Search

In order to identify suitable therapeutic targets for glioma anti-angiogenic therapy, the process of neovascularization mediated by circulating angiogenic cells (CACs) needs to be scrutinized. In the present study we compared the expression of neovascularization-related genes by three circulating CAC subsets (HPCs, CD34+ and KDR+ cells; internal controls: PBMCs and circulating endothelial cells) of treatment-naïve patients with glioblastoma (GBM) to those of patients undergoing reactive neovascularization (myocardial infarction (MI). CACs from umbilical cord (representing developmental neovascularization) and healthy subjects served as controls. Fluorescent activated cell sorting was used to isolate CACs, RT-PCR to determine the expression levels of a panel of 48 neovascularization-related genes, Luminex assays to measure plasma levels of 21 CAC-related circulating molecules. We found essential differences in gene expression between GBM and MI CACs. GBM CACs had a higher expression of pro-angiogenic factors (esp. KITL, CXCL12 and JAG1); growth factor and chemotactic receptors (IGF1R, TGFbR2, CXCR4 and CCR2); adhesion receptor monomers (ITGA5 and ITGA6) and matricellular factor POSTN. In addition, we found major differences in the levels of neovascularization-related plasma factors. A strong positive correlation between plasma MMP9 levels and expression of CXCR4 in the CAC subset of hematopoietic progenitor cells (HPCs) was found in GBM patients. Our findings indicate that CAC-mediated neovascularization in GBM is characterized by more efficient CAC homing to target tissue and a more potent pro-angiogenic response than in physiologic tissue repair in MI. Our findings can aid in selecting targets for therapeutic strategies acting against GBM-specific CACs.

The expression of the matricellular protein periostin has been associated with glioma progression. In previous work we found an association of periostin with glioma angiogenesis. Here, we screen gliomas for POSTN expression and identify the cells that express periostin in human gliomas. In addition, we study the role of periostin in an in vitro model for angiogenesis. The expression of periostin was investigated by RT-PCR and by immunohistochemistry. In addition, we used double labeling and in situ RNA techniques to identify the expressing cells. To investigate the function of periostin, we silenced POSTN in a 3D in vitro angiogenesis model. Periostin expression was elevated in pilocytic astrocytoma and glioblastoma, but not in grade II/III astrocytomas and oligodendrogliomas. The expression of periostin colocalized with PDGFRβ+ cells, but not with OLIG2+/SOX2+ glioma stem cells. Silencing of periostin in pericytes in coculture experiments resulted in attenuation of the numbers and the length of the vessels formation and in a decrease in endothelial junction formation. We conclude that pericytes are the main source of periostin in human gliomas and that periostin plays an essential role in the growth and branching of blood vessels. Therefore, periostin should be explored as a novel target for developing anti-angiogenic therapy for glioma.

Background In order to identify suitable therapeutic targets for glioma anti-angiogenic therapy, the process of neovascularization mediated by circulating angiogenic cells (CACs) needs to be scrutinized. Methods In the present study, we compared the expression of neovascularization-related genes by 3 circulating CAC subsets (hematopoietic progenitor cells [HPCs], CD34+, and KDR+ cells; internal controls: peripheral blood mononuclear cells and circulating endothelial cells) of treatment-naïve patients with glioblastoma (GBM) to those of patients undergoing reactive neovascularization (myocardial infarction (MI). CACs from umbilical cord (representing developmental neovascularization) and healthy subjects served as controls. Fluorescent-activated cell sorting was used to isolate CACs, RT-PCR to determine the expression levels of a panel of 48 neovascularization-related genes, and Luminex assays to measure plasma levels of 21 CAC-related circulating molecules. Results We found essential differences in gene expression between GBM and MI CACs. GBM CACs had a higher expression of proangiogenic factors (especially, KITL, CXCL12, and JAG1), growth factor and chemotactic receptors (IGF1R, TGFBR2, CXCR4, and CCR2), adhesion receptor monomers (ITGA5 and ITGA6), and matricellular factor POSTN. In addition, we found major differences in the levels of neovascularization-related plasma factors. A strong positive correlation between plasma MMP9 levels and expression of CXCR4 in the CAC subset of HPCs was found in GBM patients. Conclusions Our findings indicate that CAC-mediated neovascularization in GBM is characterized by more efficient CAC homing to target tissue and a more potent proangiogenic response than in physiologic tissue repair in MI. Our findings can aid in selecting targets for therapeutic strategies acting against GBM-specific CACs.

BACKGROUND The expression of the matricellular protein periostin has recently been associated with glioma progression and angiogenesis. The aim of the present study was to identify the cellular source of periostin expression in human gliomas and to study the role of periostin in an in vitro model for angiogenesis. MATERIAL AND METHODS The expression of periostin in various types and grades of brain tumors was investigated at the genetics level by RT-PCR and at the protein level by immunohistochemistry. In addition, the expression of periostin in glioma samples was studied by double labeling and in situ RNA techniques. Subsequently, by silencing periostin in a 3D in vitro angiogenesis model, its effects on angiogenesis were studied. RESULTS Periostin RNA and protein expression was found to be elevated in pilocytic astrocytoma and glioblastoma, but not in grade II/III astrocytomas. The expression of periostin co-localized with PDGFRβ + cells, but not with Olig2+ / Sox2+ glioma stem cells. Silencing of periostin in pericytes resulted in the attenuation of the number and the length of the vessels, and in decrease in the number of vessel junctions formed. CONCLUSION The results prove that pericytes are the main source of periostin in human gliomas. We demonstrate that periostin plays an essential role in the growth and branching of blood vessels. Therefore, periostin and / or its molecular interactions should be explored as a novel target for developing a new anti-angiogenic therapy for glioma.

Although extensive angiogenesis takes place in glial tumors, antiangiogenic therapies have remained without the expected success. In the peripheral circulation of glioma patients, increased numbers of endothelial precursor cells (EPCs) are present, potentially offering targets for antiangiogenic therapy. However, for an antiangiogenic therapy to be successful, the therapy should specifically target glioma-related EPC subsets and secreted factors only. Here, we compared the EPC subsets and plasma factors in the peripheral circulation of patients with gliomas to acute myocardial infarctions. We investigated the five most important EPC subsets and 21 angiogenesis-related plasma factors in peripheral blood samples of 29 patients with glioma, 14 patients with myocardial infarction, and 20 healthy people as controls, by FACS and Luminex assay. In GBM patients, all EPC subsets were elevated as compared to healthy subjects. In addition, HPC and KDR+ cell fractions were higher than in MI, while CD133+ and KDR+CD133+ cell fractions were lower. There were differences in relative EPC fractions between the groups: KDR+ cells were the largest fraction in GBM, while CD133+ cells were the largest fraction in MI. An increase in glioma malignancy grade coincided with an increase in the KDR+ fraction, while the CD133+ cell fraction decreased relatively. Most plasma angiogenic factors were higher in GBM than in MI patients. In both MI and GBM, the ratio of CD133+ HPCs correlated significantly with elevated levels of MMP9. In the GBM patients, MMP9 correlated strongly with levels of all HPCs. In conclusion, the data demonstrate that EPC traffic in patients with glioma, representing neoplasia, is different from that in myocardial infarction, representing tissue regeneration. Glioma patients may benefit from therapies aimed at lowering KDR+ cells and HPCs.

In a previous study we sought proteins that are specifically expressed in the context of glioma angiogenesis. To that aim, we compared microdissected GBM vessels with normal brain vessels and vessels of proliferating endometrium representing physiological angiogenesis. Among the proteins that were overexpressed in GBMs we most prominently found periostin. In this study we explored the expression of periostin at the protein and RNA level and sought its cellular source in the gliomas. In functional studies using gene silencing in cell cultures we studied the effects of periostin on angiogenesis and explored its regulators. We found that periostin is mainly expressed by (PDGFR+) pericytes and that these cells usually carry glioma stem cell characteristics. Co-culture experiments revealed that conditioned medium of the glioma cell line U87 increased the periostin expression by pericytes. Silencing of POSTN in pericytes in the co-cultures with endothelial cells resulted in reduction of the formation, length and junctions of the vascular tubules. We conclude that the expression of periostin is a crucial element of glioma angiogenesis. Its expression by pericytes is stimulated by the presence of glial tumor cells. The data open new clues for the development of new anti-angiogenic strategies for gliomas.

The characterization of circulating endothelial progenitor cells (EPCs) is fundamental to any study related to angiogenesis. Unfortunately, current literature lacks consistency in the definition of EPC subsets due to variations in isolation strategies and inconsistencies in the use of lineage markers. Here we address critical points in the identification of hematopoietic progenitor cells (HPCs), circulating endothelial cells (CECs), and culture-generated outgrowth endothelial cells (OECs) from blood samples of healthy adults (AB) and umbilical cord (UCB). Peripheral blood mononuclear cells (PBMCs) were enriched using a Ficoll-based gradient followed by an optimized staining and gating strategy to enrich for the target cells. Sorted EPC populations were subjected to RT-PCR for tracing the expression of markers beyond the limits of cell surface-based immunophenotyping. Using CD34, CD133 and c-kit staining, combined with FSC and SSC, we succeeded in the accurate and reproducible identification of four HPC subgroups and found significant differences in the respective populations in AB vs. UCB. Co-expression analysis of endothelial markers on HPCs revealed a complex pattern characterized by various subpopulations. CECs were identified by using CD34, KDR, CD45, and additional endothelial markers, and were subdivided according to their apoptotic state and expression of c-kit. Comparison of UCB-CECs vs. AB-CECs revealed significant differences in CD34 and KDR levels. OECs were grown from PBMC-fractions We found that viable c-kit+ CECs are a candidate circulating precursor for CECs. RT-PCR to angiogenic factors and receptors revealed that all EPC subsets expressed angiogenesis-related molecules. Taken together, the improvements in immunophenotyping and gating strategies resulted in accurate identification and comparison of better defined cell populations in a single procedure.

BACKGROUND Although extensive angiogenesis takes place in glial tumors, anti-angiogenic therapies have remained without the expected success. In the peripheral circulation of glioma patients increased numbers of endothelial precursor cells (EPCs) are present, potentially offering targets for anti-angiogenic therapy (Zheng et al., Ann Neurol, 2007). However, for an anti-angiogenic therapy to be successful, the therapy should specifically target glioma-related EPC subsets and secreted factors. Here we compared the EPC subsets and plasma factors in the peripheral circulation of patients with gliomas to acute myocardial infarctions (representing fysiologic regeneration). MATERIAL AND METHODS We investigated the five most important EPC subsets and 21 angiogenesis-related plasma factors in peripheral blood samples of 29 patients with glioma, 14 patients with myocardial infarction and 20 healthy people as controls, by an advanced FACS protocol (Huizer et al., PlosOne 2018) and Luminex assay. RESULTS In GBM patients all EPC subsets were elevated as compared to healthy subjects. In addition, HPC and KDR+ cell fractions were higher than in MI, while CD133+ and KDR+CD133+ cell fractions were lower. There were differences in relative EPC fractions between the groups: KDR+ cells were the largest fraction in GBM while CD133+ cells were the largest fraction in MI. An increase in glioma malignancy grade coincided with an increase in the KDR+ fraction while the CD133+ cell fraction decreased relatively. Most plasma angiogenic factors were higher in GBM than MI patients. In both MI and GBM, the ratio of CD133+ HPCs correlated significantly with elevated levels of MMP9. In the GBM patients MMP9 correlated strongly with levels of all HPCs. CONCLUSION In conclusion, the data demonstrate that EPC traffic in patients with glioma is different from that in normal tissue regeneration. Therefore, the effects of glioma extent beyond the brain, and future therapies aimed at lowering KDR+ cells and HPCs may add to effective treatment.

Current treatment options for glioblastoma patients have modest effects on survival. Since GBMs are one of the most vascularized tumors, treatment targeting GBM vessels has recently been introduced; clinical improvement, however, is limited. Endothelial progenitor cells are bone-marrow derived cells that home to ischemic tissue, guided by chemoattractants, where they differentiate under the influence of the microenvironment, and stimulate angiogenesis by secreting proangiogenic factors. As such, EPCs play an important role in neovascularization, including in GBMs. While changes in gene expression in EPCs once they arrive at their target location are well-established, little is known about the expression of proangiogenic factors in EPCs that are circulating in the blood. Since important factors from the GBM microenvironment also enter the bloodstream, we hypothesized that changes in gene expression could already be present in circulating EPCs from GBM patients. In order to test this hypothesis, we isolated EPCs (CD34 + CD133 + CD45dim) by FACS from blood of healthy controls (HC) and GBM patients. We determined the expression of a panel of 54 genes involved in angiogenesis by RT-PCR. The amount of circulating EPCs were similar between HC and GBM patients. Several genes, however, were differentially expressed; EPCs from GBM patients expressed both c-kit and SCF at a higher level than HC EPCs. In GBM, SCF is an important proangiogenic factor, which binds to its receptor c-kit. Both SCF and c-kit are expressed by subsets of endothelial cells in GBMs. We are currently investigating whether EPC c-kit and SCF expression correlates with tumor endothelial cell expression, and plan to do functional studies to determine the significance of our findings in GBM neovascularization. Our findings suggests that GBMs are capable of influencing gene expression in EPCs while they are still in the circulation. Influencing circulating EPCs in GBM patients could represent a novel type of antiangiogenic therapy.

The 5′untranslated region (UTR) of the FMR1 gene contains a CGG-repeat, which may become unstable upon transmission to the next generation. When repeat length exceeds 200, the FMR1 gene generally undergoes methylation-mediated transcriptional silencing. The subsequent absence of the gene product Fragile X Mental Retardation Protein (FMRP) causes the mental retardation seen in fragile X patients. A CGG-repeat length between 55 and 200 trinucleotides has been termed the premutation (PM). Predominantly elderly male PM carriers are at risk of developing a progressive neurodegenerative disorder: fragile X-associated tremor/ataxia syndrome (FXTAS). All PM carriers have elevated FMR1 mRNA levels, in spite of slightly decreased FMRP levels. The presence of intranuclear ubiquitin-positive inclusions in many brain regions is a neuropathological hallmark of FXTAS. Studies in humans attempting to correlate neuropathological outcomes with molecular measures are difficult because of the limited availability of tissue. Therefore, we have used the expanded CGG-repeat knock-in mouse model of FXTAS to examine the relationship between the molecular and neuropathological parameters in brain. We present Fmr1 mRNA and Fmrp levels and the presence of intranuclear inclusions at different repeat lengths. Contrary to existing hypotheses, our results suggest that inclusion formation may not depend on the elevation per se of Fmr1 transcript levels in aged CGG mice.

Purpose With the rapid discovery of prognostic and predictive molecular parameters for glioma, the status of histopathology in the diagnostic process should be scrutinized. Our project aimed to construct a diagnostic algorithm for gliomas based on molecular and histologic parameters with independent prognostic values. Methods The pathology slides of 636 patients with gliomas who had been included in EORTC 26951 and 26882 trials were reviewed using virtual microscopy by a panel of six neuropathologists who independently scored 18 histologic features and provided an overall diagnosis. The molecular data for IDH1, 1p/19q loss, EGFR amplification, loss of chromosome 10 and chromosome arm 10q, gain of chromosome 7, and hypermethylation of the promoter of MGMT were available for some of the cases. The slides were divided in discovery (n = 426) and validation sets (n = 210). The diagnostic algorithm resulting from analysis of the discovery set was validated in the latter. Results In 66% of cases, consensus of overall diagnosis was present. A diagnostic algorithm consisting of two molecular markers and one consensus histologic feature was created by conditional inference tree analysis. The order of prognostic significance was: 1p/19q loss, EGFR amplification, and astrocytic morphology, which resulted in the identification of four diagnostic nodes. Validation of the nodes in the validation set confirmed the prognostic value (P < .001). Conclusion We succeeded in the creation of a timely diagnostic algorithm for anaplastic glioma based on multivariable analysis of consensus histopathology and molecular parameters.