Hannah Schneider

Nogo-A is a negative regulator of CNS angiogenesis

Nogo-A is an important axonal growth inhibitor in the adult and developing CNS. In vitro, Nogo-A has been shown to inhibit migration and cell spreading of neuronal and nonneuronal cell types. Here, we studied in vivo and in vitro effects of Nogo-A on vascular endothelial cells during angiogenesis of the early postnatal brain and retina in which Nogo-A is expressed by many types of neurons. Genetic ablation or virus-mediated knock down of Nogo-A or neutralization of Nogo-A with an antibody caused a marked increase in the blood vessel density in vivo. In culture, Nogo-A inhibited spreading, migration, and sprouting of primary brain microvascular endothelial cells (MVECs) in a dose-dependent manner and induced the retraction of MVEC lamellipodia and filopodia. Mechanistically, we show that only the Nogo-A–specific Delta 20 domain exerts inhibitory effects on MVECs, but the Nogo-66 fragment, an inhibitory domain common to Nogo-A, -B, and -C, does not. Furthermore, the action of Nogo-A Delta 20 on MVECs required the intracellular activation of the Ras homolog gene family, member A (Rho-A)-associated, coiled-coil containing protein kinase (ROCK)-Myosin II pathway. The inhibitory effects of early postnatal brain membranes or cultured neurons on MVECs were relieved significantly by anti–Nogo-A antibodies. These findings identify Nogo-A as an important negative regulator of developmental angiogenesis in the CNS. They may have important implications in CNS pathologies involving angiogenesis such as stroke, brain tumors, and retinopathies.

Impairment of Tight Junctions and Glucose Transport in Endothelial Cells of Human Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) often cause hemorrhages that can result in severe clinical manifestations, including hemiparesis and seizures. The underlying mechanisms of the aggressive behavior of CCMs are undetermined to date, but alterations ofvascular matrix components may be involved. We compared the localization of the tight junction proteins (TJPs) in 12 CCM specimens and the expression of glucose transporter 1 (GLUT-1), which is sensitive to alterations in TJP levels, in 5 CCM specimens with those in 5control temporal lobectomy specimens without CCM by immunofluorescence microscopy. The TJPs occludin, claudin-5, and zonula occludens ZO-1 were downregulated at intercellular contact sites and partly redistributed within the surrounding tissue in the CCM samples; there was also a marked reduction of GLUT-1 immunoreactivity compared with that in control specimens. Corresponding analysis using quantitative real-time reverse transcription polymerase chain reactionon 8 CCM and 8 control specimens revealed significant downregulation of mRNA expression of occludin, claudin-5, ZO-1, and GLUT-1. The altered expression and localization of the TJPs at interendothelial contact sites accompanied by a reduction of GLUT-1 expression in dilated CCM microvessels likely affect vascular matrix stability and may contribute to hemorrhages of CCMs.

Autocrine VEGFR1 and VEGFR2 signaling promotes survival in human glioblastoma models in vitro and in vivo.

Background Although the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) system has become a prime target for antiangiogenic treatment, its biological role in glioblastoma beyond angiogenesis has remained controversial. Methods Using neutralizing antibodies to VEGF or placental growth factor (PlGF) or the tyrosine kinase inhibitor, cediranib, or lentiviral gene silencing, we delineated autocrine signaling in glioma cell lines. The in vivo effects of VEGFR1 and VEGFR2 depletion were evaluated in orthotopic glioma xenograft models. Results VEGFR1 and VEGFR2 modulated glioma cell clonogenicity, viability, and invasiveness in vitro in an autocrine, cell–line-specific manner. VEGFR1 silencing promoted mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling, whereas VEGFR2 silencing resulted in cell-type dependent activation of the protein kinase B (PKB)/AKT and MAPK/ERK pathways. These responses may represent specific escape mechanisms from VEGFR inhibition. The survival of orthotopic glioma-bearing mice was prolonged upon VEGFR1 silencing in the LNT-229, LN-308, and U87MG models and upon VEGFR2 silencing in LN-308 and U87MG. Disruption of VEGFR1 and VEGFR2 signaling was associated with decreased tumor size, increased tumor necrosis, or loss of matrix metalloproteinase 9 (MMP9) immunoreactivity. Neutralizing VEGF and PlGF by specific antibodies was superior to either antibody treatment alone in the VEGFR1-dependent LNT-229 model. Conclusions Differential dependence on autocrine signaling through VEGFR1 and VEGFR2 suggests a need for biomarker–stratified VEGF(R)-based therapeutic approaches to glioblastoma.

Bleeding propensity of cavernous malformations: impact of tight junction alterations on the occurrence of overt hematoma

OBJECT Endothelial tight junction (TJ) expression is mostly absent in cerebral cavernous malformations (CMs), which causes increased perilesional erythrocyte and fluid oozing. However, in a subset of CM lesions, foci of preserved TJ staining are observed along endothelial cell contacts. The clinical relevance of this finding is unclear. This study investigates the relevance of the focal TJ protein expression and its association with CM bleeding propensity. METHODS Immunohistochemical staining for the TJ proteins claudin-5, occludin, and ZO-1 was performed on 32 CM specimens that were resected during 2008-2010. The patients were allocated to 2 groups according to TJ protein expression, and the clinical and radiological parameters of aggressiveness were analyzed and compared. RESULTS Complete absence of TJ expression was identified in 20 specimens, and focal TJ protein expression in 12. CMs without TJ immunoreactivity were significantly larger (p = 0.022) and had a significantly greater propensity for development of frank hematomas (p = 0.028) and perilesional edema (p = 0.013). Symptom severity, multiplicity, developmental venous anomaly (DVA) presence, and CM location did not show a significant difference depending on TJ expression. CONCLUSIONS In a univariate analysis the authors observed significantly less propensity for frank hematomas and perilesional edema as well as smaller size in CM lesions with focal TJ expression compared with CMs without TJ expression. The observed difference in TJ protein expression might be the reason for differences in bleeding propensity of the CM lesions. Although this finding cannot be used in predictive manner at this time, it is a basis for further multivariate analyses of possible CM biological predictors.

Biological Role and Therapeutic Targeting of TGF-β 3 in Glioblastoma

Transforming growth factor (TGF)-β contributes to the malignant phenotype of glioblastoma by promoting invasiveness and angiogenesis and creating an immunosuppressive microenvironment. So far, TGF-β1 and TGF-β2 isoforms have been considered to act in a similar fashion without isoform-specific function in glioblastoma. A pathogenic role for TGF-β3 in glioblastoma has not been defined yet. Here, we studied the expression and functional role of endogenous and exogenous TGF-β3 in glioblastoma models. TGF-β3 mRNA is expressed in human and murine long-term glioma cell lines as well as in human glioma-initiating cell cultures with expression levels lower than TGF-β1 or TGF-β2 in most cell lines. Inhibition of TGF-β3 mRNA expression by ISTH2020 or ISTH2023, two different isoform-specific phosphorothioate locked nucleic acid (LNA)-modified antisense oligonucleotide gapmers, blocks downstream SMAD2 and SMAD1/5 phosphorylation in human LN-308 cells, without affecting TGF-β1 or TGF-β2 mRNA expression or protein levels. Moreover, inhibition of TGF-β3 expression reduces invasiveness in vitro. Interestingly, depletion of TGF-β3 also attenuates signaling evoked by TGF-β1 or TGF-β2. In orthotopic syngeneic (SMA-560) and xenograft (LN-308) in vivo glioma models, expression of TGF-β3 as well as of the downstream target, plasminogen-activator-inhibitor (PAI)-1, was reduced, while TGF-β1 and TGF-β2 levels were unaffected following systemic treatment with TGF-β3-specific antisense oligonucleotides. We conclude that TGF-β3 might function as a gatekeeper controlling downstream signaling despite high expression of TGF-β1 and TGF-β2 isoforms. Targeting TGF-β3 in vivo may represent a promising strategy interfering with aberrant TGF-β signaling in glioblastoma. Mol Cancer Ther; 16(6); 1177–86. ©2017 AACR.

Autocrine activation of the IFN signaling pathway may promote immune escape in glioblastoma

Background Interferons (IFNs) are cytokines typically induced upon viral infection but are constitutively expressed also in the absence of acute infection. The physiological role of autocrine and paracrine IFN signaling, however, remains poorly understood, and its function in glioblastoma has not been explored in depth. Methods Using RNA interference-mediated gene silencing, we characterized constitutive type I IFN signaling and its role in human glioma cells. Results We observed constitutive expression of phosphorylated signal transducer and activator of transcription 1 (pSTAT1) and myxovirus resistance protein A (MxA), a classical IFN-response marker, in the absence of exogenous IFN-β. In vivo, we found higher MxA expression in gliomas than in normal tissue, suggesting that IFN signaling is constitutively active in these tumors. To demonstrate the presence of an autocrine type I IFN signaling loop in glioma cells in vitro, we first confirmed the expression of the type I alpha/beta receptor (IFNAR)1/2, and its ligands, IFN-α and IFN-β. Small interfering RNA-mediated receptor gene silencing resulted in reduced expression of MxA at mRNA and protein levels, as did gene silencing of the ligands, corroborating the hypothesis of an autocrine signaling loop in which type I IFNs induce intracellular signaling through IFNAR1/2. On a functional level, following IFNAR1 or IFNAR2 gene silencing, we observed reduced programmed death ligand 1 (PD-L1) and major histocompatibility complex (MHC) class I and II expression as well as an enhanced susceptibility to natural killer immune cell lysis, suggesting that autocrine IFN signaling contributes to the immune evasion of glioma cells. Conclusions Our findings point to an important role of constitutive IFN signaling in glioma cells by modulating their interaction with the microenvironment.

Novel TIE-2 Inhibitor BAY-826 Displays In Vivo Efficacy in Experimental Syngeneic Murine Glioma Models

Targeting the vascular endothelial growth factor signaling axis in glioblastoma inevitably leads to tumor recurrence and a more aggressive phenotype. Therefore, other angiogenic pathways, like the angiopoietin/tunica interna endothelial cell kinase (TIE) signaling axis, have become additional targets for therapeutic intervention. Here, we explored whether targeting the receptor tyrosine kinase TIE‐2 using a novel, highly potent, orally available small molecule TIE‐2 inhibitor (BAY‐826) improves tumor control in syngeneic mouse glioma models. BAY‐826 inhibits TIE‐2 phosphorylation in vitro and in vivo as demonstrated by suppression of Angiopoietin‐1‐ or Na3VO4‐induced TIE‐2 phosphorylation in glioma cells or extracts of lungs from BAY‐826‐treated mice. There was a trend toward prolonged survival upon single‐agent treatment in two of four models (SMA‐497 and SMA‐540) and there was a significant survival benefit in one model (SMA‐560). Co‐treatment with BAY‐826 and irradiation was ineffective in one model (SMA‐497), but provided synergistic prolongation of survival in another (SMA‐560). Decreased vessel densities and increased leukocyte infiltration were observed, but might be independent processes as the effect was also observed in single treatment modalities. These data demonstrate that TIE‐2 inhibition may improve tumor response to treatment in highly vascularized tumors such as glioblastoma.

NKG2D-dependent anti-tumor effects of chemotherapy and radiotherapy against glioblastoma

Purpose: NKG2D is a potent activating immune cell receptor, and glioma cells express the cognate ligands (NKG2DL). These ligands are inducible by cellular stress and temozolomide (TMZ) or irradiation (IR), the standard treatment of glioblastoma, could affect their expression. However, a role of NKG2DL for the efficacy of TMZ and IR has never been addressed. Experimental Design: We assessed the effect of TMZ and IR on NKG2DL in vitro and in vivo in a variety of murine and human glioblastoma models, including glioma-initiating cells, and a cohort of paired glioblastoma samples from patients before and after therapy. Functional effects were studied with immune cell assays. The relevance of the NKG2D system for the efficacy of TMZ and IR was assessed in vivo in syngeneic orthotopic glioblastoma models with blocking antibodies and NKG2D knockout mice. Results: TMZ or IR induced NKG2DL in vitro and in vivo in all glioblastoma models, and glioblastoma patient samples had increased levels of NKG2DL after therapy with TMZ and IR. This enhanced the immunogenicity of glioma cells in a NGK2D-dependent manner, was independent from cytotoxic or growth inhibitory effects, attenuated by O6-methylguanine-DNA-methyltransferase (MGMT), and required the DNA damage response. The survival benefit afforded by TMZ or IR relied on an intact NKG2D system and was decreased upon inhibition of the NKG2D pathway. Conclusions: The immune system may influence the activity of convential cancer treatments with particular importance of the NKG2D pathway in glioblastoma. Our data provide a rationale to combine NKG2D-based immunotherapies with TMZ and IR. Clin Cancer Res; 24(4); 882–95. ©2017 AACR.

Age-associated and therapy-induced alterations in the cellular microenvironment of experimental gliomas

The poor prognosis associated with advanced age in patients with glioblastoma remains poorly understood. Glioblastoma in the elderly has been particularly associated with vascular endothelial growth factor (VEGF)-dependent angiogenesis, and early uncontrolled studies suggested that the anti-angiogenic agent bevacizumab (BEV), an antibody to VEGF, might be preferentially active in this patient population. Accordingly, we explored host age-dependent differences in survival and benefit from radiotherapy (RT) or BEV in syngeneic mouse glioma models. Survival was inferior in older mice in the SMA-540 and and less so in SMA-560, but not in the SMA-497 or GL-261 models. Detailed flow cytometric studies revealed increased myeloid and decreased effector T cell population frequencies in SMA-540 tumors of old compared to young mice, but no such difference in the SMA-497 model. Bone marrow transplantation (BMT) from young to old mice had no effect, whereas survival was reduced with BMT from old to young mice. BEV significantly decreased vessel densities in gliomas of old, but not young mice. Accordingly, old, but not young SMA-540 tumor-bearing mice benefited from BEV alone or in combination with RT. End-stage tumors of old BEV- and BEV/RT-treated mice exhibited increased infiltration of T helper and cytotoxic T cells compared to tumors of young mice. The SMA-540 model may provide a valuable tool to evaluate the influence of host age on glioblastoma progression and treatment response. The biological host factors that modulate glioma growth in old as opposed to young mice remain to be identified.

Contraceptive drugs mitigate experimental stroke-induced brain injury

AIMS Effective stroke treatments beyond reperfusion remain scant. The natural steroid hormone progesterone has shown protective effects in experimental models of brain injury and cardiovascular disease. However, unfavourable bioavailability limits its clinical use. Desogestrel and drospirenone are new generation progestins with progesterone-like properties, developed as oral contraceptives with excellent bioavailability and safety profile. We investigated the neuroprotective properties of these progestins in vivo using transient middle cerebral artery occlusion (MCAO) and in vitro using an oxygen-glucose deprivation and reoxygenation (OGD/R) model in primary neuronal cells. METHODS AND RESULTS MCAO was induced in female, female ovariectomized (modelling postmenopausal females) and male mice. Treatment with the progestins resulted in less severe strokes after MCAO and less neuronal death in OGD/R. Desogestrel and drospirenone induced higher expression levels of GABAAR α4 and delta subunits within the brain, suggesting changes in GABAAR configuration favouring tonic inhibition as potential mechanism of action. Treatment with the GABAAR blocker picrotoxin abolished the protection afforded by the progestins in vivo and in vitro. CONCLUSION For the first time, here, we delineate a potential role of desogestrel and drospirenone, both clinically approved and safe drugs in mitigating the consequences of stroke. Contraception with desogestrel and drospirenone in progestin-only preparations may be particularly beneficial for women at risk of stroke.