Hypoxic microenvironment-induced exosomes confer temozolomide resistance in glioma through transfer of pyruvate kinase M2.

OBJECTIVE: Glioma, a malignant primary brain tumor, is notorious for its high incidence rate. However, the clinical application of temozolomide (TMZ) as a treatment option for glioma is often limited due to resistance, which has been linked to hypoxic glioma cell-released exosomes. In light of this, the present study aimed to investigate the role of exosomal pyruvate kinase M2 (PKM2) in glioma cells that exhibit resistance to TMZ. METHODS: Sensitive and TMZ-resistant glioma cells were subjected to either a normoxic or hypoxic environment, and the growth patterns and enzymatic activity of glycolysis enzymes were subsequently measured. From these cells, exosomal PKM2 was isolated and the subsequent effect on TMZ resistance was examined and characterized, with a particular focus on understanding the relevant mechanisms. Furthermore, the intercellular communication between hypoxic resistant cells and tumor-associated macrophages (TAMs) via exosomal PKM2 was also assessed. RESULTS: The adverse impact of hypoxic microenvironments on TMZ resistance in glioma cells was identified and characterized. Among the three glycolysis enzymes that were examined, PKM2 was found to be a critical mediator in hypoxia-triggered TMZ resistance. Upregulation of PKM2 was found to exacerbate the hypoxia-mediated TMZ resistance. Exosomal PKM2 were identified and isolated from hypoxic TMZ-resistant glioma cells, and were found to be responsible for transmitting TMZ resistance to sensitive glioma cells. The exosomal PKM2 also contributed towards mitigating TMZ-induced apoptosis in sensitive glioma cells, while also causing intracellular ROS accumulation. Additionally, hypoxic resistant cells also released exosomal PKM2, which facilitated TMZ resistance in tumor-associated macrophages. CONCLUSION: In the hypoxic microenvironment, glioma cells become resistant to TMZ due to the delivery of PKM2 by exosomes. Targeted modulation of exosomal PKM2 may be a promising strategy for overcoming TMZ resistance in glioma.

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.

Cerebral cavernous malformation development in chronic mouse models driven by dual recombinases induced gene deletion in brain endothelial cells.

Cerebral cavernous malformation (CCM) is a brain vascular disease which can cause stroke, cerebral hemorrhage and neurological deficits in affected individuals. Loss-of-function mutations in three genes (CCM1, CCM2 and CCM3) cause CCM disease. Multiple mouse models for CCM disease have been developed although each of them are associated with various limitations. Here, we employed the Dre-Cre dual recombinase system to specifically delete Ccm genes in brain endothelial cells. In this new series of CCM mouse models, robust CCM lesions now develop in the cerebrum. The survival curve and lesion burden analysis revealed that Ccm2 deletion causes modest CCM lesions with a median life expectance of ∼10 months and Ccm3 gene deletion leads to the most severe CCM lesions with median life expectance of ∼2 months. The extended lifespan of these mutant mice enables their utility in behavioral analyses of neurologic deficits in adult mice, and allow the development of methods to quantify lesion burden in mice over time and also permit longitudinal drug testing in live animals.

Inhibitory effects of everolimus in combination with paclitaxel on adriamycin-resistant breast cancer cell line MDA-MB-231.

OBJECTIVE: We aimed to evaluate the therapeutic effects of paclitaxel in combination with mTOR inhibitor everolimus on adriamycin-resistant breast cancer cell line MDA-MB-231 (MDA-MB-231/ADR). MATERIALS AND METHODS: MDA-MB-231/ADR cells were treated with different concentrations of paclitaxel and everolimus. The IC50 values after 48 h of treatment were measured by the MTT assay. The apoptosis rate and cell cycle were detected by flow cytometry. The protein expressions of Akt, PI3K, mTOR, p-pI3K, p-AKT and p-mTOR were detected by Western blot. RESULTS: When paclitaxel at ≥1.56 µg/ml was used, the growth of MDA-MB-231/ADR cells was inhibited more significantly than that of control group (P < 0.05). After treatment with ≥6.25 µg/ml everolimus, the cell growth was also suppressed more significantly (P < 0.05). The IC50 values of everolimus and paclitaxel were 32.50 µg/ml and 7.80 µg/ml, respectively. The inhibition rate of paclitaxel plus everolimus was significantly enhanced with increasing paclitaxel concentration (P < 0.001). After treatment with 7.80 µg/ml paclitaxel, the two drugs had best synergistic inhibitory effects on proliferation. Compared with drugs alone, the combination significantly promoted apoptosis (P < 0.001). The paclitaxel + everolimus group had significantly more cells in the G0-G1 phase than those of control and individual drug groups (P < 0.001). Everolimus significantly decreased mTOR and p-mTOR expressions compared with those of control group (P < 0.001). Compared with everolimus alone, the combination reduced the expressions more significantly (P < 0.05). Paclitaxel decreased the expression levels of PI3K, p-PI3K and p-AKT. Compared with paclitaxel alone, the combination significantly promoted the reduction of PI3K, p-PI3K and p-AKT expressions (P < 0.05). CONCLUSION: Everolimus can enhance the effect of paclitaxel on MDA-MB-231/ADR cells, inhibit cell proliferation, induce apoptosis and arrest cell cycle in the G1 phase mainly by down-regulating the expressions of key proteins in the mTOR signaling pathway.

Antibodies in cerebral cavernous malformations react with cytoskeleton autoantigens in the lesional milieu.

Previous studies have reported robust inflammatory cell infiltration, synthesis of IgG, B-cell clonal expansion, deposition of immune complexes and complement within cerebral cavernous malformation (CCM) lesions. B-cell depletion has also been shown to reduce the maturation of CCM in murine models. We hypothesize that antigen(s) within the lesional milieu perpetuate the pathogenetic immune responses in CCMs. This study aims to identify those putative antigen(s) using monoclonal antibodies (mAbs) derived from plasma cells found in surgically removed human CCM lesions. We produced human mAbs from laser capture micro-dissected plasma cells from four CCM patients, and also germline-reverted versions. CCM mAbs were assayed using immunofluorescence on central nervous system (CNS) tissues and immunocytochemistry on human primary cell lines. Antigen characterization was performed using a combination of confocal microscopy, immunoprecipitation and mass spectrometry. Affinity was determined by enzyme-linked immunosorbent assay, and specificity by multi-color confocal microscopy and quantitative co-localization. CCM mAbs bound CNS tissue, especially endothelial cells and astrocytes. Non-muscle myosin heavy chain IIA (NMMHCIIA), vimentin and tubulin are three cytoskeleton proteins that were commonly targeted. Selection of cytoskeleton proteins by plasma cells was supported by a high frequency of immunoglobulin variable region somatic hypermutations, high affinity and selectivity of mAbs in their affinity matured forms, and profoundly reduced affinity and selectivity in the germline reverted forms. Antibodies produced by plasma cells in CCM lesions commonly target cytoplasmic and cytoskeletal autoantigens including NMMHCIIA, vimentin and tubulin that are abundant in endothelial cells and astrocytes. Binding to, and selection on autoantigen(s) in the lesional milieu likely perpetuates the pathogenetic immune response in CCMs. Blocking this in situ autoimmune response may yield a novel treatment for CCM.

Transcriptome clarifies mechanisms of lesion genesis versus progression in models of Ccm3 cerebral cavernous malformations.

Cerebral cavernous malformations (CCMs) are dilated capillaries causing epilepsy and stroke. Inheritance of a heterozygous mutation in CCM3/PDCD10 is responsible for the most aggressive familial form of the disease. Here we studied the differences and commonalities between the transcriptomes of microdissected lesional neurovascular units (NVUs) from acute and chronic in vivo Ccm3/Pdcd10ECKO mice, and cultured brain microvascular endothelial cells (BMECs) Ccm3/Pdcd10ECKO.We identified 2409 differentially expressed genes (DEGs) in acute and 2962 in chronic in vivo NVUs compared to microdissected brain capillaries, as well as 121 in in vitro BMECs with and without Ccm3/Pdcd10 loss (fold change ≥ |2.0|; p < 0.05, false discovery rate corrected). A functional clustered dendrogram generated using the Euclidean distance showed that the DEGs identified only in acute in vivo NVUs were clustered in cellular proliferation gene ontology functions. The DEGs only identified in chronic in vivo NVUs were clustered in inflammation and immune response, permeability, and adhesion functions. In addition, 1225 DEGs were only identified in the in vivo NVUs but not in vitro BMECs, and these clustered within neuronal and glial functions. One miRNA mmu-miR-3472a was differentially expressed (FC = - 5.98; p = 0.07, FDR corrected) in the serum of Ccm3/Pdcd10+/- when compared to wild type mice, and this was functionally related as a putative target to Cand2 (cullin associated and neddylation dissociated 2), a DEG in acute and chronic lesional NVUs and in vitro BMECs. Our results suggest that the acute model is characterized by cell proliferation, while the chronic model showed inflammatory, adhesion and permeability processes. In addition, we highlight the importance of extra-endothelial structures in CCM disease, and potential role of circulating miRNAs as biomarkers of disease, interacting with DEGs. The extensive DEGs library of each model will serve as a validation tool for potential mechanistic, biomarker, and therapeutic targets.

Biomarkers of cavernous angioma with symptomatic hemorrhage.

BACKGROUNDCerebral cavernous angiomas (CAs) with a symptomatic hemorrhage (CASH) have a high risk of recurrent hemorrhage and serious morbidity.METHODSEighteen plasma molecules with mechanistic roles in CA pathobiology were investigated in 114 patients and 12 healthy subjects. The diagnostic biomarker of a CASH in the prior year was derived as that minimizing the Akaike information criterion and validated using machine learning, and was compared with the prognostic CASH biomarker predicting bleeding in the subsequent year. Biomarkers were longitudinally followed in a subset of cases. The biomarkers were queried in the lesional neurovascular unit (NVU) transcriptome and in plasma miRNAs from CASH and non-CASH patients.RESULTSThe diagnostic CASH biomarker included a weighted combination of soluble CD14 (sCD14), VEGF, C-reactive protein (CRP), and IL-10 distinguishing CASH patients with 76% sensitivity and 80% specificity (P = 0.0003). The prognostic CASH biomarker (sCD14, VEGF, IL-1ß, and sROBO-4) was confirmed to predict a bleed in the subsequent year with 83% sensitivity and 93% specificity (P = 0.001). Genes associated with diagnostic and prognostic CASH biomarkers were differentially expressed in CASH lesional NVUs. Thirteen plasma miRNAs were differentially expressed between CASH and non-CASH patients.CONCLUSIONShared and unique biomarkers of recent symptomatic hemorrhage and of future bleeding in CA are mechanistically linked to lesional transcriptome and miRNA. The biomarkers may be applied for risk stratification in clinical trials and developed as a tool in clinical practice.FUNDINGNIH, William and Judith Davis Fund in Neurovascular Surgery Research, Be Brave for Life Foundation, Safadi Translational Fellowship, Pritzker School of Medicine, and Sigrid Jusélius Foundation.

Phenotypic characterization of murine models of cerebral cavernous malformations.

Cerebral cavernous malformations (CCMs) are clusters of dilated capillaries that affect around 0.5% of the population. CCMs exist in two forms, sporadic and familial. Mutations in three documented genes, KRIT1(CCM1), CCM2, and PDCD10(CCM3), cause the autosomal dominant form of the disease, and somatic mutations in these same genes underlie lesion development in the brain. Murine models with constitutive or induced loss of respective genes have been applied to study disease pathobiology and therapeutic manipulations. We aimed to analyze the phenotypic characteristic of two main groups of models, the chronic heterozygous models with sensitizers promoting genetic instability, and the acute neonatal induced homozygous knockout model. Acute model mice harbored a higher lesion burden than chronic models, more localized in the hindbrain, and largely lacking iron deposition and inflammatory cell infiltrate. The chronic model mice showed a lower lesion burden localized throughout the brain, with significantly greater perilesional iron deposition, immune B- and T-cell infiltration, and less frequent junctional protein immunopositive endothelial cells. Lesional endothelial cells in both models expressed similar phosphorylated myosin light chain immunopositivity indicating Rho-associated protein kinase activity. These data suggest that acute models are better suited to study the initial formation of the lesion, while the chronic models better reflect lesion maturation, hemorrhage, and inflammatory response, relevant pathobiologic features of the human disease.

Plasma Biomarkers of Inflammation and Angiogenesis Predict Cerebral Cavernous Malformation Symptomatic Hemorrhage or Lesional Growth.

RATIONALE: The clinical course of cerebral cavernous malformations is highly unpredictable, with few cross-sectional studies correlating proinflammatory genotypes and plasma biomarkers with prior disease severity. OBJECTIVE: We hypothesize that a panel of 24 candidate plasma biomarkers, with a reported role in the physiopathology of cerebral cavernous malformations, may predict subsequent clinically relevant disease activity. METHODS AND RESULTS: Plasma biomarkers were assessed in nonfasting peripheral venous blood collected from consecutive cerebral cavernous malformation subjects followed for 1 year after initial sample collection. A first cohort (N=49) was used to define the best model of biomarker level combinations to predict a subsequent symptomatic lesional hemorrhagic expansion within a year after the blood sample. We generated the receiver operating characteristic curves and area under the curve for each biomarker individually and each weighted linear combination of relevant biomarkers. The best model to predict lesional activity was selected as that minimizing the Akaike information criterion. In this cohort, 11 subjects experienced symptomatic lesional hemorrhagic expansion (5 bleeds and 10 lesional growths) within a year after the blood draw. Subjects had lower soluble CD14 (cluster of differentiation 14; P=0.05), IL (interleukin)-6 (P=0.04), and VEGF (vascular endothelial growth factor; P=0.0003) levels along with higher plasma levels of IL-1ß (P=0.008) and soluble ROBO4 (roundabout guidance receptor 4; P=0.03). Among the 31 weighted linear combinations of these 5 biomarkers, the best model (with the lowest Akaike information criterion value, 25.3) was the weighted linear combination including soluble CD14, IL-1ß, VEGF, and soluble ROBO4, predicting a symptomatic hemorrhagic expansion with a sensitivity of 86% and specificity of 88% (area under the curve, 0.90; P<0.0001). We then validated our best model in the second sequential independent cohort (N=28). CONCLUSIONS: This is the first study reporting a predictive association between plasma biomarkers and subsequent cerebral cavernous malformation disease clinical activity. This may be applied in clinical prognostication and stratification of cases in clinical trials.

Plasma Biomarkers of Inflammation Reflect Seizures and Hemorrhagic Activity of Cerebral Cavernous Malformations.

The clinical course of cerebral cavernous malformations (CCMs) is highly variable. Based on recent discoveries implicating angiogenic and inflammatory mechanisms, we hypothesized that serum biomarkers might reflect chronic or acute disease activity. This single-site prospective observational cohort study included 85 CCM patients, in whom 24 a priori chosen plasma biomarkers were quantified and analyzed in relation to established clinical and imaging parameters of disease categorization and severity. We subsequently validated the positive correlations in longitudinal follow-up of 49 subjects. Plasma levels of matrix metalloproteinase-2 and intercellular adhesion molecule 1 were significantly higher (P = 0.02 and P = 0.04, respectively, FDR corrected), and matrix metalloproteinase-9 was lower (P = 0.04, FDR corrected) in patients with seizure activity at any time in the past. Vascular endothelial growth factor and endoglin (both P = 0.04, FDR corrected) plasma levels were lower in patients who had suffered a symptomatic bleed in the prior 3 months. The hierarchical clustering analysis revealed a cluster of four plasma inflammatory cytokines (interleukin 2, interferon gamma, tumor necrosis factor alpha, and interleukin 1 beta) separating patients into what we designated "high" and "low" inflammatory states. The "high" inflammatory state was associated with seizure activity (P = 0.02) and more than one hemorrhagic event during a patient's lifetime (P = 0.04) and with a higher rate of new hemorrhage, lesion growth, or new lesion formation (P < 0.05) during prospective follow-up. Peripheral plasma biomarkers reflect seizure and recent hemorrhagic activity in CCM patients. In addition, four clustered inflammatory biomarkers correlate with cumulative disease aggressiveness and predict future clinical activity.

RhoA Kinase Inhibition With Fasudil Versus Simvastatin in Murine Models of Cerebral Cavernous Malformations.

BACKGROUND AND PURPOSE: We sought to compare the effect of chronic treatment with commonly tolerated doses of Fasudil, a specific RhoA kinase (ROCK) inhibitor, and simvastatin (with pleiotropic effects including ROCK inhibition) on cerebral cavernous malformation (CCM) genesis and maturation in 2 models that recapitulate the human disease. METHODS: Two heterozygous murine models, Ccm1+/-Msh2-/- and Ccm2+/-Trp53-/-, were treated from weaning to 4 to 5 months of age with Fasudil (100 mg/kg per day), simvastatin (40 mg/kg per day) or with placebo. Mouse brains were blindly assessed for CCM lesion burden, nonheme iron deposition (as a quantitative measure of chronic lesional hemorrhage), and ROCK activity. RESULTS: Fasudil, but not simvastatin, significantly decreased mature CCM lesion burden in Ccm1+/-Msh2-/- mice, and in meta-analysis of both models combined, when compared with mice receiving placebo. Fasudil and simvastatin both significantly decreased the integrated iron density per mature lesion area in Ccm1+/-Msh2-/- mice, and in both models combined, compared with mice given placebo. ROCK activity in mature lesions of Ccm1+/-Msh2-/- mice was similar with both treatments. Fasudil, but not simvastatin, improved survival in Ccm1+/-Msh2-/- mice. Fasudil and simvastatin treatment did not affect survival or lesion development significantly in Ccm2+/-Trp53-/- mice alone, and Fasudil benefit seemed limited to males. CONCLUSIONS: ROCK inhibitor Fasudil was more efficacious than simvastatin in improving survival and blunting the development of mature CCM lesions. Both drugs significantly decreased chronic hemorrhage in CCM lesions. These findings justify the development of ROCK inhibitors and the clinical testing of commonly used statin agents in CCM.

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.

Micro-computed tomography in murine models of cerebral cavernous malformations as a paradigm for brain disease.

BACKGROUND: Cerebral cavernous malformations (CCMs) are hemorrhagic brain lesions, where murine models allow major mechanistic discoveries, ushering genetic manipulations and preclinical assessment of therapies. Histology for lesion counting and morphometry is essential yet tedious and time consuming. We herein describe the application and validations of X-ray micro-computed tomography (micro-CT), a non-destructive technique allowing three-dimensional CCM lesion count and volumetric measurements, in transgenic murine brains. NEW METHOD: We hereby describe a new contrast soaking technique not previously applied to murine models of CCM disease. Volumetric segmentation and image processing paradigm allowed for histologic correlations and quantitative validations not previously reported with the micro-CT technique in brain vascular disease. RESULTS: Twenty-two hyper-dense areas on micro-CT images, identified as CCM lesions, were matched by histology. The inter-rater reliability analysis showed strong consistency in the CCM lesion identification and staging (K=0.89, p<0.0001) between the two techniques. Micro-CT revealed a 29% greater CCM lesion detection efficiency, and 80% improved time efficiency. COMPARISON WITH EXISTING METHOD: Serial integrated lesional area by histology showed a strong positive correlation with micro-CT estimated volume (r(2)=0.84, p<0.0001). CONCLUSIONS: Micro-CT allows high throughput assessment of lesion count and volume in pre-clinical murine models of CCM. This approach complements histology with improved accuracy and efficiency, and can be applied for lesion burden assessment in other brain diseases.

Dual-Channel Minimally Invasive Endoscopic Port for Evacuation of Deep-Seated Spontaneous Intracerebral Hemorrhage with Obstructive Hydrocephalus.

BACKGROUND: In minimally invasive endoscopic port surgery, the medium is air, and the image is clearer than in fluid. The most commonly used port is a single-channel port, which accommodates the rod lens of the endoscope and 2 microsurgical instruments. This setup decreases the freedom of movement of the 3 instruments, making the bimanual procedure difficult. We describe a novel "dual-channel" endoscopic port to facilitate a bimanual refinement procedure for removing deep-seated spontaneous intracerebral hematomas, and we demonstrate the feasibility of this method. METHODS: The small channel accommodates a 0° endoscope lens, and the large channel accommodates 2 microsurgical instruments. This method was used in 8 patients with deep-seated spontaneous intracerebral hematomas with obstructive hydrocephalus. It was necessary to evacuate the deep-seated hematomas in these patients as soon as possible to recover the circulation of cerebrospinal fluid. RESULTS: Dual-channel port surgery was performed in 8 patients with an average age of 55 years (range, 44-79 years). The time from ictus to surgery ranged from 4 hours to 12 days. The duration of drainage tube placement was 2-5 days. The hematomas in all patients, in the third ventricle or thalamus, were evacuated thoroughly. In each patient, improvements in Glasgow Coma Scale scores were observed from admission to discharge. CONCLUSIONS: The dual-channel endoscopic port facilitated bimanual refinement microsurgery during the evacuation of deep-seated intracerebral hematomas, and it prevented the disturbance of the 3 instruments without restraining the scope of the operation during the microsurgical procedure.

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.

Peripheral plasma vitamin D and non-HDL cholesterol reflect the severity of cerebral cavernous malformation disease.

AIM: To correlate cerebral cavernous malformations (CCMs) disease aggressiveness with peripheral blood biomarkers hypothesized mechanistically. PATIENTS & METHODS: A prospective case-control study enrolled 43 CCM patients, where 25-(OH) vitamin D, HDL and non-HDL cholesterol, CRP plasma levels and leukocyte ROCK activity were correlated with parameters of disease aggressiveness reflecting chronic and acute domains. RESULTS: Patients with one or more features of chronically aggressive disease (early age at symptom onset, two or more symptomatic bleeds, high lesion burden) had significantly lower 25-(OH) vitamin D and non-HDL cholesterol levels in comparison to patients without these features. CONCLUSION: Validation of these biomarkers and their potential treatment modulation may influence the clinical care of patients with CCM disease.

Inhibition of autophagy induced by quercetin at a late stage enhances cytotoxic effects on glioma cells.

Glioma is the most common primary brain tumor in the central nervous system (CNS) with high morbidity and mortality in adults. Although standardized comprehensive therapy has been adapted, the prognosis of glioma patients is still frustrating and thus novel therapeutic strategies are urgently in need. Quercetin (Quer), an important flavonoid compound found in many herbs, is shown to be effective in some tumor models including glioma. Recently, it is reported that adequate regulation of autophagy can strengthen cytotoxic effect of anticancer drugs. However, it is not yet fully clear how we should modulate autophagy to achieve a satisfactory therapeutic effect. 3-Methyladenine (3-MA) and Beclin1 short hairpin RNA (shRNA) were used to inhibit the early stage of autophage while chloroquine (CQ) to inhibit the late stage. MTT assay was implemented to determine cell viability. Transmission electron microscopy, western blot, and immunohistochemistry were adopted to evaluate autophagy. Western blot, flow cytometry, and immunohistochemistry were used to detect apoptosis. C6 glioma xenograft models were established to assess the therapeutic effect (the body weight change, the median survival time, and tumor volume) in vivo. Quercetin can inhibit cell viability and induce autophagy of U87 and U251 glioma cells in a dose-dependent manner. Inhibition of early-stage autophagy by 3-MA or shRNA against Beclin1 attenuated the quercetin-induced cytotoxicity. In contrast, suppression of autophagy at a late stage by CQ enhanced the anti-glioma efficiency of quercetin. Therapeutic effect of quercetin for malignant glioma can be strengthened by inhibition of autophagy at a late stage, not initial stage, which may provide a novel opportunity for glioma therapy.

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.

Long-term seizure outcome for international consensus classification of hippocampal sclerosis: a survival analysis.

PURPOSE: Surgery is regarded as a common treatment option for patients with mesial temporal lobe epilepsy (MTLE) as a result of hippocampal sclerosis (HS). However, approximately one-third of patients with intractable epilepsy did not become seizure-free after tailored resection strategies. It would be compelling to identify predictive factors of postoperative seizure outcomes. Our aim was to assess the correlation between HS classification and long-term postoperative seizure outcome in patients with MTLE due to HS. METHODS: To investigate HS classification, semi-quantitative analysis and immunohistochemical staining of neuronal nuclei (NeuN) were performed on 100 postoperative hippocampal specimens. All patients had a 1-7 year postoperative follow-up. The postoperative seizure outcome was evaluated using International League Against Epilepsy (ILAE) outcome classification. RESULTS: Three types of HS were recognized. The highest incidence of initial precipitating injury (IPI) was noted in the HS ILAE type 1 group (53.1%). The most favorable long-term seizure outcome was also noted in the HS ILAE type 1 group. The shortest epilepsy duration was recorded in the HS ILAE type 2 group (mean epilepsy duration=6.64 ± 5.83 years). The completely seizure free rate of patients in all groups declined with an increase in time. CONCLUSIONS: Our study for the first time demonstrated a significant correlation between HS ILAE types and long-term postoperative seizure outcome in patients with MTLE due to HS. Therefore, HS ILAE types have predictive value in long-term seizure outcome following epilepsy surgery.

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

PURPOSE: The phenotypic manifestations of cerebral cavernous malformation disease caused by rare PDCD10 mutations have not been systematically examined, and a mechanistic link to Rho kinase-mediated hyperpermeability, a potential therapeutic target, has not been established. METHODS: We analyzed PDCD10 small interfering RNA-treated endothelial cells for stress fibers, Rho kinase activity, and permeability. Rho kinase activity was assessed in cerebral cavernous malformation lesions. Brain permeability and cerebral cavernous malformation lesion burden were quantified, and clinical manifestations were assessed in prospectively enrolled subjects with PDCD10 mutations. RESULTS: We determined that PDCD10 protein suppresses endothelial stress fibers, Rho kinase activity, and permeability in vitro. Pdcd10 heterozygous mice have greater lesion burden than other Ccm genotypes. We demonstrated robust Rho kinase activity in murine and human cerebral cavernous malformation vasculature and increased brain vascular permeability in humans with PDCD10 mutation. Clinical phenotype is exceptionally aggressive compared with the more common KRIT1 and CCM2 familial and sporadic cerebral cavernous malformation, 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. CONCLUSION: These findings define a unique cerebral cavernous malformation disease with exceptional aggressiveness, and they inform preclinical therapeutic testing, clinical counseling, and the design of trials.Genet Med 17 3, 188-196.