Glioma Blood Vessels Research Articles

Non-Coding RNAs in Glioma Microenvironment and Angiogenesis.

Glioma, especially glioblastoma, is the most common and lethal brain tumor. In line with the complicated vascularization processes and the strong intratumoral heterogeneity, tumor-associated blood vessels in glioma are regulated by multiple types of cells through a variety of molecular mechanisms. Components of the tumor microenvironment, including tumor cells and tumor-associated stromata, produce various types of molecular mediators to regulate glioma angiogenesis. As critical regulatory molecules, non-coding RNAs (ncRNAs) inside cells or secreted to the tumor microenvironment play essential roles in glioma angiogenesis. In this review, we briefly summarize recent studies about the production, delivery, and functions of ncRNAs in the tumor microenvironment, as well as the molecular mechanisms underlying the regulation of angiogenesis by ncRNAs. We also discuss the ncRNA-based therapeutic strategies in the anti-angiogenic therapy for glioma treatment.

A Dual Receptor Targeting- and BBB Penetrating- Peptide Functionalized Polyethyleneimine Nanocomplex for Secretory Endostatin Gene Delivery to Malignant Glioma.

PurposeVascular endothelial growth factor receptor 2 (VEGFR-2) and neuropilin-1 (NRP-1) are two prominent synergistic receptors overexpressed on new blood vessels in glioma and may be promising targets for antiglioma therapy. The aim of this study was to design a dual receptor targeting and blood-brain barrier (BBB) penetrating peptide-modified polyethyleneimine (PEI) nanocomplex that can efficiently deliver the angiogenesis-inhibiting secretory endostatin gene (pVAXI-En) to treat glioma.Materials and MethodsWe first constructed the tandem peptide TAT-AT7 by conjugating AT7 to TAT and evaluated its binding affinity to VEGFR-2 and NRP-1, vasculature-targeting ability and BBB crossing capacity. Then, TAT-AT7-modified PEI polymer (PPTA) was synthesized, and a pVAXI-En-loaded PPTA nanocomplex (PPTA/pVAXI-En) was prepared. The physicochemical properties, cytotoxicity, transfection efficiency, capacities to cross the BBB and BTB (blood-tumor barrier) and glioma-targeting properties of PPTA/pVAXI-En were investigated. Moreover, the in vivo anti-angiogenic behaviors and anti-glioma effects of PPTA/pVAXI-En were evaluated in nude mice.ResultsThe binding affinity of TAT-AT7 to VEGFR-2 and NRP-1 was approximately 3 to 10 times greater than that of AT7 or TAT. The cellular uptake of TAT-AT7 in endothelial cells was 5-fold and 119-fold greater than that of TAT and AT7 alone, respectively. TAT-AT7 also displayed remarkable efficiency in penetrating the BBB and glioma tissue in vivo. PPTA/pVAXI-En exhibited lower cytotoxicity, stronger BBB and BTB traversing abilities, higher selective glioma targeting and better gene transfection efficiency than PEI/pVAXI-En. More importantly, PPTA/pVAXI-En significantly suppressed the tube formation and migration of endothelial cells, inhibited glioma growth, and reduced the microvasculature in orthotopic U87 glioma-bearing nude mice.ConclusionOur study demonstrates that PPTA/pVAXI-En can be exploited as an efficient dual-targeting nanocomplex to cross the BBB and BTB, and hence it represents a feasible and promising nonviral gene delivery system for effective glioma therapy.

SUMOylation of PUM2 promotes the vasculogenic mimicry of glioma cells via regulating CEBPD.

Glioma is the most common form of primary central nervous malignant tumors. Vasculogenic mimicry (VM) is a blood supply channel that is different from endothelial blood vessels in glioma. VM is related to tumor invasion and metastasis. Therefore, it plays an important role to target therapy for glioma VM. Our experimental results showed abnormal expression of UBE2I, PUM2, CEBPD, and DSG2 in glioma cells. The Co‐IP and Immunofluorescence staining were used to detect that PUM2 can be modified by SUMO2/3. The interaction between PUM2 and CEBPD mRNA was detected by the RIP assays. The interaction between transcription factor CEBPD and promoter region of DSG2 was detected by the ChIP assays and luciferase assays. The capacity for migration in glioma cells was observed by the laser holographic microscope. The capacity for invasion in glioma cells was detected by Transwell method. The VM in glioma cells was detected by three‐dimensional cell culture method. The experimental results found that the upregulation of UBE2I in glioma tissues and cells promotes the SUMOylation of PUM2, which decreases not only the stability of PUM2 protein but also decreases the inhibitory effect of PUM2 on CEBPD mRNA. The upregulation of CEBPD promotes the binding to the upstream promoter region of DSG2 gene, further upregulates the expression of DSG2, and finally promotes the development of glioma VM. In conclusion, this study found that the UBE2I/PUM2/CEBPD/DSG2 played crucial roles in regulating glioma VM. It also provides potential targets and alternative strategies for combined treatment of glioma.

Automatic and label-free identification of blood vessels in gliomas using the combination of multiphoton microscopy and image analysis.

Currently, the targeted treatment of tumor based on the tumor microenvironment is newly developed. Blood vessels are the key parts in the tumor microenvironment, which is taken as a new visible target for tumor therapy. Multiphoton microscopy (MPM), based on the second harmonic generation and two-photon excited fluorescence, is available to make the label-free analysis on the blood vessels in human gliomas. MPM can reveal the vascular morphological characteristics in gliomas, including vascular malformation, intense vascular proliferation, perivascular collagen deposition, perivascular lymphocytes aggregation and microvascular proliferation. In addition, the image analysis algorithms were developed to automatically calculate the perivascular collagen content, vascular cavity area, lumen area, wall area and vessel number. Thus, the vascular morphology, the perivascular collagen deposition and intense vascular proliferation degree can be further quantitatively characterized. Compared with the pathological analysis, the combination of MPM and image analysis has potential advantages in making a quantitative and qualitative analyzing on vascular morphology in glioma microenvironment. As micro-endoscope and two-photon fiberscope are technologically improved, this combined method will be a useful imaging way to make the real-time research on the targeting tumor microenvironment in gliomas.

EGFL7 enhances surface expression of integrin α5β1 to promote angiogenesis in malignant brain tumors

Glioblastoma (GBM) is a typically lethal type of brain tumor with a median survival of 15 months postdiagnosis. This negative prognosis prompted the exploration of alternative treatment options. In particular, the reliance of GBM on angiogenesis triggered the development of anti‐VEGF (vascular endothelial growth factor) blocking antibodies such as bevacizumab. Although its application in human GBM only increased progression‐free periods but did not improve overall survival, physicians and researchers still utilize this treatment option due to the lack of adequate alternatives. In an attempt to improve the efficacy of anti‐VEGF treatment, we explored the role of the egfl7 gene in malignant glioma. We found that the encoded extracellular matrix protein epidermal growth factor‐like protein 7 (EGFL7) was secreted by glioma blood vessels but not glioma cells themselves, while no major role could be assigned to the parasitic miRNAs miR‐126/126*. EGFL7 expression promoted glioma growth in experimental glioma models in vivo and stimulated tumor vascularization. Mechanistically, this was mediated by an upregulation of integrin α5β1 on the cellular surface of endothelial cells, which enhanced fibronectin‐induced angiogenic sprouting. Glioma blood vessels that formed in vivo were more mature as determined by pericyte and smooth muscle cell coverage. Furthermore, these vessels were less leaky as measured by magnetic resonance imaging of extravasating contrast agent. EGFL7‐inhibition using a specific blocking antibody reduced the vascularization of experimental gliomas and increased the life span of treated animals, in particular in combination with anti‐VEGF and the chemotherapeutic agent temozolomide. Data allow for the conclusion that this combinatorial regimen may serve as a novel treatment option for GBM.

Pericytes promote abnormal tumor angiogenesis in a rat RG2 glioma model.

In glioma angiogenesis, tumor vessels cause morphological and functional abnormalities associated with malignancy and tumor progression. We hypothesized that certain structural changes or scantiness of functional pericytes may be involved in the formation of dysfunctional blood vessels in gliomas. In this study, we performed morphological examinations to elucidate the possible involvement of pericytes in brain tumor vessel abnormalities using a rat RG2 glioma model. After implantation of RG2 glioma cells in the syngeneic rat brain, gliomas were formed as early as day 7. In immunohistochemical examinations, desmin-positive pericytes, characterized by morphological abnormalities, were abundantly found on leaky vessels, as assessed by extravasation of lectin and high-molecular-weight dextrans. Interestingly, desmin-positive pericytes seemed to be characteristic of gliomas in rats. These pericytes were also found to express heat-shock protein 47, which plays an important role in the formation of the basement membrane, suggesting that RG2 pericytes promoted angiogenesis by producing basement membrane as a scaffold for newly forming blood vessels and caused functional abnormalities. We concluded that RG2 pericytes may be responsible for abnormal tumor angiogenesis lacking the functional ability to maintain the blood-brain barrier.

The role of extracellular-5'-nucleotidase/CD73 in glioma peritumoural brain edema.

During pathological conditions, extracellular-5'-nucleotidase/CD73 can protect neurons by reducing the permeability of the blood brain barrier. In recent years, it has been demonstrated that CD73 can negatively contribute to the growth of gliomas; however, the function of CD73 in glioma blood vessels is not clear. We analysed the expression of CD73 in 72 glioma patients using immunohistochemistry and correspondingly compared the results with the Edema index (EI). We established an in vitro model of the blood-tumour barrier and analysed the expression of CD73 in vascular endothelial cells. Lastly, CD73 expression was inhibited in endothelial cells, and the effects of this inhibition on tight junction structure and transendothelial resistance were observed. Compared to normal brains, the expression of CD73 in blood vessels of glioma patients was significantly decreased, and the amount was lower in the centre of the tumour than the periphery. The proportion of CD73-positive blood vessels had a positive correlation with the EI. The expression of CD73 in the in vitro endothelial cell blood-tumour barrier model was decreased. Lastly, inhibiting CD73 was found to decrease the expression of tight junction related proteins in endothelial cells and to decrease the value of transendothelial electric resistance. The expression of CD73 in glioma blood vessels was significantly decreased, which may play a multi-functional role in decreasing the expression of tight junction related proteins of brain microvascular endothelial cells and may also increase blood-tumour barrier permeability and accelerate the formation of PTBE.

Growing blood vessels in gliomas

Cancer Aggressive gliomas have a high density of abnormal blood vessels that enables tumor growth and damages the brain. Zhang et al. analyzed patient data and correlated increased levels of a secreted factor called pleiotrophin with more aggressive grades of glioma and decreased survival. When implanted in mice, glioma cells that released pleiotrophin formed larger tumors with more blood vessels. Mice developed smaller gliomas and survived longer when treated with inhibitors of ALK, a receptor for pleiotrophin. Sci. Signal. 8 , ra125 (2015).

Discrimination between two different grades of human glioma based on blood vessel infrared spectral imaging.

Gliomas are brain tumours classified into four grades with increasing malignancy from I to IV. The development and the progression of malignant glioma largely depend on the tumour vascularization. Due to their tissue heterogeneity, glioma cases can be difficult to classify into a specific grade using the gold standard of histological observation, hence the need to base classification on a quantitative and reliable analytical method for accurately grading the disease. Previous works focused specifically on vascularization study by Fourier transform infrared (FTIR) spectroscopy, proving this method to be a way forward to detect biochemical changes in the tumour tissue not detectable by visual techniques. In this project, we employed FTIR imaging using a focal plane array (FPA) detector and globar source to analyse large areas of glioma tumour tissue sections via molecular fingerprinting in view of helping to define markers of the tumour grade. Unsupervised multivariate analysis (hierarchical cluster analysis and principal component analysis) of blood vessel spectral data, retrieved from the FPA images, revealed the fine structure of the borderline between two areas identified by a pathologist as grades III and IV. Spectroscopic indicators are found capable of discriminating different areas in the tumour tissue and are proposed as biomolecular markers for potential future use of grading gliomas.Graphical Infrared imaging of glioma blood vessels provides a means to revise the pathologists’ line of demarcation separating grade III (GIII) from grade IV (GIV) parts.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-015-8891-z) contains supplementary material, which is available to authorized users.

Investigation of blood vessels in glioblastoma at a micrometric scale: a comparative study by synchrotron and conventional micro-FTIR

Glioblastoma, the most malignant brain tumor in humans, is characterized by being severely angiogenic and an increase in vascularization generally worsens the prognosis of patients. Finding the best approach to characterize glioma blood vessels (BVs) is very important in view of helping to determine any specific biomolecular markers of these tumors. In previous work by conventional FTIR spectroscopy we were able to discriminate some molecular markers in order to differentiate between normal and tumor BVs in glioma tissue sections. The aim of the present study was to assess whether FTIR microspectroscopy using a synchrotron radiation (SR) source could provide advantages over a classical globar IR source for detailed spectral analysis on such small features like micro-BVs. Using chemometric analysis such as PCA and HCA, the results show that a high brilliant SR beam provides a very satisfying quality signal compared with the globar source to study spectral images for relevant analysis of glioma big and micro-BVs and determination of subtle molecular markers characterizing them from the surrounding tissue.

Bevacizumab Has Differential and Dose-Dependent Effects on Glioma Blood Vessels and Tumor Cells

Bevacizumab targets VEGF-A and has proved beneficial in glioma patients, improving clinical symptoms by the reduction of tumor edema. However, it remains controversial whether or not bevacizumab exerts antitumor effects in addition to (and potentially independent of) its effects on tumor vessels, and it is unknown what doses are needed to achieve this. We established a novel orthotopic glioma mouse model that allowed us to simultaneously study the kinetics of the morphologic and functional vascular changes, tumor growth, and the viability of individual tumor cells during the course of anti-VEGF therapy in the same microscopic tumor region in real-time. Three doses of bevacizumab were compared, a subclinical dose and two clinical doses (medium and high). Low (subclinical) doses of bevacizumab led to a significant reduction of the total vascular volume without affecting tumor cell viability or the overall tumor growth rates. Medium and high doses triggered a similar degree of vascular regression but significantly decreased tumor growth and prolonged survival. Remaining vessels revealed morphologic features of vascular normalization, reduced permeability, and an increase in blood flow velocity; the latter was dose dependent. We observed an uncoupling of the antitumoral and the antivascular effects of bevacizumab with the high dose only, which showed the potential to cause microregional glioma cell regression. In some tumor regions, pronounced glioma cell regression occurred even without vascular regression. In vitro, there was no effect of bevacizumab on glioma cell proliferation. Regression of glioma cells can occur independently from vascular regression, suggesting that high doses of bevacizumab have indirect anticancer cell properties in vivo.

Abstract 1156: MicroRNA-21 in gliomas: An in situ hybridization study

Abstract High-grade gliomas are some of the most lethal forms of human cancers. In glioblastomas, which are the most frequent and malignant type of glioma, microRNA-21 (miR21) has recently been suggested to be strongly upregulated. Until now, miR21 expression analysis has mainly been carried out using PCR methods, hence little is known about the cellular localization of miR21 in gliomas. Studies of other cancers have demonstrated miR21's prognostic value as well as roles in invasion, growth and anti-apoptosis. This suggests that miR21 is a potential therapeutic target as well as a promising novel prognostic marker in gliomas. The aim of the present study was therefore to investigate the expression of miR21 in gliomas and correlate it with tumor grade and overall survival using the new possibilities of locked nucleic acid (LNA) based in situ hybridization. We used 193 formalin-fixed paraffin embedded astrocytic and oligodendroglial brain tumors of increasing grade (WHO I-IV). Sections from these tumors were stained by a robust in situ hybridization assay using high-affinity LNA probes that specifically detect miR21. The expression of miR21 was revealed as a blue chromogenic staining reaction. Using advanced image analysis, the area of the staining reaction was measured by a trained pixel classifier on images recorded by stereological principles. The optimal sampling fraction was estimated to be 10%. The results showed that the miR21 signal was localized to blood vessels and populations of dispersed single cells. Moreover, the characteristic gemistocytic tumor cells seemed to display a distinct staining reaction. The miR21 signal increased with tumor grade and a very high individual signal variation was observed, especially among glioblastomas. When comparing the miR21 signal with overall survival, a high signal was associated with shorter survival, but in multivariate analysis this was not independent of grade. In conclusion miR21 is localized in both tumor cells and blood vessels in gliomas. A high miR21 expression appears to be associated with tumor grade and shorter survival underlining the importance of miR21 in glioma biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1156. doi:10.1158/1538-7445.AM2011-1156

Expression Sites of Colligin 2 in Glioma Blood Vessels

In a previous study using state‐of‐the‐art proteomic techniques, we identified colligin 2 (HSP47) as a glioma blood vessel‐specific protein. In the present study we precisely localized the expression of colligin 2 in the blood vessels of diffusely infiltrating gliomas and relate the expression to the distinct cellular components of the vessels by using multiple immunolabeling and confocal microscopy. We grouped the glioma blood vessels into morphological categories ranging from normal looking capillaries to vessels with hypertrophic and sclerotic changes. The expression patterns of various markers of endothelial and pericytic differentiation were correlated with the position of the cells in the vessels and the expression of colligin 2. We found that colligin 2 is expressed in all categories of glioma blood vessels in cells with endothelial and pericytic lineage. Expression of colligin 2 was also found in cells scattered around blood vessels and in few glial fibrillary acidic protein‐positive cells within the blood vessels. There is overlap in the expression of colligin 2 and the collagens type I and IV for which colligin 2 is a chaperon. We conclude that colligin 2 is expressed in all cellular components of glioma blood vessels and may serve as a general marker for active angiogenesis.

Identification of Glioma Neovascularization-related Proteins by Using MALDI-FTMS and Nano-LC Fractionation to Microdissected Tumor Vessels

The identification of angiogenesis-related proteins is important for the development of new antiangiogenic therapies, and such proteins are potential new biomarkers for gliomas. The aim of this study was to identify proteins that are exclusively present in glioma neovasculature and not in the vasculature of normal brain. We combined advanced proteomics techniques to compare the expression profiles of microdissected blood vessels from glioma with blood vessels of normal control brain samples. We measured the enzymatic generated peptide profiles from these microdissected samples by MALDI-FTMS. Subsequently, the samples were fractionated by nano-LC prior to MALDI-TOF/TOF. This combined approach enabled us to identify four proteins that appeared to be exclusively expressed in the glioma blood vessels. Two of these proteins, fibronectin and colligin 2, were validated on tissue sections using specific antibodies. We found that both proteins are present in active angiogenesis in glioma, other neoplasms, and reactive conditions in which neoangiogenesis takes place. This work proves that gel-free mass spectrometric techniques can be used on relatively small numbers of cells generated by microdissection procedures to successfully identify differentially expressed proteins.

Differential expression of Hela‐type caldesmon in tumour neovascularization: a new marker of angiogenic endothelial cells

Caldesmon (CaD) is a major actomyosin-binding protein found in various cell types. There are at least two high-molecular-weight isoforms (h-CaD) and four low-molecular-weight isoforms (l-CaD) produced by alternative splicing. The alternatively spliced variants of the l-CaD class are further differentiated by inclusion (Hela l-CaD) or exclusion (WI-38 l-CaD) of exon 1. Currently, nothing is known about differential expression of the Hela l-CaD in tumour neovascularization. In a previous study, expression of the Hela-type transcripts was found in glioma blood vessels but not in the normal cerebral vasculature. To investigate whether the differentially expressed transcripts are translated into protein, a specific antibody against the peptide encoded by exon 1 was raised. Initially, exclusive expression of the protein in glioma vasculature was confirmed. To determine further whether these findings are generalizable to neovascularization in a wide variety of other tumour types, a large cohort of cancers derived from various organs, including breast, lung, kidney, colon, stomach, ovary, uterus, prostate, thyroid, liver, giving a total of 180 cases, were examined. Expression of the Hela l-CaD was restricted to tumour vasculature and was not found in normal blood vessels. Hela l-CaD was preferentially expressed in the early stage of tumour neovascularization and the Hela l-CaD+ endothelial cells (ECs) were frequently enlarged, multinucleated, and developed elongated cell projections or free fragments of cytoplasm, correlating with the features of motile cells. In the Hela l-CaD+ ECs, disassembly of focal adhesion and the formation of podosome-like structures was observed. Therefore, the findings support the notion that quiescent ECs undergo activation of motility, necessary for ubiquitous tumour-associated neovascularization. The data indicate that Hela l-CaD can be considered as a marker for angiogenic ECs during the early stages of tumour neovascularization.

Expression of integrin alpha v beta 3 in small blood vessels of glioblastoma tumors.

Angiogenesis, which promotes tumor proliferation and invasiveness, is mediated by integrin-dependent cell adhesion mechanisms and requires expression of integrin alpha v beta 3. To determine whether integrin alpha v beta 3 is expressed in the small blood vessel hyperplasia characteristic of glioblastoma tumors, we explored the mRNA and protein expression of integrin alpha v, beta 3, beta 5, and beta 1 subunits in small blood vessels in gliomas of various grades and nontumorous brain biopsies. Antisense beta 3 and beta 5 riboprobes hybridized to small blood vessels (endothelial and adjacent mesenchymal cells) in 11 of 11 glioblastomas and 2 of 4 anaplastic astrocytomas, but failed to hybridize to small blood vessels in 12 non-neoplastic specimens and 4 low-grade astrocytomas. In contrast, antisense alpha v and beta 1 riboprobes hybridized to small blood vessels in all of the biopsies. The expression of integrin alpha v beta 3 protein in these vessels was determined by immunohistochemical analysis using anti-integrin subunit-specific antibodies. The findings were similar to those obtained using in situ hybridization: beta 3 subunit protein was detected on small blood vessels in 9 of 12 glioblastoma and 2 of 4 anaplastic astrocytomas, whereas it was not detected in small blood vessels of 10 non-neoplastic brains and 4 low-grade astrocytomas. In contrast, the alpha v and beta 1 subunit proteins were detected in small blood vessels in all of the brain biopsies, whereas the beta 5 subunit protein could not be detected. These data suggest that integrin alpha v beta 3 is expressed in small blood vessels of glioblastoma tumors.

Glomeruloid blood vessels in ethylnitrosourea-induced rat gliomas. Histological and immunohistochemical studies.

Glomeruloid blood vessels (GBVs), a characteristic histological feature of most human malignant gliomas, were recognized with high incidence in autochthonous rat gliomas induced by transplacental administration of ethylnitrosourea. To evaluate some of the biological properties of these GBVs, we carried out a study using histological methods and immunohistochemical staining for glial fibrillary acidic protein, factor VIII-related antigen (VIII Ag) and bromodeoxyuridine (BrdUrd). Of 22 animals with large, massively growing gliomas in the CNS, GBVs including conglomerate aggregations of small blood vessels with endothelial hyperplasia and strong VIII Ag expression were observed in 13 large gliomas histologically consisting of primitive neuroepithelial neoplasms (PNN; so called ependymoma) and mixed-type gliomas in combination with astrocytoma and PNN or anaplastic astrocytoma. The anaplastic gliomas in our series were devoid of GBVs. These findings indicate that GBV formation takes place in a histological variety of experimental gliomas. Furthermore, the GBVs were frequently associated with the vasculo-mesenchymal stroma in the parent gliomas, suggesting an intimate relationship with the morphogenesis of GBVs. In addition, it was shown that the GBVs had a higher BrdUrd-labelling index than that of other blood vessels in gliomas and also that of neoplastic cells in most parent gliomas, except for anaplastic gliomas. Based on these results, the possible mechanism of GBV morphogenesis is discussed with regard to the roles of macromolecules in the induction and regulation of GBVs.