Convergence of coronary artery disease genes onto endothelial cell programs.

Linking variants from genome-wide association studies (GWAS) to underlying mechanisms of disease remains a challenge1-3. For some diseases, a successful strategy has been to look for cases in which multiple GWAS loci contain genes that act in the same biological pathway1-6. However, our knowledge of which genes act in which pathways is incomplete, particularly for cell-type-specific pathways or understudied genes. Here we introduce a method to connect GWAS variants to functions. This method links variants to genes using epigenomics data, links genes to pathways de novo using Perturb-seq and integrates these data to identify convergence of GWAS loci onto pathways. We apply this approach to study the role of endothelial cells in genetic risk for coronary artery disease (CAD), and discover 43 CAD GWAS signals that converge on the cerebral cavernous malformation (CCM) signalling pathway. Two regulators of this pathway, CCM2 and TLNRD1, are each linked to a CAD risk variant, regulate other CAD risk genes and affect atheroprotective processes in endothelial cells. These results suggest a model whereby CAD risk is driven in part by the convergence of causal genes onto a particular transcriptional pathway in endothelial cells. They highlight shared genes between common and rare vascular diseases (CAD and CCM), and identify TLNRD1 as a new, previously uncharacterized member of the CCM signalling pathway. This approach will be widely useful for linking variants to functions for other common polygenic diseases.

PIK3CA and CCM mutations fuel cavernomas through a cancer-like mechanism.

Vascular malformations are thought to be monogenic disorders that result in dysregulated growth of blood vessels. In the brain, cerebral cavernous malformations (CCMs) arise owing to inactivation of the endothelial CCM protein complex, which is required to dampen the activity of the kinase MEKK31-4. Environmental factors can explain differences in the natural history of CCMs between individuals5, but why single CCMs often exhibit sudden, rapid growth, culminating in strokes or seizures, is unknown. Here we show that growth of CCMs requires increased signalling through the phosphatidylinositol-3-kinase (PI3K)-mTOR pathway as well as loss of function of the CCM complex. We identify somatic gain-of-function mutations in PIK3CA and loss-of-function mutations in the CCM complex in the same cells in a majority of human CCMs. Using mouse models, we show that growth of CCMs requires both PI3K gain of function and CCM loss of function in endothelial cells, and that both CCM loss of function and increased expression of the transcription factor KLF4 (a downstream effector of MEKK3) augment mTOR signalling in endothelial cells. Consistent with these findings, the mTORC1 inhibitor rapamycin effectively blocks the formation of CCMs in mouse models. We establish a three-hit mechanism analogous to cancer, in which aggressive vascular malformations arise through the loss of vascular 'suppressor genes' that constrain vessel growth and gain of a vascular 'oncogene' that stimulates excess vessel growth. These findings suggest that aggressive CCMs could be treated using clinically approved mTORC1 inhibitors.

mTORC1 Signaling in Brain Endothelial Progenitors Contributes to CCM Pathogenesis.

BACKGROUND: Cerebral vascular malformations (CCMs) are primarily found within the brain, where they result in increased risk for stroke, seizures, and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier formed by the brain neurovascular unit. Recent studies suggest that loss of CCM genes causes disruptions of blood-brain barrier integrity as the inciting events for CCM development. CCM lesions are proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling events in the subclass of brain ECs/EPCs for CCM lesion initiation and progression are unclear. METHODS: Brain EC-specific CCM3-deficient (Pdcd10BECKO) mice were generated by crossing Pdcd10fl/fl mice with Mfsd2a-CreERT2 mice. Single-cell RNA-sequencing analyses were performed by the chromium single-cell platform (10× genomics). Cell clusters were annotated into EC subtypes based on visual inspection and GO analyses. Cerebral vessels were visualized by 2-photon in vivo imaging and tissue immunofluorescence analyses. Regulation of mTOR (mechanistic target of rapamycin) signaling by CCM3 and Cav1 (caveolin-1) was performed by cell biology and biochemical approaches. RESULTS: Single-cell RNA-sequencing analyses from P10 Pdcd10BECKO mice harboring visible CCM lesions identified upregulated CCM lesion signature and mitotic EC clusters but decreased blood-brain barrier-associated EC clusters. However, a unique EPC cluster with high expression levels of stem cell markers enriched with mTOR signaling was identified from early stages of the P6 Pdcd10BECKO brain. Indeed, mTOR signaling was upregulated in both mouse and human CCM lesions. Genetic deficiency of Raptor (regulatory-associated protein of mTOR), but not of Rictor (rapamycin-insensitive companion of mTOR), prevented CCM lesion formation in the Pdcd10BECKO model. Importantly, the mTORC1 (mTOR complex 1) pharmacological inhibitor rapamycin suppressed EPC proliferation and ameliorated CCM pathogenesis in Pdcd10BECKO mice. Mechanistic studies suggested that Cav1/caveolae increased in CCM3-depleted EPC-mediated intracellular trafficking and complex formation of the mTORC1 signaling proteins. CONCLUSIONS: CCM3 is critical for maintaining blood-brain barrier integrity and CCM3 loss-induced mTORC1 signaling in brain EPCs initiates and facilitates CCM pathogenesis.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

Immunothrombosis and vascular heterogeneity in cerebral cavernous malformation.

Cerebral cavernous malformation (CCM) is a neurovascular disease that results in various neurological symptoms. Thrombi have been reported in surgically resected CCM patient biopsies, but the molecular signatures of these thrombi remain elusive. Here, we investigated the kinetics of thrombi formation in CCM and how thrombi affect the vasculature and contribute to cerebral hypoxia. We used RNA sequencing to investigate the transcriptome of mouse brain endothelial cells with an inducible endothelial-specific Ccm3 knock-out (Ccm3-iECKO). We found that Ccm3-deficient brain endothelial cells had a higher expression of genes related to the coagulation cascade and hypoxia when compared with wild-type brain endothelial cells. Immunofluorescent assays identified key molecular signatures of thrombi such as fibrin, von Willebrand factor, and activated platelets in Ccm3-iECKO mice and human CCM biopsies. Notably, we identified polyhedrocytes in Ccm3-iECKO mice and human CCM biopsies and report it for the first time. We also found that the parenchyma surrounding CCM lesions is hypoxic and that more thrombi correlate with higher levels of hypoxia. We created an in vitro model to study CCM pathology and found that human brain endothelial cells deficient for CCM3 expressed elevated levels of plasminogen activator inhibitor-1 and had a redistribution of von Willebrand factor. With transcriptomics, comprehensive imaging, and an in vitro CCM preclinical model, this study provides experimental evidence that genes and proteins related to the coagulation cascade affect the brain vasculature and promote neurological side effects such as hypoxia in CCMs. This study supports the concept that antithrombotic therapy may be beneficial for patients with CCM.

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. Video Abstract.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Somatic variants of MAP3K3 are sufficient to cause cerebral and spinal cord cavernous malformations.

Cerebral cavernous malformations (CCMs) and spinal cord cavernous malformations (SCCMs) are common vascular abnormalities of the CNS that can lead to seizure, haemorrhage and other neurological deficits. Approximately 85% of patients present with sporadic (versus congenital) CCMs. Somatic mutations in MAP3K3 and PIK3CA were recently reported in patients with sporadic CCM, yet it remains unknown whether MAP3K3 mutation is sufficient to induce CCMs. Here we analysed whole-exome sequencing data for patients with CCM and found that ∼40% of them have a single, specific MAP3K3 mutation [c.1323C>G (p.Ile441Met)] but not any other known mutations in CCM-related genes. We developed a mouse model of CCM with MAP3K3I441M uniquely expressed in the endothelium of the CNS. We detected pathological phenotypes similar to those found in patients with MAP3K3I441M. The combination of in vivo imaging and genetic labelling revealed that CCMs were initiated with endothelial expansion followed by disruption of the blood-brain barrier. Experiments with our MAP3K3I441M mouse model demonstrated that CCM can be alleviated by treatment with rapamycin, the mTOR inhibitor. CCM pathogenesis has usually been attributed to acquisition of two or three distinct genetic mutations involving the genes CCM1/2/3 and/or PIK3CA. However, our results demonstrate that a single genetic hit is sufficient to cause CCMs.

Plasma water T2 detects age-stratified differences in cardiometabolic health among familial CCM patients with Hispanic CCM1 mutation.

Cerebral cavernous malformations (CCMs) are abnormal clusters of capillaries in the nervous system. This pilot study analyzed the cardiometabolic health status of individuals with familial CCMs caused by a rare mutation in the CCM1 gene (fCCM1). The aim was to compare plasma water T2 values from individuals with fCCM1 with values from metabolically unhealthy and healthy individuals with no known CCM mutations. This observational, cross-sectional study included 75 participants: 11 fCCM1 patients, 24 metabolically unhealthy and 40 metabolically healthy individuals. Plasma water T2, an early, global and practical marker of cardiometabolic health, was measured in the time domain using benchtop magnetic resonance relaxometry. The results were stratified by age (equal to or less than 45 vs. older than 45 years). Group means were compared using Welch's one-way ANOVA and post hoc Tukey-Kramer tests. Multivariable linear regression, with T2 as the outcome variable, was used to explore associations with age, gender, Hispanic ethnicity and fCCM1 status. In the younger age stratum, the fCCM1 group had a mean plasma water T2 value comparable to the metabolically healthy group (p = 0.6388), but higher than the unhealthy group (p < 0.0001). By contrast, in the older stratum, the mean plasma water T2 value for the fCCM1 group was comparable to the metabolically unhealthy group (p = 0.7819) and lower than the healthy group (p = 0.0005). Multivariable linear regression revealed that age and the interaction between age and fCCM1 status were significant predictors of T2, even after adjusting for gender and Hispanic ethnicity. Plasma water T2 shows potential as a biomarker for assessing the health status of individuals with fCCM1. Further research is needed to validate these preliminary observations and elucidate the association between CCMs and cardiometabolic health.

mTORC1 Inhibitor Rapamycin Inhibits Growth of Cerebral Cavernous Malformation in Adult Mice.

BACKGROUND: Cerebral cavernous malformations (CCMs) are vascular malformations that frequently cause stroke. CCMs arise due to loss of function in one of the genes that encode the CCM complex, a negative regulator of MEKK3-KLF2/4 signaling in vascular endothelial cells. Gain-of-function mutations in PIK3CA (encoding the enzymatic subunit of the PI3K (phosphoinositide 3-kinase) pathway associated with cell growth) synergize with CCM gene loss-of-function to generate rapidly growing lesions. METHODS: We recently developed a model of CCM formation that closely reproduces key events in human CCM formation through inducible CCM loss-of-function and PIK3CA gain-of-function in mature mice. In the present study, we use this model to test the ability of rapamycin, a clinically approved inhibitor of the PI3K effector mTORC1, to treat rapidly growing CCMs. RESULTS: We show that both intraperitoneal and oral administration of rapamycin arrests CCM growth, reduces perilesional iron deposition, and improves vascular perfusion within CCMs. CONCLUSIONS: Our findings further establish this adult CCM model as a valuable preclinical model and support clinical testing of rapamycin to treat rapidly growing human CCMs.

A novel KRIT1/CCM1 mutation accompanied by a NOTCH3 mutation in a Chinese family with multiple cerebral cavernous malformations.

Family cerebral cavernous malformations (FCCMs) are mainly inherited through the mutation of classical CCM genes, including CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10. FCCMs can cause severe clinical symptoms, including epileptic seizures, intracranial hemorrhage (ICH), or functional neurological deficits (FNDs). In this study, we reported a novel mutation in KRIT1 accompanied by a NOTCH3 mutation in a Chinese family. This family consists of 8 members, 4 of whom had been diagnosed with CCMs using cerebral MRI (T1WI, T2WI, SWI). The proband (II-2) and her daughter (III-4) had intracerebral hemorrhage and refractory epilepsy, respectively. Based on whole-exome sequencing (WES) data and bioinformatics analysis from 4 patients with multiple CCMs and 2 normal first-degree relatives, a novel KRIT1 mutation, NG_012964.1 (NM_194456.1): c.1255-1G > T (splice-3), in intron 13 was considered a pathogenic gene in this family. Furthermore, based on 2 severe and 2 mild CCM patients, we found an SNV missense mutation, NG_009819.1 (NM_000435.2): c.1630C > T (p.R544C), in NOTCH3. Finally, the KRIT1 and NOTCH3 mutations were validated in 8 members using Sanger sequencing. This study revealed a novel KRIT1 mutation, NG_012964.1 (NM_194456.1): c.1255-1G > T (splice-3), in a Chinese CCM family, which had not been reported previously. Moreover, the NOTCH3 mutation NG_009819.1 (NM_000435.2): c.1630C > T (p.R544C) might be a second hit and associated with the progression of CCM lesions and severe clinical symptoms.

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.

Genetics of brain arteriovenous malformations and cerebral cavernous malformations.

Cerebrovascular malformations comprise abnormal development of cerebral vasculature. They can result in hemorrhagic stroke due to rupture of lesions as well as seizures and neurological defects. The most common forms of cerebrovascular malformations are brain arteriovenous malformations (bAVMs) and cerebral cavernous malformations (CCMs). They occur in both sporadic and inherited forms. Rapidly evolving molecular genetic methodologies have helped to identify causative or associated genes involved in genesis of bAVMs and CCMs. In this review, we highlight the current knowledge regarding the genetic basis of these malformations.

Discovery and Characterization of Ephrin B2 and EphB4 Dysregulation and Novel Mutations in Cerebral Cavernous Malformations: In Vitro and Patient-Derived Evidence of Ephrin-Mediated Endothelial Cell Pathophysiology.

Intracranial vascular malformations manifest on a continuum ranging from predominantly arterial to predominantly venous in pathology. Cerebral cavernous malformations (CCMs) are capillary malformations that exist at the midpoint of this continuum. The axon guidance factor Ephrin B2 and its receptor EphB4 are critical regulators of vasculogenesis in the developing central nervous system. Ephrin B2/EphB4 dysregulation has been implicated in the pathogenesis of arterial-derived arteriovenous malformations and vein-based vein of Galen malformations. Increasing evidence supports the hypothesis that aberrant Ephrin B2/EphB4 signaling may contribute to developing vascular malformations, but their role in CCMs remains largely uncharacterized. Evidence of Ephrin dysregulation in CCMs would be important to establish a common link in the pathogenic spectrum of EphrinB2/Ephb4 dysregulation. By studying patient-derived primary CCM endothelial cells (CCMECs), we established that CCMECs are functionally distinct from healthy endothelial cell controls; CCMECs demonstrated altered patterns of migration, motility, and impaired tube formation. In addition to the altered phenotype, the CCMECs also displayed an increased ratio of EphrinB2/EphB4 compared to the healthy endothelial control cells. Furthermore, whole exome sequencing identified mutations in both EphrinB2 and EphB4 in the CCMECs. These findings identify functional alterations in the EphrinB2/EphB4 ratio as a feature linking pathophysiology across the spectrum of arterial, capillary, and venous structural malformations in the central nervous system while revealing a putative therapeutic target.

Targeting miR-27a/VE-cadherin interactions rescues cerebral cavernous malformations in mice.

Cerebral cavernous malformations (CCMs) are vascular lesions predominantly developing in the central nervous system (CNS), with no effective treatments other than surgery. Loss-of-function mutation in CCM1/krev interaction trapped 1 (KRIT1), CCM2, or CCM3/programmed cell death 10 (PDCD10) causes lesions that are characterized by abnormal vascular integrity. Vascular endothelial cadherin (VE-cadherin), a major regulator of endothelial cell (EC) junctional integrity is strongly disorganized in ECs lining the CCM lesions. We report here that microRNA-27a (miR-27a), a negative regulator of VE-cadherin, is elevated in ECs isolated from mouse brains developing early CCM lesions and in cultured ECs with CCM1 or CCM2 depletion. Furthermore, we show miR-27a acts downstream of kruppel-like factor (KLF)2 and KLF4, two known key transcription factors involved in CCM lesion development. Using CD5-2 (a target site blocker [TSB]) to prevent the miR-27a/VE-cadherin mRNA interaction, we present a potential therapy to increase VE-cadherin expression and thus rescue the abnormal vascular integrity. In CCM1- or CCM2-depleted ECs, CD5-2 reduces monolayer permeability, and in Ccm1 heterozygous mice, it restores dermal vessel barrier function. In a neonatal mouse model of CCM disease, CD5-2 normalizes vasculature and reduces vascular leakage in the lesions, inhibits the development of large lesions, and significantly reduces the size of established lesions in the hindbrain. Furthermore, CD5-2 limits the accumulation of inflammatory cells in the lesion area. Our work has established that VE-cadherin is a potential therapeutic target for normalization of the vasculature and highlights that targeting miR-27a/VE-cadherin interaction by CD5-2 is a potential novel therapy for the devastating disease, CCM.

Cerebral Cavernous Malformation: From Mechanism to Therapy.

Cerebral cavernous malformations are acquired vascular anomalies that constitute a common cause of central nervous system hemorrhage and stroke. The past 2 decades have seen a remarkable increase in our understanding of the pathogenesis of this vascular disease. This new knowledge spans genetic causes of sporadic and familial forms of the disease, molecular signaling changes in vascular endothelial cells that underlie the disease, unexpectedly strong environmental effects on disease pathogenesis, and drivers of disease end points such as hemorrhage. These novel insights are the integrated product of human clinical studies, human genetic studies, studies in mouse and zebrafish genetic models, and basic molecular and cellular studies. This review addresses the genetic and molecular underpinnings of cerebral cavernous malformation disease, the mechanisms that lead to lesion hemorrhage, and emerging biomarkers and therapies for clinical treatment of cerebral cavernous malformation disease. It may also serve as an example for how focused basic and clinical investigation and emerging technologies can rapidly unravel a complex disease mechanism.

Natural history of familial cerebral cavernous malformation syndrome in children: a multicenter cohort study.

PURPOSE: There is limited data concerning neuroimaging findings and longitudinal evaluation of familial cerebral cavernous malformations (FCCM) in children. Our aim was to study the natural history of pediatric FCCM, with an emphasis on symptomatic hemorrhagic events and associated clinical and imaging risk factors. METHODS: We retrospectively reviewed all children diagnosed with FCCM in four tertiary pediatric hospitals between January 2010 and March 2022. Subjects with first available brain MRI and [Formula: see text] 3 months of clinical follow-up were included. Neuroimaging studies were reviewed, and clinical data collected. Annual symptomatic hemorrhage risk rates and cumulative risks were calculated using survival analysis and predictors of symptomatic hemorrhagic identified using regression analysis. RESULTS: Forty-one children (53.7% males) were included, of whom 15 (36.3%) presenting with symptomatic hemorrhage. Seven symptomatic hemorrhages occurred during 140.5 person-years of follow-up, yielding a 5-year annual hemorrhage rate of 5.0% per person-year. The 1-, 2-, and 5-year cumulative risks of symptomatic hemorrhage were 7.3%, 14.6%, and 17.1%, respectively. The latter was higher in children with prior symptomatic hemorrhage (33.3%), CCM2 genotype (33.3%), and positive family history (20.7%). Number of brainstem (adjusted hazard ratio [HR] = 1.37, P = 0.005) and posterior fossa (adjusted HR = 1.64, P = 0.004) CCM at first brain MRI were significant independent predictors of prospective symptomatic hemorrhage. CONCLUSION: The 5-year annual and cumulative symptomatic hemorrhagic risk in our pediatric FCCM cohort equals the overall risk described in children and adults with all types of CCM. Imaging features at first brain MRI may help to predict potential symptomatic hemorrhage at 5-year follow-up.

Contact-dependent signaling triggers tumor-like proliferation of CCM3 knockout endothelial cells in co-culture with wild-type cells.

Cerebral cavernous malformations (CCM) are low-flow vascular lesions prone to cause severe hemorrhage-associated neurological complications. Pathogenic germline variants in CCM1, CCM2, or CCM3 can be identified in nearly 100% of CCM patients with a positive family history. In line with the concept that tumor-like mechanisms are involved in CCM formation and growth, we here demonstrate an abnormally increased proliferation rate of CCM3-deficient endothelial cells in co-culture with wild-type cells and in mosaic human iPSC-derived vascular organoids. The observation that NSC59984, an anticancer drug, blocked the abnormal proliferation of mutant endothelial cells further supports this intriguing concept. Fluorescence-activated cell sorting and RNA sequencing revealed that co-culture induces upregulation of proangiogenic chemokine genes in wild-type endothelial cells. Furthermore, genes known to be significantly downregulated in CCM3-/- endothelial cell mono-cultures were upregulated back to normal levels in co-culture with wild-type cells. These results support the hypothesis that wild-type ECs facilitate the formation of a niche that promotes abnormal proliferation of mutant ECs. Thus, targeting the cancer-like features of CCMs is a promising new direction for drug development.

Inflammation and neutrophil extracellular traps in cerebral cavernous malformation.

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 (Ccm3iECKO), we show that endothelial cells from Ccm3iECKO 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 Ccm3iECKO 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 Ccm3iECKO 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.