MicroRNA-132/212 family enhances arteriogenesis after hindlimb ischaemia through modulation of the Ras-MAPK pathway.

Arteriogenesis is a complicated process induced by increased local shear-and radial wall-stress, leading to an increase in arterial diameter. This process is enhanced by growth factors secreted by both inflammatory and endothelial cells in response to physical stress. Although therapeutic promotion of arteriogenesis is of great interest for ischaemic diseases, little is known about the modulation of the signalling cascades via microRNAs. We observed that miR-132/212 expression was significantly upregulated after occlusion of the femoral artery. miR-132/212 knockout (KO) mice display a slower perfusion recovery after hind-limb ischaemia compared to wildtype (WT) mice. Immunohistochemical analysis demonstrates a clear trend towards smaller collateral arteries in KO mice. Although Ex vivo aortic ring assays score similar number of branches in miR-132/212 KO mice compared to WT, it can be stimulated with exogenous miR-132, a dominant member of the miR-132/212 family. Moreover, in in vitro pericyte-endothelial co-culture cell assays, overexpression of miR-132 and mir-212 in endothelial cells results in enhanced vascularization, as shown by an increase in tubular structures and junctions. Our results suggested that miR-132/212 may exert their effects by enhancing the Ras-Mitogen-activated protein kinases MAPK signalling pathway through direct inhibition of Rasa1, and Spred1. The miR-132/212 cluster promotes arteriogenesis by modulating Ras-MAPK signalling via direct targeting of its inhibitors Rasa1 and Spred1.

Endothelial cells require miR-214 to secrete exosomes that suppress senescence and induce angiogenesis in human and mouse endothelial cells.

Signaling between endothelial cells, endothelial progenitor cells, and stromal cells is crucial for the establishment and maintenance of vascular integrity and involves exosomes, among other signaling pathways. Exosomes are important mediators of intercellular communication in immune signaling, tumor survival, stress responses, and angiogenesis. The ability of exosomes to incorporate and transfer messenger RNAs (mRNAs) encoding for "acquired" proteins or micro RNAs (miRNAs) repressing "resident" mRNA translation suggests that they can influence the physiological behavior of recipient cells. We demonstrate that miR-214, an miRNA that controls endothelial cell function and angiogenesis, plays a dominant role in exosome-mediated signaling between endothelial cells. Endothelial cell-derived exosomes stimulated migration and angiogenesis in recipient cells, whereas exosomes from miR-214-depleted endothelial cells failed to stimulate these processes. Exosomes containing miR-214 repressed the expression of ataxia telangiectasia mutated in recipient cells, thereby preventing senescence and allowing blood vessel formation. Concordantly, specific reduction of miR-214 content in exosome-producing endothelial cells abolishes the angiogenesis stimulatory function of the resulting exosomes. Collectively, our data indicate that endothelial cells release miR-214-containing exosomes to stimulate angiogenesis through the silencing of ataxia telangiectasia mutated in neighboring target cells.

Myc-associated zinc finger protein (MAZ) is regulated by miR-125b and mediates VEGF-induced angiogenesis in glioblastoma.

In patients with glioblastomas, vascular endothelial growth factor (VEGF) is a key mediator of tumor-associated angiogenesis. Glioblastomas are notorious for their capacity to induce neovascularization, driving continued tumor growth. Here we report that miR-125b is down-regulated in glioblastoma-associated endothelial cells, resulting in increased expression of its target, myc-associated zinc finger protein (MAZ), a transcription factor that regulates VEGF. The down-regulation of miR-125b was also observed on exposure of endothelial cells to glioblastoma-conditioned medium or VEGF, resulting in increased MAZ expression. Further analysis revealed that inhibition of MAZ accumulation by miR-125b, or by MAZ-specific shRNAs, attenuated primary human brain endothelial cell migration and tubule formation in vitro, phenomena considered to mimick angiogenic processes in vitro. Moreover, MAZ expression was elevated in brain blood vessels of glioblastoma patients. Altogether these results demonstrate a functional feed-forward loop in glioblastoma-related angiogenesis, in which VEGF inhibits the expression of miR-125b, resulting in increased expression of MAZ, which in its turn causes transcriptional activation of VEGF. This loop is functionally impeded by the VEGF receptor inhibitor vandetanib, and our results may contribute to the further development of inhibitors of tumor-angiogenesis.

Trimethylation of H3K27 during human cerebellar development in relation to medulloblastoma.

Medulloblastoma (MB), the most common malignant childhood brain tumor, encompasses a collection of four clinically and molecularly distinct tumor subgroups, i.e. WNT, SHH, Group 3 and Group 4. These tumors are believed to originate from precursor cells during cerebellar development. Although the exact etiology of these brain tumors is not yet known, histone modifications are increasingly recognized as key events during cerebellum development and MB tumorigenesis. Recent studies show that key components involved in post-translational modifications of histone H3 lysine 27 (H3K27) are commonly deregulated in MB. In this descriptive study, we have investigated the trimethylation status of H3K27, as well as the expression of the H3K27 methylase EZH2 and demethylases KDM6A and KDM6B, during human cerebellum development in relation to MB. H3K27 Trimethylation status differed between the MB subgroups. Moreover, trimethylation of H3K27 and expression of its modifiers EZH2, KDM6A and KDM6B were detected in a spatio-temporal manner during development of the human cerebellum, with consistent high occurrence in the four proliferative zones, which are believed to harbor the precursor cells of the different MB subgroups. Our results suggest that H3K27 trimethylation in MB is deregulated by EZH2, KDM6A and KDM6B. Moreover, we provide evidence that during development of the human cerebellum H3K27me3 and its regulators are expressed in a spatio-temporal manner.

EZH2-regulated DAB2IP is a medulloblastoma tumor suppressor and a positive marker for survival.

PURPOSE: Medulloblastoma is the most common malignant brain tumor in children. Despite recent improvements, the molecular mechanisms driving medulloblastoma are not fully understood and further elucidation could provide cues to improve outcome prediction and therapeutic approaches. EXPERIMENTAL DESIGN: Here, we conducted a meta-analysis of mouse and human medulloblastoma gene expression data sets, to identify potential medulloblastoma tumor suppressor genes. RESULTS: We identified DAB2IP, a member of the RAS-GTPase-activating protein family (RAS GAP), and showed that DAB2IP expression is repressed in medulloblastoma by EZH2-induced trimethylation. Moreover, we observed that reduced DAB2IP expression correlates significantly with a poor overall survival of patients with medulloblastoma, independent of metastatic stage. Finally, we showed that ectopic DAB2IP expression enhances stress-induced apoptosis in medulloblastoma cells and that reduced expression of DAB2IP in medulloblastoma cells conveys resistance to irradiation-induced cell death. CONCLUSION: These results suggest that repression of DAB2IP may at least partly protect medulloblastoma cells from apoptotic cell death. Moreover, DAB2IP may represent a molecular marker to distinguish patients with medulloblastoma at high risk from those with a longer survival prognosis.

Down-regulation of miR-101 in endothelial cells promotes blood vessel formation through reduced repression of EZH2.

Angiogenesis is a balanced process controlled by pro- and anti-angiogenic molecules of which the regulation is not fully understood. Besides classical gene regulation, miRNAs have emerged as post-transcriptional regulators of angiogenesis. Furthermore, epigenetic changes caused by histone-modifying enzymes were shown to modulate angiogenesis as well. However, a possible interplay between miRNAs and histone-modulating enzymes during angiogenesis has not been described. Here we show that VEGF-mediated down-regulation of miR-101 caused pro-angiogenic effects. We found that the pro-angiogenic effects are partly mediated through reduced repression by miR-101 of the histone-methyltransferase EZH2, a member of the Polycomb group family, thereby increasing methylation of histone H3 at lysine 27 and transcriptome alterations. In vitro, the sprouting and migratory properties of primary endothelial cell cultures were reduced by inhibiting EZH2 through up-regulation of miR-101, siRNA-mediated knockdown of EZH2, or treatment with 3-Deazaneplanocin-A (DZNep), a small molecule inhibitor of EZH2 methyltransferase activity. In addition, we found that systemic DZNep administration reduced the number of blood vessels in a subcutaneous glioblastoma mouse model, without showing adverse toxicities. Altogether, by identifying a pro-angiogenic VEGF/miR-101/EZH2 axis in endothelial cells we provide evidence for a functional link between growth factor-mediated signaling, post-transcriptional silencing, and histone-methylation in the angiogenesis process. Inhibition of EZH2 may prove therapeutic in diseases in which aberrant vascularization plays a role.

miR-101 is down-regulated in glioblastoma resulting in EZH2-induced proliferation, migration, and angiogenesis.

BACKGROUND: Glioblastoma (GBM) is a malignant brain tumor with dismal prognosis. GBM patients have a median survival of less than 2 years. GBM is characterized by fast cell proliferation, infiltrative migration, and by the induction of angiogenesis. MicroRNAs and polycomb group (PcG) proteins have emerged as important regulators of gene expression. METHODS: Here we determined that miR-101 is down-regulated in GBM, resulting in overexpression of the miR-101 target PcG protein EZH2, a histone methyltransferase affecting gene expression profiles in an epigenetic manner. RESULTS: Inhibition of EZH2 in vitro by pre-miR-101, EZH2 siRNA, or small molecule DZNep, attenuated GBM cell growth, migration/invasion, and GBM-induced endothelial tubule formation. In addition, for each biological process we identified ontology-associated transcripts that significantly correlate with EZH2 expression. Inhibition of EZH2 in vivo by systemic DZNep administration in a U87-Fluc-mCherry GBM xenograft mouse imaging model resulted in reduced tumor growth. CONCLUSION: Our results indicate that EZH2 has a versatile function in GBM progression and that its overexpression is at least partly due to decreased miR-101 expression. Inhibition of EZH2 may be a potential therapeutic strategy to target GBM proliferation, migration, and angiogenesis.