ( with , with and with ) was identified in three patients. Regulation of sma- and mad-related proteins (SMADs) (involved in bone morphogenic protein (BMP)/transforming growth factor beta (TGF-β) signalling) and vascular endothelial growth factor (VEGF)/vascular endotheliual growth factor recepter 2 (VEGFR2) binding and activity (affecting the VEGF signalling pathway) were the most significantly affected biological process involved in the pathogenesis of BAVM. CONCLUSIONS:Our study highlights the specific role of BMP/TGF-β and VEGF/VEGFR signalling in the aetiology of BAVM and the efficiency of intensive parallel sequencing in the challenging context of genetically heterogeneous paradigm.
Whole-exome sequencing reveals known and novel variants in a cohort of intracranial vertebral-basilar artery dissection (IVAD).
Wang Kun,Zhao Sen,Zhang Qianqian,Yuan Jian,Liu Jiaqi,Ding Xinghuan,Song Xiaofei,Lin Jiachen,Du Renqian,Zhou Yangzhong,Sugimoto Michihiko,Chen Weisheng,Yuan Bo,Liu Jian,Yan Zihui,Liu Bowen,Zhang Yisen,Li Xiaoxin,Niu Yuchen,Long Bo,Shen Yiping,Zhang Shuyang,Abe Kuniya,Su Jianzhong,Wu Zhihong,Wu Nan,Liu Pengfei,Yang Xinjian,
Journal of human genetics
Intracranial vertebral-basilar artery dissection (IVAD) is an arterial disorder leading to life-threatening consequences. Genetic factors are known to be causative to certain syndromic forms of IVAD. However, systematic study of the molecular basis of sporadic and isolated IVAD is lacking. To identify genetic variants contributing to the etiology of IVAD, we enrolled a cohort of 44 unrelated cases with a clinical diagnosis of isolated IVAD and performed whole-exome sequencing (WES) for all the participants; a trio exome sequencing approach was used when samples from both parents were available. Four previously reported disease-causing heterozygous variants (three in COL3A1 and one in FBN1) and seven novel heterozygous variants in IVAD-related genes were identified. In addition, six variants in novel IVAD genes including two de novo heterozygous nonsynonymous variants (each in VPS52 and CDK18), two stop-gain variants (each in MYH9 and LYL1), and two heterozygous biallelic variants in TNXB were considered to be possibly contributing to the phenotype, with unknown significance according to the existing knowledge. A significantly higher mutational rate of IVAD candidate genes was observed in patients versus our in-house controls (P = 0.002) (DISCO study, http://www.discostudy.org/ , n = 2248). Our study provided a mutational landscape for patients with isolated IVAD.
KRAS Activating Signaling Triggers Arteriovenous Malformations.
Cheng Feixiong,Nussinov Ruth
Trends in biochemical sciences
The underlying genetic causes and altered signaling pathways of brain arteriovenous malformations remain unknown. A study published in The New England Journal of Medicine reported that KRAS somatic mutations (p.Gly12Val/Asp) were identified in brain arteriovenous malformations of human subjects and endothelial cell-enriched cultures, which might specifically activate the MAPK (mitogen-activated protein kinase)-ERK (extracellular signal-regulated kinase) signaling pathway in brain endothelial cells.
Somatic Activating KRAS Mutations in Arteriovenous Malformations of the Brain.
Nikolaev Sergey I,Vetiska Sandra,Bonilla Ximena,Boudreau Emilie,Jauhiainen Suvi,Rezai Jahromi Behnam,Khyzha Nadiya,DiStefano Peter V,Suutarinen Santeri,Kiehl Tim-Rasmus,Mendes Pereira Vitor,Herman Alexander M,Krings Timo,Andrade-Barazarte Hugo,Tung Takyee,Valiante Taufik,Zadeh Gelareh,Tymianski Mike,Rauramaa Tuomas,Ylä-Herttuala Seppo,Wythe Joshua D,Antonarakis Stylianos E,Frösen Juhana,Fish Jason E,Radovanovic Ivan
The New England journal of medicine
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 (KRAS) 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.).
Angiopoietin-Like Proteins 4 (ANGPTL4) Gene Polymorphisms and Risk of Brain Arteriovenous Malformation.
Erkinova Sarafroz A,Sokolova Ekaterina A,Orlov Kirill Y,Kiselev Vitaly S,Strelnikov Nikolay V,Dubovoy Andrey V,Voronina Elena N,Filipenko Maxim L
Journal of stroke and cerebrovascular diseases : the official journal of National Stroke Association
BACKGROUND:Brain arteriovenous malformations (BAVMs) are formed by hypertrophied arterial vessels (afferents, feeders), a large number of arteriovenous shunts which become tangled to form a body (nidus) of malformation, which then expands draining proximal veins. The aim of this study was a replication of single nucleotide polymorphism (SNP) rs11672433 association with BAVM development with the subsequent meta-analysis of published data. METHODS:A total of 252 Russian patients with brain BAVMs and 480 control subjects were included in the present study. Genotyping was performed using real-time polymerase chain reaction with competitive hydrolysis probes. RESULTS:In our case-control study, we found no significant association with brain arteriovenous malformation for the SNP rs11672433 of ANGPTL4 gene (odds ratio .82, 95% confidence interval = .57-1.17 P value = .27) as well as in meta-analysis (odds ratio 1.18, 95% confidence interval = .81-1.73, P value = .39). CONCLUSIONS:Our data showed that SNP rs11672433 was not associated with the BAVM Russian population and the following meta-analysis did not detect an association in total. Thus, in spite of the fact that ANGPTL4 (protein) participates in the angiogenesis regulation processes, we consider that SNP rs11672433, a high-frequency locus in the ANGPTL4 gene, does not influence the predisposition to BAVM or its effect is too small to be detected in the present size sample set.
Focal neurons: another source of vascular endothelial growth factor in brain arteriovenous malformation tissues?
Li Pengchen,Zhang Longbo,Chen Deshun,Zeng Ming,Chen Fenghua
Background Brain arteriovenous malformations (bAVMs) are devastating, hemorrhage-prone, cerebrovascular entities characterized by well-defined feeding arteries, draining veins, and the absence of a capillary bed. The endothelial cells that comprise bAVMs exhibit a loss of arterial and venous specification. The role of abnormal angiogenesis in the formation and progression of bAVMs is still unclear. This study aimed to investigate the expression of vascular endothelial growth factor (VEGF) in neurons and glial cells in bAVMs to try to uncover the multiple cell origin of VEGF. Methods A total of 25 bAVM specimens and 25 control tissues were obtained. Western blot and immunohistochemical analyses were used to evaluate the expression of VEGF. The distribution of VEGF in neurons and glial cells in these bAVMs were observed by double-label immunofluorescence staining and subsequent imaging. Results Western blot analysis revealed a significant overexpression of VEGF in bAVM tissues (P < 0.05). Immunohistochemistry showed that the amount of cells that overexpressed VEGF in bAVM tissues was significantly greater compared to that in normal tissues (P < 0.05). Double-label immunofluorescence staining showed no significant difference between the mean amounts of VEGF-positive cells in astrocytes and in neurons (P < 0.05). Conclusions The formation and progression of bAVMs is related to the local overexpression of VEGF. Similar levels of VEGF overexpression are found in astrocytes, neurons, and vascular endothelial cells, which suggest that VEGF may be derived from astrocytes and neurons. It implied that focal neurons may play a certain role in the pathophysical process of bAVMs, however, identification of the production and functional mechanisms of VEGF in the neurons still requires further investigation.
The roles of endoglin gene in cerebrovascular diseases.
Zhu Wan,Ma Li,Zhang Rui,Su Hua
Neuroimmunology and neuroinflammation
Endoglin (ENG, also known as CD105) is a transforming growth factor β (TGFβ) associated receptor and is required for both vasculogenesis and angiogenesis. Angiogenesis is important in the development of cerebral vasculature and in the pathogenesis of cerebral vascular diseases. ENG is an essential component of the endothelial nitric oxide synthase activation complex. Animal studies showed that ENG deficiency impairs stroke recovery. ENG deficiency also impairs the regulation of vascular tone, which contributes to the pathogenesis of brain arteriovenous malformation (bAVM) and vasospasm. In human, functional haploinsufficiency of gene causes type I hereditary hemorrhagic telangiectasia (HHT1), an autosomal dominant disorder. Compared to normal population, HHT1 patients have a higher prevalence of AVM in multiple organs including the brain. Vessels in bAVM are fragile and tend to rupture, causing hemorrhagic stroke. High prevalence of pulmonary AVM in HHT1 patients are associated with a higher incidence of paradoxical embolism in the cerebral circulation causing ischemic brain injury. Therefore, HHT1 patients are at risk for both hemorrhagic and ischemic stroke. This review summarizes the possible mechanism of ENG in the pathogenesis of cerebrovascular diseases in experimental animal models and in patients.
Association of ACVRL1 Genetic Polymorphisms with Arteriovenous Malformations: A Case-Control Study and Meta-Analysis.
Ge Mingxu,Du Chigang,Li Zhaona,Liu Yingchao,Xu Shangchen,Zhang Liyong,Pang Qi
OBJECTIVE:To investigate the association between polymorphisms in the gene encoding activin receptorlike kinase 1 (ACVRL1) with brain arteriovenous malformations (BAVMs) using a case-control study in a Chinese Han population, followed by a meta-analysis of the published literature. METHODS:This study focused on the genotypic analysis of 4 single nucleotide polymorphisms (SNPs; rs2071219, rs706819, rs2293094, and rs11169953) in 50 patients with BAVM and 120 healthy volunteers attending Provincial Hospital in China. A meta-analysis was subsequently conducted involving an extensive literature search for relevant studies. RESULTS:Our cohort study showed a significant association between ACVRL1 rs706819 and increased risk for BAVM. Reduced BAVM risk was correlated with the G allele of rs2293094 and the C allele of rs11169953. However, neither the genotype nor allele frequencies of rs2071219 were found to be significantly different between the BAVM and control groups. Meta-analysis further confirmed that no significant evidence of association was found between rs2071219 and BAVM risk. Haplotype analysis of rs706819, rs2293094, and rs11169953 showed that the GGT haplotype could reduce the risk of BAVM, whereas the GAC haplotype may increase the risk of BAVM. CONCLUSIONS:The present study indicates an association between 3 susceptibility SNPs, rs706819, rs2293094, and rs11169953, in the ACVRL1 gene and BAVM. Follow-up functional studies on the ACVRL1 gene are required to better understand its roles in BAVM development.
DNA Methylation of the PDGFD Gene Promoter Increases the Risk for Intracranial Aneurysms and Brain Arteriovenous Malformations.
Zhou Shengjun,Gao Xiang,Sun Jie,Lin Zhiqing,Huang Yi
DNA and cell biology
The aim of this study was to determine the role of DNA methylation of the platelet-derived growth factor-D (PDGFD) gene promoter in the development of intracranial aneurysms (IAs) and brain arteriovenous malformations (BAVMs). A total of 70 patients with IAs or BAVMs and 26 control individuals were enrolled for this study. The PDGFD level in the plasma was determined using enzyme-linked immunosorbent assay. DNA methylation levels of seven cytosine-phosphate-guanine (CpG) dinucleotides present in the PDGFD gene promoter were measured using bisulfite pyrosequencing technology. The plasma PDGFD levels in IA and BAVM were significantly lower than those in the control group (p = 0.0008 and 0.002, respectively). CpG1 methylation levels of the PDGFD gene promoter were significantly higher in IA patients (4.63 ± 0.35, p = 0.017) than in the control group (3.36 ± 0.35). CpG1 methylation of the PDGFD gene promoter in BAVM patients (6.00 ± 0.86, p = 0.003) was also significantly higher than that in the control group, although these differences were seen in both male and female patients (p = 2.81E-04 and p = 0.017, respectively). In addition, CpG1 methylation of the PDGFD promoter was associated with apolipoprotein E (APOE) levels in IA patients (p = 0.013). In conclusion, our study has demonstrated significant correlations between DNA methylation of the PDGFD gene promoter and the risk of developing either IA or BAVM. Furthermore, PDGFD gene promoter CpG1 methylation shows a significant correlation with APOE in IAs. Further functional studies on these relationships and correlations are warranted.
Soluble FLT1 Gene Therapy Alleviates Brain Arteriovenous Malformation Severity.
Zhu Wan,Shen Fanxia,Mao Lei,Zhan Lei,Kang Shuai,Sun Zhengda,Nelson Jeffrey,Zhang Rui,Zou Dingquan,McDougall Cameron M,Lawton Michael T,Vu Thiennu H,Wu Zhijian,Scaria Abraham,Colosi Peter,Forsayeth John,Su Hua
BACKGROUND AND PURPOSE:Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current therapies are associated with high morbidities. Excessive vascular endothelial growth factor has been implicated in bAVM pathophysiology. Because soluble FLT1 binds to vascular endothelial growth factor with high affinity, we tested intravenous delivery of an adeno-associated viral vector serotype-9 expressing soluble FLT1 (AAV9-sFLT1) to alleviate the bAVM phenotype. METHODS:Two mouse models were used. In model 1, bAVM was induced in R26CreER; mice through global gene deletion and brain focal angiogenic stimulation; AAV2-sFLT02 (an AAV expressing a shorter form of sFLT1) was injected into the brain at the time of model induction, and AAV9-sFLT1, intravenously injected 8 weeks after. In model 2, SM22αCre; mice had a 90% occurrence of spontaneous bAVM at 5 weeks of age and 50% mortality at 6 weeks; AAV9-sFLT1 was intravenously delivered into 4- to 5-week-old mice. Tissue samples were collected 4 weeks after AAV9-sFLT1 delivery. RESULTS:AAV2-sFLT02 inhibited bAVM formation, and AAV9-sFLT1 reduced abnormal vessels in model 1 (GFP versus sFLT1: 3.66±1.58/200 vessels versus 1.98±1.29, <0.05). AAV9-sFLT1 reduced the occurrence of bAVM (GFP versus sFLT1: 100% versus 36%) and mortality (GFP versus sFLT1: 57% [12/22 mice] versus 24% [4/19 mice], <0.05) in model 2. Kidney and liver function did not change significantly. Minor liver inflammation was found in 56% of AAV9-sFLT1-treated model 1 mice. CONCLUSIONS:By applying a regulated mechanism to restrict sFLT1 expression to bAVM, AAV9-sFLT1 can potentially be developed into a safer therapy to reduce the bAVM severity.
Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase 2 Expression in Brain Arteriovenous Malformations and its Association with Brain Arteriovenous Malformation Size.
Neyazi Belal,Tanrikulu Levent,Wilkens Ludwig,Hartmann Christian,Stein Klaus-Peter,Dumitru Claudia A,Sandalcioglu I Erol
BACKGROUND AND OBJECTIVE:Brain arteriovenous malformations (bAVM) are severe conditions that can cause severe neurologic deficits and mortality. The underlying cellular and molecular mechanisms associated with bAVM growth and rupture remain unclear. The objective of this study was to explore the potential role of PLOD2 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2) in the pathophysiology of bAVM. METHODS:Expression and localization of PLOD2 were analyzed on tissue microarrays from patients with bAVM (n = 60) by immunohistochemistry. Correlations between PLOD2 levels and clinical parameters were examined with a Pearson test or Spearman rank correlation coefficient. Comparison between different clinical parameters was performed using a t test or nonparametric Mann-Whitney U test. A Fisher exact test was used for categorical data. RESULTS:PLOD2 was mainly expressed within the tunica media of the blood vessels. High levels of PLOD2 expression correlated with bAVM size (linear regression, P = 0.0083, R=0.158). Small bAVM showed a higher frequency of hemorrhage compared with large ones (P = 0.001). Although PLOD2 was not directly associated with bAVM hemorrhage, high PLOD2-expressing bAVM had a lower frequency of hemorrhage compared with low or medium PLOD2-expressing bAVM (25% vs. 63% and 75%, respectively). CONCLUSIONS:This study reports for the first time the expression of PLOD2 in bAVM and suggests a potential role of PLOD2 in bAVM pathophysiology. These findings contribute to an better understanding of the microenvironment of bAVM and may foster the development of improved therapeutic strategies for this disease.
Dysregulation of cell-cell interactions in brain arteriovenous malformations: A quantitative proteomic study.
Wang Xia,Hao Qiang,Zhao Yuanli,Guo Yi,Ge Wei
Proteomics. Clinical applications
PURPOSE:Detailed and exact mechanisms underlying brain arteriovenous malformations (bAVM) are still clinically confusing. Understanding the quantitative changes in proteins and signaling pathways would provide useful information for clinicians to understand the formation and development of bAVM, guiding individualized treatment strategies. This study was performed to establish a large human bAVM proteome database using tandem mass tag labeling and to detect changes of protein expression and pathways in human bAVM. EXPERIMENTAL DESIGN:This study used quantitative 6-plex tandem mass tag labeling to profile protein changes in bAVM lesions. Integrated bioinformatics analysis was used to classify and identify the altered proteins and relating signaling pathways. Western blot analyzes were used to validate the proteomic data. RESULTS:Our work established the first human bAVM proteome databases to date. A total of 1264 proteins were identified, and the expression of 316 proteins was significantly differentially expressed, with 249 upregulated proteins. Bioinformatics analysis demonstrated that the altered proteins had close functional correlations, including integrin cell surface interactions, extracellular matrix organization, and smooth muscle contraction. Three signaling pathways (focal adhesions, tight junctions, and gap junctions), which represent an important arena of cell-cell interactions, were found to be activated in bAVM. The proteomics data are available via ProteomeXchange with identifier PXD003289. CONCLUSION AND CLINICAL RELEVANCE:Cell-cell interactions, including focal adhesions, tight junctions, and gap junctions, were significantly influenced in human bAVM. Understanding the molecular mechanisms that underlie bAVM would provide useful information for the development of future therapeutic approaches, guiding possible precise and individual treatment strategies.
Integrin β8 Deletion Enhances Vascular Dysplasia and Hemorrhage in the Brain of Adult Alk1 Heterozygous Mice.
Ma Li,Shen Fanxia,Jun Kristine,Bao Chen,Kuo Robert,Young William L,Nishimura Stephen L,Su Hua
Translational stroke research
Brain arteriovenous malformation (bAVM), characterized by tangled dysplastic vessels, is an important cause of intracranial hemorrhage in young adults, and its pathogenesis and progression are not fully understood. Patients with haploinsufficiency of transforming growth factor-β (TGF-β) receptors, activin receptor-like kinase 1 (ALK1) or endoglin (ENG) have a higher incidence of bAVM than the general population. However, bAVM does not develop effectively in mice with the same haploinsufficiency. The expression of integrin β8 subunit (ITGB8), another member in the TGF-β superfamily, is reduced in sporadic human bAVM. Brain angiogenic stimulation results at the capillary level of vascular malformation in adult Alk1 haploinsufficient (Alk1 ) mice. We hypothesized that deletion of Itgb8 enhances bAVM development in adult Alk1 mice. An adenoviral vector expressing Cre recombinase (Ad-Cre) was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brain of Alk1 ;Itgb8-floxed mice to induce focal Itgb8 gene deletion and angiogenesis. We showed that compared with Alk mice (4.75 ± 1.38/mm), the Alk1 ;Itgb8-deficient mice had more dysplastic vessels in the angiogenic foci (7.14 ± 0.68/mm, P = 0.003). More severe hemorrhage was associated with dysplastic vessels in the brain of Itgb8-deleted Alk1 , as evidenced by larger Prussian blue-positive areas (1278 ± 373 pixels/mm vs. Alk1 : 320 ± 104 pixels/mm; P = 0.028). These data indicate that both Itgb8 and Alk1 are important in maintaining normal cerebral angiogenesis in response to VEGF. Itgb8 deficiency enhances the formation of dysplastic vessels and hemorrhage in Alk1 mice.
Persistent infiltration and pro-inflammatory differentiation of monocytes cause unresolved inflammation in brain arteriovenous malformation.
Zhang Rui,Han Zhenying,Degos Vincent,Shen Fanxia,Choi Eun-Jung,Sun Zhengda,Kang Shuai,Wong Michael,Zhu Wan,Zhan Lei,Arthur Helen M,Oh S Paul,Faughnan Marie E,Su Hua
An abnormally high number of macrophages are present in human brain arteriovenous malformations (bAVM) with or without evidence of prior hemorrhage, causing unresolved inflammation that may enhance abnormal vascular remodeling and exacerbate the bAVM phenotype. The reasons for macrophage accumulation at the bAVM sites are not known. We tested the hypothesis that persistent infiltration and pro-inflammatory differentiation of monocytes in angiogenic tissues increase the macrophage burden in bAVM using two mouse models and human monocytes. Mouse bAVM was induced through deletion of AVM causative genes, Endoglin (Eng) globally or Alk1 focally, plus brain focal angiogenic stimulation. An endothelial cell and vascular smooth muscle cell co-culture system was used to analyze monocyte differentiation in the angiogenic niche. After angiogenic stimulation, the Eng-deleted mice had fewer CD68(+) cells at 2 weeks (P = 0.02), similar numbers at 4 weeks (P = 0.97), and more at 8 weeks (P = 0.01) in the brain angiogenic region compared with wild-type (WT) mice. Alk1-deficient mice also had a trend toward more macrophages/microglia 8 weeks (P = 0.064) after angiogenic stimulation and more RFP(+) bone marrow-derived macrophages than WT mice (P = 0.01). More CD34(+) cells isolated from peripheral blood of patients with ENG or ALK1 gene mutation differentiated into macrophages than those from healthy controls (P < 0.001). These data indicate that persistent infiltration and pro-inflammatory differentiation of monocytes might contribute to macrophage accumulation in bAVM. Blocking macrophage homing to bAVM lesions should be tested as a strategy to reduce the severity of bAVM.
Sex-dichotomous effects of NOS1AP promoter DNA methylation on intracranial aneurysm and brain arteriovenous malformation.
Wang Zhepei,Zhao Jikuang,Sun Jie,Nie Sheng,Li Keqing,Gao Feng,Zhang Tiefeng,Duan Shiwei,Di Yazhen,Huang Yi,Gao Xiang
The goal of this study was to investigate the contribution of NOS1AP-promoter DNA methylation to the risk of intracranial aneurysm (IA) and brain arteriovenous malformation (BAVM) in a Han Chinese population. A total of 48 patients with IAs, 22 patients with BAVMs, and 26 control individuals were enrolled in the study. DNA methylation was tested using bisulfite pyrosequencing technology. We detected significantly higher DNA methylation levels in BAVM patients than in IA patients based on the multiple testing correction (CpG4-5 methylation: 5.86±1.04% vs. 4.37±2.64%, P=0.006). In women, CpG4-5 methylation levels were much lower in IA patients (3.64±1.97%) than in BAVM patients (6.11±1.20%, P<0.0001). However, in men, CpG1-3 methylation levels were much higher in the controls (6.92±0.78%) than in BAVM patients (5.99±0.70%, P=0.008). Additionally, there was a gender-based difference in CpG1 methylation within the controls (men vs. women: 5.75±0.50% vs. 4.99±0.53%, P=0.003) and BAVM patients (men vs. women: 4.70±0.74% vs. 5.50±0.87%, P=0.026). A subgroup analysis revealed significantly higher CpG3 methylation in patients who smoked than in those who did not (P=0.041). Our results suggested that gender modulated the interaction between NOS1AP promoter DNA methylation in IA and BAVM patients. Our results also confirmed that regular tobacco smoking was associated with increased NOS1AP methylation in humans. Additional studies with larger sample sizes are required to replicate and extend these findings.
Replication Study of Polymorphisms Associated With Brain Arteriovenous Malformation in a Population From South of Brazil.
Franciscatto André Cerutti,Ludwig Fernanda S,Matte Ursula S,Mota Simone,Stefani Marco A
INTRODUCTION:The aim of this study was to reproducibly determine if any of the polymorphisms were associated with the susceptibility to brain arteriovenous malformations (BAVM) or the risk of intracranial hemorrhage (ICH) presentation. METHODS:We recruited 63 BAVM patients and 96 controls. The polymorphisms selected for evaluation were apolipoprotein E (APOE), tumor necrosis factor alpha (TNF 238G>A - rs361525), interleukin 1 beta (IL1B 511C>T - rs16944 and IL1B -31T>C - rs1143627), activin-like kinase 1 (ACVRL1 IVS3-35A>G - rs2071219), endoglin (ENG 207G>A - rs11545664), and interleukin 6 (IL6 174G>C - rs1800795). RESULTS:In the single analysis, we observed statistically signiﬁcant differences in the allele distributions for IL1B -31T>C (rs1143627) between the BAVM patients and control subjects (P = 0.02). There was a trend toward significance for the association between the IL1B 511C>T (rs16944) allele and BAVM risk (P = 0.07). In further logistic regression analysis, no polymorphism was significantly associated with the risk of BAVM. No polymorphisms were associated with hemorrhage presentation according to both single and multivariable analyses. CONCLUSIONS:In our sample from a south Brazil population, we found no association between the risks of BAVM and ICH presentation with any of the selected polymorphisms.
Genome-wide association study of sporadic brain arteriovenous malformations.
Weinsheimer Shantel,Bendjilali Nasrine,Nelson Jeffrey,Guo Diana E,Zaroff Jonathan G,Sidney Stephen,McCulloch Charles E,Al-Shahi Salman Rustam,Berg Jonathan N,Koeleman Bobby P C,Simon Matthias,Bostroem Azize,Fontanella Marco,Sturiale Carmelo L,Pola Roberto,Puca Alfredo,Lawton Michael T,Young William L,Pawlikowska Ludmila,Klijn Catharina J M,Kim Helen,
Journal of neurology, neurosurgery, and psychiatry
BACKGROUND:The pathogenesis of sporadic brain arteriovenous malformations (BAVMs) remains unknown, but studies suggest a genetic component. We estimated the heritability of sporadic BAVM and performed a genome-wide association study (GWAS) to investigate association of common single nucleotide polymorphisms (SNPs) with risk of sporadic BAVM in the international, multicentre Genetics of Arteriovenous Malformation (GEN-AVM) consortium. METHODS:The Caucasian discovery cohort included 515 BAVM cases and 1191 controls genotyped using Affymetrix genome-wide SNP arrays. Genotype data were imputed to 1000 Genomes Project data, and well-imputed SNPs (>0.01 minor allele frequency) were analysed for association with BAVM. 57 top BAVM-associated SNPs (51 SNPs with p<10(-05) or p<10(-04) in candidate pathway genes, and 6 candidate BAVM SNPs) were tested in a replication cohort including 608 BAVM cases and 744 controls. RESULTS:The estimated heritability of BAVM was 17.6% (SE 8.9%, age and sex-adjusted p=0.015). None of the SNPs were significantly associated with BAVM in the replication cohort after correction for multiple testing. 6 SNPs had a nominal p<0.1 in the replication cohort and map to introns in EGFEM1P, SP4 and CDKAL1 or near JAG1 and BNC2. Of the 6 candidate SNPs, 2 in ACVRL1 and MMP3 had a nominal p<0.05 in the replication cohort. CONCLUSIONS:We performed the first GWAS of sporadic BAVM in the largest BAVM cohort assembled to date. No GWAS SNPs were replicated, suggesting that common SNPs do not contribute strongly to BAVM susceptibility. However, heritability estimates suggest a modest but significant genetic contribution.
Vascular Integrity in the Pathogenesis of Brain Arteriovenous Malformation.
Zhang Rui,Zhu Wan,Su Hua
Acta neurochirurgica. Supplement
Brain arteriovenous malformation (bAVM) is an important cause of intracranial hemorrhage (ICH), particularly in the young population. ICH is the first clinical symptom in about 50 % of bAVM patients. The vessels in bAVM are fragile and prone to rupture, causing bleeding into the brain. About 30 % of unruptured and non-hemorrhagic bAVMs demonstrate microscopic evidence of hemosiderin in the vascular wall. In bAVM mouse models, vascular mural cell coverage is reduced in the AVM lesion, accompanied by vascular leakage and microhemorrhage. In this review, we discuss possible signaling pathways involved in abnormal vascular development in bAVM.
Immunohistochemical Analysis of Sox17 Associated Pathway in Brain Arteriovenous Malformations.
Hermanto Yulius,Takagi Yasushi,Ishii Akira,Yoshida Kazumichi,Kikuchi Takayuki,Funaki Takeshi,Mineharu Yohei,Miyamoto Susumu
BACKGROUND:Sox17 has emerged as an important factor in vascular remodeling because of the potential linkage with Wnt/β-catenin, Notch, and the inflammatory pathway. Brain arteriovenous malformation (BAVM), as an angiogenic and inflammatory disorder, might possess an aberrant regulation of the Sox17 associated pathway. We sought to investigate the expression of the Sox17 associated pathway in BAVMs. METHODS:Using immunohistochemical methods, 16 paraffin specimens of BAVM nidus were analyzed. Specimens were obtained from patients during surgical procedures. RESULTS:Expression of Sox17, Hey1, and β-catenin was observed in all specimens. Large veins possessed a distinct pattern of expression; thick-walled veins had a stronger intensity, whereas thin-walled veins had a weaker intensity, of Sox17, Hey1, and β-catenin (P < 0.001). Thick-walled veins also had a higher expression of Sox17, Hey1, and β-catenin compared with large arteries (P < 0.05). Hey1 and β-catenin expression was also higher in thick-walled veins compared with brain microvessels (P < 0.01). In addition, the difference in expression of the Sox17 associated pathway (Hey1 and β-catenin) was observed in medium and small arteries compared with large arteries in BAVM nidus and brain microvessels (P < 0.01). CONCLUSIONS:The Sox17 associated pathway was activated in the BAVM nidus. Our results indicate that arterial identity is gained in thick-walled veins; this might reflect the process of arterialization of the veins as a result of hemodynamic stress. In addition, high expression of the Sox17 associated pathway in medium and small arteries indicates that BAVM vessels are intrinsically active.
The Role of Macrophage in the Pathogenesis of Brain Arteriovenous Malformation.
Ma Li,Guo Yi,Zhao Yuan-Li,Su Hua
International journal of hematology research
Brain arteriovenous malformation (BAVM) is an important risk factor for intracranial hemorrhage, especially in children and young adults. Inflammation has been implicated in BAVM lesion progression. Among various inflammatory components, macrophage is one of the major inflammatory cells present in human ruptured and unruptured BAVM and in the BAVM lesions of animal models. The role of macrophage in BAVM pathogenesis is not fully understood. In this review, we summarize recent studies on macrophages and introduce a non-invasive imaging protocol as a potential tool for detecting macrophage in BAVM and predicting the risk of BAVM rupture.
RASA1 functions in EPHB4 signaling pathway to suppress endothelial mTORC1 activity.
Kawasaki Jun,Aegerter Sandrine,Fevurly R Dawn,Mammoto Akiko,Mammoto Tadanori,Sahin Mustafa,Mably John D,Fishman Steven J,Chan Joanne
The Journal of clinical investigation
Vascular malformations are linked to mutations in RAS p21 protein activator 1 (RASA1, also known as p120RasGAP); however, due to the global expression of this gene, it is unclear how these mutations specifically affect the vasculature. Here, we tested the hypothesis that RASA1 performs a critical effector function downstream of the endothelial receptor EPHB4. In zebrafish models, we found that either RASA1 or EPHB4 deficiency induced strikingly similar abnormalities in blood vessel formation and function. Expression of WT EPHB4 receptor or engineered receptors with altered RASA1 binding revealed that the ability of EPHB4 to recruit RASA1 is required to restore blood flow in EPHB4-deficient animals. Analysis of EPHB4-deficient zebrafish tissue lysates revealed that mTORC1 is robustly overactivated, and pharmacological inhibition of mTORC1 in these animals rescued both vessel structure and function. Furthermore, overexpression of mTORC1 in endothelial cells exacerbated vascular phenotypes in animals with reduced EPHB4 or RASA1, suggesting a functional EPHB4/RASA1/mTORC1 signaling axis in endothelial cells. Tissue samples from patients with arteriovenous malformations displayed strong endothelial phospho-S6 staining, indicating increased mTORC1 activity. These results indicate that deregulation of EPHB4/RASA1/mTORC1 signaling in endothelial cells promotes vascular malformation and suggest that mTORC1 inhibitors, many of which are approved for the treatment of certain cancers, should be further explored as a potential strategy to treat patients with vascular malformations.
Common variants on 9p21.3 are associated with brain arteriovenous malformations with accompanying arterial aneurysms.
Bendjilali Nasrine,Nelson Jeffrey,Weinsheimer Shantel,Sidney Stephen,Zaroff Jonathan G,Hetts Steven W,Segal Mark,Pawlikowska Ludmila,McCulloch Charles E,Young William L,Kim Helen
Journal of neurology, neurosurgery, and psychiatry
OBJECTIVE:To investigate whether previously reported 9p21.3 single nucleotide polymorphisms (SNPs) are associated with risk of brain arteriovenous malformations (BAVM), which often have accompanying arterial aneurysms. Common variants in the 9p21.3 locus have been reported to be associated with multiple cardiovascular phenotypes, including coronary artery disease and intracranial aneurysms (rs10757278 and rs1333040). METHODS:We used data from 338 BAVM cases participating in the University of California, San Francisco (UCSF)-Kaiser Brain AVM Study Project and 504 healthy controls to evaluate genotypes for seven common SNPs (minor allele frequency>0.05) that were imputed using 1000 Genomes Phase 1 European data (R(2)>0.87). Association with BAVM was tested using logistic regression adjusting for age, sex and the top three principal components of ancestry. Subgroup analysis included 205 BAVM cases with aneurysm data: 74 BAVM with aneurysm versus 504 controls and 131 BAVM without aneurysm versus 504 controls. RESULTS:We observed suggestive association with BAVM and rs10757278-G (OR=1.23, 95% CI 0.99 to 1.53, p=0.064) and rs1333040-T (OR=1.27, 95% CI 1.01 to 1.58, p=0.04). For rs10757278-G, the association was stronger in BAVM cases with aneurysm (OR=1.52, 95% CI 1.03 to 2.22, p=0.032) than in BAVM without aneurysm (OR=0.98, 95% CI 0.72 to 1.34, p=0.91). Similar patterns of effects were observed for rs1333040 and for other SNPs in linkage disequilibrium (r(2)>0.8) with rs10757278. CONCLUSIONS:Common 9p21.3 variants showed similar effect sizes for association with BAVM as previously reported for aneurysmal disease. The association with BAVM appears to be explained by known associations with aneurysms, suggesting that BAVM-associated aneurysms share similar vascular pathology mechanisms with other aneurysm types.
Brain arteriovenous malformation modeling, pathogenesis, and novel therapeutic targets.
Chen Wanqiu,Choi Eun-Jung,McDougall Cameron M,Su Hua
Translational stroke research
Patients harboring brain arteriovenous malformation (bAVM) are at life-threatening risk of rupture and intracranial hemorrhage (ICH). The pathogenesis of bAVM has not been completely understood. Current treatment options are invasive, and ≈ 20 % of patients are not offered interventional therapy because of excessive treatment risk. There are no specific medical therapies to treat bAVMs. The lack of validated animal models has been an obstacle for testing hypotheses of bAVM pathogenesis and testing new therapies. In this review, we summarize bAVM model development and bAVM pathogenesis and potential therapeutic targets that have been identified during model development.
Receptors of the Notch signaling pathway are associated with hemorrhage of brain arteriovenous malformations.
Li Shu,Wang Rong,Wang Yajie,Li Haowen,Zheng Jian,Duan Ran,Zhao Jizong
Molecular medicine reports
Brain arteriovenous malformation (bAVM) is currently one of the most common cerebral vascular diseases, which result in severe clinical outcomes. The Notch signaling pathway is involved in vasculogenesis and angiogenesis, as well as vascular remodeling and arteriovenous differentiation in multiple diseases. Although there are previous studies on the correlation between bAVM and the Notch signaling pathway, none of these studies have elucidated whether abnormal expression levels of the key factors in this pathway are associated with hemorrhage of bAVMs. The present study compared the expression levels of NOTCH1, NOTCH4 and two of their binding ligand genes, DLL4 and JAGGED1, in bAVM nidus and normal superficial temporal arteries (STAs) by quantitative polymerase chain reaction and immunohistochemical staining. The bAVM patient group was further stratified into hemorrhage and non-hemorrhage groups to determine the expression levels of the four genes. It was observed that the expression levels of NOTCH1 and NOTCH4 were significantly increased in the bAVM cohort as compared with that of the control group. DLL4 and JAGGED1 exhibited the same expression levels in bAVMs and STAs. In addition, increased expression levels of NOTCH1 were observed in the hemorrhage group compared with that of the non-hemorrhage group. However, the expression levels of NOTCH4, DLL4 and JAGGED1 showed no significant differences between the hemorrhage and non-hemorrhage groups. Abnormal NOTCH1 expression was detected in the hemorrhage group, but other ligands of the Notch signaling pathway remained the same, suggesting that, although NOTCH1 was upregulated in patients with bAVM, other ligands in this signaling pathway may be irrelevant to hemorrhage.
Distinctive distribution of lymphocytes in unruptured and previously untreated brain arteriovenous malformation.
Guo Yi,Tihan Tarik,Kim Helen,Hess Christopher,Lawton Michael T,Young William L,Zhao Yuanli,Su Hua
Neuroimmunology and neuroinflammation
AIM:To test the hypothesis that lymphocyte infiltration in brain arteriovenous malformation (bAVM) is not associated with iron deposition (indicator of microhemorrhage). METHODS:Sections of unruptured, previously untreated bAVM specimens (n=19) were stained immunohistochemically for T-lymphocytes (CD3), B-lymphocytes (CD20), plasma cells (CD138) and macrophages (CD68). Iron deposition was assessed by hematoxylin and eosin and Prussian blue stains. Superficial temporal arteries (STA) were used as control. RESULTS:Both T lymphocytes and macrophages were present in unruptured, previously untreated bAVM specimens, whereas few B cells and plasma cells were detected. Iron deposition was detected in 8 specimens (42%; 95% confidence interval =20-67%). The samples with iron deposition tended to have more macrophages than those without (666±313 vs 478±174 cells/mm; P=0.11). T-cells were clustered on the luminal side of the endothelial surface, on the vessel-wall, and in the perivascular regions. There was no correlation between T lymphocyte load and iron deposition (P=0.88). No macrophages and lymphocytes were detected in STA controls. CONCLUSIONS:T-lymphocytes were present in bAVM specimens. Unlike macrophages, the load and location of T-lymphocytes were not associated with iron deposition, suggesting the possibility of an independent cell-mediated immunological mechanism in bAVM pathogenesis.
A genome-wide investigation of copy number variation in patients with sporadic brain arteriovenous malformation.
Bendjilali Nasrine,Kim Helen,Weinsheimer Shantel,Guo Diana E,Kwok Pui-Yan,Zaroff Jonathan G,Sidney Stephen,Lawton Michael T,McCulloch Charles E,Koeleman Bobby P C,Klijn Catharina J M,Young William L,Pawlikowska Ludmila
BACKGROUND:Brain arteriovenous malformations (BAVM) are clusters of abnormal blood vessels, with shunting of blood from the arterial to venous circulation and a high risk of rupture and intracranial hemorrhage. Most BAVMs are sporadic, but also occur in patients with Hereditary Hemorrhagic Telangiectasia, a Mendelian disorder caused by mutations in genes in the transforming growth factor beta (TGFβ) signaling pathway. METHODS:To investigate whether copy number variations (CNVs) contribute to risk of sporadic BAVM, we performed a genome-wide association study in 371 sporadic BAVM cases and 563 healthy controls, all Caucasian. Cases and controls were genotyped using the Affymetrix 6.0 array. CNVs were called using the PennCNV and Birdsuite algorithms and analyzed via segment-based and gene-based approaches. Common and rare CNVs were evaluated for association with BAVM. RESULTS:A CNV region on 1p36.13, containing the neuroblastoma breakpoint family, member 1 gene (NBPF1), was significantly enriched with duplications in BAVM cases compared to controls (P = 2.2×10(-9)); NBPF1 was also significantly associated with BAVM in gene-based analysis using both PennCNV and Birdsuite. We experimentally validated the 1p36.13 duplication; however, the association did not replicate in an independent cohort of 184 sporadic BAVM cases and 182 controls (OR = 0.81, P = 0.8). Rare CNV analysis did not identify genes significantly associated with BAVM. CONCLUSION:We did not identify common CNVs associated with sporadic BAVM that replicated in an independent cohort. Replication in larger cohorts is required to elucidate the possible role of common or rare CNVs in BAVM pathogenesis.
Angioarchitectural characteristics associated with initial hemorrhagic presentation in supratentorial brain arteriovenous malformations.
Pan Jianwei,Feng Lei,Vinuela Fernando,He Hongwei,Wu Zhongxue,Zhan Renya
European journal of radiology
OBJECTIVE:The difference in arterial supply, venous drainage, functional localization in supratentorial and infratentorial compartments may contribute to the conflicting results about risk factors for hemorrhage in published case series of brain arteriovenous malformation (bAVM). Further investigation focused on an individual brain compartment is thus necessary. This retrospective study aims to identify angioarchitectural characteristics associated with the initial hemorrhagic event of supratentorial bAVMs. MATERIALS AND METHODS:The clinical and angiographic features of 152 consecutive patients with supratentorial bAVMs who presented to our hospital from 2005 to 2008 were retrospectively reviewed. All these patients had new diagnosis of bAVM. Univariate (χ(2) test) and multivariate analyses were conducted to assess the angiographic features in patients with and without initial hemorrhagic presentations. A probability value of less than 0.05 was considered statistically significant in each analysis. RESULTS:In 152 patients with supratentorial AVMs, 70.6% of deep and 52.5% of superficial sbAVMs presented with hemorrhage. The deep location was correlated with initial hemorrhagic presentation in univariate analysis (χ(2)=3.499, p=0.046) but not in the multivariate model (p=0.144). There were 44 sbAVMs with perforating feeders, 39 (88.6%) of which bled at a significantly higher rate than those with terminal feeders (χ(2)=25.904, p=0.000). 87.5% (21/24) of exclusive deep venous drainage presented with hemorrhage, a significantly higher rate than those of the other type of venous drainage (χ(2)=11.099, p=0.004). All 10 patients with both perforating feeders and exclusive deep draining vein presented with initial hemorrhage. Hemorrhagic presentation was correlated with perforating feeders (p=0.000) and exclusive deep draining vein (p=0.007) in multivariate analysis as well. CONCLUSIONS:Supratentorial bAVMs with perforating feeders and deep venous drainage have a higher risk of hemorrhage. In contrast with many previous reports, AVM location was not associated with hemorrhagic presentation in adjusted analyses. The correlation between deep location and initial hemorrhage in univariate analysis might be caused by the involved perforating feeders and deep draining vein in the deep located AVMs.
Association between the rs1333040 polymorphism on the chromosomal 9p21 locus and sporadic brain arteriovenous malformations.
Sturiale Carmelo Lucio,Gatto Ilaria,Puca Alfredo,D'Arrigo Sonia,Giarretta Igor,Albanese Alessio,Di Rocco Concezio,Maira Giulio,Pola Roberto
Journal of neurology, neurosurgery, and psychiatry
BACKGROUND:Single nucleotide polymorphisms (SNPs) on chromosome 9p21 have been recently associated with intracranial aneurysms and stroke. In this study, we tested the association between the rs1333040C>T polymorphism on the 9p21 locus and sporadic brain arteriovenous malformations (BAVMs). METHODS:We studied 78 patients with sporadic BAVMs and 103 unaffected controls. Genomic DNA was isolated from peripheral blood and the rs1333040C>T polymorphism was assessed by PCR-restriction fragment length polymorphism using the BsmI restriction endonuclease. RESULTS:We found that the distribution of the three genotypes (TT/TC/CC) of the rs1333040 polymorphism was significantly different between cases and controls (p=0.02). Using dominant, recessive and additive genetic models, we found that the TT genotype and the T allele were significantly more common in the BAVM group than in controls. We also evaluated whether the rs1333040 polymorphism was associated with prototypical angio-architectural features of BAVMs (such as nidus size, venous drainage pattern and Spetzler-Martin grading) and with the occurrence of seizures and bleeding. We detected a significant association between the homozygous T allele in the recessive model and BAVMs with a nidus >4 cm in diameter. Deep venous drainage was significantly more frequent among subjects carrying at least one T allele in the dominant model. Patients with seizures showed a significant association with the TT genotype and the T allele in all genetic models examined whereas those who experienced intracranial bleeding showed a significant association with the T allele in the trend model. CONCLUSIONS:This is the first study demonstrating an association between an SNP of the 9p21 region and sporadic BAVMs. Our results emphasise the relevance of this chromosomal locus as a common risk factor for various forms of cerebrovascular diseases.
Allelic variation of the MMP3 promoter affects transcription activity through the transcription factor C-MYB in human brain arteriovenous malformations.
Huai Cong,Song Jianping,Ma Zengyi,Qin Xuanfeng,Li Peiliang,Chen Hongyan,Zhao Fan,Lu Daru,Song Donglei,Mao Ying,Song Xiao,Zhao Yao
MMPs comprise a family of proteolytic enzymes that degrade pericellular substances, which may result in the destabilization of vessels and related to the development of brain arteriovenous malformations (BAVM). MMP3 is a key member of this family, overexpressed in BAVM tissues, and a single nucleotide polymorphism within MMP3, -709A>G (rs522616), is significantly associated with the risk of BAVM. In this study, we aimed to investigate the mechanism through which the polymorphism rs522616 regulates the expression of MMP3. Our results showed that -709A led to a over 2-fold higher transcriptional activity compared with the G allele (P<0.05) and this transcriptional activity can be depressed by co-transfecting cells with competitive DNA fragments containing -709A but not -709G. Bioinformatics analyses suggested that the transcription factor C-MYB might bind to the area around rs522616. Overexpressed C-MYB significantly increased the transcriptional activity of -709A compared with -709G or controls that did not overexpress c-myb (P<0.01) in HEK293 and HUVEC cells. ChIP assays indicated that C-MYB bound to the SNP region in the two cell lines and three BAVM tissue samples. Together, these data indicated that C-MYB can bind to the -709A allele of the MMP3 promoter, activate its transcription and lead to a higher expression of this gene. This novel hypothesis, supported by molecular evidence, explains how this SNP affects MMP3 promoter function and results in a risk of BAVM development.
Human brain arteriovenous malformations are associated with interruptions in elastic fibers and changes in collagen content.
Guo Yi,Qumu Shi-Wei,Nacar Osman Arikan,Yang Jin-Yan,Du Jiang,Belen Deniz,Pan Lin,Zhao Yuan-Li
AIM:We hypothesized that the loss of structural integrity in vessels of brain arteriovenous malformations (bAVM) is associated with abnormal expression of collagen subtypes. MATERIAL AND METHODS:We retrospectively obtained the nidus tissue specimens from 24 bAVM patients and structurally normal cerebral cortex from six surgical epilepsy patients. The samples were paraffin-embedded and stained with Victoria blue and picrosirius red and examined by light microscopy. RESULTS:Light microscopy showed that collagen fibers became disorganized, and the internal elastic lamina was duplicated or interrupted in the arteries from the nidus tissue of bAVM. In some vessels in the nidus, Col I was the only collagen component and the interstitial tissue was mainly comprised of Col I. bAVM patients showed significantly higher Col I levels (86.25% ±13.92) and lower Col III levels (13.75% ±13.92) in the vasculature in the nidus tissue than those of otherwise normal surgical epilepsy patients (Col I, 80.78% ±18.03; Col III, 19.22% ±18.03) (p < 0.001). Additionally, bAVM patients showed a higher Col I / Col III ratio (24.71±40.47) than that of normal controls (16.04 ±31.28) (p < 0.001). CONCLUSION:bAVM patients exhibit changes in the Col I/Col III ratio and elastic fibers in the vasculature, which may compromise the structural integrity of cerebral vessels.
Single nucleotide polymorphisms associated with sporadic brain arteriovenous malformations: where do we stand?
Sturiale Carmelo Lucio,Puca Alfredo,Sebastiani Paola,Gatto Ilaria,Albanese Alessio,Di Rocco Concezio,Maira Giulio,Pola Roberto
Brain : a journal of neurology
Brain arteriovenous malformations are characterized by a tangle of abnormal vessels directly shunting blood from the arterial to venous circulation. They are known to occur either sporadically or in the context of well-defined genetic disorders. Haemorrhage represents the most severe clinical manifestation, whereas other common symptoms include headache, seizures and neurological deficits. Although sporadic forms do not recognize a specific genetic cause, in recent years, it has been hypothesized that genes involved in angiogenesis and inflammation or coding for proteins, such as fibronectins, laminins and integrins, may play a role in the pathophysiology of brain arteriovenous malformations. More recently, a new trend of genetic studies has investigated the association between sporadic arteriovenous malformations and single nucleotide polymorphisms, single base variations between genomes within members of a biological species or between paired chromosomes in an individual, which may determine the susceptibility to develop complex diseases and influence their natural history. Several polymorphisms in two different families of genes have been associated with disease susceptibly and increased haemorrhagic risk. These genes are mainly involved in the inflammatory cascade and in the regulation of angiogenesis. However, most of the investigated polymorphisms have been selected on the basis of candidate genes because of their potential functional role in the pathogenesis of brain arteriovenous malformations or in other cerebrovascular diseases. Only one hypothesis-free genome-wide association study in a small number of patients has been performed so far, but it was unable to identify significant associations between brain arteriovenous malformations and specific genetic loci. In this article, we review and analyse the polymorphisms investigated to date in association with sporadic brain arteriovenous malformations in the medical literature. We discuss the biological, pathophysiological and clinical implications of these studies, with particular attention to the prediction of haemorrhagic risk and the possibility of building genetic profiles capable of defining the architectural features of the malformations and predict their evolution and natural history. We also present a joint analysis of the risk estimates found by the studies in literature that have evaluated the association between single nucleotide polymorphisms and brain arteriovenous malformation susceptibility and risk of bleeding. This analysis shows a statistically significant association between the interleukin 6 -174G>C (odds ratio = 1.97; 95% confidence interval: 1.15-3.38) and the tumour necrosis factor α -238G>A (odds ratio = 2.19; 95% confidence interval: 1.25-3.83) gene polymorphisms and risk of intracranial haemorrhage and between the activin-like kinase 1 (also known as ACVRL1) intervening sequence 3 -35A>G (odds ratio = 2.42; 95% confidence interval: 1.54-3.8) gene polymorphism and disease susceptibility.
G Protein-Coupled Receptor 124 (GPR124) Gene Polymorphisms and Risk of Brain Arteriovenous Malformation.
Weinsheimer Shantel,Brettman Ari D,Pawlikowska Ludmila,Wu D Christine,Mancuso Michael R,Kuhnert Frank,Lawton Michael T,Sidney Stephen,Zaroff Jonathan G,McCulloch Charles E,Young William L,Kuo Calvin,Kim Helen
Translational stroke research
Abnormal endothelial proliferation and angiogenesis may contribute to brain arteriovenous malformation (BAVM) formation. G protein-coupled receptor 124 (GPR124) mediates embryonic CNS angiogenesis; thus we investigated the association of single nucleotide polymorphisms (SNPs) and haplotypes in GPR124 with risk of BAVM. Ten tagging SNPs spanning 39 kb of GPR124 were genotyped in 195 Caucasian BAVM patients and 243 Caucasian controls. SNP and haplotype association with risk of BAVM was screened using χ(2) analysis. Associated variants were further evaluated using multivariable logistic regression, adjusting for age and sex. The minor alleles of 3 GPR124 SNPs adjacent to exon 2 and localized to a 16 kb region of high linkage disequilibrium were associated with reduced risk of BAVM (rs7015566 A, P=0.001; rs7823249 T, P=0.014; rs12676965 C, P=0.007). SNP rs7015566 (intron 1) remained associated after permutation testing (additive model P=0.033). Haplotype analysis revealed a significant overall association (χ(2)=12.55, 4 df, P=0.014); 2 haplotypes (ATCC, P=0.006 and GGCT, P=0.008) were associated with risk of BAVM. We genotyped a known synonymous SNP (rs16887051) in exon 2, however genotype frequency did not differ between cases and controls. Sequencing of conserved GPR124 regions revealed a novel indel polymorphism in intron 2. Immunohistochemistry confirmed GPR124 expression in the endothelium with no qualitative difference in expression between BAVM cases and controls. SNP rs7015566 mapping to intron 1 of GPR124 was associated with BAVM susceptibility among Caucasians. Future work is focused on investigating this gene region.
The rs9509 polymorphism of MMP-9 is associated with risk of hemorrhage in brain arteriovenous malformations.
Sun Bing,Qiu Huijia,Zhao Fan,Qiao Nidan,Fan Weiwei,Lu Daru,Chen Hongyan,Hu Jin,Fu Chaowei,Zhou Liangfu,Gu Yuxiang,Zhao Yao,Mao Ying
Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia
We examined whether single nucleotide polymorphisms (SNP) of the matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 4 (TIMP-4) genes are associated with risk of intracranial hemorrhage (ICH) among patients with brain arteriovenous malformation (BAVM). For 311 Chinese patients with BAVM, we performed genotyping analysis for 11 selected SNP of MMP-9 and TIMP-4 using the MassARRAY genotyping system (Sequenom, San Diego, CA, USA). Associations between each genotype and risk of hemorrhage were evaluated using logistic regression analysis. Multivariate logistic regression analysis revealed that MMP-9_rs9509 was significantly associated with ICH among patients with BAVM with adjustments for BAVM size, venous drainage type, age and sex (adjusted odds ratio [OR]=0.19; 95% confidence interval [CI]=0.05-0.66; p=0.009 for CC compared with TT genotype). However, the association was not significant (p=0.072) after Bonferroni correction and was not significant (p=0.064) in the univariate model. The TIMP-4_rs3755724 polymorphism did not have a statistically significant effect in the multivariate model (adjusted OR=0.57; 95% CI=0.32-1.01; p=0.055 for CT compared with TT genotype). The global score test did not reveal any statistically significant differences in haplotype frequency distributions for these two genes. Our findings suggest that the MMP-9_rs9509 polymorphism may be associated with ICH in patients with BAVM.
Perlecan domain V is upregulated in human brain arteriovenous malformation and could mediate the vascular endothelial growth factor effect in lesional tissue.
Kahle Michael P,Lee Boyeon,Pourmohamad Tony,Cunningham Austin,Su Hua,Kim Helen,Chen Yongmei,McCulloch Charles E,Barbaro Nicholas M,Lawton Michael T,Young William L,Bix Gregory J
Brain arteriovenous malformation (BAVM), a rare but important cause of intracranial hemorrhage, has increased angiogenesis and inflammation as key components of the nidus of abnormal vessels and stroma that form the resected surgical specimen. Accordingly, both vascular endothelial growth factor (VEGF) and transforming growth factor-β have been implicated in the pathology of BAVM for their proangiogenic and vascular-regulating roles. The C-terminal fragment of the extracellular matrix component perlecan (domain V, DV) has been shown to be increased and through the α5β1 integrin, to increase VEGF levels in and around areas of cerebral ischemic injury, another proangiogenic condition. We aimed to determine whether the concentrations of DV, DV's proangiogenic receptor α5β1 integrin, or DV's antiangiogenic receptor α2β1 integrin are elevated in human BAVM tissue. DV levels were increased in BAVM compared with control brain tissue from epileptic resection, as was α5β1 integrin. In addition, α5β1 integrin was preferentially increased and localized to endothelial cells compared with α2β1 integrin. VEGF and transforming growth factor-β levels were also increased in BAVM compared with control tissue. Furthermore, increases in all components were strongly correlated. Excessive generation of proangiogenic DV in BAVM suggests that DV may participate in its pathology and may represent a future therapeutic target.
Hemorrhage risk after partial endovascular NBCA and ONYX embolization for brain arteriovenous malformation.
Lv Xianli,Wu Zhongxue,Li Youxiang,Yang Xinjian,Jiang Chuhan
OBJECTIVE:To estimate for hemorrhage risk of partially endovascularly embolized brain arteriovenous malformation (BAVM). METHODS:We retrospectively reviewed 147 consecutive patients with BAVM-treated mainly with endovascular N-butyl cyanoacrylate (NBCA) and ONYX embolization. In Kaplan-Meier survival analysis, the index date was the dated of initial endovascular embolization; cases were censored at time of subsequent intracranial hemorrhage (ICH), or loss to follow-up, and multivariate proportional-hazards regression models included age of presentation (≤30 years old), clinical presentations, and other potential confounders. RESULTS:We reviewed 147 patients with BAVM (58.8% male; mean age±SD at treatment: 27.5±11.1 years) treated with endovascular embolization. One hundred and forty-four NBCA and 76 ONYX embolizations were performed. Complete obliteration of BAVMs was achieved in 29 patients (19.7%). Thirty-two (21.8%) patients were treated with additional Gamma-knife radiosurgery. During 499.5 years of follow-up, 15 partially treated patients suffered a further hemorrhage, which caused four deaths. The crude annual risk of hemorrhage was 3.0% and the annual death rate was 0.8%. Among partially treated patients with hemorrhage at initial presentation, the risk of hemorrhage was 3.8%, while the risk of hemorrhage for patients without hemorrhage at initial presentation was 2.5%. The annual rate of subsequent hemorrhage was 2.6% for non-ICH and 4.2% for ICH in the partial NBCA embolization group compared with 2.4% for non-ICH and 2.4% for ICH in the partial ONYX embolization group. CONCLUSIONS:The effect of partial AVM embolization with liquid embolic agents may still be unclear as for risk reduction of annual hemorrhage rate of cerebral AVM.
Minimal homozygous endothelial deletion of Eng with VEGF stimulation is sufficient to cause cerebrovascular dysplasia in the adult mouse.
Choi Eun-Jung,Walker Espen J,Shen Fanxia,Oh S Paul,Arthur Helen M,Young William L,Su Hua
Cerebrovascular diseases (Basel, Switzerland)
BACKGROUND:Brain arteriovenous malformations (bAVMs) represent a high risk for hemorrhagic stroke, leading to significant neurological morbidity and mortality in young adults. The etiopathogenesis of bAVM remains unclear. Research progress has been hampered by the lack of animal models. Hereditary Hemorrhagic Telangiectasia (HHT) patients with haploinsufficiency of endoglin (ENG, HHT1) or activin receptor-like kinase 1 (ALK1, HHT2) have a higher incidence of bAVM than the general population. We previously induced cerebrovascular dysplasia in the adult mouse that resembles human bAVM through Alk1 deletion plus vascular endothelial growth factor (VEGF) stimulation. We hypothesized that Eng deletion plus VEGF stimulation would induce a similar degree of cerebrovascular dysplasia as the Alk1-deleted brain. METHODS:Ad-Cre (an adenoviral vector expressing Cre recombinase) and AAV-VEGF (an adeno-associated viral vector expressing VEGF) were co-injected into the basal ganglia of 8- to 10-week-old Eng(2f/2f) (exons 5 and 6 flanked by loxP sequences), Alk1(2f/2f) (exons 4-6 flanked by loxP sequences) and wild-type (WT) mice. Vascular density, dysplasia index, and gene deletion efficiency were analyzed 8 weeks later. RESULTS:AAV-VEGF induced a similar degree of angiogenesis in the brain with or without Alk1- or Eng-deletion. Abnormally patterned and dilated dysplastic vessels were found in the viral vector-injected region of Alk1(2f/2f) and Eng(2f/2f) brain sections, but not in WT. Alk1(2f/2f) mice had about 1.8-fold higher dysplasia index than Eng(2f/2f) mice (4.6 ± 1.9 vs. 2.5 ± 1.1, p < 0.05). However, after normalization of the dysplasia index with the gene deletion efficiency (Alk1(2f/2f): 16% and Eng(2f/2f): 1%), we found that about 8-fold higher dysplasia was induced per copy of Eng deletion (2.5) than that of Alk1 deletion (0.3). ENG-negative endothelial cells were detected in the Ad-Cre-treated brain of Eng(2f/2f) mice, suggesting homozygous deletion of Eng in the cells. VEGF induced more severe vascular dysplasia in the Ad-Cre-treated brain of Eng(2f/2f) mice than that of Eng(+/-) mice. CONCLUSIONS:(1) Deletion of Eng induces more severe cerebrovascular dysplasia per copy than that of Alk1 upon VEGF stimulation. (2) Homozygous deletion of Eng with angiogenic stimulation may be a promising strategy for development of a bAVM mouse model. (3) The endothelial cells that have homozygous causal gene deletion in AVM could be crucial for lesion development.
Bevacizumab attenuates VEGF-induced angiogenesis and vascular malformations in the adult mouse brain.
Walker Espen J,Su Hua,Shen Fanxia,Degos Vincent,Amend Gregory,Jun Kristine,Young William L
BACKGROUND AND PURPOSE:Vascular endothelial growth factor (VEGF) expression is elevated in human brain arteriovenous malformations (bAVM). We have developed a bAVM model in the adult mouse by focal Alk1 gene deletion and human VEGF stimulation. We hypothesized that once the abnormal vasculature has been established, tonic VEGF stimulation is necessary to maintain the abnormal phenotype, and VEGF antagonism by bevacizumab (Avastin) would reduce vessel density and attenuate the dysplastic vascular phenotype. METHODS:Angiogenesis and bAVM were induced by injection of adeno-associated viral vector expressing human VEGF alone into the brain of wild-type mice or with adenoviral vector expressing Cre recombinase (Ad-Cre) into Alk1(2f/2f) mice. Six weeks later, bevacizumab or trastuzumab (Herceptin, bevacizumab control) was administered. Vessel density, dysplasia index, vascular cell proliferation and apoptosis, and human IgG were assessed (n=6/group). RESULTS:Compared with trastuzumab (15 mg/kg), administration of 5, 10, and 15 mg/kg of bevacizumab to adeno-associated viral vector expressing human VEGF treated wild-type mice reduced focal vessel density (P<0.05); administration of 5 mg/kg bevacizumab decreased proliferating vascular cells (P=0.04) and increased TUNEL-positive vascular cells (P=0.03). More importantly, bevacizumab (5 mg/kg) treatment reduced both vessel density (P=0.01) and dysplasia index (P=0.02) in our bAVM model. Human IgG was detected in the vessel wall and in the parenchyma in the angiogenic foci of bevacizumab-treated mice. CONCLUSIONS:We provide proof-of-principle that, once abnormal AVM vessels have formed, VEGF antagonism may reduce the number of dysplastic vessels and should be evaluated further as a therapeutic strategy for the human disease.
Brain arteriovenous malformations are associated with interleukin-1 cluster gene polymorphisms.
Fontanella Marco,Rubino Elisa,Crobeddu Emanuela,Gallone Salvatore,Gentile Salvatore,Garbossa Diego,Ducati Alessandro,Pinessi Lorenzo,Rainero Innocenzo
BACKGROUND:Brain arteriovenous malformations (BAVMs) are a rare but important cause of hemorrhagic stroke in young adults. Functional polymorphisms in proinflammatory cytokines have been associated with various cerebrovascular phenotypes, including ischemic stroke, aneurysmal subarachnoid hemorrhage, and BAVM. OBJECTIVE:To investigate whether functional polymorphisms in the IL-1α, IL-1β, and IL-1RN genes are associated with both susceptibility and clinical characteristics in BAVM patients. METHODS:Allelic and genotypic frequencies of IL-1α (-889 C>T), IL-1β (-511 C>T), and IL-1RN (VNTR) polymorphisms were analyzed in 101 unrelated BAVM patients and in 210 healthy subjects. Main clinical characteristics of the disease were compared according to different genotypes. RESULTS:Both allelic and genotypic frequencies of IL-1α -889 C>T showed a significant association with BAVM (P < .001). The carriage of the T allele was related to a 2.47 increased risk of BAVM (odds ratio, 2.47; 95% confidence interval: 1.72-3.56). Allelic and genotypic frequencies of IL-1RN VNTR were different between cases and controls (P = .009). Allele 1 was associated with about a twofold increased disease risk (95% confidence interval: 2.01-5.58). Haplotype analyses confirmed these findings. Several clinical characteristics of the disease were significantly modified by IL-1α and IL-1β genotypes. CONCLUSION:Our data suggest that functional polymorphisms within the IL-1 complex gene are associated with BAVMs and influence the clinical characteristics of the disease, supporting a role for proinflammatory cytokines in disease etiopathogenesis.
Matrix Gla protein deficiency causes arteriovenous malformations in mice.
Yao Yucheng,Jumabay Medet,Wang Anthony,Boström Kristina I
The Journal of clinical investigation
Arteriovenous malformations (AVMs) in organs, such as the lungs, intestine, and brain, are characteristic of hereditary hemorrhagic telangiectasia (HHT), a disease caused by mutations in activin-like kinase receptor 1 (ALK1), which is an essential receptor in angiogenesis, or endoglin. Matrix Gla protein (MGP) is an antagonist of BMPs that is highly expressed in lungs and kidneys and is regulated by ALK1. The objective of this study was to determine the role of MGP in the vasculature of the lungs and kidneys. We found that Mgp gene deletion in mice caused striking AVMs in lungs and kidneys, where overall small organ size contrasted with greatly increased vascularization. Mechanistically, MGP deficiency increased BMP activity in lungs. In cultured lung epithelial cells, BMP-4 induced VEGF expression through induction of ALK1, ALK2, and ALK5. The VEGF secretion induced by BMP-4 in Mgp-/- epithelial cells stimulated proliferation of ECs. However, BMP-4 inhibited proliferation of lung epithelial cells, consistent with the increase in pulmonary vasculature at the expense of lung tissue in the Mgp-null mice. Similarly, BMP signaling and VEGF expression were increased in Mgp-/- mouse kidneys. We therefore conclude that Mgp gene deletion is what we believe to be a previously unidentified cause of AVMs. Because lack of MGP also causes arterial calcification, our findings demonstrate that the same gene defect has drastically different effects on distinct vascular beds.
Susceptible gene single nucleotide polymorphism and hemorrhage risk in patients with brain arteriovenous malformation.
Jiang Nan,Li Xuesong,Qi Tiewei,Guo Shaolei,Liang Feng,Huang Zhengsong
Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia
The relationship between single nucleotide polymorphism (SNP) of interleukin-17 (IL-17A), transforming growth factor β (TGF-β), as well as its receptor (TGFR-β2) and susceptibility to intracerebral hemorrhage in patients with brain arteriovenous malformation (BAVM) was investigated in the present study. A total of 53 patients with BAVM and 120 healthy controls were recruited, all of whom were Han Chinese from South China. There were no statistically significant differences in the IL-17A-197 guanine/adenine (G/A) or TGF-β1-509 cytosine/thymine (C/T) genotypes or gene frequencies between BAVM patients and controls (p>0.05), but the gene frequency of the TGFR-β2-875 A/G genotype in patients with BAVM was significantly higher (p<0.05). Furthermore, the frequencies of the G allele of IL-17A-197 G/A and TGFR-β2-875 A/G in BAVM patients with hemorrhage were higher than those without hemorrhage. TGFR-β2-875 G/G genotype is a risk factor for BAVM, and the IL-17A-197 G/A and TGFR-β2-875 A/G genotype is closely related to hemorrhage risk for patients with BAVM.
Polymorphisms of VEGFA gene and susceptibility to hemorrhage risk of brain arteriovenous malformations in a Chinese population.
Gong Zhi-ping,Qiao Ni-dan,Gu Yu-xiang,Song Jian-ping,Li Pei-liang,Qiu Hui-jia,Fan Wei-wei,Mao Ying,Chen Hong-yan,Zhao Yao
Acta pharmacologica Sinica
AIM:To evaluate the influence of the vascular endothelial growth factor A (VEGFA) polymorphisms on risk of presentation with intracerebral hemorrhage (ICH). METHODS:Nine selected VEGFA single-nucleotide polymorphisms (SNPs) were genotyped in 311 patients with brain arteriovenous malformations (BAVM) in a Chinese population. Associations between individual SNPs/haplotypes and the hemorrhage risk of BAVMs were evaluated using logistic regression analysis. RESULTS:In the single-locus analysis, rs1547651 was associated with increased risk of ICH (adjusted OR=2.11, 95% CI=1.01-4.42 compared with the AA genotype). In particular, an increased risk for ICH was associated with this variant in female patients (adjusted OR=3.21, and 95% CI=0.99-10.36). Haplotype-based analyses revealed that haplotype 'GC' in block 1 and haplotype 'ACC' in block 2 were associated with a 30%-38% reduction in the risk of ICH in patients with BAVMs compared to the most common haplotype (P(sim)=0.033 and P(sim)=0.005, respectively). The protective effect of haplotype 'ACC' in block 2 was more evident in male patients and subjects with BAVMs of a size ≥3 cm (adjusted OR=0.57, 95% CI=0.34-0.97 and adjusted OR=0.57, 95% CI=0.31-0.86, respectively). CONCLUSION:The results suggest that VEGFA gene variants may contribute to ICH risk of BAVM.
Polymorphisms of the vascular endothelial growth factor A gene and susceptibility to sporadic brain arteriovenous malformation in a Chinese population.
Chen Hongyan,Gu Yuxiang,Wu Wenting,Chen Dan,Li Peiliang,Fan Weiwei,Lu Daru,Zhao Fan,Qiao Nidan,Qiu Huijia,Fu Chaowei,Mao Ying,Zhao Yao
Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia
Human brain arteriovenous malformation (BAVM) tissue contains increased levels of vascular endothelial growth factor A (VEGFA). We carried out a case-control study to determine whether polymorphisms in the VEGFA gene are associated with sporadic BAVM. Nine selected VEGFA single-nucleotide polymorphisms (SNP) were genotyped in 319 patients with BAVM and 333 controls from a Chinese population using the MassARRAY genotyping system. We found four single variants in the VEGFA gene (rs1547651, rs2010963, rs833069 and rs3025010), with one haplotype, ACT, possibly associated with the risk of developing BAVM.
Angiopoietin-like 4 (ANGPTL4) gene polymorphisms and risk of brain arteriovenous malformations.
Mikhak Bahar,Weinsheimer Shantel,Pawlikowska Ludmila,Poon Annie,Kwok Pui-Yan,Lawton Michael T,Chen Yongmei,Zaroff Jonathan G,Sidney Stephen,McCulloch Charles E,Young William L,Kim Helen
Cerebrovascular diseases (Basel, Switzerland)
BACKGROUND:Brain arteriovenous malformations (BAVM) are high-flow vascular lesions prone to intracranial hemorrhage (ICH). Abnormal angiogenesis is a key characteristic of BAVM tissue. Angiopoietin-like 4 (ANGPTL4), a secreted glycoprotein, is thought to be involved in angiogenesis and required for proper postnatal blood vessel partitioning. We investigated whether common single nucleotide polymorphisms (SNPs) in ANGPTL4 were associated with risk of BAVM or ICH. METHODS AND RESULTS:We conducted a case-control study of 216 Caucasian BAVM cases and 246 healthy controls, and a secondary case-only analysis, comparing 83 ruptured (ICH) with 133 unruptured BAVM cases at presentation. Four tagSNPs in ANGPTL4 captured variation over a 10-kb region (rs2278236, rs1044250, rs11672433, and rs1808536) and were tested for association with BAVM or ICH. The minor allele (A) of rs11672433 (exon 6, Pro389Pro) was associated with an increased risk of BAVM (p = 0.006), which persisted after adjusting for multiple comparisons (p = 0.03). After adjustments for age and sex, carriers of the minor allele (A) remained at higher risk for BAVM compared to noncarriers (odds ratio, OR = 1.56; 95% confidence interval, CI = 1.01-2.41; p = 0.046) and risk of BAVM was increased with increasing copy of the minor A allele (OR = 1.49, 95% CI = 1.03-2.15; p(trend) = 0.03). Five common haplotypes (frequency >1%) were inferred; overall haplotype distribution differed between BAVM cases and controls (χ(2) = 12.2, d.f. = 4, p = 0.02). Neither SNPs (p > 0.05) nor haplotype distribution (χ(2) = 1.1, d.f. = 4, p = 0.89) were associated with risk of ICH among BAVM cases. CONCLUSION:A synonymous SNP in ANGPTL4 and haplotypes carrying it are associated with risk of BAVM but not with ICH presentation in BAVM cases.
The rs522616 polymorphism in the matrix metalloproteinase-3 (MMP-3) gene is associated with sporadic brain arteriovenous malformation in a Chinese population.
Zhao Yao,Li Peiliang,Fan Weiwei,Chen Dan,Gu Yuxiang,Lu Daru,Zhao Fan,Hu Jin,Fu Chaowei,Chen Xiancheng,Zhou Liangfu,Mao Ying
Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia
In this study, we investigated the association between common variants in the matrix metalloproteinase-3 (MMP-3) gene and the risk of developing sporadic brain arteriovenous malformation (BAVM). We performed genotyping analyses for five single nucleotide polymorphisms (SNPs) in MMP-3 in a case-control study involving 319 Chinese patients with BAVM and 333 Chinese controls. The association between MMP-3 genotypes and the risk of developing BAVM was evaluated using logistic regression analyses. We found that the genotype frequencies were significantly different between patients and controls for the rs522616 A > G variant of MMP-3 (p = 0.02). Logistic regression analysis revealed that the variant genotype of this polymorphism was associated with a significantly decreased risk of BAVM (adjusted odds ratio = 0.62, 95% confidence interval = 0.44-0.87, p = 0.006 for the AG compared with the AA genotype; adjusted odds ratio=0.68, 95% confidence interval = 0.49-0.94, p = 0.019 for the AG + GG compared with the AA genotype). These findings indicate for the first time that the MMP-3 rs522616 polymorphism may contribute to the etiology of sporadic BAVM in the Chinese population.
VEGF Induces More Severe Cerebrovascular Dysplasia in Endoglin than in Alk1 Mice.
Hao Qi,Zhu Yiqian,Su Hua,Shen Fanxia,Yang Guo-Yuan,Kim Helen,Young William L
Translational stroke research
Brain arteriovenous malformations (BAVMs) are an important cause of intracranial hemorrhage (ICH) in young adults. A small percent of BAVMs is due to hereditary hemorrhagic telangiectasia 1 and 2 (HHT1 and 2), which are caused by mutations in two genes involved in TGF-β signaling: endoglin (ENG) and activin-like kinase 1 (ALK1). The BAVM phenotype is an incomplete penetrant in HHT patients, and the mechanism is unknown. We tested the hypothesis that a "response-to-injury" triggers abnormal vascular (dysplasia) development, using Eng and Alk1 haploinsufficient mice. Adeno-associated virus (AAV) expressing vascular endothelial growth factor (VEGF) was used to mimic the injury conditions. VEGF overexpression caused a similar degree of angiogenesis in the brain of all groups, except that the cortex of Alk1(+/-) mice had a 33% higher capillary density than other groups. There were different levels of cerebrovascular dysplasia in haploinsufficient mice (Eng(+/)>Alk1(+/-)), which simulates the relative penetrance of BAVM in HHT patients (HHT1>HHT2). Few dysplastic capillaries were observed in AAV-LacZ-injected mice. Our data indicate that both angiogenic stimulation and genetic alteration are necessary for the development of dysplasia, suggesting that anti-angiogenic therapies might be adapted to slow the progression of the disease and decrease the risk of spontaneous ICH.
Reduced expression of integrin alphavbeta8 is associated with brain arteriovenous malformation pathogenesis.
Su Hua,Kim Helen,Pawlikowska Ludmila,Kitamura Hideya,Shen Fanxia,Cambier Stephanie,Markovics Jennifer,Lawton Michael T,Sidney Stephen,Bollen Andrew W,Kwok Pui-Yan,Reichardt Louis,Young William L,Yang Guo-Yuan,Nishimura Stephen L
The American journal of pathology
Brain arteriovenous malformations (BAVMs) are a rare but potentially devastating hemorrhagic disease. Transforming growth factor-beta signaling is required for proper vessel development, and defective transforming growth factor-beta superfamily signaling has been implicated in BAVM pathogenesis. We hypothesized that expression of the transforming growth factor-beta activating integrin, alphavbeta8, is reduced in BAVMs and that decreased beta8 expression leads to defective neoangiogenesis. We determined that beta8 protein expression in perivascular astrocytes was reduced in human BAVM lesional tissue compared with controls and that the angiogenic response to focal vascular endothelial growth factor stimulation in adult mouse brains with local Cre-mediated deletion of itgb8 and smad4 led to vascular dysplasia in newly formed blood vessels. In addition, common genetic variants in ITGB8 were associated with BAVM susceptibility, and ITGB8 genotypes associated with increased risk of BAVMs correlated with decreased beta8 immunostaining in BAVM tissue. These three lines of evidence from human studies and a mouse model suggest that reduced expression of integrin beta8 may be involved in the pathogenesis of sporadic BAVMs.
Notch-1 signalling is activated in brain arteriovenous malformations in humans.
ZhuGe Qichuan,Zhong Ming,Zheng WeiMing,Yang Guo-Yuan,Mao XiaoOu,Xie Lin,Chen Gourong,Chen Yongmei,Lawton Michael T,Young William L,Greenberg David A,Jin Kunlin
Brain : a journal of neurology
A role for the Notch signalling pathway in the formation of arteriovenous malformations during development has been suggested. However, whether Notch signalling is involved in brain arteriovenous malformations in humans remains unclear. Here, we performed immunohistochemistry on surgically resected brain arteriovenous malformations and found that, compared with control brain vascular tissue, Notch-1 signalling was activated in smooth muscle and endothelial cells of the lesional tissue. Western blotting showed an activated form of Notch-1 in brain arteriovenous malformations, irrespective of clinical presentation and with or without preoperative embolization, but not in normal cerebral vessels from controls. In addition, the Notch-1 ligands Jagged-1 and Delta-like-4 and the downstream Notch-1 target Hes-1 were increased in abundance and activated in human brain arteriovenous malformations. Finally, increased angiogenesis was found in adult rats treated with a Notch-1 activator. Our findings suggest that activation of Notch-1 signalling is a phenotypic feature of brain arteriovenous malformations, and that activation of Notch-1 in normal vasculature induces a pro-angiogenic state, which may contribute to the development of vascular malformations.
EPHB4 gene polymorphisms and risk of intracranial hemorrhage in patients with brain arteriovenous malformations.
Weinsheimer Shantel,Kim Helen,Pawlikowska Ludmila,Chen Yongmei,Lawton Michael T,Sidney Stephen,Kwok Pui-Yan,McCulloch Charles E,Young William L
Circulation. Cardiovascular genetics
BACKGROUND:Brain arteriovenous malformations (BAVMs) are a tangle of abnormal vessels directly shunting blood from the arterial to venous circulation and an important cause of intracranial hemorrhage (ICH). EphB4 is involved in arterial-venous determination during embryogenesis; altered signaling could lead to vascular instability resulting in ICH. We investigated the association of single-nucleotide polymorphisms (SNPs) and haplotypes in EPHB4 with risk of ICH at clinical presentation in patients with BAVM. METHODS AND RESULTS:Eight haplotype-tagging SNPs spanning approximately 29 kb were tested for association with ICH presentation in 146 white patients with BAVM (phase I: 56 ICH, 90 non-ICH) using allelic, haplotypic, and principal components analysis. Associated SNPs were then genotyped in 102 additional cases (phase II: 37 ICH, 65 non-ICH), and data were combined for multivariable logistic regression. Minor alleles of 2 SNPs were associated with reduced risk of ICH presentation (rs314313_C, P=0.005; rs314308_T, P=0.0004). Overall, haplotypes were also significantly associated with ICH presentation (chi(2)=17.24, 6 df, P=0.008); 2 haplotypes containing the rs314308 T allele (GCCTGGGT, P=0.003; GTCTGGGC, P=0.036) were associated with reduced risk. In principal components analysis, 2 components explained 91% of the variance and complemented haplotype results by implicating 4 SNPs at the 5' end, including rs314308 and rs314313. These 2 SNPs were replicated in the phase II cohort, and combined data resulted in greater significance (rs314313, P=0.0007; rs314308, P=0.00008). SNP association with ICH presentation persisted after adjusting for age, sex, BAVM size, and deep venous drainage. CONCLUSIONS:EPHB4 polymorphisms are associated with risk of ICH presentation in patients with BAVM, warranting further study.
Endothelial Notch signaling is upregulated in human brain arteriovenous malformations and a mouse model of the disease.
Murphy Patrick A,Lu Gloria,Shiah Steven,Bollen Andrew W,Wang Rong A
Laboratory investigation; a journal of technical methods and pathology
Brain arteriovenous malformations (BAVMs) can cause lethal hemorrhagic stroke and have no effective treatment. The cellular and molecular basis for this disease is largely unknown. We have previously shown that expression of constitutively-active Notch4 receptor in the endothelium elicits and maintains the hallmarks of BAVM in mice, thus establishing a mouse model of the disease. Our work suggested that Notch pathway could be a critical molecular mediator of BAVM pathogenesis. Here, we investigated the hypothesis that upregulated Notch activation contributes to the pathogenesis of human BAVM. We examined the expression of the canonical Notch downstream target Hes1 in the endothelium of human BAVMs by immunofluorescence, and showed increased levels relative to either autopsy or surgical biopsy controls. We then analyzed receptor activity using an antibody to the activated form of the Notch1 receptor, and found increased levels of activity. These findings suggest that Notch activation may promote the development and even maintenance of BAVM. We also detected increases in Hes1 and activated Notch1 expression in our mouse model of BAVM induced by constitutively active Notch4, demonstrating molecular similarity between the mouse model and the human disease. Our work suggests that activation of Notch signaling is an important molecular candidate in BAVM pathogenesis and further validates that our animal model provides a platform to study the progression as well as the regression of the disease.
Common variants in interleukin-1-Beta gene are associated with intracranial hemorrhage and susceptibility to brain arteriovenous malformation.
Kim Helen,Hysi Pirro G,Pawlikowska Ludmila,Poon Annie,Burchard Esteban González,Zaroff Jonathan G,Sidney Stephen,Ko Nerissa U,Achrol Achal S,Lawton Michael T,McCulloch Charles E,Kwok Pui-Yan,Young William L
Cerebrovascular diseases (Basel, Switzerland)
BACKGROUND:Polymorphisms in the proinflammatory cytokine interleukin (IL)-1beta gene have been associated with systemic atherogenesis, thrombosis and rupture. The aim of this study was to investigate associations between single nucleotide polymorphisms (SNPs) in IL-1beta and intracranial hemorrhage (ICH) in the natural course of brain arteriovenous malformation (BAVM) patients. METHOD:Two IL-1beta promoter SNPs (-511C-->T, -31T-->C) and 1 synonymous coding SNP in exon 5 at +3953C-->T (Phe) were genotyped in 410 BAVM patients. We performed a survival analysis of time to subsequent ICH, censoring cases at first treatment, death or last follow-up. A Cox regression analysis was performed to obtain hazard ratios (HRs) for genotypes adjusted for age, sex, Caucasian race/ethnicity and hemorrhagic presentation. RESULTS:Subjects with the -31 CC genotype (HR = 2.7; 95% CI 1.1-6.6; p = 0.029) or the -511 TT genotype (HR = 2.6; 95% CI 1.1-6.5; p = 0.039) had a greater risk of subsequent ICH compared with reference genotypes, adjusting for covariates. The +3953C-->T SNP was not significantly associated with an increased ICH risk (p = 0.22). The IL-1beta promoter polymorphisms were also associated with BAVM susceptibility among a subset of 235 BAVM cases and 255 healthy controls of Caucasian race/ethnicity (p < 0.001). CONCLUSION:IL-1beta promoter polymorphisms were associated with an increased risk of ICH in BAVM clinical course and with BAVM susceptibility. These results suggest that inflammatory pathways, including the IL-1beta cytokine, may play an important role in ICH.
Endothelial Notch4 signaling induces hallmarks of brain arteriovenous malformations in mice.
Murphy Patrick A,Lam Michael T Y,Wu Xiaoqing,Kim Tyson N,Vartanian Shant M,Bollen Andrew W,Carlson Timothy R,Wang Rong A
Proceedings of the National Academy of Sciences of the United States of America
Brain arteriovenous malformations (BAVMs) can cause devastating stroke in young people and contribute to half of all hemorrhagic stroke in children. Unfortunately, the pathogenesis of BAVMs is unknown. In this article we show that activation of Notch signaling in the endothelium during brain development causes BAVM in mice. We turned on constitutively active Notch4 (int3) expression in endothelial cells from birth by using the tetracycline-regulatable system. All mutants developed hallmarks of BAVMs, including cerebral arteriovenous shunting and vessel enlargement, by 3 weeks of age and died by 5 weeks of age. Twenty-five percent of the mutants showed signs of neurological dysfunction, including ataxia and seizure. Affected mice exhibited hemorrhage and neuronal cell death within the cerebral cortex and cerebellum. Strikingly, int3 repression resolved ataxia and reversed the disease progression, demonstrating that int3 is not only sufficient to induce, but also required to sustain the disease. We show that int3 expression results in widespread enlargement of the microvasculature, which coincided with a reduction in capillary density, linking vessel enlargement to Notch's known function of inhibiting vessel sprouting. Our data suggest that the Notch pathway is a molecular regulator of BAVM pathogenesis in mice, and offer hope that their regression might be possible by targeting the causal molecular lesion.
Leptin plays a role in ruptured human brain arteriovenous malformations.
Xie Q,Chen X C,Gong Y,Gu Y X
Acta neurochirurgica. Supplement
INTRODUCTION:Intracerebral hemorrhage (ICH) is one of the most common clinical manifestations of human brain arteriovenous malformation (BAVM). However, the hemorrhagic mechanism of BAVM is still unclear. Leptin, first discovered in obesity research, has not been systematically studied in BAVM and ICH. We investigated expression and effect of leptin on human BAVM. METHODS:Specimens were obtained from 6 BAVM patients, who had been divided into either hemorrhagic or non-hemorrhagic groups. Leptin, leptin receptor, and signal transducers and activators of transcription-3 (STAT3) were analyzed by different methods, such as gene chips, reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot. Perinidal brain tissue around each BAVM served as control. RESULTS:Gene chips and RT-PCR found transcriptional leptin raised at least 2 levels in hemorrhagic BAVM. Immunohistochemical slices also showed higher expression of leptin, leptin receptor, and STAT3 on nidus part of hemorrhagic BAVM than non-hemorrhagic ones. On Western blot analysis, hemorrhagic BAVMs had higher levels of leptin (p < 0.01). CONCLUSIONS:The transcriptional and translational levels of leptin, leptin receptor, and STAT3 were higher in hemorrhagic BAVM, suggesting that leptin may play an important role in the hemorrhagic mechanism of BAVM.
[Expression of TGFbeta1 and its type I receptors ALK1 and ALK5 mRNA in brain arteriovenous malformation].
Chen Guang-zhong,Li Tie-lin,Quan Wei,Huang Tao,Zhao Qing-ping,Wang Jian-qi,Duan Chuan-zhi,Wang Qiu-jing,Jiang Xiao-dan
Nan fang yi ke da xue xue bao = Journal of Southern Medical University
OBJECTIVE:To explore the expression of transforming growth factor beta1 (TGFbeta1) and its type I receptors activin-like kinase 1 (ALK1) and ALK5 mRNA in the development of brain arteriovenous malformation (BAVM). METHODS:The mRNA expressions of TGFbeta1, ALK1and ALK5 were detected with semiquantitative RT-PCR in patients with BAVM. RESULTS:The expressions of TGFbeta1 and ALK5 mRNA increased significantly in BAVM, and their relative expression quantity were 0.777-/+0.047 and 0.585-/+0.074, respectively. However, ALK1 mRNA expression declined significantlies with a relative expression of 0.173-/+0.044 in comparison with the control group (0.720-/+0.098, P<0.01). CONCLUSION:The balance of TGFbeta1 and its type I receptors ALK1 and ALK5 mRNA expressions may play important role in the development of BAVM.
Dose-response effect of tetracyclines on cerebral matrix metalloproteinase-9 after vascular endothelial growth factor hyperstimulation.
Lee Chanhung Z,Yao Jianhua S,Huang Yong,Zhai Wenwu,Liu Weizhong,Guglielmo B Joseph,Lin Emil,Yang Guo-Yuan,Young William L
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism
Brain arteriovenous malformations (BAVMs) are a potentially life-threatening disorder. Matrix metalloproteinase (MMP)-9 activity was greatly increased in BAVM tissue specimens. Doxycycline was shown to decrease cerebral MMP-9 activities and angiogenesis induced by vascular endothelial growth factor (VEGF). In the present study, we determined the dose-response effects of doxycycline and minocycline on cerebral MMP-9 using our mouse model with VEGF focal hyperstimulation delivered with adenoviral vector (AdVEGF) in the brain. Mice were treated with doxycycline or minocycline, respectively, at 1, 5, 10, 30, 50, or 100 mg/kg/day through drinking water for 1 week. Our results have shown that MMP-9 messenger ribonucleic acid (mRNA) expression was inhibited by doxycycline starting at 10 mg/kg/day (P<0.02). Minocycline showed more potent inhibition on MMP-9 mRNA expression, starting at 1 (P<0.005) and further at more than 30 (P<0.001) mg/kg/day. At the enzymatic activity level, doxycycline started to suppress MMP-9 activity at 5 mg/kg/day (P<0.001), while minocycline had an effect at a lower dose, 1 mg/kg/day (P<0.02). The inhibition of cerebral MMP-9 mRNA and activity were highly correlated with drug levels in the brain tissue. We also assessed the potential relevant signaling pathway in vitro to elucidate the mechanisms underlying the MMP-9 inhibition by tetracyclines. In vitro, minocycline, but not doxycycline, inhibits MMP-9, at least in part, via the extracellular signaling-related kinase 1/2 (ERK1/2)-mediated pathway. This study provided the evidence that the tetracyclines inhibit stimulated cerebral MMP-9 at multiple levels and are effective at very low doses, offering great potential for therapeutic use.
Tumor necrosis factor-alpha-238G>A promoter polymorphism is associated with increased risk of new hemorrhage in the natural course of patients with brain arteriovenous malformations.
Achrol Achal S,Pawlikowska Ludmila,McCulloch Charles E,Poon K Y Trudy,Ha Connie,Zaroff Jonathan G,Johnston S Claiborne,Lee Chanhung,Lawton Michael T,Sidney Stephen,Marchuk Douglas A,Kwok Pui-Yan,Young William L,
BACKGROUND AND PURPOSE:Identification of single-nucleotide polymorphisms (SNPs) associated with increased risk of new intracranial hemorrhage (ICH) after brain arteriovenous malformation (BAVM) diagnosis would facilitate risk stratification and identify potential targets for therapeutic intervention. METHODS:Patients with BAVM were longitudinally followed. Primary outcome was new ICH after diagnosis; censoring events were last follow-up or any BAVM treatment. We genotyped 4 promoter SNPs in 2 inflammatory cytokine genes: interleukin-6 (IL-6-174G>C; IL-6-572G>C) and tumor necrosis factor-alpha (TNF-alpha-238G>A; TNF-alpha-308G>A). Association of genotype with risk of new ICH was screened using chi2; SNPs associated with new ICH were further characterized using Cox proportional hazards. RESULTS:We genotyped 280 patients (50% female; 59% white, mean+/-SD age at diagnosis 37+/-17 years; 40% presenting with ICH). TNF-alpha-238G>A was associated with increased risk of new ICH after diagnosis (chi2; P=0.003). After adjusting for age, race/ethnicity, and clinical presentation, the risk of new ICH was increased for patients with TNF-alpha-238 AG genotype (hazard ratio, 4.01; P=0.015). No other SNP was found to be associated with new ICH. CONCLUSIONS:A TNF-alpha SNP was associated with increased risk of new ICH in the natural course of BAVMs. The role of inflammatory cytokines in the pathogenesis of BAVM hemorrhage merits further study.
Coexpression of angiogenic factors in brain arteriovenous malformations.
Hashimoto Tomoki,Wu Yongqin,Lawton Michael T,Yang Guo-Yuan,Barbaro Nicholas M,Young William L
OBJECTIVE:Brain arteriovenous malformations (BAVMs) are structurally unstable blood vessels that display an angiogenic phenotype, possibly maintained by concerted effects of key angiogenic factors. Therefore, we hypothesized that there are close correlations among key angiogenic factors in BAVMs and that levels of key angiogenic factors are associated with BAVM clinical characteristics that are linked with vascular instability. METHODS:We measured the expression of angiopoietin-2 (Ang-2), matrix metalloproteinase-9, vascular endothelial growth factor (VEGF), and platelet-derived growth factor-AA and -BB by use of enzyme-linked immunosorbent assay in 27 BAVM surgical specimens. Tissues were also collected from 14 structurally normal brain specimens obtained during epilepsy surgery. RESULTS:Ang-2, VEGF, and matrix metalloproteinase-9 were highly expressed in BAVM samples (11.9 +/- 15.5 ng/mg protein, 137 +/- 102 pg/mg protein, and 379 +/- 455 pg/mg protein, respectively). Platelet-derived growth factor-BB was detectable in 7 of 21 BAVM samples. There were close correlations between Ang-2 and VEGF (R2 = 0.79). Higher Ang-2 and VEGF levels seemed to be associated with draining vein characteristics linked with BAVM hemorrhage but not with feeding artery pressure or BAVM size. In the structurally normal brain specimens, levels of Ang-2, VEGF, and matrix metalloproteinase-9 were low (0.8 +/- 2.3 ng/mg protein, 38 +/- 25 pg/mg protein, and 52 +/- 43 pg/mg protein, respectively). CONCLUSION:There were close correlations among angiogenic factors in BAVMs. Concerted effects of angiogenic factors may maintain the angiogenic phenotype in BAVMs and thereby determine the clinical course of BAVMs.
Suppression of MMP-9 by doxycycline in brain arteriovenous malformations.
Hashimoto Tomoki,Matsumoto Melissa M,Li Jenny F,Lawton Michael T,Young William L,
BACKGROUND:The primary aim of this study is to demonstrate the feasibility of utilizing doxycycline to suppress matrix metalloproteinase-9 (MMP-9) in brain arteriovenous malformations (AVMs). METHODS:Ex-vivo treatment of AVM tissues: Intact AVM tissues were treated with doxycycline for 48 hours. Active and total MMP-9 in the medium were measured. Pilot trial: AVM patients received either doxycycline (100 mg) or placebo twice a day for one week prior to AVM resection. Active and total MMP-9 in BVM tissues were measured. RESULTS:Ex-vivo treatment of AVM tissues: Doxycycline at 10 and 100 microg/ml significantly decreased MMP-9 levels in AVM tissues ex-vivo (total: control vs 10 vs 100 microg/ml = 100 +/- 6 vs 60 +/- 16 vs 61 +/- 9%; active: 100 +/- 8 vs 48 +/- 16 vs 59 +/- 10%). Pilot trial: 10 patients received doxycycline, and 4 patients received placebo. There was a trend for both MMP-9 levels to be lower in the doxycycline group than in the placebo group (total: 2.18 +/- 1.94 vs 3.26 +/- 3.58, P = .50; active: 0.48 +/- 0.48 vs 0.95 +/- 1.01 ng/100 microg protein, P = .25). CONCLUSIONS:A clinically relevant concentration of doxycycline decreased MMP-9 in ex-vivo AVM tissues. Furthermore, there was a trend that oral doxycycline for as short as one week resulted in a decrease in MMP-9 in AVM tissues. Further studies are warranted to justify a clinical trial to test effects of doxycycline on MMP-9 expression in AVM tissues.
Polymorphisms in genes involved in inflammatory and angiogenic pathways and the risk of hemorrhagic presentation of brain arteriovenous malformations.
Pawlikowska Ludmila,Tran Mary N,Achrol Achal S,McCulloch Charles E,Ha Connie,Lind Denise L,Hashimoto Tomoki,Zaroff Jonathan,Lawton Michael T,Marchuk Douglas A,Kwok Pui-Yan,Young William L,
BACKGROUND AND PURPOSE:Accurate estimates of intracranial hemorrhage (ICH) risk in patients harboring brain arteriovenous malformation (BAVM) are needed to evaluate interventional strategies and to help guide clinical management. Identification of genetic polymorphisms associated with ICH would facilitate risk stratification in BAVM patients. METHODS:We identified patients with BAVM and documented clinical presentation, demographic data, venous drainage pattern, and BAVM size. Patients were genotyped for 5 polymorphisms in 3 inflammatory cytokine genes, and 9 polymorphisms in 5 angiogenesis-related genes. Association of genotype with risk of hemorrhagic BAVM presentation was evaluated using logistic regression analysis. RESULTS:We genotyped 180 patients with BAVM (53% female, 57% white, mean age at diagnosis 35+/-17 years, 41% presenting with ICH). BAVM patients homozygous for the interleukin 6 (IL6)-174G allele had a greater risk of ICH presentation (OR, 2.62, P=0.003) than IL6-174C carriers. In a multivariate logistic regression model, IL6-174G>C genotype, small BAVM size, and exclusively deep venous drainage were independent predictors of ICH presentation. A similar univariate trend was noted for the TNFalpha-308 GG genotype (P=0.055). The other polymorphisms genotyped were not associated with ICH. CONCLUSIONS:A polymorphism in the inflammatory cytokine IL6, but not polymorphisms in angiogenesis-related genes, was associated with ICH presentation of BAVM. Further studies are needed to define the role of inflammatory cytokines in the pathogenesis of BAVM hemorrhage.
Gene microarray analysis of human brain arteriovenous malformations.
Hashimoto Tomoki,Lawton Michael T,Wen Gen,Yang Guo-Yuan,Chaly Thomas,Stewart Campbell L,Dressman Holly K,Barbaro Nicholas M,Marchuk Douglas A,Young William L
OBJECTIVE:Human brain arteriovenous malformations (BAVMs) display abnormal expression of various angiogenesis-related genes and their products. We examined gene expression patterns in BAVMs by the gene microarray technique. METHODS:We analyzed BAVM and control brain samples obtained by temporal lobectomy for medically intractable seizure by Affymetrix Human Gene Set U95Av2 (Affymetrix, Inc., Santa Clara, CA). The gene microarray data were compared with new and previously published data that used conventional molecular biology techniques. RESULTS:We analyzed six BAVM and five control brain samples. From 12,625 gene probes assayed, 1781 gene probes showed differential expression between BAVMs and controls. BAVM samples had a gene expression pattern that was distinct from those of control brain samples. Increased messenger ribonucleic acid expression of vascular endothelial growth factor A was accompanied by increased expression of its protein product. A majority of the gene data was in agreement with previously published data. The gene microarray data generated a new testable hypothesis regarding integrin, and we found increased expression of integrin alphavbeta3 protein in BAVMs. CONCLUSION:The gene expression pattern of BAVMs was distinct from those of control brain samples. We verified the gene microarray data by demonstrating that increased gene expression levels for angiogenesis-related molecules were accompanied by increased levels of their protein product expression. The gene microarray technique may be a useful tool to study multiple pathways simultaneously in BAVM specimens.
Induction of Brain Arteriovenous Malformation Through CRISPR/Cas9-Mediated Somatic Alk1 Gene Mutations in Adult Mice.
Zhu Wan,Saw Daniel,Weiss Miriam,Sun Zhengda,Wei Meng,Shaligram Sonali,Wang Sen,Su Hua
Translational stroke research
Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. The pathogenesis of bAVM has not been fully understood. Animal models are important tools for dissecting bAVM pathogenesis and testing new therapies. We have developed several mouse bAVM models using genetically modified mice. However, due to the body size, mouse bAVM models have some limitations. Recent studies identified somatic mutations in sporadic human bAVM. To develop a feasible tool to create sporadic bAVM in rodent and animals larger than rodent, we made tests using the CRISPR/Cas9 technique to induce somatic gene mutations in mouse brain in situ. Two sequence-specific guide RNAs (sgRNAs) targeting mouse Alk1 exons 4 and 5 were cloned into pAd-Alk1e4sgRNA + e5sgRNA-Cas9 plasmid. These sgRNAs were capable to generate mutations in Alk1 gene in mouse cell lines. After packaged into adenovirus, Ad-Alk1e4sgRNA + e5sgRNA-Cas9 was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brains of wild-type C57BL/6J mice. Eight weeks after viral injection, bAVMs were detected in 10 of 12 mice. Compared to the control (Ad-GFP/AAV-VEGF-injected) brain, 13% of Alk1 alleles were mutated and Alk1 expression was reduced by 26% in the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF-injected brains. Around the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF injected site, Alk1-null endothelial cells were detected. Our data demonstrated that CRISPR/Cas9 is a feasible tool for generating bAVM model in animals.