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.

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.

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.

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.

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.

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.

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.

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.

Dynamic permeability and quantitative susceptibility: related imaging biomarkers in cerebral cavernous malformations.

BACKGROUND AND PURPOSE: Hyperpermeability and iron deposition are 2 central pathophysiological phenomena in human cerebral cavernous malformation (CCM) disease. Here, we used 2 novel MRI techniques to establish a relationship between these phenomena. METHODS: Subjects with CCM disease (4 sporadic and 17 familial) underwent MRI imaging using the dynamic contrast-enhanced quantitative perfusion and quantitative susceptibility mapping techniques that measure hemodynamic factors of vessel leak and iron deposition, respectively, previously demonstrated in CCM disease. Regions of interest encompassing the CCM lesions were analyzed using these techniques. RESULTS: Susceptibility measured by quantitative susceptibility mapping was positively correlated with permeability of lesions measured using dynamic contrast-enhanced quantitative perfusion (r=0.49; P≤0.0001). The correlation was not affected by factors, including lesion volume, contrast agent, and the use of statin medication. Susceptibility was correlated with lesional blood volume (r=0.4; P=0.0001) but not with lesional blood flow. CONCLUSIONS: The correlation between quantitative susceptibility mapping and dynamic contrast-enhanced quantitative perfusion suggests that the phenomena of permeability and iron deposition are related in CCM; hence, more leaky lesions also manifest a more cumulative iron burden. These techniques might be used as biomarkers to monitor the course of this disease and the effect of therapy.

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.

A novel mouse model of cerebral cavernous malformations based on the two-hit mutation hypothesis recapitulates the human disease.

Cerebral cavernous malformations (CCMs) are vascular lesions of the central nervous system appearing as multicavernous, blood-filled capillaries, leading to headache, seizure and hemorrhagic stroke. CCM occurs either sporadically or as an autosomal dominant disorder caused by germline mutation of one of the three genes: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10. Surgically resected human CCM lesions have provided molecular and immunohistochemical evidence for a two-hit (germline plus somatic) mutation mechanism. In contrast to the equivalent human genotype, mice heterozygous for a Ccm1- or Ccm2-null allele do not develop CCM lesions. Based on the two-hit hypothesis, we attempted to improve the penetrance of the model by crossing Ccm1 and Ccm2 heterozygotes into a mismatch repair-deficient Msh2(-/-) background. Ccm1(+/-)Msh2(-/-) mice exhibit CCM lesions with high penetrance as shown by magnetic resonance imaging and histology. Significantly, the CCM lesions range in size from early-stage, isolated caverns to large, multicavernous lesions. A subset of endothelial cells within the CCM lesions revealed somatic loss of CCM protein staining, supporting the two-hit mutation mechanism. The late-stage CCM lesions displayed many of the characteristics of human CCM lesions, including hemosiderin deposits, immune cell infiltration, increased endothelial cell proliferation and increased Rho-kinase activity. Some of these characteristics were also seen, but to a lesser extent, in early-stage lesions. Tight junctions were maintained between CCM lesion endothelial cells, but gaps were evident between endothelial cells and basement membrane was defective. In contrast, the Ccm2(+/-)Msh2(-/-) mice lacked cerebrovascular lesions. The CCM1 mouse model provides an in vivo tool to investigate CCM pathogenesis and new therapies.

Increased expression of ABCB6 enhances protoporphyrin IX accumulation and photodynamic effect in human glioma.

BACKGROUND: Glioma recurrence usually occurs close to the tumor resection margins as a result of residual infiltrating glioma cells. 5-aminolevulinic acid (ALA) fluorescence-guided resection of gliomas has been demonstrated to enhance discrimination of tumor tissue and to improve survival. ALA-based photodynamic therapy is an effective albeit still experimental adjuvant treatment option for gliomas. However, insufficient protoporphyrin IX (PpIX) accumulation may limit the benefits of fluorescence-guided resection and photodynamic therapy. METHODS: We investigated the expression of the ATP-binding cassette transporter ABCB6, which regulates porphyrin synthesis, in surgical specimens from human gliomas and manipulated ABCB6 in human glioma cell lines. RESULTS: Our findings demonstrated that expression levels of ABCB6 were greatly elevated in human gliomas compared with normal brain tissues and correlated with World Health Organization histologic grade. A previously undescribed finding was that ABCB6 mRNA expression in solidly fluorescing tumor tissues was higher than that in vaguely fluorescing tumors, suggesting that ABCB6 may be at least in part responsible for PpIX accumulation in glioma cells. Accordingly, ABCB6 overexpression in glioma cell lines caused a marked increase in intracellular levels of PpIX, and was more sensitive to ALA-induced photodynamic therapy-events that could be prevented by silencing ABCB6 via siRNA treatment. CONCLUSIONS: Our findings indicate a crucial role of ABCB6 in ALA metabolism and accumulation of PpIX in glioma. ABCB6 overexpression is a potential approach to enhance accumulation of PpIX for optimizing the subjective discrimination of vague fluorescence and improving the efficacy of ALA-based photodynamic therapy.

Inflammatory Mechanisms in a Neurovascular Disease: Cerebral Cavernous Malformation.

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

Fasudil decreases lesion burden in a murine model of cerebral cavernous malformation disease.

BACKGROUND AND PURPOSE: Cerebral cavernous malformations (CCMs) are characterized by grossly dilated capillaries, associated with vascular leak and hemorrhage, and occur in sporadic or inherited (autosomal-dominant) forms with mutations in 1 of 3 gene loci (CCM 1, 2 or 3). We previously reported that the CCM1 protein (KRIT1) localizes to endothelial cell-cell junctions and loss of KRIT1 leads to junctional instability associated with activation of RhoA and its effector Rho kinase. Although Rho kinase inhibition has been proposed as potential therapy for CCM, there has been no demonstration of a therapeutic effect on CCM lesion genesis in vivo. METHODS: Our recently generated a model of CCM1 disease (Ccm1(+/-)Msh2(-/-)) was treated with the Rho kinase inhibitor fasudil (100 mg/kg/day administered in drinking water from weaning to 5 months of age), or placebo, and blindly assessed CCM lesion burden by systematic survey of animals' brains. For comparison, we also assessed therapeutic effect in previously described Ccm2(+/-)Trp53(-/-) mice treated with the same dose and duration of fasudil and placebo. RESULTS: Fasudil-treated Ccm1(+/-)Msh2(-/-) mice had a significantly decreased prevalence of CCM lesions compared with placebo controls. Lesions in treated animals were smaller and less likely associated with hemorrhage, inflammation, and endothelial proliferation and exhibited decreased expression of Rho kinase activation biomarkers. A therapeutic effect was also documented in Ccm2(+/-)Trp53(-/-) mice. CONCLUSIONS: This represents the first report of therapeutic benefit of pharmacological therapy in development and progression of CCMs and indicates that Rho kinase activation is a critical step in CCM lesion genesis and maturation.

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.

Role of pericytes in the development of cerebral cavernous malformations.

Cerebral cavernous malformation (CCM) is caused by loss-of-function mutations in CCM1, CCM2, or CCM3 genes of endothelial cells. It is characterized by pericyte deficiency. However, the role of pericytes in CCMs is not yet clarified. We found pericytes in Cdh5Cre ERT2 ;Ccm1 fl/fl (Ccm1 ECKO ) mice had a high expression of PDGFRß. The inhibition of pericyte function by CP-673451 aggravated the CCM lesion development. RNA-sequencing analysis revealed the molecular traits of pericytes, such as highly expressed ECM-related genes, especially Fn1. Furthermore, KLF4 coupled with phosphorylated SMAD3 (pSMAD3) promoted the transcription of fibronectin in the pericytes of CCM lesions. RGDS peptide, an inhibitor of fibronectin, decreased the lesion area in the cerebella and retinas of Ccm1 ECKO mice. Also, human CCM lesions had abundant fibronectin deposition, and pSMAD3- and KLF4-positive pericytes. These findings indicate that pericytes are essential for CCM lesion development, and fibronectin intervention may provide a novel target for therapeutic intervention in such patients.

Inhibition of heme oxygenase-1 enhances anti-cancer effects of arsenic trioxide on glioma cells.

We have previously reported that arsenic trioxide (ATO) could inhibit glioma growth both in vitro and in vivo, and demonstrated its potent therapeutic effects on gliomas. In this study we showed that ATO induced cell damage and heme oxygenase-1 (HO-1) expression in glioma cells via ROS generation. HO-1 inducer clearly protected from ATO-induced cell death and ROS generation, and HO-1 inhibitor led to a significant increase in cell death and ROS generation induced by ATO. In addition, knockdown of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) strongly inhibited HO-1 expression induced by ATO, and significantly enhanced ATO-induced oxidative damage. Our results demonstrated, for the first time, that HO-1 inhibition or Nrf2 knockdown significantly potentiated ATO's effects on glioma cells. Considering that HO-1 is highly expressed in glioma tissues, administration of ATO in combination with either HO-1 inhibitor or Nrf2 knockdown may act as a new approach to the treatment of glioma.

Cerebral cavernous malformations as a disease of vascular permeability: from bench to bedside with caution.

Tremendous insight into the molecular and genetic pathogenesis of cerebral cavernous malformations (CCMs) has been gained over the past 2 decades. This includes the identification of 3 distinct genes involved in familial CCMs. Still, a number of unanswered questions regarding the process from gene mutation to vascular malformation remain. It is becoming more evident that the disruption of interendothelial junctions and ensuing vascular hyperpermeability play a principal role. The purpose of this review is to summarize the current understanding of CCM genes, associated proteins, and functional pathways. Promising molecular and genetic therapies targeted at identified molecular aberrations are discussed as well.

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.