Circulating angiogenic cells in glioblastoma: toward defining crucial functional differences in CAC-induced neoplastic versus reactive neovascularization.

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.

Periostin Is Expressed by Pericytes and Is Crucial for Angiogenesis in Glioma.

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 PDGFRß+ 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.

Circulating Proangiogenic Cells and Proteins in Patients with Glioma and Acute Myocardial Infarction: Differences in Neovascularization between Neoplasia and Tissue Regeneration.

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.

CGG-repeat length and neuropathological and molecular correlates in a mouse model for fragile X-associated tremor/ataxia syndrome.

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.

Improving the characterization of endothelial progenitor cell subsets by an optimized FACS protocol.

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.

Evidence-Based Diagnostic Algorithm for Glioma: Analysis of the Results of Pathology Panel Review and Molecular Parameters of EORTC 26951 and 26882 Trials.

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.