Somatic MAP3K3 mutation defines a subclass of cerebral cavernous malformation.

Cerebral cavernous malformations (CCMs) are vascular disorders that affect up to 0.5% of the total population. About 20% of CCMs are inherited because of familial mutations in CCM genes, including CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10, whereas the etiology of a majority of simplex CCM-affected individuals remains unclear. Here, we report somatic mutations of MAP3K3, PIK3CA, MAP2K7, and CCM genes in CCM lesions. In particular, somatic hotspot mutations of PIK3CA are found in 11 of 38 individuals with CCMs, and a MAP3K3 somatic mutation (c.1323C>G [p.Ile441Met]) is detected in 37.0% (34 of 92) of the simplex CCM-affected individuals. Strikingly, the MAP3K3 c.1323C>G mutation presents in 95.7% (22 of 23) of the popcorn-like lesions but only 2.5% (1 of 40) of the subacute-bleeding or multifocal lesions that are predominantly attributed to mutations in the CCM1/2/3 signaling complex. Leveraging mini-bulk sequencing, we demonstrate the enrichment of MAP3K3 c.1323C>G mutation in CCM endothelium. Mechanistically, beyond the activation of CCM1/2/3-inhibited ERK5 signaling, MEKK3 p.Ile441Met (MAP3K3 encodes MEKK3) also activates ERK1/2, JNK, and p38 pathways because of mutation-induced MEKK3 kinase activity enhancement. Collectively, we identified several somatic activating mutations in CCM endothelium, and the MAP3K3 c.1323C>G mutation defines a primary CCM subtype with distinct characteristics in signaling activation and magnetic resonance imaging appearance.

Endothelial hyperactivation of mutant MAP3K3 induces cerebral cavernous malformation enhanced by PIK3CA GOF mutation.

Cerebral cavernous malformations (CCMs) refer to a common vascular abnormality that affects up to 0.5% of the population. A somatic gain-of-function mutation in MAP3K3 (p.I441M) was recently reported in sporadic CCMs, frequently accompanied by somatic activating PIK3CA mutations in diseased endothelium. However, the molecular mechanisms of these driver genes remain elusive. In this study, we performed whole-exome sequencing and droplet digital polymerase chain reaction to analyze CCM lesions and the matched blood from sporadic patients. 44 of 94 cases harbored mutations in KRIT1/CCM2 or MAP3K3, of which 75% were accompanied by PIK3CA mutations (P = 0.006). AAV-BR1-mediated brain endothelial-specific MAP3K3I441M overexpression induced CCM-like lesions throughout the brain and spinal cord in adolescent mice. Interestingly, over half of lesions disappeared at adulthood. Single-cell RNA sequencing found significant enrichment of the apoptosis pathway in a subset of brain endothelial cells in MAP3K3I441M mice compared to controls. We then demonstrated that MAP3K3I441M overexpression activated p38 signaling that is associated with the apoptosis of endothelial cells in vitro and in vivo. In contrast, the mice simultaneously overexpressing PIK3CA and MAP3K3 mutations had an increased number of CCM-like lesions and maintained these lesions for a longer time compared to those with only MAP3K3I441M. Further in vitro and in vivo experiments showed that activating PI3K signaling increased proliferation and alleviated apoptosis of endothelial cells. By using AAV-BR1, we found that MAP3K3I441M mutation can provoke CCM-like lesions in mice and the activation of PI3K signaling significantly enhances and maintains these lesions, providing a preclinical model for the further mechanistic and therapeutic study of CCMs.

Unruptured brain arteriovenous malformations causing seizures localize to one common brain network.

Seizures are a frequent symptom of unruptured brain arteriovenous malformations (bAVMs). However, the brain regions responsible for these seizures remain unclear. To identify the brain regions causally involved in bAVM-related seizures, we retrospectively reviewed 220 patients with unruptured bAVMs. Using voxel-based lesion-symptom mapping (VLSM) analyses, we tested whether individual brain regions were associated with unruptured bAVM-related seizures. The result revealed that unruptured bAVMs causing seizures are anatomically heterogeneous at the voxel level. Subsequently, lesion network mapping (LNM) analyses was performed to determine whether bAVMs causing seizures belonged to a distributed brain network. LNM analyses indicated that these lesions were located in a functional network characterized by connectivity to the left caudate and precuneus. Moreover, the discrimination performance of the identified seizure network was evaluated in discovery set by calculating the individualized network damage score and was tested in validation set. Based on the calculated network damage scores, patients were divided into low-, medium-, and high-risk groups. The prevalence of seizures significantly differed among the three risk categories in both discovery (p = .003) and validation set (p = .004). Finally, we calculated the percentage of voxels in the canonical resting-state networks that overlapped with the seizure-susceptible brain regions to investigate the involvement of resting-state networks. With an involvement percentage over 50%, the frontoparietal control (82.9%), limbic function (76.7%), and default mode network (69.3%) were considered to be impacted in bAVM-related seizures. Our study identified the seizure-susceptible brain regions for unruptured bAVMs, which could be a plausible neuroimaging biomarker in predicting possible seizures.

De Novo Germline and Somatic Variants Convergently Promote Endothelial-to-Mesenchymal Transition in Simplex Brain Arteriovenous Malformation.

[Figure: see text].

Mesenchymal Behavior of the Endothelium Promoted by SMAD6 Downregulation Is Associated With Brain Arteriovenous Malformation Microhemorrhage.

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. TGFß (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: TGFß/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.

Aspirin and Growth of Small Unruptured Intracranial Aneurysm: Results of a Prospective Cohort Study.

BACKGROUND AND PURPOSE: The role of aspirin in unruptured intracranial aneurysm (UIA) growth remains largely unknown. We aim to identify whether aspirin is associated with a lower rate of UIA growth in patients with UIA <7 mm. METHODS: This prospective cohort study consecutively enrolled patients with UIAs <7 mm with ischemic cerebrovascular disease between January 2016 and December 2019. Baseline and follow-up patient information, including the use of aspirin and blood pressure level, were recorded. Patients were considered aspirin users if they took aspirin, including standard- and low-dose aspirin, ≥3× per week. The primary end point was aneurysm growth in any direction or an indisputable change in aneurysm shape. RESULTS: Among the 315 enrolled patients, 272 patients (86.3%) underwent imaging examinations during follow-up (mean follow-up time, 19.6±12.7 months). A total of 113 patients were continuously treated with aspirin. UIA growth occurred in 31 (11.4%) patients. In the multivariate Cox analysis, specific aneurysm locations (anterior communicating artery, posterior communicating artery, or middle cerebral artery; hazard ratio, 2.89 [95% CI, 1.22-6.88]; P=0.016) and a UIA size of 5 to <7 mm (hazard ratio, 7.61 [95% CI, 3.02-19.22]; P<0.001) were associated with a high risk of UIA growth, whereas aspirin and well-controlled blood pressure were associated with a low risk of UIA growth (hazard ratio, 0.29 [95% CI, 0.11-0.77]; P=0.013 and hazard ratio, 0.25 [95% CI, 0.10-0.66]; P=0.005, respectively). The cumulative annual growth rates were as high as 40.0 and 53.3 per 100 person-years in the high-risk patients (>1 risk factor) with and without aspirin, respectively. CONCLUSIONS: Aspirin therapy and well-controlled blood pressure are associated with a low risk of UIA growth; the incidence of UIA growth in high-risk patients in the first year is high, warranting intensive surveillance in this patient group. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02846259.

Surgical management of complex brain arteriovenous malformations with hybrid operating technique: study protocol of a prospective registry and a pragmatic clinical trial.

BACKGROUND: Complex brain arteriovenous malformations (bAVMs) in ≥3 Spetzler-Martin grades have long been challenges among cerebrovascular diseases. None of the traditional methods, such as microsurgical operation, endovascular intervention, or stereotactic radiotherapy, can completely eliminate complex bAVMs without a risk of neural function deterioration. The multistaged hybrid operation solved part of the challenge but remained risky in the installment procedures and intervals. The one-staged hybrid operation was applied in the surgical treatment of cerebrovascular diseases and proved to be a potentially safe and effective method for curing complex bAVMs. However, lacking the support of high-level evidence, its advantages remain unclear. This study was proposed to validate the benefits and risks of one-staged hybrid operation in the treatment of complex bAVMs, as well as its indications, key technologies, and workflows. METHODS: The study is being conducted from Jan 2016 to Dec 2020 with 20 cooperation centers. It consists of 2 sets. The registry set is designed as a prospective real-world registry. The trial set is designed as a prospective pragmatic clinical trial, specifically for the patients with perforating arterial feeders. The two sets share a common grouping: the traditional operation group and the one-staged hybrid operation group. The assignment is based on the clinical condition in the registry set and is randomized in the trial set. End points will be evaluated at scheduled time points. The safety and efficiency of one-staged hybrid operation in treating complex bAVMs will be validated. DISCUSSION: The study is designed for a real-world exploration of benefits and risks of one-staged hybrid operation in the treatment of complex bAVMs. The two-set design reduces the compromise of clinical practice due to the study and improves the statistical power and research quality with a practical sample size. In the study, advantages of the one-staged hybrid operation will be evaluated and compared to those of traditional operation. A spanning development of neurosurgical operation might be facilitated by the study, which means a higher cure rate and lower disability rate in patients with complex bAVMs. TRIAL REGISTRATION: The study was retrospectively registered in ClinicalTrials.gov ( NCT03774017 ) on 11th Dec, 2018.

Clinical features and long-term outcomes of pediatric intraventricular meningiomas: data from a single neurosurgical center.

The purpose of this study is to investigate the clinical characteristics and long-term outcomes of pediatric patients with intraventricular meningioma. We retrospectively analyzed a total of 30 pediatric patients with intraventricular meningiomas who were surgically treated at our department between January 2005 and June 2016 and analyzed their clinical characteristics and surgical outcomes. Among the 160 pediatric patients with intracranial meningioma, 33 (20.6%) had intraventricular lesions. However, only 30 patients had complete demographic and clinical data. A male predilection (male/female = 1.5:1) was observed, and the mean age of our patient cohort was 12.6 years. The lateral ventricle was the most common lesion site (88.6%). In addition, the most common initial symptom was headache or dizziness, and the average interval from symptom onset to admission was 19.17 months (0.25-72 months). Twenty-six patients (86.7%) achieved a Simpson grade of I. Based on the WHO classification, 28 (93.3%) meningiomas were classified as grade I, and the remaining two cases were grades II and III. During the follow-up period (0.67-10.08 years), 3 patients experienced tumor recurrence (15, 18, and 83 months, respectively), and 1 patient died of recurrence. Pediatric and adult intraventricular meningiomas present similar clinical characteristics and surgical outcomes; however, intraventricular meningiomas compose a higher percentage of pediatric meningiomas and have a male predilection. Compared with general pediatric meningiomas, pediatric intraventricular meningiomas tend to have higher incidence of benign subtypes. They are also more likely to be completely resected and have lower recurrence and mortality rates.

Risk factors for neurological deficits after surgical treatment of brain arteriovenous malformations supplied by deep perforating arteries.

The treatment of brain arteriovenous malformations supplied by deep perforating arteries (PA) (P-BAVM) remains challenging. The aims of this study were to determine the outcomes after surgical treatment in patients with P-BAVMs and to identify the risk factors associated with postoperative neurological deficits. We retrospectively reviewed the medical charts and imaging records of 228 consecutive patients with BAVMs who underwent microsurgical resection of their BAVMs at Beijing Tiantan Hospital between September 2012 and March 2016. Patients were included if the BAVMs were totally or partially supplied by PA. All patients had undergone preoperative diffusion tensor imaging (DTI), MRI, 3D time-of-flight MRA (3D TOF-MRA) and digital subtraction angiography (DSA) followed by resection. Both functional and angioarchitectural factors were analysed with respect to the postoperative neurological deficits, including motor deficits, visual field deficits and aphasia. Statistical analysis was performed using the statistical package SPSS (version 20.0.0, IBM Corp.). Fifty-nine patients with P-BAVMs were enrolled. Radical obliteration was achieved in all P-BAVMs according to postoperative DSA. Forty-five (76.3%) patients obtained neurological deficits 1 week after surgery. At a mean follow-up of 14.7 ± 9.4 (3-30) months after surgery, 34 patients (57.6%) had long-term neurological deficits. Multivariable logistic regression analyses showed that a shorter lesion-to-eloquent fibre tracts distance (LFD) was an independent risk factor for short- (P = 0.014) and long-term (P = 0.013) neurological deficits. The cut-off point of LFD for long-term neurological deficits was 5.20 mm. The predominant supply of the PA (P = 0.008) was an independent risk factor for long-term neurological deficits. This study identified a high risk of surgical morbidity for P-BAVMs. The predominant supply of the PA and a shorter LFD are crucial risk factors for postoperative neurological deficits in patients with P-BAVMs.

A comparison of clinicopathological features and surgical outcomes between pediatric skull base and non-skull base meningiomas.

OBJECT: The purpose of this study is to analyze and compare the clinicopathological characteristics and surgical outcomes between skull base and non-skull base meningiomas in pediatric population. METHODS: We retrospectively analyzed a total of 140 cases of pediatric meningiomas surgically treated in our department from January 2005 to July 2015 and compared the clinicopathological characteristics and surgical outcomes between skull base and non-skull base meningiomas. RESULTS: Of all the pediatric meningiomas, 50 (35.8 %) were located at the skull base and 90 (64.2 %) were located at the non-skull base. Skull base and non-skull base meningiomas had a similar sex distribution (male/female = 1:1 in skull base meningiomas and male/female = 1.5:1 in non-skull base meningiomas) (P = 0.288) and high-grade meningioma occurrence rate (P = 0.569). In addition, the mean age of non-skull base meningiomas was 12.5 years which was younger than that of skull base meningiomas (14.2 years) (P = 0.019), and the preoperative tumor size was smaller in skull base (mean size = 4.7 cm in skull base meningiomas and mean size = 5.7 cm in non-skull base meningiomas) (P = 0.020). Gross total resection was achieved in 64 non-skull base patients (73.9 %) and 26 skull base patients (52 %) (P = 0.046). Patients with gross total resection had better progression free survival (PFS) than those with subtotal resection. CONCLUSION: Pediatric skull base and non-skull base meningiomas are similar in sex distribution and high-grade meningioma occurrence rate. In comparison with non-skull base ones, pediatric skull base meningiomas occur at elder age and are smaller in size and they are more likely to be incompletely resected. Gross total resection and early treatment are recommended to prolong PFS of pediatric patients.

Pediatric intracranial clear cell meningioma: a clinicopathological study of seven cases and literature review.

BACKGROUND: In this study, we reported seven cases of pediatric intracranial clear cell meningiomas (CCMs) in our institution and reviewed the relevant literature to investigate the clinicopathological characteristics, treatment options, and prognosis of these rare tumors. METHODS: From January 2005 to June 2016, we retrospectively reviewed seven pediatric intracranial CCMs in terms of their clinical data, preoperative MRI features, and prognosis. Moreover, a critical review of the English language literature was also conducted. RESULTS: The patients consisted of two males and five females with a median age of 10.5 years (range 6-15 years) at initial surgery. Petroclival and cerebellopontine angle area was the most common location site (5/7). Accordingly, the most common initial manifestation was hearing loss (3/7), and the mean interval from onset of symptoms to admission was 6.8 months (1.5-24 months). Gross total resection was achieved in five patients. Of the six tumors with immunohistochemical records, MIB-1 labeling index varied from 3 to 20 % (mean 8.1 %). During the follow-up period (mean 76.9 months, range 16-180 months), four patients had experienced tumor recurrences and three patients died due to recurrences. CONCLUSIONS: Pediatric intracranial CCMs have a tendency to recur. There is a significant relationship between MIB-1 labeling index and recurrence. Gross total resection is recommended; if not available, adjuvant radiotherapy should be used to reduce the recurrent rate. In addition, postoperative MRI follow-up should be monitored at an interval time after resection.

Zabramski classification in predicting the occurrence of symptomatic intracerebral hemorrhage in sporadic cerebral cavernous malformations.

OBJECTIVE: The authors aimed to investigate the evolutionary characteristics of the Zabramski classification of cerebral cavernous malformations (CCMs) and the value of the Zabramski classification in predicting clinical outcome in patients with sporadic CCM. METHODS: This retrospective study consecutively included cases of sporadic CCM that had been untreated from January 2001 through December 2021. Baseline and follow-up patient information was recorded. The evolution of the Zabramski classification of a sporadic CCM was defined as the initial lesion type changing into another type for the first time on MRI follow-up. The primary outcome was the occurrence of a hemorrhage event, which was defined as a symptomatic event with radiological evidence of overt intracerebral hemorrhage. RESULTS: Among the 255 included cases, 55 (21.6%) were classified as type I CCM, 129 (50.6%) as type II CCM, and 71 (27.8%) as type III CCM, based on initial MRI. During a mean follow-up of 58.8 ± 33.6 months, 51 (20.0%) patients had lesion classification transformation, whereas 204 (80.0%) patients maintained their initial type. Among the 51 transformed lesions, 29 (56.9%) were type I, 11 (21.6%) were type II, and 11 (21.6%) were type III. Based on all follow-up imaging, of the initial 55 type I lesions, 26 (47.3%) remained type I and 27 (49.1%) regressed to type III because of hematoma absorption; 91.5% of type II and 84.5% of type III lesions maintained their initial type during MRI follow-up. The classification change rate of type I lesions was statistically significantly higher than those of type II and III lesions. After a total follow-up of 1157.7 patient-years, new clinical hemorrhage events occurred in 40 (15.7%) patients. The annual cumulative incidence rate for symptomatic hemorrhage in all patients was 3.4 (95% CI 2.5-4.7) per 100 person-years. Kaplan-Meier survival analysis showed that the annual cumulative incidence rate for symptomatic hemorrhage of type I CCM (15.3 per 100 patient-years) was significantly higher than those of type II (0.6 per 100 patient-years) and type III (2.3 per 100 patient-years). CONCLUSIONS: This study suggests that the Zabramski classification is helpful in estimating clinical outcome and can assist with surgical decision-making in patients with sporadic CCM.

Therapy management and outcome of acute hydrocephalus secondary to intraventricular hemorrhage in adults.

BACKGROUND: Intraventricular hemorrhage (IVH) refers to bleeding within the brain's ventricular system, and hydrocephalus is a life-threatening complication of IVH characterized by increased cerebrospinal fluid accumulation in the ventricles resulting in elevated intracranial pressure. IVH poses significant challenges for healthcare providers due to the complexity of the underlying pathophysiology and lack of standardized treatment guidelines. Herein, we performed a systematic review of the treatment strategies for hydrocephalus secondary to IVH. METHODS: This systematic review was prospectively registered with PROSPERO (CRD42023450786). The search was conducted in PubMed, Cochrane Library, and Web of Science on July 15, 2023. We included original studies containing valid information on therapy management and outcome of hydrocephalus secondary to primary, spontaneous, and subarachnoid or intracranial hemorrhage following IVH in adults that were published between 2000 and 2023. Glasgow Outcome Scale (GOS) or modified Ranking Scale (mRS) scores during follow-up were extracted as primary outcomes. The risk of bias was assessed using the Newcastle-Ottawa Scale for Cohort Studies or Cochrane Risk of Bias 2.0 Tool. RESULTS: Two hundred and seven patients from nine published papers, including two randomized controlled trials, were included in the analysis. The GOS was used in five studies, while the mRS was used in four. Seven interventions were applied, including craniotomy for removal of hematoma, endoscopic removal of hematoma with/without endoscopic third ventriculostomy (ETV), traditional external ventricular drainage (EVD), and various combinations of EVD, lumbar drainage (LD), and intraventricular fibrinolysis (IVF). Endoscopic removal of hematoma was performed in five of nine studies. Traditional EVD had no obvious benefit compared with new management strategies. Three different combinations of EVD, LD, and IVF demonstrated satisfactory outcomes, although more studies are required to confirm their reliability. Removal of hematoma through craniotomy generated reliable result. Generally, endoscopic removal of hematoma with ETV, removal of hematoma through craniotomy, EVD with IVF, and EVD with early continuous LD were useful. CONCLUSION: EVD is still crucial for the management of IVH and hydrocephalus. Despite a more reliable result from the removal of hematoma through craniotomy, a trend toward endoscopic approach was observed due to a less invasive profile.

Exosomal miR-3131 derived from endothelial cells with KRAS mutation promotes EndMT by targeting PICK1 in brain arteriovenous malformations.

AIMS: To explore the underlying mechanism by which low-frequency KRAS mutations result in extensive EndMT occurrence. METHODS: Exosomes derived from primarily cultured brain arteriovenous malformation (bAVMs) and human umbilical vein endothelial cells (HUVECs) transfected with KRASG12D , KRASWT , or KRASNC lentiviruses were isolated, and their effects on HUVECs were identified by western blotting and immunofluorescence staining. The expression levels of exosomal microRNAs (miRNAs) were evaluated by miRNA microarray, followed by functional experiments on miR-3131 and detection of its downstream target, and miR-3131 inhibitor in reversing the EndMT process induced by KRASG12D -transfected HUVECs and bAVM endothelial cells (ECs) were explored. RESULTS: Exosomes derived from KRASG12D bAVM ECs and KRASG12D -transfected HUVECs promoted EndMT in HUVECs. MiR-3131 levels were highest in the exosomes of KRASG12D -transfected HUVECs, and HUVECs transfected with the miR-3131 mimic acquired mesenchymal phenotypes. RNA-seq and dual-luciferase reporter assays revealed that PICK1 is the direct downstream target of miR-3131. Exosomal miR-3131 was highly expressed in KRASG12D bAVMexos compared with non-KRAS-mutant bAVMexos or HUVECexos . Finally, a miR-3131 inhibitor reversed EndMT in HUVECs treated with exosomes or the supernatant of KRASG12D -transfected HUVECs and KRASG12D bAVM ECs. CONCLUSION: Exosomal miR-3131 promotes EndMT in KRAS-mutant bAVMs, and miR-3131 might be a potential biomarker and therapeutic target in KRASG12D -mutant bAVMs.

KRAS mutation-induced EndMT of brain arteriovenous malformation is mediated through the TGF-ß/BMP-SMAD4 pathway.

OBJECTIVE: Somatic KRAS mutations have been identified in the majority of brain arteriovenous malformations (bAVMs), and subsequent in vivo experiments have confirmed that KRAS mutation in endothelial cells (ECs) causes AVMs in mouse and zebrafish models. Our previous study demonstrated that the KRASG12D mutant independently induced the endothelial-mesenchymal transition (EndMT), which was reversed by treatment with the lipid-lowering drug lovastatin. However, the underlying mechanisms of action were unclear. METHODS: We used human umbilical vein ECs (HUVECs) overexpressing the KRASG12D mutant for Western blotting, quantitative real-time PCR, and immunofluorescence and wound healing assays to evaluate the EndMT and determine the activation of downstream pathways. Knockdown of SMAD4 by RNA interference was performed to explore the role of SMAD4 in regulating the EndMT. BAVM ECs expressing the KRASG12D mutant were obtained to verify the SMAD4 function. Finally, we performed a coimmunoprecipitation assay to probe the mechanism by which lovastatin affects SMAD4. RESULTS: HUVECs infected with KRASG12D adenovirus underwent the EndMT. Transforming growth factor beta (TGF-ß) and bone morphogenetic protein (BMP) signalling pathways were activated in the KRASG12D-mutant HUVECs and ECs in bAVM tissue. Knocking down SMAD4 expression in both KRASG12D-mutant HUVECs and ECs in bAVM tissues inhibited the EndMT. Lovastatin attenuated the EndMT by downregulating p-SMAD2/3, p-SMAD1/5 and acetylated SMAD4 expression in KRASG12D-mutant HUVECs. CONCLUSIONS: Our findings suggest that the KRASG12D mutant induces the EndMT by activating the ERK-TGF-ß/BMP-SMAD4 signalling pathway and that lovastatin inhibits the EndMT by suppressing TGF-ß/BMP pathway activation and SMAD4 acetylation.

Somatic GJA4 mutation in intracranial extra-axial cavernous hemangiomas.

OBJECTIVE: Extra-axial cavernous hemangiomas (ECHs) are sporadic and rare intracranial occupational lesions that usually occur within the cavernous sinus. The aetiology of ECHs remains unknown. METHODS: Whole-exome sequencing was performed on ECH lesions from 12 patients (discovery cohort) and droplet digital polymerase-chain-reaction (ddPCR) was used to confirm the identified mutation in 46 additional cases (validation cohort). Laser capture microdissection (LCM) was carried out to capture and characterise subgroups of tissue cells. Mechanistic and functional investigations were carried out in human umbilical vein endothelial cells and a newly established mouse model. RESULTS: We detected somatic GJA4 mutation (c.121G>T, p.G41C) in 5/12 patients with ECH in the discovery cohort and confirmed the finding in the validation cohort (16/46). LCM followed by ddPCR revealed that the mutation was enriched in lesional endothelium. In vitro experiments in endothelial cells demonstrated that the GJA4 mutation activated SGK-1 signalling that in turn upregulated key genes involved in cell hyperproliferation and the loss of arterial specification. Compared with wild-type littermates, mice overexpressing the GJA4 mutation developed ECH-like pathological morphological characteristics (dilated venous lumen and elevated vascular density) in the retinal superficial vascular plexus at the postnatal 3 weeks, which were reversed by an SGK1 inhibitor, EMD638683. CONCLUSIONS: We identified a somatic GJA4 mutation that presents in over one-third of ECH lesions and proposed that ECHs are vascular malformations due to GJA4-induced activation of the SGK1 signalling pathway in brain endothelial cells.

RNA sequencing analysis between ruptured and un-ruptured brain AVM.

BACKGROUND: A brain arteriovenous malformation (BAVM) is a tangle of abnormal blood vessels connecting the arteries and veins in the brain and is associated with a higher risk for intracerebral hemorrhage (ICH). RNA sequencing technology has been recently used to investigate the mechanism of diseases owing to its ability to identify the gene changes on a transcriptome-wide level. This study aims to gain insights into the potential mechanism involved in BAVM rupture. METHODS: Sixty-five BAVM nidus samples were collected, among which 28 were ruptured and 37 were un-ruptured. Then, next-generation RNA sequencing was performed on all of them to obtain differential expressed genes (DEGs) between the two groups. In addition, bioinformatics analysis was performed to evaluate the involved biological processes and pathways by GO and KEGG analysis. Finally, we performed a univariate Cox regression analysis to obtain the early rupture-prone DEGs. RESULTS: A total of 951 genes were differentially expressed between the ruptured and un-ruptured BAVM groups, of which 740 genes were upregulated and 211 genes were downregulated in ruptured BAVMs. Then, bioinformatics analysis showed the biological processes and pathways related to the inflammatory processes and extracellular matrix organization were significantly enriched. Meanwhile, some downregulated genes are involved in cell adhesion and genes participating in response to muscle activity and the terms of nervous system development. Finally, one hundred twenty-five genes, many were involved in inflammation, were correlated with the early rupture of BAVMs. CONCLUSIONS: The upregulated genes in the ruptured BAVM group were involved in inflammatory processes and extracellular matrix organization. Some of the downregulated genes participated in cell adhesion and myofibril assembly, indicating the role of enhanced inflammation and reduced inflammation vessel strength in BAVMs rupture.

Grading scale based on arcuate fasciculus segmentation to predict postoperative language outcomes of brain arteriovenous malformations.

OBJECTIVE: The long-term postoperative language outcomes for brain arteriovenous malformations (bAVMs) have not been well characterised. With fibres scattered in the Broca's, Wernicke's and Geschwind's area, the arcuate fasciculus (AF) is considered as a crucial structure of language function. This study aimed to observe the language outcomes, determine the risk factors and construct a grading system for long-term postoperative language deficits (LDs) in patients with bAVMs involving the AF (AF-bAVMs). METHODS: We retrospectively reviewed 135 patients with AF-bAVMs. Based on the course of the AF and our clinical experience, three boundary lines were drawn to divide the AF into segments I, II, III and IV in spatial order from the frontal lobe to the temporal lobe. Surgery-related LD evaluations were performed 1 week (short term) and at the last follow-up (long term) after surgery. Finally, based on multivariable logistic regression analysis, a grading system was constructed to predict long-term postoperative LD. The predictive accuracy was assessed using the area under the receiver operating characteristic curve (AUC). RESULTS: Sixty-two (45.9%) patients experienced short-term postoperative LD. After a mean follow-up of 50.2±24.9 months, long-term LD was found in 14 (10.4%) patients. Nidus size (p=0.007), LD history (p=0.009) and segment II involvement (p=0.030) were independent risk factors for short-term LD. Furthermore, segment II involvement (p=0.002), anterior choroidal artery (AChA) feeding (p=0.001), patient age (p=0.023) and LD history (p=0.001) were independent risk factors for long-term LD. A grading system was developed by combining the risk factors for long-term LD; its predictive accuracy was 0.921. CONCLUSIONS: The involvement of the trunk of the AF between Broca's area and the inferior parietal lobule, a nidus supplied by the AChA, older patient age and history of LD were associated with long-term postoperative LD. The grading system combining these factors demonstrated favourable predictive accuracy for long-term language outcomes.

Machine Learning-Enabled Determination of Diffuseness of Brain Arteriovenous Malformations from Magnetic Resonance Angiography.

The diffuseness of brain arteriovenous malformations (bAVMs) is a significant factor in surgical outcome evaluation and hemorrhagic risk prediction. However, there are still predicaments in identifying diffuseness, such as the judging variety resulting from different experience and difficulties in quantification. The purpose of this study was to develop a machine learning (ML) model to automatically identify the diffuseness of bAVM niduses using three-dimensional (3D) time-of-flight magnetic resonance angiography (TOF-MRA) images. A total of 635 patients with bAVMs who underwent TOF-MRA imaging were enrolled. Three experienced neuroradiologists delineated the bAVM lesions and identified the diffuseness on TOF-MRA images, which were considered the ground-truth reference. The U-Net-based segmentation model was trained to segment lesion areas. Eight mainstream ML models were trained through the radiomic features of segmented lesions to identify diffuseness, based on which an integrated model was built and yielded the best performance. In the test set, the Dice score, F2 score, precision, and recall for the segmentation model were 0.80 [0.72-0.84], 0.80 [0.71-0.86], 0.84 [0.77-0.93], and 0.82 [0.69-0.89], respectively. For the diffuseness identification model, the ensemble-based model was applied with an area under the Receiver-operating characteristic curves (AUC) of 0.93 (95% CI 0.87-0.99) in the training set. The AUC, accuracy, precision, recall, and F1 score for the diffuseness identification model were 0.95, 0.90, 0.81, 0.84, and 0.83, respectively, in the test set. The ML models showed good performance in automatically detecting bAVM lesions and identifying diffuseness. The method may help to judge the diffuseness of bAVMs objectively, quantificationally, and efficiently.

Simplex cerebral cavernous malformations with MAP3K3 mutation have distinct clinical characteristics.

Objectives: To investigate the clinical characteristics of cerebral cavernous malformations (CCMs) with MAP3K3 somatic mutation. Methods: We performed a retrospective review of our CCMs database between May 2017 and December 2019. The patients with simplex CCMs identified to harbor a MAP3K3 or CCM gene somatic mutation were included. Clinical characteristics were recorded. Univariate and multivariate logistic analyses were used to assess the risk factors associated with hemorrhage events of CCMs. To explore the underlying mechanism, we transfected MEKK3-I441M-overexpressing and CCM2-knockdown lentiviruses into human umbilical vein endothelial cells (HUVECs) and investigated thrombomodulin (TM) and tight junctions (TJs) protein expression by western blotting and immunofluorescence. Finally, immunohistochemistry was used to validate TM and TJs protein expression in surgical samples. Results: Fifty simplex CCMs patients were included, comprising 38 MAP3K3 mutations and 12 CCM gene mutations. Nine (23.7%) patients with MAP3K3 mutations and 11(91.7%) patients with CCM gene mutations exhibited overt hemorrhage, respectively. Multivariate logistic analyses revealed that MAP3K3 mutation was associated with a lower risk of hemorrhage events. In the vitro experiments, ZO-1 expression was not reduced in MEKK3-I441M-overexpressing HUVECs compared with wild type, whereas it was significantly decreased in CCM2-knockdown HUVECs compared with control. In the MEKK3-I441M-overexpressing HUVECs, TM expression was increased, and the NF-κB pathway was significantly activated. After treatment with an NF-κB signaling inhibitor, TM expression was further upregulated. Meanwhile, TM expression was increased, but the NF-κB pathway was not activated in CCM2-knockdown HUVECs. Accordingly, immunohistochemistry showed that ZO-1 expression in the MAP3K3-mutant samples was significantly higher than that in the CCM-mutant samples. TM expression in the MAP3K3-mutant lesions was significantly lower than that in the CCM-mutant samples. Conclusion: Simplex CCMs with MAP3K3 mutation occasionally present with overt hemorrhage, which is associated with the biological function of MAP3K3 mutation.