Cerebral Cavernous Malformations Lesions Research Articles

Oxidative stress and inflammation in cerebral cavernous malformation disease pathogenesis: Two sides of the same coin

Cerebral Cavernous Malformation (CCM) is a vascular disease of proven genetic origin, which may arise sporadically or is inherited as an autosomal dominant condition with incomplete penetrance and highly variable expressivity. CCM lesions exhibit a range of different phenotypes, including wide inter-individual differences in lesion number, size, and susceptibility to intracerebral hemorrhage (ICH). Lesions may remain asymptomatic or result in pathological conditions of various type and severity at any age, with symptoms ranging from recurrent headaches to severe neurological deficits, seizures, and stroke. To date there are no direct therapeutic approaches for CCM disease besides the surgical removal of accessible lesions. Novel pharmacological strategies are particularly needed to limit disease progression and severity and prevent de novo formation of CCM lesions in susceptible individuals.Useful insights into innovative approaches for CCM disease prevention and treatment are emerging from a growing understanding of the biological functions of the three known CCM proteins, CCM1/KRIT1, CCM2 and CCM3/PDCD10. In particular, accumulating evidence indicates that these proteins play major roles in distinct signaling pathways, including those involved in cellular responses to oxidative stress, inflammation and angiogenesis, pointing to pathophysiological mechanisms whereby the function of CCM proteins may be relevant in preventing vascular dysfunctions triggered by these events. Indeed, emerging findings demonstrate that the pleiotropic roles of CCM proteins reflect their critical capacity to modulate the fine-tuned crosstalk between redox signaling and autophagy that govern cell homeostasis and stress responses, providing a novel mechanistic scenario that reconciles both the multiple signaling pathways linked to CCM proteins and the distinct therapeutic approaches proposed so far. In addition, recent studies in CCM patient cohorts suggest that genetic susceptibility factors related to differences in vascular sensitivity to oxidative stress and inflammation contribute to inter-individual differences in CCM disease susceptibility and severity.This review discusses recent progress into the understanding of the molecular basis and mechanisms of CCM disease pathogenesis, with specific emphasis on the potential contribution of altered cell responses to oxidative stress and inflammatory events occurring locally in the microvascular environment, and consequent implications for the development of novel, safe, and effective preventive and therapeutic strategies.

Vascular permeability and iron deposition biomarkers in longitudinal follow-up of cerebral cavernous malformations.

OBJECTIVE Vascular permeability and iron leakage are central features of cerebral cavernous malformation (CCM) pathogenesis. The authors aimed to correlate prospective clinical behavior of CCM lesions with longitudinal changes in biomarkers of dynamic contrast-enhanced quantitative permeability (DCEQP) and quantitative susceptibility mapping (QSM) assessed by MRI. METHODS Forty-six patients with CCMs underwent 2 or more permeability and/or susceptibility studies in conjunction with baseline and follow-up imaging and clinical surveillance during a mean 12.05 months of follow-up (range 2.4-31.27 months). Based on clinical and imaging features, cases/lesions were classified as stable, unstable, or recovering. Associated and predictive changes in quantitative permeability and susceptibility were investigated. RESULTS Lesional mean permeability and QSM values were not significantly different in stable versus unstable lesions at baseline. Mean lesional permeability in unstable CCMs with lesional bleeding or growth increased significantly (+85.9% change; p = 0.005), while mean permeability in stable and recovering lesions did not significantly change. Mean lesional QSM values significantly increased in unstable lesions (+44.1% change; p = 0.01), decreased slightly with statistical significance in stable lesions (-3.2% change; p = 0.003), and did not significantly change in recovering lesions. Familial cases developing new lesions during the follow-up period showed a higher background brain permeability at baseline (p = 0.001), as well as higher regional permeability (p = 0.003) in the area that would later develop a new lesion as compared with the homologous contralateral brain region. CONCLUSIONS In vivo assessment of vascular permeability and iron deposition on MRI can serve as objective and quantifiable biomarkers of disease activity in CCMs. This may be applied in natural history studies and may help calibrate clinical trials. The 2 techniques are likely applicable in other disorders of vascular integrity and iron leakage such as aging, hemorrhagic microangiopathy, and traumatic brain injury.

B-Cell Depletion Reduces the Maturation of Cerebral Cavernous Malformations in Murine Models.

Cerebral cavernous malformations (CCMs) are relatively common vascular malformations, characterized by increased Rho kinase (ROCK) activity, vascular hyper-permeability and the presence of blood degradation products including non-heme iron. Previous studies revealed robust inflammatory cell infiltration, selective synthesis of IgG, in situ antigen driven B-cell clonal expansion, and deposition of immune complexes and complement proteins within CCM lesions. We aimed to evaluate the impact of suppressing the immune response on the formation and maturation of CCM lesions, as well as lesional iron deposition and ROCK activity. Two murine models of heterozygous Ccm3 (Pdcd10), which spontaneously develop CCM lesions with severe and milder phenotypes, were either untreated or received anti-mouse BR3 to deplete B cells. Brains from anti-mouse BR3-treated mice exhibited significantly fewer mature CCM lesions and smaller lesions compared to untreated mice. B cell depletion halted the progression of lesions into mature stage 2 lesions but did not prevent their genesis. Non-heme iron deposition and ROCK activity was decreased in lesions of B cell depleted mice. This represents the first report of the therapeutic benefit of B-cell depletion in the development and progression of CCMs, and provides a proof of principle that B cells play a critical role in CCM lesion genesis and maturation. These findings add biologics to the list of potential therapeutic agents for CCM disease. Future studies would characterize the putative antigenic trigger and further define the mechanism of immune response in the lesions.

Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling.

Cerebral cavernous malformations (CCMs) are common inherited and sporadic vascular malformations that cause stroke and seizures in younger individuals1. CCMs arise from endothelial cell loss of KRIT1, CCM2, or PDCD10, non-homologous proteins that form an adaptor complex2. How disruption of the CCM complex results in disease remains controversial, with numerous signaling pathways (including Rho3,4, SMAD5 and Wnt/β-catenin6) and processes such as endothelial-mesenchymal transition (EndMT)5 proposed to play causal roles. CCM2 binds MEKK37–11, and we have recently demonstrated that CCM complex regulation of MEKK3 is essential during vertebrate heart development12. Here, we investigate this mechanism in CCM disease pathogenesis. Using a neonatal mouse model of CCM disease, we find that expression of the MEKK3 target genes KLF2 and KLF4, as well as Rho and ADAMTS protease activity, are increased in the endothelial cells of early CCM lesions. In contrast, we find no evidence of EndMT or increased SMAD or Wnt signaling during early CCM formation. Endothelial-specific loss of Mekk3, Klf2, or Klf4 dramatically prevents lesion formation, reverses the increase in Rho activity, and rescues lethality. Consistent with these findings in mice, we demonstrate that endothelial expression of KLF2 and KLF4 is elevated in human familial and sporadic CCM lesions, and that a disease-causing human CCM2 mutation abrogates MEKK3 interaction without affecting CCM complex formation. These studies identify gain of MEKK3 signaling and KLF2/4 function as causal mechanisms for CCM pathogenesis that may be targeted to develop new CCM therapeutics.

Quantitative Susceptibility Mapping in Cerebral Cavernous Malformations: Clinical Correlations.

Quantitative susceptibility mapping has been shown to assess iron content in cerebral cavernous malformations. In this study, our aim was to correlate lesional iron deposition assessed by quantitative susceptibility mapping with clinical and disease features in patients with cerebral cavernous malformations. Patients underwent routine clinical scans in addition to quantitative susceptibility mapping on 3T systems. Data from 105 patients met the inclusion criteria. Cerebral cavernous malformation lesions identified on susceptibility maps were cross-verified by T2-weighted images and differentiated on the basis of prior overt hemorrhage. Mean susceptibility per cerebral cavernous malformation lesion (χ̄lesion) was measured to correlate with lesion volume, age at scanning, and hemorrhagic history. Temporal rates of change in χ̄lesion were evaluated in 33 patients. Average χ̄lesion per patient was positively correlated with patient age at scanning (P < .05, 4.1% change with each decade of life). Cerebral cavernous malformation lesions with prior overt hemorrhages exhibited higher χ̄lesion than those without (P < .05). Changes in χ̄lesion during 3- to 15-month follow-up were small in patients without new hemorrhage between the 2 scans (bias = -0.0003; 95% CI, -0.06-0.06). The study revealed a positive correlation between mean quantitative susceptibility mapping signal and patient age in cerebral cavernous malformation lesions, higher mean quantitative susceptibility mapping signal in hemorrhagic lesions, and minimum longitudinal quantitative susceptibility mapping signal change in clinically stable lesions. Quantitative susceptibility mapping has the potential to be a novel imaging biomarker supplementing conventional imaging in cerebral cavernous malformations. The clinical significance of such measures merits further study.

Endothelial Cells Lining Sporadic Cerebral Cavernous Malformation Cavernomas Undergo Endothelial-to-Mesenchymal Transition.

Cerebral cavernous malformation (CCM) is characterized by multiple lumen vascular malformations in the central nervous system that can cause neurological symptoms and brain hemorrhages. About 20% of CCM patients have an inherited form of the disease with ubiquitous loss-of-function mutation in any one of 3 genes CCM1, CCM2, and CCM3. The rest of patients develop sporadic vascular lesions histologically similar to those of the inherited form and likely mediated by a biallelic acquired mutation of CCM genes in the brain vasculature. However, the molecular phenotypic features of endothelial cells in CCM lesions in sporadic patients are still poorly described. This information is crucial for a targeted therapy. We used immunofluorescence microscopy and immunohistochemistry to analyze the expression of endothelial-to-mesenchymal transition markers in the cavernoma of sporadic CCM patients in parallel with human familial cavernoma as a reference control. We report here that endothelial cells, a cell type critically involved in CCM development, undergo endothelial-to-mesenchymal transition in the lesions of sporadic patients. This switch in endothelial phenotype has been described only in genetic CCM patients and in murine models of the disease. In addition, TGF-β/p-Smad- and β-catenin-dependent signaling pathways seem activated in sporadic cavernomas as in familial ones. Our findings support the use of common therapeutic strategies for both sporadic and genetic CCM malformations.

Two cases of familial cerebral cavernous malformation caused by mutations in the CCM1 gene.

Cerebral cavernous malformation (CCM) is a vascular malformation characterized by abnormally enlarged capillary cavities without any intervening neural tissue. We report 2 cases of familial CCMs diagnosed with the CCM1 mutation by using a genetic assay. A 5-year-old boy presented with headache, vomiting, and seizure-like movements. Brain magnetic resonance imaging (MRI) revealed multiple CCM lesions in the cerebral hemispheres. Subsequent mutation analysis of his father and other family members revealed c.940_943 del (p.Val314 Asn315delinsThrfsX3) mutations of the CCM1 gene. A 10-month-old boy who presented with seizure-like movements was reported to have had no perinatal event. His aunt was diagnosed with cerebral angioma. Brain and spine MRI revealed multiple angiomas in the cerebral hemisphere and thoracic spinal cord. Mutation analysis of his father was normal, although that of the patient and his mother revealed c.535C>T (p.Arg179X) mutations of the CCM1 gene. Based on these studies, we suggest that when a child with a familial history of CCMs exhibits neurological symptoms, the physician should suspect familial CCMs and consider brain imaging or a genetic assay.

Defective autophagy is a key feature of cerebral cavernous malformations.

Cerebral cavernous malformation (CCM) is a major cerebrovascular disease affecting approximately 0.3–0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss-of-function mutations have been identified in three genes, KRIT1 (CCM1), CCM2 (MGC4607), and PDCD10 (CCM3), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/SQSTM1, defective quality control systems, and increased intracellular stress. KRIT1 loss-of-function activates the mTOR-ULK1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the mole-cular and cellular phenotypes associated with CCM. Insufficient autophagy is also evident in CCM2-silenced human endothelial cells and in both cells and tissues from an endothelial-specific CCM3-knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial-to-mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions.

Vascular permeability in cerebral cavernous malformations.

Patients with the familial form of cerebral cavernous malformations (CCMs) are haploinsufficient for the CCM1, CCM2, or CCM3 gene. Loss of corresponding CCM proteins increases RhoA kinase-mediated endothelial permeability in vitro, and in mouse brains in vivo. A prospective case-controlled observational study investigated whether the brains of human subjects with familial CCM show vascular hyperpermeability by dynamic contrast-enhanced quantitative perfusion magnetic resonance imaging, in comparison with CCM cases without familial disease, and whether lesional or brain vascular permeability correlates with CCM disease activity. Permeability in white matter far (WMF) from lesions was significantly greater in familial than in sporadic cases, but was similar in CCM lesions. Permeability in WMF increased with age in sporadic patients, but not in familial cases. Patients with more aggressive familial CCM disease had greater WMF permeability compared to those with milder disease phenotype, but similar lesion permeability. Subjects receiving statin medications for routine cardiovascular indications had a trend of lower WMF, but not lesion, permeability. This is the first demonstration of brain vascular hyperpermeability in humans with an autosomal dominant disease, as predicted mechanistically. Brain permeability, more than lesion permeability, may serve as a biomarker of CCM disease activity, and help calibrate potential drug therapy.

The Pathological Modifications of the Blood Brain Barrier and Cerebral Cavernous Malformations

Brain microvasculature constitutes a highly specialized and selective vascular barrier between blood and the central nervous system, called blood brain barrier (BBB). In these vessels endothelial cells present a highly developed system of tight junctions (TJs), absence of fenestration and low pinocytotic activity. Cells of the brain parenchyma, the astrocytes, contribute to the BBB-coverage with their foot processes, which constitute about the eighty percent of the basal aspect of the vesselsThree proteins, CCM1, CCM2 and CCM3, expressed by brain endothelial cells, are emerging as key modulators of the organization and function of the BBB. Indeed, mutations occurring in any of the genes encoding these proteins, leads to Cerebral Cavernous Malformation (CCM), a pathology characterized by brain vascular malformations. The endothelium in the lesions presents very few tight junctions and gaps are observed between endothelial cells. In addition, the vascular basal lamina is disorganized and the astrocytes do not take contact with the endothelial wall. The structural alterations of the BBB observed in CCM lesions are associated with severe clinical manifestations, such as cerebral hemorrhages and stroke. Additional data point to a possible link between inflammatory cytokines and the function of CCM proteins in the regulation of BBB stability. These data open new therapeutic opportunities for this so far incurable disease

Abstract T P231: B- Cell Depletion Decreases Lesion Burden in a Murine Model of Cerebral Cavernous Malformation Disease

Introduction: The cerebral cavernous malformation (CCM), a common cerebrovascular anomaly, predisposes patients to a lifetime risk of seizures, hemorrhagic stroke, and other neurological sequelae. Previous studies revealed robust inflammatory cell infiltration and selective synthesis of IgG within the lesions. Recently, in situ antigen-driven B- cell clonal expansion and immune complex deposits were demonstrated in CCM lesions. However, we do not know whether B cells indeed could play a role in CCM genesis and maturation. Therefore we hypothesize B-cell depletion could inhibit CCM development in a murine model of CCMs. Methods: Based on the two-hit hypothesis, we crossed Ccm3 heterozygotes into a Trp53 background, a mouse model that demonstrated 100% penetrance with severe lesion burden. Three weeks after birth, 5 CCM3+/-Trp53-/- mice were assigned into treatment group, receiving anti-mouse BR3 (Genentech) to deplete B cells with IP injection once a week (5mg/kg/week) for a total of 12-15 weeks. At 4 months of age, the mouse brains were harvested and fixed. B cell depletion was demonstrated by FACS analysis of peripheral blood and spleen cell suspensions, further confirmed in the spleens via immunohistochemistry. CCM lesion burden was assessed using either poisson regression (number) or student t test (area). Results: CD19+, CD22+, and CD45R/B220+ B cells of peripheral blood and spleen cell suspensions was significantly decreased in all 5 CCM3+/-Trp53-/- mice treated with anti-mouse BR3, as compared with those in the control group. B cell depletion was further confirmed in spleen. Anti-mouse BR3-treated mice exhibited a significantly lower CCM burden, including the number of ballooned capillaries (stage 1) and multicavernous lesions (stage 2), and the areas of stage 2 lesions per animal, as compared to 15 placebo mice. Infiltrated B cells in CCM3+/-Trp53-/- CCM lesions were also depleted. Conclusions: This represented the first report of therapeutic benefit of B- cell depletion therapy in the development and progression of CCMs and provide proof of principle that B cells is a critical step in CCM lesion genesis and maturation.

Abstract W P415: A Comparison between Fasudil and Simvastatin Treatment in Two Murine Models with Cerebral Cavernous Malformation

Objective: To compare therapeutic effect on cerebral cavernous malformation (CCM) genesis and maturation of fasudil (specific RhoA kinase inhibitor) and simvastatin (with pleiotropic effects including Rho inhibition) in two heterozygous murine models. Methods: The murine models Ccm1 +/- Msh2 -/- and Ccm2 +/- Trp53 -/- were treated from weaning to 5 months of age with fasudil (100 mg/kg/day administered in drinking water), simvastatin (40 mg/kg/day administered in chow) or with placebo. Brains were removed, fixed, embedded and blindly analyzed for lesion count and size, and for iron leak by Perls staining. Results: Fasudil, but not simvastatin, improved survival in Ccm1 +/- Msh2 -/- mice ( P =0.05). Both drugs did not affect attrition in Ccm2 +/- Trp53 -/- mice. Fasudil decreased mature CCM lesion burden and total lesional area per animal in Ccm1 +/- Msh2 -/- mice ( P =0.007, P =0.18, respectively), more in males ( P =0.04, P =0.05, respectively) than in females ( P =0.10, P =0.81, respectively) when compared to mice given placebo. In the same model, simvastatin decreased total lesion count less significantly ( P =0.09), with no decrease in lesional area. Integrated iron density per area of CCM lesions was decreased in mice treated with fasudil ( P &lt;0.01) or simvastatin ( P =0.09) compared to mice given placebo. In contrast, both fasudil and simvastatin treatment of Ccm2 +/- Trp53 -/- mice (with less aggressive phenotype) did not significantly affect attrition, lesion count or lesional area. No decrease in iron density was observed in Ccm2 +/- Trp53 -/- mice given fasudil. Conclusions: The ROCK inhibitor fasudil was more efficacious than simvastatin in the longitudinal treatment of Ccm1 +/- Msh2 -/- mice, decreasing lesion formation, maturation, and iron leak. The favorable, although weaker benefit of simvastatin, including decreased iron leak, would suggest a need for higher statin dose to accomplish a same effect as fasudil. There was no effect by either drug in Ccm2 +/- Trp53 -/- mice, likely in part because of milder phenotype in this model. Treatment with higher statin dose and effects on more aggressive Ccm3 +/ - models are being assessed in ongoing research.

Strategy for identifying repurposed drugs for the treatment of cerebral cavernous malformation.

Cerebral cavernous malformation (CCM) is a hemorrhagic stroke disease affecting up to 0.5% of North Americans that has no approved nonsurgical treatment. A subset of patients have a hereditary form of the disease due primarily to loss-of-function mutations in KRIT1, CCM2, or PDCD10. We sought to identify known drugs that could be repurposed to treat CCM. We developed an unbiased screening platform based on both cellular and animal models of loss of function of CCM2. Our discovery strategy consisted of 4 steps: an automated immunofluorescence and machine-learning-based primary screen of structural phenotypes in human endothelial cells deficient in CCM2, a secondary screen of functional changes in endothelial stability in these same cells, a rapid in vivo tertiary screen of dermal microvascular leak in mice lacking endothelial Ccm2, and finally a quaternary screen of CCM lesion burden in these same mice. We screened 2100 known drugs and bioactive compounds and identified 2 candidates, cholecalciferol (vitamin D3) and tempol (a scavenger of superoxide), for further study. Each drug decreased lesion burden in a mouse model of CCM vascular disease by ≈50%. By identifying known drugs as potential therapeutics for CCM, we have decreased the time, cost, and risk of bringing treatments to patients. Each drug also prompts additional exploration of biomarkers of CCM disease. We further suggest that the structure-function screening platform presented here may be adapted and scaled to facilitate drug discovery for diverse loss-of-function genetic vascular disease.

EHMTI-0257. Intractable refractory unilateral hemicrania with autonomic symptoms – a case study of an undiagnosed pontine cavernous malformation

Cerebral Cavernous malformations (CCM) occur in less than 1% of the general population, and one-third may have more than one lesion. Headaches alone may accompany CCM in up to 10% patients although micro-haemorrhage with accompanying headache is more common. A 67-year old male presented with a 15-year history of episodes of sudden onset left facial pain with dysesthesia in the fifth cranial nerve V1 distribution and left sided headache. The frequency of facial pain has increased with daily episodes of occurrence, exacerbated by movements. His headache impact score HIT-6 was 71. The patient’s family history is a sister died of a stroke, another from multiple sclerosis and a nephew died at 15-years-old of cerebral haemorrhage. On examination there were signs consistent with a brainstem lesion. Extensive scanning demonstrated no haemorrhage into any lesion and confirming multiple foci including a pontine lesion. Genetic testing confirmed a CCM2 mutation heterozygous deletion of two-nucleotides and 50% risk of inheritance of the c76_77del to family members. Pontine CCM can precipitate in migrainous-type headache and atypical facial nerve distribution symptoms due to disruption of the trigeminovascular system (TGVS). The Periaqueductal Grey area (PAG) is involved in modulating pain through both ascending and descending projections, the latters connections to the TGVS that inhibits nociceptive afferent information. CCM lesion is also disrupting the Nucleus Raphe Magnus (NRM), acting as a migraine generator. The diagnosis for the cause of this intractable facial pain and headache is the large pontine CCM lesion disinhibiting fibres of the TGVS. No conflict of interest.

Exceptional aggressiveness of cerebral cavernous malformation disease associated with PDCD10 mutations.

PurposeThe phenotypic manifestations of cerebral cavernous malformation (CCM) disease caused by rare PDCD10 mutations have not been systematically examined, and a mechanistic link to Rho kinase (ROCK) mediated hyperpermeability, a potential therapeutic target, has not been established.MethodsWe analyze PDCD10-siRNA treated endothelial cells for stress fibers, ROCK activity and permeability. ROCK activity is assessed in CCM lesions. Brain permeability and CCM lesion burden is quantified, and clinical manifestations are assessed in prospectively enrolled subjects with PDCD10 mutations.ResultsWe determine that PDCD10 protein suppresses endothelial stress fibers, ROCK activity and permeability in vitro. Pdcd10 heterozygous mice have greater lesion burden than other Ccm genotypes. We demonstrate robust ROCK activity in murine and human CCM vasculature, and increased brain vascular permeability in humans with PDCD10 mutation. Clinical phenotype is exceptionally aggressive compared to the more common KRIT1 and CCM2 familial and sporadic CCM, with greater lesion burden and more frequent hemorrhages earlier in life. We first report other phenotypic features including scoliosis, cognitive disability and skin lesions, unrelated to lesion burden or bleeding.ConclusionThese findings define a unique CCM disease with exceptional aggressiveness, and they inform preclinical therapeutic testing, clinical counseling and the design of trials.

Lack of CCM1 induces hypersprouting and impairs response to flow.

Cerebral cavernous malformation (CCM) is a disease of vascular malformations known to be caused by mutations in one of three genes: CCM1, CCM2 or CCM3. Despite several studies, the mechanism of CCM lesion onset remains unclear. Using a Ccm1 knockout mouse model, we studied the morphogenesis of early lesion formation in the retina in order to provide insight into potential mechanisms. We demonstrate that lesions develop in a stereotypic location and pattern, preceded by endothelial hypersprouting as confirmed in a zebrafish model of disease. The vascular defects seen with loss of Ccm1 suggest a defect in endothelial flow response. Taken together, these results suggest new mechanisms of early CCM disease pathogenesis and provide a framework for further study.

Evaluation of Iron Content in Human Cerebral Cavernous Malformation Using Quantitative Susceptibility Mapping

The aims of this study were to investigate and validate quantitative susceptibility mapping (QSM) for lesional iron quantification in cerebral cavernous malformations (CCMs). Magnetic resonance imaging studies were performed in phantoms and 16 patients on a 3-T scanner. Susceptibility weighted imaging, QSM, and R2* maps were reconstructed from in vivo data acquired with a 3-dimensional, multi-echo, and T2*-weighted gradient echo sequence. Magnetic susceptibility measurements were correlated to susceptibility weighted imaging and R2* results. In addition, iron concentrations from surgically excised CCM lesion specimens were determined using inductively coupled plasma mass spectrometry and correlated with QSM measurements. The QSM images demonstrated excellent image quality for depicting CCM lesions in both sporadic and familial cases. Susceptibility measurements revealed a positive linear correlation with R2* values (R(2) = 0.99 for total, R(2) = 0.69 for mean; P < 0.01). Quantitative susceptibility mapping values of known iron-rich brain regions matched closely with those of previous studies and in interobserver consistency. A strong correlation was found between QSM and the concentration of iron phantoms (0.925; P < 0.01), as well as between QSM and mass spectroscopy estimation of iron deposition (0.999 for total iron, 0.86 for iron concentration; P < 0.01) in 18 fragments of 4 excised human CCM lesion specimens. The ability of QSM to evaluate iron deposition in CCM lesions was illustrated via phantom, in vivo, and ex vivo validation studies. Quantitative susceptibility mapping may be a potential biomarker for monitoring CCM disease activity and response to treatments.

Immune complex formation and in situ B-cell clonal expansion in human cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) represent clusters of dilated vascular channels, predisposing to hemorrhagic stroke and seizures. They are associated with defective blood brain barrier, hemorrhages of different ages and a robust inflammatory cell infiltrate. We report for the first time evidence of co-localized IgG and complement membrane attack complexes in CCM lesions. CD4+ and CD8+ T-cells are aggregated with CD20+ B-cells. And IgG repertoire analyses demonstrate in situ B-cell clonal expansion and antigen-driven affinity maturation in CCMs. These results suggest an organ-intrinsic adaptive immune response in CCMs that should be further characterized as a potential therapeutic target.

Lesions from patients with sporadic cerebral cavernous malformations harbor somatic mutations in the CCM genes: evidence for a common biochemical pathway for CCM pathogenesis

Cerebral cavernous malformations (CCMs) are vascular lesions affecting the central nervous system. CCM occurs either sporadically or in an inherited, autosomal dominant manner. Constitutional (germline) mutations in any of three genes, KRIT1, CCM2 and PDCD10, can cause the inherited form. Analysis of CCM lesions from inherited cases revealed biallelic somatic mutations, indicating that CCM follows a Knudsonian two-hit mutation mechanism. It is still unknown, however, if the sporadic cases of CCM also follow this genetic mechanism. We extracted DNA from 11 surgically excised lesions from sporadic CCM patients, and sequenced the three CCM genes in each specimen using a next-generation sequencing approach. Four sporadic CCM lesion samples (36%) were found to contain novel somatic mutations. Three of the lesions contained a single somatic mutation, and one lesion contained two biallelic somatic mutations. Herein, we also describe evidence of somatic mosaicism in a patient presenting with over 130 CCM lesions localized to one hemisphere of the brain. Finally, in a lesion regrowth sample, we found that the regrown CCM lesion contained the same somatic mutation as the original lesion. Together, these data bolster the idea that all forms of CCM have a genetic underpinning of the two-hit mutation mechanism in the known CCM genes. Recent studies have found aberrant Rho kinase activation in inherited CCM pathogenesis, and we present evidence that this pathway is activated in sporadic CCM patients. These results suggest that all CCM patients, including those with the more common sporadic form, are potentially amenable to the same therapy.

MAGI1 signaling in cerebral cavernous malformations (664.11)

Cerebral Cavernous Malformations (CCMs) are vascular brain lesions affecting 0.5% of humans. Late stage lesions are clusters of grossly dilated capillaries lacking smooth muscle cell or pericyte support, and can cause hemorrhagic stroke. No treatment exists other than surgical removal of symptomatic lesions. CCMs occur sporadically through spontaneous mutations, or by familial autosomal dominant transmission of mutations in three genes : KRIT1, CCM2 or CCM3 (PDCD10). Our lab’s previous work showed that KRIT1 and CCM2 normally regulate endothelial permeability through regulation of RhoA and ROCK1/2. We now used proteomics and CHO cell co‐immunoprecipitations to identify a signalosome consisting of a unique brain microvascular endothelial cell‐specific splice variant of Rap1‐interacting junctional protein MAGI, “MAGI1C‐beta2”, that binds to KRIT1 and CCM2 through a PDZ domain to mediate translocation of two CCM proteins to endothelial junctions, and is required to maintain barrier function. We further identified a MAGI‐1 interacting protein , AMOT (angiomotin) as a convergence point for all three CCM protein signaling pathways, by showing that PDCD10 interacts directly with AMOT at junctional complex , while KRIT1 and CCM2 are localized there through MAGI1C‐beta2. Further, this intact CCM1‐2‐3/AMOT complex suppresses RhoA activation, providing a mechanistic explanation for some aspects of CCM lesion development.Grant Funding Source: NIH