Since glioma cells have a high capacity to induce angiogenesis (Brem et al., 1972
; Folkerth, 2000
), we used them as a means to stimulate this process in normal endothelial cells in a co-culture system. Primary human microvascular endothelial cells isolated from normal human brain (HBMVECs; Cell Systems, ACBRI-376) were cultured in the presence or absence of human U87 glioma cells expressing the fluorescent protein Cerulean (CFP) in endothelial basal medium lacking additional angiogenic factors (EBM; Cambrex). Elongation of the endothelial cells was induced by the cancer cells as a first step in the activation of angiogenesis, as previously described (Khodarev et al., 2003
) (). After 24 hr of either culturing the endothelial cells alone or co-culturing them with human U87 glioma cells, the endothelial cells were isolated using CD31 magnetic beads (Dynal Biotech). The purity (>99%) of the endothelial cell preparation was confirmed by the absence of glioma cells expressing the CFP marker (data not shown). Total RNA was isolated from endothelial cells, and the small RNA fraction was hybridized to miRNA arrays containing probes for 407 mature miRNAs (as in (Krichevsky et al., 2003
)) in order to identify differentially expressed miRNAs. Analysis of array hybridizations revealed eighty miRNAs expressed in HBMVECs at detectable levels ( and Supplementary Fig. S1
), and confirmed the expression of a number of previously described miRNAs in endothelial cells (Kuehbacher et al., 2007
; Poliseno et al., 2006
; Suarez et al., 2007
; Tuccoli et al., 2006
). After exposure of HBMVECs to U87 glioma cells the expression levels of a number of miRNAs changed significantly. This suggests that glioma cells can influence miRNA expression in endothelial blood vessel cells (). Most of the differentially expressed miRNAs were found to be down-regulated. One miRNA, miR-296, was identified and further confirmed by quantitative RT-PCR (qRT-PCR) analysis as up-regulated. We used miR-186 as a control miRNA and GAPDH as a normalization control, both of which were uniformly expressed in endothelial cells in the presence or absence of tumor cells (). In this study we further investigated miR-296, since it was the only significantly up-regulated miRNA in the glioma-induced endothelial cells.
Fig. 1 Glioma-induced disregulation of miRNAs in human brain endothelial cells (A) Primary human brain microvascular endothelial cells (HBMVEC) were cultured in the absence (left) or presence (right) of human U87-CFP glioma cells. Images were produced by using (more ...)
In order to determine what factors are responsible for the induction of miR-296, HBMVECs were cultured using various conditions after which RNA was isolated and the miR-296 expression levels were determined. Of note, miR-296 was not only induced in HBMVECs upon exposure of these cells to U87 glioma cells, but also by culture medium derived from U87 glioma cells and by EBM supplemented with angiogenic cocktail (EGM, containing hydrocortisone, EGF, FGF, VEGF, IGF, ascorbic acid, FBS, and heparin; Singlequots from Cambrex). Similar ~2-fold induction of miR-296 in HBMVECs was achieved by EGM and glioma cell stimulation. Since U87-conditioned medium and EGM both contain many growth factors, HBMVECs were also stimulated with basal medium supplemented with VEGF or EGF, each resulting in significant miR-296 upregulation, in contrast to exposure to hydrocortisone and heparin which did not change levels ().
Since VEGF enhanced miR-296 expression in endothelial cells, we addressed VEGF function in angiogenesis in endothelial cell culture. First, HBMVECs were cultured In the presence of different amounts of VEGF in EBM on a Matrigel substrate to promote tubule formation for 24 hr. A clear dose-response effect of VEGF on endothelial tubule formation and branching was observed (). In order to determine whether endothelial cell migration was also affected by VEGF, scratch assays were performed. HBMVECs were first cultured in EGM, then a spatula was used to scratch the monolayer, after which the cells were washed and cultured in EBM containing different amounts of VEGF, or in EGM, for 24 hr. VEGF significantly induced the migration of HBMVECs in a dose-response manner (). To determine a correlation between VEGF-induced phenotype and miR-296 expression, HBMVECs were cultured for 24 hr in EBM containing different amounts of VEGF, or in EGM, after which RNA was isolated and the miR-296 expression levels were determined by qRT-PCR. Notably, 10 ng/ml of VEGF were sufficient to coordinately up-regulate miR-296 levels and induce endothelial cell migration and tubule formation in HBMVEC cultures ().
Fig. 2 VEGF-mediated induction of tubule formation, migration, and miR-296 expression (A) HBMVECs cells were cultured on Matrigel-coated plates in basal medium (EBM; Cambrex) only, basal medium supplemented with a cocktail of angiogenic factors (EGM), or different (more ...)
To address miR-296 function in angiogenesis, we examined effects of its inhibition on glioma-induced angiogenesis in cell culture. In these experiments, HBMVECs were cultured on Matrigel either in EBM alone, or with human glioma cells, or in EGM. Tubules were visualized by a combination of light and fluorescence microscopy. Endothelial cells were discriminated from CFP-expressing U87 glioma cells by fluorescence microscopy (). miR-296 in endothelial cells was blocked by transfecting endothelial cells with a modified anti-sense miR-296 inhibitor (Ambion) prior to mixing the endothelial cells with U87 cells, with transfection efficiency being >99% as determined by transfection of siRNA-Cy3 (Alnylam) (). Levels of miR-296 decreased 4-fold in HBMVECs in the presence of this inhibitor, as determined by qRT-PCR (). At 48 hr after transfection of the miR-296 inhibitor or a non-related oligonucleotide of similar chemistry and 24 hr of culturing on Matrigel with or without U87-CFP cells, endothelial cells were analyzed for effects on tubule length and tubule branching (). Downregulation of miR-296 resulted in a significant decrease in tubule branching () and total tubule length () in HBMVECs exposed to an angiogenic cocktail or U87 glioma cells as compared to unexposed cells, but had no significant effect on the viability of endothelial cells (Supplementary Fig. S2
). U87 glioma cells induced tubule elongation to a greater extent than EGM, which may be due to other glioma cell induced effects, in addition to elevation of miR-296 (). Similar inhibitory effects on tubule formation were observed when miR-296 was blocked in human umbilical vein endothelial cells exposed to an angiogenic cocktail (Supplementary Fig. S3
), suggesting a general miR-296-mediated angiogenic mechanism operating in endothelial cells of different origins. In order to determine the effect of miR-296 inhibition on glioma-induced endothelial cell migration, scratch assays were performed. HBMVECs were transfected with the miR-296 inhibitor or control oligonucleotide. Twenty-four hr after transfection a scratch was made in the monolayer of HBMVECs cultured in U87-conditioned medium. Twenty-four hr after that the migration distance was analyzed by light microscopy and MetaVue software. Downregulation of miR-296 resulted in a significant decrease in U87-induced HBMVEC migration ().
Fig. 3 Angiogenesis co-culture assay: miR-296-mediated inhibition and induction of angiogenesis (A) HBMVECs cells were cultured on Matrigel-coated plates in basal medium (EBM; Cambrex) only, or basal medium supplemented with a cocktail of angiogenic factors (more ...)
To investigate whether miR-296 acts as a dominant determinant of angiogenesis miR-296 precursor molecules or control precursor-like molecules (Ambion) were transfected into HBMVECs cultured in EBM medium. Transfection efficiency was >99% as analyzed by fluorescence microscopy using a precursor control molecule conjugated to a Cy3 fluorophor (Ambion) (data not shown). Overexpression of miR-296 in cells transfected with miR-296 precursors was confirmed by qRT-PCR (). Twenty-four hr after transfection HBMVECs were plated on Matrigel in the absence of angiogenic stimuli. Pre-miR-296 stimulated tubule elongation and branching, in contrast to the control miRNA precursor (). These effects were observed for up to at least 4 days after transfection of pre-miR-296 molecules into HBMVECs (Supplementary Fig. S4
). Together these data support a positive regulatory role for miR-296 in the induction of angiogenesis, i.e. miR-296 inhibition reduces the angiogenic phenotype, while miR-296 overexpression increases it.
To identify target genes regulated by miR-296 and involved in control of angiogenesis, we next evaluated targets computationally predicted by publicly available algorithms (Griffiths-Jones et al., 2006
; John et al., 2004
; Krek et al., 2005
; Lewis et al., 2005
; Miranda et al., 2006
). Interestingly, hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) was one of the highest-scored (according to miRBase, http://microrna.sanger.ac.uk
(Griffiths-Jones et al., 2006
)) among the predicted miR-296 targets, with two potential conserved binding sites within its 3' UTR (). The HGS protein is involved in the regulation of levels of growth factors receptors, such as PDGFR-β (Takata et al., 2000
) and VEGFR2 (Ewan et al., 2006
). Upon ligand-stimulation, phosphorylation, ubiquitination, and internalization of growth factor receptors, HGS mediates the sorting of these ligand/receptor complexes to lysosomes, where they are degraded (Bache et al., 2003
; Ewan et al., 2006
; Haglund et al., 2003
; Raiborg et al., 2002
; Stern et al., 2007
; Takata et al., 2000
). Elevated levels of growth factor receptors on the surface of endothelial cells, usually observed during tumor-induced angiogenesis, suggest a diminished role for HGS. In fact, silencing of HGS by siRNAs results in decreased degradation of growth factor receptors and hence their increased cellular levels (Bache et al., 2003
; Stern et al., 2007
). We hypothesized that miR-296 may down-regulate synthesis of HGS by direct binding to sites within the 3' UTR of its message, thereby facilitating accumulation of angiogenic growth factor receptors. To test this hypothesis, we constructed luciferase reporter vectors encoding the complete wild type 3' UTR of the HGS mRNA (HGS WT), as well as parallel control vectors containing mismatches in the predicted miR-296 binding sites (HGS mut1 and HGS mut2) or a luciferase vector containing no HGS UTR (MCS), and transfected them into miR-296-expressing HEK 293T cells (Miranda et al., 2006
). Transfection of the HGS WT plasmid resulted in a decrease in luciferase expression (mock and control inhibitor) as compared to transfection of the MCS control (same conditions), supporting a negative effect of the endogenous miR-296 molecules on the HGS 3' UTR (). Blockage of endogenous miR-296 molecules using anti-sense inhibitor (Ambion) resulted in a significant increase in HGS WT luciferase expression (). Mutations in the predicted miR-296 binding site 1 (nt 114-134; HGS mut 1, ) caused a loss of luciferase sensitivity to miR-296 inhibition as compared to HGS WT, while HGS mut 2 with a mutation in miR-296 binding site 2 (nt 352-372) did not. These experiments validated the regulatory potential of miR-296 via the binding site 1, which has a better predicted base-pairing and is more conserved evolutionary relative to binding site 2, within the HGS 3'UTR.
Fig. 4 HGS is a direct target of miR-296 (A) Alignment of potential miR-296-binding sites in the 3' UTR of the HGS mRNA of different species. (B) pMir-Report vectors containing no 3'UTR (MCS), or containing the 3' UTR of the HGS mRNA (HGS WT) or mutated miR-296 (more ...)
In order to confirm miR-296-mediated modulation of endogenous HGS in endothelial cells, HBMVECs were transfected with miR-296 inhibitor or control molecules and cultured in EGM or U87-conditioned medium. After 48 hr the cell lysates were analyzed for HGS expression levels by Western blotting. Inhibition of miR-296 resulted in increased levels of HGS (). To determine whether HGS protein levels were reduced in angiogenic endothelial cells, HBMVECs were cultured in EBM, EGM, or U87-conditioned medium. After 24 hr, the HGS, PDGFR-β and VEGFR2 protein expression levels were determined by Western blotting and normalized using β-Actin levels. HBMVECs cultured in the angiogenesis-inducing (and miR-296-inducing) EGM or U87-conditioned medium exhibited decreased HGS expression levels, and increased PDGFR-β and VEGFR2 expression levels (). In parallel, HGS, as well as PDGFR-β and VEGFR2 protein expression levels were determined under normal (EBM) and angiogenic (EGM) conditions by immunostaining. Immunostaining () and quantitation of the fluorescence intensities () showed that the HGS protein level is decreased at least 2-fold by angiogenic factors, whereas the PDGFR-β and VEGFR2 protein levels are increased about 3-fold. These results show that HGS is down-regulated under angiogenic conditions (in which miR-296 was up-regulated) with this reduction being prevented by blocking miR-296, and with HGS expression levels being inversely correlated with PDGFR-β and VEGFR2 levels. To further confirm the functional effect of HGS on angiogenesis, HGS siRNAs (Qiagen) or control siRNAs were transfected in HBMVECs. After 24 hr the transfected HBMVECs were cultured in EBM on Matrigel. Silencing of HGS resulted in increased tubule formation and branching (), supporting critical involvement of HGS in the angiogenic process.
Importantly, anti-sense inhibition of miR-296 resulted in a moderate but significant increase in levels of endogenous HGS in HBMVECs and the effect was abolished by co-transfection with an siRNA directed against HGS, as shown by immunofluorescence visualization () and quantification of average fluorescence intensities (). In addition, overexpression of miR-296 resulted in moderate but also significant downregulation of HGS expression (). The knock-down of HGS by siRNA or pre-miR-296 was confirmed by Western blots (). Notable, when HGS levels were decreased by either siRNA or pre-miR-296 transfection, a significant increase in PDGFR-β and VEGFR2 protein levels was observed by immunofluorescence staining (), and confirmed by Western blots (), consistent with downregulation of HGS by miR-296 which, in turn, reduces degradation of the growth factor receptors. Both down- and upregulation of miR-296 is considered to be physiologically relevant, since they affected the levels of downstream proteins (HGS, VEGFR2 and PDGFR-β) within the physiologically relevant range observed in angiogenic versus non-angiogenic conditions.
Fig. 5 miR-296 affects HGS-modulated PDGFR-β and VEGFR2 expression (A) HGS immunostaining of HBMVECs after treatment with miR-296 inhibitors, anti-HGS siRNAs, and pre-miR-296 molecules, size bar 20 μm. (B) Immunostaining of PDGFR-β and (more ...)
In order to confirm that HGS-regulated growth factor receptors are functional in glioma-induced angiogenesis, VEGFR2 receptor antibody blocking experiments were performed. HBMVECs were cultured in U87-conditioned medium. After 24 hr the HBMVECs were transferred onto Matrigel and cultured in U87-conditioned medium in the presence or absence of VEGFR2 blocking antibody (1 μg/ml). After 24 hr, a significant decrease in tubule length and branching was observed in the presence, as compared to the absence of these antibodies (). In addition, scratch motility assays were performed using HBMVECs cultured in the presence or absence of VEGFR2 blocking antibody. After 24 hr a significant reduction in the migration distance was observed upon incubation of the HBMVECs in the presence of VEGFR2 antibody (). These results indicate that functional VEGFR2 expression contributes to the angiogenic phenotype of HBMVECs stimulated by U87-conditioned medium.
Fig. 6 Blocking of VEGFR2 reduces angiogenesis in vitro (A) HBMVECs were stimulated using U87-conditioned medium and cultured on Matrigel-coated plates in the presence or absence of VEGFR2 blocking antibody, size bar 300 μm. (B) After 24 hr VEGFR2 blocking (more ...)
To analyze whether miR-296 affects glioma angiogenesis in vivo
, a synthetic cholesterol-conjugated antagomir-296 antisense oligonucleotide was designed. U87 glioma cells were injected in the back flanks of nude mice. After 7 days of tumor growth, antagomir-296 or mismatch control antagomir were injected intravenously into these mice (100 μl of a 20 mg/ml stock diluted in PBS). At 4 days after injection of the antagomirs, AngioSense 750 (Visen Medical) was injected intravenously in order to quantify the neo-vascularization by fluorescence mediated tomography (Montet et al., 2007
). The mice injected with the antagomir-296 showed a significant decrease in mean fluorescence intensity in the tumor as compared to the mice injected with the mismatch control (). T1-weighted MR images were also acquired to show the tumor volume (). These results demonstrate that inhibition of miR-296 affects glioma angiogenesis in vivo
Fig. 7 In vivo analysis of miR-296 inhibition on tumor neo-vascularization (A) Mice with subcutaneous U87 tumors (N = 6) were injected intravenously with miR-296 antagomirs or mismatch control antagomirs and 4 days later, the same mice were injected with Angiosense (more ...)
In order to establish the clinical relevance of miR-296 regulation in glioma angiogenesis, we performed experiments on human endothelial cells isolated from tumor tissue obtained from neurosurgical resections. Endothelial cells were isolated from blood vessels dissected from six glioma patients [three grade-IV gliomas (highly malignant) and three grade-II gliomas (less malignant)], all of which were highly angiogenic as confirmed by CD31 immunohistochemical analysis in pathological specimens (data not shown), as well as from three normal non-neoplastic human brain samples (). miR-296 expression levels, determined by qRT-PCR, were elevated in tumor blood vessels relative to quiescent normal brain vessels (), while control miR-186 showed no difference in expression levels. We further examined expression of HGS, PDGFR-β and VEGFR2 proteins in endothelial cells from human non-neoplastic brain and malignant gliomas. Immunohistochemical staining revealed that HGS expression was low and PDGFR- β and VEGFR2 were up-regulated in angiogenic glioma blood vessels relative to normal control vessels (). Finally, using qRT-PCR, we determined mRNA levels of VEGF in endothelial cells isolated from non-neoplastic human brain and glioma tissues, as well as in HBMVECs cultured in the presence or absence of U87 glioma cells. A clear increase in VEGF mRNA was observed in tumor endothelial cells, and in U87-exposed HBMVEC cultures as compared to these controls (), paralleling miR-296 expression levels. These data suggest that growth factor induced miR-296 expression observed in cultured brain endothelial cells parallels changes in miR-296 levels in angiogenic endothelial cells in brain tumors in vivo. Further, miR-296 downregulation of HGS signaling increases functional PDGFR-β and VEGFR2 in tumors, which is complemented by increased expression of VEGF.
Fig. 8 Analysis of expression of miR-296 and its targets in tumor endothelial cells isolated from human gliomas (A) RNA was isolated from primary endothelial cell cultures (passage 0) prepared from normal human brain (NNB; N = 3), grade II astrocytomas (N = (more ...)