Transcriptional profiling of human glioblastoma vessels indicates a key role of VEGF‐A and TGFβ2 in vascular abnormalization

Abstract

Glioblastoma are aggressive astrocytic brain tumours characterized by microvascular proliferation and an abnormal vasculature, giving rise to brain oedema and increased patient morbidity. Here, we have characterized the transcriptome of tumour-associated blood vessels and describe a gene signature clearly associated with pleomorphic, pathologically altered vessels in human glioblastoma (grade IV glioma). We identified 95 genes differentially expressed in glioblastoma vessels, while no significant differences in gene expression were detected between vessels in non-malignant brain and grade II glioma. Differential vascular expression of ANGPT2, CD93, ESM1, ELTD1, FILIP1L and TENC1 in human glioblastoma was validated by immunohistochemistry, using a tissue microarray. Through qPCR analysis of gene induction in primary endothelial cells, we provide evidence that increased VEGF-A and TGFβ2 signalling in the tumour microenvironment is sufficient to invoke many of the changes in gene expression noted in glioblastoma vessels. Notably, we found an enrichment of Smad target genes within the distinct gene signature of glioblastoma vessels and a significant increase of Smad signalling complexes in the vasculature of human glioblastoma in situ. This indicates a key role of TGFβ signalling in regulating vascular phenotype and suggests that, in addition to VEGF-A, TGFβ2 may represent a new target for vascular normalization therapy.

Multiplex mapping of chromatin accessibility and DNA methylation within targeted single molecules identifies epigenetic heterogeneity in neural stem cells and glioblastoma

“…However, additional studies are required to determine the functional significance of DNA methylation, if any, at these genes. Our finding of differentially methylated/accessible genes previously shown to harbor mutations in GBM-COL19A1 (Sumiyoshi et al 1997), CD93 (Dieterich et al 2012), AGAP2 (Knobbe et al 2005), and ACSL5 (Mashima et al 2009)-supports the validity of this approach in identifying GBM-relevant epigenetic perturbations. Interestingly, most of the genes that were identified as differentially methylated between GBM and NSC were classified as variably methylated in NSC (Table 4, lower).…”

Elevated Expression of the C-Type Lectin CD93 in the Glioblastoma Vasculature Regulates Cytoskeletal Rearrangements That Enhance Vessel Function and Reduce Host Survival

“…In line with our initial finding on elevated CD93 expression in glioblastoma vasculature, a subsequent study identified CD93 as one of the top genes of a core human primary tumor angiogenesis signature, highly expressed in head and neck squamous cell carcinomas, breast cancers, and clear cell renal cell carcinomas (7,15). These studies are the first ones to report CD93 as a tumor vascular marker.…”

“…Previously, we reported that these pathologically altered vessels in grade IV glioma express high levels of CD93, a transmembrane protein from the C-type superfamily of lectins with a calcium-dependent carbohydrate-binding domain (7). CD93 was initially suggested to play a role in angiogenesis due to its high expression in areas of blood vessel remodeling in the developing vasculature of mouse embryos (24).…”

“…We previously demonstrated that high vascular density, vascular abnormalization, and formation of glomeruloid microvascular proliferations are characteristic features of grade IV glioma, and that these aberrant vessels produce high levels of CD93 mRNA and protein (7). In order to extend our analysis of CD93 protein expression in tumor vessels in a larger material, we scored the fraction of blood vessels staining positive for CD93 in several tumor tissue microarrays that contained a total of 235 biopsies of human control brain and grade II, grade III, and grade IV glioma tissues of various histologic subtypes.…”

“…Resistance mechanisms leading to upregulation of other proangiogenic factors or toxicities arising due to the important role of VEGF/VEGFR2 signaling in normal physiology may contribute to these disappointing results. An alternative strategy is to exploit the heterogeneity of gene expression in tumor endothelial cells as compared with normal endothelial cells, and target molecules specifically expressed in the tumor vasculature that contribute to angiogenesis and vascular malformation in glioblastoma (6)(7)(8)(9). Through laser capture microdissection followed by microarray analysis of gene expression, we have recently characterized the transcriptome of blood vessels in human glioblastoma, low-grade glioma, and control brain tissue.…”