Cerebral Cavernous Malformations Lesions Research Articles

Except for Robust Outliers, Rapamycin Increases Lesion Burden in a Murine Model of Cerebral Cavernous Malformations.

Cerebral cavernous malformation (CCM) is a hemorrhagic cerebrovascular disease where lesions develop in the setting of endothelial mutations of CCM genes, with many cases also harboring somatic PIK3CA gain of function (GOF) mutations. Rapamycin, an mTORC1 inhibitor, inhibited progression of murine CCM lesions driven by Ccm gene loss and Pik3ca GOF, but it remains unknown if rapamycin is beneficial in the absence of induction of Pik3ca GOF. We investigated the effect of rapamycin at three clinically relevant doses on lesion development in the Ccm3-/-PDGFb-icreERPositive murine model of familial CCM disease, without induction of Pik3ca GOF. Lesion burden, attrition, and acute and chronic hemorrhaging were compared between placebo and rapamycin-treated mice. Plasma miRNome was compared to identify potential biomarkers of rapamycin response. Outlier, exceptionally large CCM lesions (> 2 SD above the mean lesion burden) were exclusively observed in the placebo group. Rapamycin, across all dosages, may have prevented the emergence of large outlier lesions. Yet rapamycin also appeared to exacerbate mean lesion burden of surviving mice when outliers were excluded, increased attrition, and did not alter hemorrhage. miR-30c-2-3p, decreased in rapamycin-treated mouse plasma, has gene targets in PI3K/AKT and mTOR signaling. Progression of outlier lesions in a familial CCM model may have been halted by rapamycin treatment, at the potential expense of increased mean lesion burden and increased attrition. If confirmed, this can have implications for potential rapamycin treatment of familial CCM disease, where lesion development may not be driven by PIK3CA GOF. Further studies are necessary to determine specific pathways that mediate potential beneficial and detrimental effects of rapamycin treatment, and whether somatic PIK3CA mutations drive particularly aggressive lesions.

Mild Hypoxia Accelerates Cerebral Cavernous Malformation Disease Through CX3CR1-CX3CL1 Signaling.

Heterogeneity in the severity of cerebral cavernous malformations (CCMs) disease, including brain bleedings and thrombosis that cause neurological disabilities in patients, suggests that environmental, genetic, or biological factors act as disease modifiers. Still, the underlying mechanisms are not entirely understood. Here, we report that mild hypoxia accelerates CCM disease by promoting angiogenesis, neuroinflammation, and vascular thrombosis in the brains of CCM mouse models. We used genetic studies, RNA sequencing, spatial transcriptome, micro-computed tomography, fluorescence-activated cell sorting, multiplex immunofluorescence, coculture studies, and imaging techniques to reveal that sustained mild hypoxia via the CX3CR1-CX3CL1 (CX3C motif chemokine receptor 1/chemokine [CX3C motif] ligand 1) signaling pathway influences cell-specific neuroinflammatory interactions, contributing to heterogeneity in CCM severity. Histological and expression profiles of CCM neurovascular lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) in male and female mice found that sustained mild hypoxia (12% O2, 7 days) accelerates CCM disease. Our findings indicate that a small reduction in oxygen levels can significantly increase angiogenesis, neuroinflammation, and thrombosis in CCM disease by enhancing the interactions between endothelium, astrocytes, and immune cells. Our study indicates that the interactions between CX3CR1 and CX3CL1 are crucial in the maturation of CCM lesions and propensity to CCM immunothrombosis. In particular, this pathway regulates the recruitment and activation of microglia and other immune cells in CCM lesions, which leads to lesion growth and thrombosis. We found that human CX3CR1 variants are linked to lower lesion burden in familial CCMs, proving it is a genetic modifier in human disease and a potential marker for aggressiveness. Moreover, monoclonal blocking antibody against CX3CL1 or reducing 1 copy of the Cx3cr1 gene significantly reduces hypoxia-induced CCM immunothrombosis. Our study reveals that interactions between CX3CR1 and CX3CL1 can modify CCM neuropathology when lesions are accelerated by environmental hypoxia. Moreover, a hypoxic environment or hypoxia signaling caused by CCM disease influences the balance between neuroinflammation and neuroprotection mediated by CX3CR1-CX3CL1 signaling. These results establish CX3CR1 as a genetic marker for patient stratification and a potential predictor of CCM aggressiveness.

Hyaluronic acid turnover controls the severity of cerebral cavernous malformations in bioengineered human micro-vessels.

Cerebral cavernous malformations (CCMs) are vascular lesions that predominantly form in blood vessels of the central nervous system upon loss of the CCM multimeric protein complex. The endothelial cells within CCM lesions are characterized by overactive MEKK3 kinase and KLF2/4 transcription factor signaling, leading to pathological changes such as increased endothelial cell spreading and reduced junctional integrity. Concomitant to aberrant endothelial cell signaling, non-autonomous signals from the extracellular matrix (ECM) have also been implicated in CCM lesion growth and these factors might explain why CCM lesions mainly develop in the central nervous system. Here, we adapted a three-dimensional microfluidic system to examine CCM1 deficient human micro-vessels in distinctive extracellular matrices. We validate that pathological hallmarks are maintained in this model. We further show that key genes responsible for homeostasis of hyaluronic acid, a major extracellular matrix component of the central nervous system, are dysregulated in CCM. Supplementing the matrix in our model with distinct forms of hyaluronic acid inhibits pathological cell spreading and rescues barrier function. Hyaluronic acid acts by dampening cell-matrix adhesion signaling in CCM, either downstream or in parallel of KLF2/4. This study provides a proof-of-principle that ECM embedded 3D microfluidic models are ideally suited to identify how changes in ECM structure and signaling impact vascular malformations.

Transcriptomic signatures of individual cell types in cerebral cavernous malformation

Cerebral cavernous malformation (CCM) is a hemorrhagic neurovascular disease with no currently available therapeutics. Prior evidence suggests that different cell types may play a role in CCM pathogenesis. The contribution of each cell type to the dysfunctional cellular crosstalk remains unclear. Herein, RNA-seq was performed on fluorescence-activated cell sorted endothelial cells (ECs), pericytes, and neuroglia from CCM lesions and non-lesional brain tissue controls. Differentially Expressed Gene (DEG), pathway and Ligand-Receptor (LR) analyses were performed to characterize the dysfunctional genes of respective cell types within CCMs. Common DEGs among all three cell types were related to inflammation and endothelial-to-mesenchymal transition (EndMT). DEG and pathway analyses supported a role of lesional ECs in dysregulated angiogenesis and increased permeability. VEGFA was particularly upregulated in pericytes. Further pathway and LR analyses identified vascular endothelial growth factor A/ vascular endothelial growth factor receptor 2 signaling in lesional ECs and pericytes that would result in increased angiogenesis. Moreover, lesional pericytes and neuroglia predominantly showed DEGs and pathways mediating the immune response. Further analyses of cell specific gene alterations in CCM endorsed potential contribution to EndMT, coagulation, and a hypoxic microenvironment. Taken together, these findings motivate mechanistic hypotheses regarding non-endothelial contributions to lesion pathobiology and may lead to novel therapeutic targets.Ddgp6MUP9uoWsxMj-KUiCyVideo

Long-Term Outcomes of Gamma Knife Radiosurgery for Cerebral Cavernous Malformations: 10 Years and Beyond.

We aimed to evaluate long-term outcomes of gamma knife radiosurgery (GKS) for cerebral cavernous malformations (CCMs). Among the 233 CCM patients who underwent GKS, 79 adult patients (96 lesions) followed for over 10 years were included and analyzed retrospectively. Annual hemorrhage rate (AHR) was analyzed the entire cohort of 233 patients and the subset of 79 enrolled patients by dividing lesions into overall CCM lesions and brainstem lesions. AHR, neurologic outcome, adverse radiation effect (ARE), and changes of lesions in magnetic resonance imaging (MRI) were compared before and after GKS. Cox-regression analysis was performed to identify risk factors for hemorrhage following GKS. Mean follow-up duration of 79 enrolled patients was 14 years (range, 10-23 years). The AHR of all CCMs for entire cohort at each time point was 17.8% (pre-GKS), 5.9% (≤ 2 years post-GKS), 1.8% (≤ 10 years post-GKS). The AHR of all CCM for 79 enrolled patients was 21.4% (pre-GKS), 3.8% (2 years post-GKS), 1.4% (10 years post-GKS), and 2.3% (> 10 years post-GKS). The AHR of brainstem cavernous malformation (CM) for entire cohort at each time point was 22.4% (pre-GKS), 10.1% (≤ 2 years post-GKS), 3.2% (≤ 10 years post-GKS). The AHR of brainstem CM for 79 enrolled patients was 27.2% (pre-GKS), 5.8% (2 years post-GKS), 3.4% (10 years post-GKS), and 3.5% (> 10 years post-GKS). Out of the 79 enrolled patients, 35 presented with focal neurologic deficits at the initial clinical visit. Among these patients, 74.3% showed recovery at the last follow-up. Symptomatic ARE occurred in five (6.4%) patients. No mortality occurred. Most lesions were decreased in size at the last follow-up MRI. Previous hemorrhage history (hazard ratio [HR], 8.38; 95% confidence interval [CI], 1.07-65.88; P = 0.043), and brainstem location (HR, 3.10; 95% CI, 1.26-7.64; P = 0.014) were significant risk factors for hemorrhage event. GKS for CCM showed favorable long-term outcomes. GKS should be considered for CCM, especially when it has a previous hemorrhage history and brainstem location.

Single-nucleus DNA sequencing reveals hidden somatic loss-of-heterozygosity in Cerebral Cavernous Malformations

Cerebral Cavernous Malformations (CCMs) are vascular malformations of the central nervous system which can lead to moderate to severe neurological phenotypes in patients. A majority of CCM lesions are driven by a cancer-like three-hit mutational mechanism, including a somatic, activating mutation in the oncogene PIK3CA, as well as biallelic loss-of-function mutations in a CCM gene. However, standard sequencing approaches often fail to yield a full complement of pathogenic mutations in many CCMs. We suggest this reality reflects the limited sensitivity to identify low-frequency variants and the presence of mutations undetectable with bulk short-read sequencing. Here we report a single-nucleus DNA-sequencing approach that leverages the underlying biology of CCMs to identify lesions with somatic loss-of-heterozygosity, a class of such hidden mutations. We identify an alternative genetic mechanism for CCM pathogenesis and establish a method that can be repurposed to investigate the genetic underpinning of other disorders with multiple somatic mutations.

Role of Genetics and Surgical Interventions for the Management of Cerebral Cavernous Malformations (CMM).

Cerebral cavernous malformations (CCMs) are comprised of tissue matter within the brain possessing anomalous vascular architecture. In totality, the dilated appearance of the cavernomatakes on a mulberry-like shape contributed by the shape and relation to vascular and capillary elements. Analyzing its pathophysiology along with its molecular and genetic pathways plays a vital role in whether or not a patient receives GKRS, medical management, or Surgery, the most invasive of procedures. To avoid neurological trauma, microsurgical resection of cavernomas canbe guided by the novel clinical application of a 3D Slicer with Sina/MosoCam. When cavernomas present in deep lesions with poor accessibility, gamma knife stereotactic radiosurgery (GKSR) is recommended. For asymptomatic and non-multilobal lesions, medical and symptom management is deemed standard, such as antiepileptic therapy. The two-hit hypothesis serves to explain the mutations in three key genes that are most pertinent to the progression of cavernomas: CCM1/KRIT1, CCM2/Malcavernin, and CCM3/PDCD10. Various exon deletions and frameshift mutations can cause dysfunction in vascular structure through loss and gain of function mutations. MEKK3 and KLF2/4 are involved in a protein kinase signaling cycle that promotes abnormal angiogenesis and cavernoma formation. In terms of potential treatments, RhoKinase inhibitors have shown to decrease endothelial to mesenchymal transition and CCM lesion development in mice models. All in all, understanding the research behind the molecular genetics in CCMs can foster personalized medicine and potentially create new neurosurgical and medicative treatments.

Inflammatory Mechanisms in a Neurovascular Disease: Cerebral Cavernous Malformation.

Cerebral cavernous malformation (CCM) is a common cerebrovascular malformation causing intracranial hemorrhage, seizures, and focal neurologic deficits. A unique CCM lesional inflammatory microenvironment has been shown to influence the clinical course of the disease. This review addresses the inflammatory cell infiltrate in the CCM lesion and the role of a defined antigen-driven immune response in pathogenicity. We summarize immune mechanisms associated with the loss of the CCM gene and disease progression, including the potential role of immunothrombosis. We also review evidence of circulating inflammatory biomarkers associated with CCM disease and its clinical activity. We articulate future directions for this research, including the role of individual cell type contributions to the immune response in CCM, single cell transcriptomics of inflammatory cells, biomarker development, and therapeutic implications. The concepts are applicable for developing diagnostic and treatment strategies for CCM and for studying other neurovascular diseases.

Clinical characteristics of familial and sporadic pediatric cerebral cavernous malformations and outcomes.

Cerebral cavernous malformation (CCM) is a subtype of the vascular malformations found within the cerebral cortex. Although rare and usually discovered incidentally, these vascular abnormalities can predispose patients to spontaneous cerebral hemorrhage and subsequently lead to a myriad of neurological symptoms at presentation such as seizures and other focal neurological deficits. Although the symptoms and presentations of CCM have been adequately described in the adult population, disease characteristics and outcomes have not been extensively described in the pediatric population. Furthermore, the etiology of CCM-e.g., familial versus sporadic disease, as well as the risk factors for hemorrhage and neurological deficits and predictors of clinical and surgical outcomes-has not been adequately explored in the pediatric population. The current study attempts to classify and characterize differences in the clinical presentation, characteristics, and outcomes of CCMs between familial and sporadic cases within the pediatric population. A retrospective review identified 131 pediatric patients with radiographically confirmed diagnosis of CCM. All pertinent demographic and clinical variables were collected. CCM lesions were characterized using T2-weighted and susceptibility-weighted angiography (SWAN) MRI. Statistical analysis was conducted using the t-test for continuous variables, whereas categorical variables were analyzed with the Fisher exact test or chi-square test. Multivariate analysis was performed using a Cox proportional hazards model with R version 4.2.0. This retrospective study identified 131 pediatric CCM patients with a mean age of 8.4 years, and 54% (n = 71) were male. Twenty-seven percent (n = 35) were identified as cases with familial CCM, with the remainder classified as sporadic. The most common symptoms at presentation included generalized symptoms (headaches, nausea, and vomiting) or seizures, with a large proportion of patients also presenting as asymptomatic. No significant differences were observed in severity of symptoms between patients harboring different forms of the disease. Patients with familial CCM were noted to have a larger lesion size on average (5.26 cm3 vs 1.6 cm3, p = 0.047). These patients also had a shorter progression-free follow-up interval, with 50% of patients showing progression by 888 days, compared with only 15% of sporadic CCM patients during the same period (p = 0.0019). Familial etiology of the disease and larger average lesion volume were independent, significant predictors of disease progression (p = 0.001, HR 3.29, 95% CI 1.65-6.54) and future hemorrhage (p = 0.023, HR 1.1, 95% CI 1.01-1.10), respectively. Familial and sporadic CCMs tend to present with similar characteristics within the pediatric population. Patients with the familial form of the disease have an increased risk of progressive disease in terms of further hemorrhagic events.

Magnetic Resonance Imaging of Mouse Cerebral Cavernomas Reveal Differential Lesion Progression and Variable Permeability to Gadolinium.

Cerebral cavernous malformations, also known as cavernous angiomas, are blood vessel abnormalities comprised of clusters of grossly enlarged and hemorrhage-prone capillaries. The prevalence in the general population, including asymptomatic cases, is estimated to be 0.5%. Some patients develop severe symptoms, including seizures and focal neurological deficits, whereas others remain asymptomatic. The causes of this remarkable presentation heterogeneity within a primarily monogenic disease remain poorly understood. We established a chronic mouse model of cerebral cavernous malformations, induced by postnatal ablation of Krit1 with Pdgfb-CreERT2, and examined lesion progression in these mice with T2-weighted 7T magnetic resonance imaging (MRI). We also established a modified protocol for dynamic contrast-enhanced MRI and produced quantitative maps of gadolinium tracer gadobenate dimeglumine. After terminal imaging, brain slices were stained with antibodies against microglia, astrocytes, and endothelial cells. These mice develop cerebral cavernous malformations lesions gradually over 4 to 5 months of age throughout the brain. Precise volumetric analysis of individual lesions revealed nonmonotonous behavior, with some lesions temporarily growing smaller. However, the cumulative lesional volume invariably increased over time and after about 2 months followed a power trend. Using dynamic contrast-enhanced MRI, we produced quantitative maps of gadolinium in the lesions, indicating a high degree of heterogeneity in lesional permeability. MRI properties of the lesions were correlated with cellular markers for endothelial cells, astrocytes, and microglia. Multivariate comparisons of MRI properties of the lesions with cellular markers for endothelial and glial cells revealed that increased cell density surrounding lesions correlates with stability, whereas denser vasculature within and surrounding the lesions may correlate with high permeability. Our results lay a foundation for better understanding individual lesion properties and provide a comprehensive preclinical platform for testing new drug and gene therapies for controlling cerebral cavernous malformations.

Familial CCM Genes Might Not Be Main Drivers for Pathogenesis of Sporadic CCMs-Genetic Similarity between Cancers and Vascular Malformations.

Cerebral cavernous malformations (CCMs) are abnormally dilated intracranial capillaries that form cerebrovascular lesions with a high risk of hemorrhagic stroke. Recently, several somatic "activating" gain-of-function (GOF) point mutations in PIK3CA (phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit p110α) were discovered as a dominant mutation in the lesions of sporadic forms of cerebral cavernous malformation (sCCM), raising the possibility that CCMs, like other types of vascular malformations, fall in the PIK3CA-related overgrowth spectrum (PROS). However, this possibility has been challenged with different interpretations. In this review, we will continue our efforts to expound the phenomenon of the coexistence of gain-of-function (GOF) point mutations in the PIK3CA gene and loss-of-function (LOF) mutations in CCM genes in the CCM lesions of sCCM and try to delineate the relationship between mutagenic events with CCM lesions in a temporospatial manner. Since GOF PIK3CA point mutations have been well studied in reproductive cancers, especially breast cancer as a driver oncogene, we will perform a comparative meta-analysis for GOF PIK3CA point mutations in an attempt to demonstrate the genetic similarities shared by both cancers and vascular anomalies.

A feasibility study for quantitative assessment of cerebrovascular malformations using flutriciclamide ([18F]GE-180) PET/MRI.

Neuroinflammation plays a key role in both the pathogenesis and the progression of cerebral cavernous malformations (CCM). Flutriciclamide ([18F]GE-180) is a translocator protein (TSPO) targeting positron emission tomography (PET) tracer, developed for imaging neuroinflammation. The objectives of this study were to describe characteristics of flutriciclamide uptake in different brain tissue regions in CCM patients compared to controls, and to evaluate flutriciclamide uptake and iron deposition within CCM lesions. Five patients with CCM and six controls underwent a 60 or 90 min continuous PET/MRI scan following 315 ± 68.9 MBq flutriciclamide administration. Standardized uptake value (SUV) and standardized uptake value ratio (SUVr) were obtained using the striatum as a pseudo-reference. Quantitative susceptibility maps (QSM) were used to define the location of the vascular malformation and calculate the amount of iron deposition in each lesion. Increased flutriciclamide uptake was observed in all CCM lesions. The temporal pole demonstrated the highest radiotracer uptake; the paracentral lobule, cuneus and hippocampus exhibited moderate uptake; while the striatum had the lowest uptake, with average SUVs of 0.66, 0.55, 0.63, 0.55, and 0.33 for patient with CCM and 0.57, 0.50, 0.48, 0.42, and 0.32 for controls, respectively. Regional SUVr showed similar trends. The average SUV and QSM values in CCM lesions were 0.58 ± 0.23 g/ml and 0.30 ± 0.10 ppm. SUVs and QSM were positively correlated in CCM lesions (r = 0.53, p = 0.03). The distribution of flutriciclamide ([18F]GE-180) in the human brain and CCM lesions demonstrated the potential of this TSPO PET tracer as a marker of neuroinflammation that may be relevant for characterizing CCM disease progression along with QSM.

Impaired retinoic acid signaling in cerebral cavernous malformations

The capillary-venous pathology cerebral cavernous malformation (CCM) is caused by loss of CCM1/Krev interaction trapped protein 1 (KRIT1), CCM2/MGC4607, or CCM3/PDCD10 in some endothelial cells. Mutations of CCM genes within the brain vasculature can lead to recurrent cerebral hemorrhages. Pharmacological treatment options are urgently needed when lesions are located in deeply-seated and in-operable regions of the central nervous system. Previous pharmacological suppression screens in disease models of CCM led to the discovery that treatment with retinoic acid improved CCM phenotypes. This finding raised a need to investigate the involvement of retinoic acid in CCM and test whether it has a curative effect in preclinical mouse models. Here, we show that components of the retinoic acid synthesis and degradation pathway are transcriptionally misregulated across disease models of CCM. We complemented this analysis by pharmacologically modifying retinoic acid levels in zebrafish and human endothelial cell models of CCM, and in acute and chronic mouse models of CCM. Our pharmacological intervention studies in CCM2-depleted human umbilical vein endothelial cells (HUVECs) and krit1 mutant zebrafish showed positive effects when retinoic acid levels were increased. However, therapeutic approaches to prevent the development of vascular lesions in adult chronic murine models of CCM were drug regiment-sensitive, possibly due to adverse developmental effects of this hormone. A treatment with high doses of retinoic acid even worsened CCM lesions in an adult chronic murine model of CCM. This study provides evidence that retinoic acid signaling is impaired in the CCM pathophysiology and suggests that modification of retinoic acid levels can alleviate CCM phenotypes.

Release of STK24/25 suppression on MEKK3 signaling in endothelial cells confers cerebral cavernous malformation.

Loss-of-function mutations in cerebral cavernous malformation (CCM) genes and gain-of-function mutation in the MAP3K3 gene encoding MEKK3 cause CCM. Deficiency of CCM proteins leads to the activation of MEKK3-KLF2/4 signaling, but it is not clear how this occurs. Here, we demonstrate that deletion of the CCM3 interacting kinases STK24/25 in endothelial cells causes defects in vascular patterning during development as well as CCM lesion formation during postnatal life. While permanent deletion of STK24/25 in endothelial cells caused developmental defects of the vascular system, inducible postnatal deletion of STK24/25 impaired angiogenesis in the retina and brain. More importantly, deletion of STK24/25 in neonatal mice led to the development of severe CCM lesions. At the molecular level, a hybrid protein consisting of the STK kinase domain and the MEKK3 interacting domain of CCM2 rescued the vascular phenotype caused by the loss of ccm gene function in zebrafish. Our study suggests that CCM2/3 proteins act as adapters to allow recruitment of STK24/25 to limit the constitutive MEKK3 activity, thus contributing to vessel stability. Loss of STK24/25 causes MEKK3 activation, leading to CCM lesion formation.

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.

Clinical characteristics and risk factors of cerebral cavernous malformation-related epilepsy

PurposeThis study aimed to summarize the clinical characteristics and explore the risk factors for cerebral cavernous malformation (CCM)-related epilepsy (CRE). MethodsWe retrospectively analyzed the clinical data of patients with CCM in our cerebral vascular malformations database. Descriptive statistics were used to present the clinical characteristics of CRE patients. Patients were divided into a CRE and a non-CRE group according to clinical presentation. Binary logistic regression analysis was used to analyze the risk factors of CRE. ResultsA total of 199 patients with CCM confirmed by postoperative pathological examination were enrolled, 93 of whom were diagnosed with CRE, and 34 patients had drug-resistant epilepsy. The most common seizure type of CRE patients was focal to bilateral tonic-clonic seizure (FBTCS), followed by focal impaired awareness motor seizure. All CCM lesions were supratentorial, 97.8% of which involved the cerebral cortex, 86.0% of lesions had hemosiderin rim, and 50.5% of lesions were located in the temporal lobe. Binary logistic regression analysis indicated that CCM diagnosis age ≤ 44 years (odds ratio [OR] 2.79, p = 0.010), temporal lobe lesion location (OR = 9.07, p = 0.042), medial temporal lobe lesion (OR = 14.09, p = 0.002), cortical involvement of the lesion (OR = 32.77, p = 0.010), and hemosiderin rim around the lesion (OR = 16.48, p = 0.001) significantly increased the risk of CRE. ConclusionsThe most common seizure type of CRE was FBTCS. Those whose CCM diagnosis age was ≤ 44 years, having a temporal lobe lesion location, especially the medial temporal lobe lesion, cortical involvement, and hemosiderin rim around the lesion had a higher risk of developing CRE.

Distant Recurrence of a Cerebral Cavernous Malformation in the Vicinity of a Developmental Venous Anomaly: Case Report of Local Oxy-Inflammatory Events

Cerebral cavernous malformations (CCMs) are a major type of cerebrovascular lesions of proven genetic origin that occur in either sporadic (sCCM) or familial (fCCM) forms, the latter being inherited as an autosomal dominant condition linked to loss-of-function mutations in three known CCM genes. In contrast to fCCMs, sCCMs are rarely linked to mutations in CCM genes and are instead commonly and peculiarly associated with developmental venous anomalies (DVAs), suggesting distinct origins and common pathogenic mechanisms. A hemorrhagic sCCM in the right frontal lobe of the brain was surgically excised from a symptomatic 3 year old patient, preserving intact and pervious the associated DVA. MRI follow-up examination performed periodically up to 15 years after neurosurgery intervention demonstrated complete removal of the CCM lesion and no residual or relapse signs. However, 18 years after surgery, the patient experienced acute episodes of paresthesia due to a distant recurrence of a new hemorrhagic CCM lesion located within the same area as the previous one. A new surgical intervention was, therefore, necessary, which was again limited to the CCM without affecting the pre-existing DVA. Subsequent follow-up examination by contrast-enhanced MRI evidenced a persistent pattern of signal-intensity abnormalities in the bed of the DVA, including hyperintense gliotic areas, suggesting chronic inflammatory conditions. This case report highlights the possibility of long-term distant recurrence of hemorrhagic sCCMs associated with a DVA, suggesting that such recurrence is secondary to focal sterile inflammatory conditions generated by the DVA.

β1 integrin monoclonal antibody treatment ameliorates cerebral cavernous malformations.

β1 integrins are important in blood vessel formation and function, finely tuning the adhesion of endothelial cells to each other and to the extracellular matrix. The role of integrins in the vascular disease, cerebral cavernous malformation (CCM) has yet to be explored in vivo. Endothelial loss of the gene KRIT1 leads to brain microvascular defects, resulting in debilitating and often fatal consequences. We tested administration of a monoclonal antibody that enforces the active β1 integrin conformation, (clone 9EG7), on a murine neonatal CCM mouse model, Krit1flox/flox ;Pdgfb-iCreERT2 (Krit1ECKO ), and on KRIT1-silenced human umbilical vein endothelial cells (HUVECs). In addition, endothelial deletion of the master regulator of integrin activation, Talin 1 (Tln1), in Krit1ECKO mice was performed to assess the effect of completely blocking endothelial integrin activation on CCM. Treatment with 9EG7 reduced lesion burden in the Krit1ECKO model and was accompanied by a strong reduction in the phosphorylation of the ROCK substrate, myosin light chain (pMLC), in both retina and brain endothelial cells. Treatment of KRIT1-silenced HUVECs with 9EG7 in vitro stabilized cell-cell junctions. Overnight treatment of HUVECs with 9EG7 resulted in significantly reduced total surface expression of β1 integrin, which was associated with reduced pMLC levels, supporting our in vivo findings. Genetic blockade of integrin activation by Tln1ECKO enhanced bleeding and did not reduce CCM lesion burden in Krit1ECKO mice. In sum, targeting β1 integrin with an activated-specific antibody reduces acute murine CCM lesion development, which we found to be associated with suppression of endothelial ROCK activity.

Neuroinflammation Plays a Critical Role in Cerebral Cavernous Malformation Disease.

Cerebral cavernous malformations (CCMs) are neurovascular lesions caused by loss of function mutations in 1 of 3 genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3). CCMs affect ≈1 out of 200 children and adults, and no pharmacologic therapy is available. CCM lesion count, size, and aggressiveness vary widely among patients of similar ages with the same mutation or even within members of the same family. However, what determines the transition from quiescent lesions into mature and active (aggressive) CCM lesions is unknown. We use genetic, RNA-sequencing, histology, flow cytometry, and imaging techniques to report the interaction between CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils (CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils interaction) during the pathogenesis of CCMs in the brain tissue. Expression profile of astrocytes in adult mouse brains using translated mRNAs obtained from the purification of EGFP (enhanced green fluorescent protein)-tagged ribosomes (Aldh1l1-EGFP/Rpl10a) in the presence or absence of CCM lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) identifies a novel gene signature for neuroinflammatory astrocytes. CCM-induced reactive astrocytes have a neuroinflammatory capacity by expressing genes involved in angiogenesis, chemotaxis, hypoxia signaling, and inflammation. RNA-sequencing analysis on RNA isolated from brain endothelial cells in chronic Pdcd10BECKO mice (CCM endothelium), identified crucial genes involved in recruiting inflammatory cells and thrombus formation through chemotaxis and coagulation pathways. In addition, CCM endothelium was associated with increased expression of Nlrp3 and Il1b. Pharmacological inhibition of NLRP3 (NOD [nucleotide-binding oligomerization domain]-' LRR [leucine-rich repeat]- and pyrin domain-containing protein 3) significantly decreased inflammasome activity as assessed by quantification of a fluorescent indicator of caspase-1 activity (FAM-FLICA [carboxyfluorescein-fluorochrome-labeled inhibitors of caspases] caspase-1) in brain endothelial cells from Pdcd10BECKO in chronic stage. Importantly, our results support the hypothesis of the crosstalk between astrocytes and CCM endothelium that can trigger recruitment of inflammatory cells arising from brain parenchyma (microglia) and the peripheral immune system (leukocytes) into mature active CCM lesions that propagate lesion growth, immunothrombosis, and bleedings. Unexpectedly, partial or total loss of brain endothelial NF-κB (nuclear factor κB) activity (using Ikkbfl/fl mice) in chronic Pdcd10BECKO mice does not prevent lesion genesis or neuroinflammation. Instead, this resulted in a trend increase in the number of lesions and immunothrombosis, suggesting that therapeutic approaches designed to target inflammation through endothelial NF-κB inhibition may contribute to detrimental side effects. Our study reveals previously unknown links between neuroinflammatory astrocytes and inflamed CCM endothelium as contributors that trigger leukocyte recruitment and precipitate immunothrombosis in CCM lesions. However, therapeutic approaches targeting brain endothelial NF-κB activity may contribute to detrimental side effects.

Cerebral Cavernous Malformation: Immune and Inflammatory Perspectives.

Cerebral cavernous malformation (CCM) is a type of vascular anomaly that arises due to the dyshomeostasis of brain capillary networks. In the past two decades, many advances have been made in this research field. Notably, as a more reasonable current view, the CCM lesions should be attributed to the results of a great number of additional events related to the homeostasis disorder of the endothelial cell. Indeed, one of the most fascinating concerns in the research field is the inflammatory perturbation in the immune microenvironment, which would affect the disease progression as well as the patients’ outcomes. In this work, we focused on this topic, and underlined the immune-related factors’ contribution to the CCM pathologic progression.