miR-210 activates notch signaling pathway in ... - Springer Link

Mol Cell Biochem (2012) 370:45–51 DOI 10.1007/s11010-012-1396-6

miR-210 activates notch signaling pathway in angiogenesis induced by cerebral ischemia Yuan-Lei Lou • Fei Guo • Fen Liu • Fa-Liang Gao Peng-Qi Zhang • Xin Niu • Shang-Chun Guo • Jun-Hui Yin • Yang Wang • Zhi-Feng Deng

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Received: 3 May 2012 / Accepted: 7 July 2012 / Published online: 26 July 2012 Ó Springer Science+Business Media, LLC. 2012

Abstract The compensatory angiogenesis that occurs after cerebral ischemia increases blood flow to the injured area and limits extension of the ischemic penumbra. In this way, it improves the local blood supply. Fostering compensatory angiogenesis is an effective treatment for ischemic cerebrovascular disease. However, angiogenesis in the adult organism is a complex, multi-step process, and the mechanisms underlying the regulation of angiogenesis are not well understood. Although Notch signaling reportedly regulates the vascularization process that occurs in ischemic tissues, little is known about the role of Notch signaling in the regulation of ischemia-induced angiogenesis after ischemic stroke. Recent research has indicated that miR-210, a hypoxia-induced microRNA, plays a crucial role in regulating the biological processes that occur in blood vessel endothelial cells under hypoxic conditions. This study was undertaken to investigate the role of miR-210 in regulating angiogenesis in response to brain ischemia injury and the role of the Notch pathway in the body’s response. We found

Y.-L. Lou F. Guo F. Liu Y. Wang Institute of Urology, Nanchang University, Nanchang 330006, China F.-L. Gao Z.-F. Deng Department of Neurosurgery, The Second Affiliated Hospital of Nanchang University, Nanchang 330006, China P.-Q. Zhang Z.-F. Deng (&) Department of Neurosurgery, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai 200233, China e-mail: [email protected] X. Niu S.-C. Guo J.-H. Yin Y. Wang (&) Institute of Orthopaedic Surgery, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai 200233, China e-mail: [email protected]

miR-210 to be significantly up-regulated in adult rat ischemic brain cortexes in which the expression of Notch1 signaling molecules was also increased. Hypoxic models of human umbilical vein endothelial cells (HUVE-12) were used to assess changes in miR-210 and Notch1 expression in endothelial cells. Results were consistent with in vivo findings. To determine the molecular mechanisms behind these phenomena, we transfected HUVE-12 cells with miR-210 recombinant lentiviral vectors. We found that miR-210 overexpression caused up-regulation of Notch1 signaling molecules and induced endothelial cells to migrate and form capillary-like structures on Matrigel. These data suggest that miR-210 is involved in the regulation of angiogenesis in response to ischemic injury to the brain. Up-regulation of miR-210 can activate the Notch signaling pathway, which may contribute to angiogenesis after cerebral ischemia. Keywords miR-210 Angiogenesis Notch1 Cerebral ischemia

Introduction Ischemic stroke is a common clinical disorder. The neuronal degeneration and necrosis that can result from cerebral ischemia and anoxia often leads to serious neurological deficits. The restoration of blood supply to the affected area is essential to the treatment of ischemic stroke. It may not only rescue the dying neurons in the ischemic penumbra but also provide the conditions necessary for the survival, proliferation, differentiation, and functional remodeling of endogenous and transplanted stem cells so that they may restore neurological function. Previous studies have demonstrated that reactive angiogenesis can take place after ischemia. Based on animal

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studies and clinical observations, vascular endothelial cells proliferate and differentiate hours after an ischemic event [1–4]. Angiogenesis involves a series of interrelated and mutually interactive pathophysiological processes. VEGF and other vascular growth factors are involved in the signaling pathway that regulates angiogenesis, but the process also involves many other key players, some of which have yet to be identified. Notch signaling plays an important regulatory role in normal vascular development. For Notch1, normal expression of the ligand Dll4 in the endothelia of new blood vessels is required for the formation of functional vasculature [5–7]. However, the regulatory mechanism of Notch1 in angiogenesis after cerebral ischemic injury remains to be determined. MicroRNA (miRNA) is a class of endogenous, noncoding, single-stranded small RNA molecules comprising 18–28 nucleotides. miRNA plays an important role in the developmental biology, angiogenesis, tumor progression, and stem cell proliferation and differentiation [8, 9]. Recent evidence suggests that miRNA is expressed in vascular endothelial cells and that a subset of miRNA is involved in the regulation of angiogenesis [10, 11]. However, it is not currently clear whether miRNAs are involved in the regulation of angiogenesis after cerebral ischemia. In this study, we show that hypoxia-specific miR-210 is involved in angiogenesis after cerebral ischemia and provide potential mechanisms by which miR-210 may up-regulate Notch signaling for capillary formation in vitro.

Materials and methods Ethics statements All animal procedures were conducted after approval from the Medical Ethics Review Board of Nanchang University (License No. 2010002). All experiments were performed in compliance with the principles of the Institutional Animal Care and Use Committee of the University of Nanchang.

Mol Cell Biochem (2012) 370:45–51

its tip rounded by heating near a flame (filaments 280–305 lm in diameter were used for occlusion) was gently advanced within the lumen of the internal carotid artery until it reached to the origin of the middle cerebral artery (MCA). After 2 h MCAO, reperfusion was effected by withdrawal of the suture until the tip cleared the lumen of the external carotid artery. The rats were killed 1, 3, and, 7 days after MCAO, and the impaired cortical areas were extracted for subsequent biochemical analysis. Sham control animals were subjected to an identical operation without MCAO. TTC staining TTC (2,3,5-triphenyl-2h-tetrazolium chloride) was used to estimate the extent of brain infarct. After 24 h MCA occlusion, each animal was deeply anesthetized with 4 % chloral hydrate. After cardiac perfusion with 200 ml 0.1 % phosphate-buffered saline (PBS), the brains were quickly removed and coronally sectioned into slices 2 mm thick. Each slice was then immersed in 1 % TTC at 37 °C for 30 min for vital staining. Immunohistochemistry Tissue was fixed by perfusion with 4 % paraformaldehyde. Coronal brain slices from bregma 1–2 mm in thickness were embedded into OCT and sectioned into 8.0 lm slices. The following primary antibodies were used in the study: rabbit anti-Factor VIII-related antigen (1:100, Santa Cruz), mouse anti-Notch1 (1:100, Chemicon). The secondary antibodies used TRITC-conjugated bovine anti-rabbit IgG (Santa Cruz) and FITC-conjugated goat anti-mouse IgG (Sigma). Tissue sections were blocked with 1 % albumin from bovine serum in PBS for 30 min at 37 °C and then incubated with primary antibodies overnight at 4 °C. Sections were rinsed three times with PBS and then incubated with secondary antibodies for 1 h at 37 °C. Nuclei were counterstained with 40 ,60 -diamidino-2-phenylindole (DAPI, Sigma).

Induction of cerebral ischemia Cell culture and hypoxia Experiments were performed on adult male Sprague– Dawley rats weighing 250–270 g. These rats were purchased from Nanchang University (Nanchang, China). All animal procedures were conducted after approval from the Animal Committee of Nanchang University. All experiments were performed in compliance with international guidelines for the ethical use of animals. The model of right middle cerebral artery occlusion (MCAO) was created in rats by intraluminal of vascular occlusion Longa et al. (1989). In brief, rats were initially anesthetized with 4 % chloral hydrate. A 4-0 monofilament nylon suture with

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HUVE-12 cells were maintained in a RPMI1640 supplemented with 10 % fetal bovine serum at 37 °C with 5 % CO2 in a humidified incubator. The fresh culture medium was replaced every 2 days and cells were passaged by trypsinase when confluent. For hypoxia, HUVE-12 cells were cultured in RPMI1640 medium without fetal bovine serum in a hypoxia incubator (Sanyo, Japan) under hypoxic conditions (5 % CO2, 94 % N2, and 1 % O2,) for 12 h. Cells cultured under normoxic conditions were used as controls.


Recombinant lentivirus-miR210 expression vector production and transfection Homo sapiens pre-miR-210 stem-loop sequence was obtained from the Sanger microRNA database (miRBase) and cloned into eukaryotic expression vector pGCSIL-GFP to construct a recombinant plasmid. Human embryonic kidney (HEK) 293T cells were transduced with recombinant plasmid–pre-miR210-GFP and empty carrier pGCSIL-GFP along with the packaging plasmids pHelper 1.0 and pHelper 2.0 using Lipofectamine 2000 reagent. All kits and reagents were used in accordance with the manufacturers’ instructions. Viral supernatant was collected at 48 h post co-transfection. HUVE-12 was transfected with recombinant lentivirus-miR210 expression vector (LVmiR210-GFP) in complete medium using polybrene. Cells transfected with lentiviral pGCSIL-GFP vector without clones of pre-miR210 (LV-GFP) were used as controls. After 24–48 h infection, cells were monitored for GFP expression using fluorescence microscopy. miRNA real-time quantitative PCR Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was performed to evaluate miR210 expression in HUVE-12 cells and rat brain samples. MicroRNA was isolated using a mirVanaTM miRNA Isolation Kit (Ambion, AM1560), and complementary DNA was reverse-transcribed using a TaqMan MicroRNA Reverse Transcription Kit (ABI, 4366596) according to the manufacturer’s protocols. Target gene expression was normalized to the expression of the housekeeping gene miR-16 for each sample. The RT primers, specific PCR primers, and probes of miR-210 and miR-16 were taken from TaqMan MicroRNA assay kits (ABI, 4373089, ABI, 4373121). Relative real-time quantitative PCR analysis was performed using TaqMan Universal PCR Master Mix (ABI, 4324018) and a 7,300 real-time PCR system (Applied Biosystems) according to a PCR protocol (95 °C for 10 min, then 40 cycles of 95 °C for 15 s, and 60 °C for 1 min). All real-time RT-PCR results are expressed as fold changes in target gene expression relative to the control group. Data were analyzed using the comparative CT method (2-DDCT). All reactions were performed in triplicate. Western blot analysis Western blotting was performed to detect Notch1 and HIF1alpha (HIF-1a) protein expression in the brain tissues and HUVE-12 cells. The protein samples (30 lg/lane) were separated by 10 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose

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membranes using a semi-dry blotter (Bio-rad). After blocking in Tris-buffered saline containing 0.1 % Tween 20 (TBS-T) and 5 % (w/v) non-fat milk at room temperature for 1 h, the membranes were incubated with primary antibody (HIF1alpha, H1alpha67, Abcam; Notch1, 5B5, Cell Signaling Technology and b-actin, Santa Cruz) diluted in blocking buffer (1:500) overnight at 4 °C. The membranes were washed three times with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody (1:2,000 dilution; Santa Cruz) for 1 h at room temperature. The membranes were washed more than three times and then visualized using a chemiluminescence reagent (Pierce Biotechnology, US). The results were recorded on X-ray films. Proteins were quantified by analysis of digitized scans of the autoradiograph. Both the size and grayscale density of each protein band were accounted for. Angiogenesis experiments For angiogenesis experiments, HUVE-12 cells transfected with miR-210 or control were cultured on Matrigel in 96-well plates. After 24 h, capillary-like structures were randomly counted in five different fields at 109 magnification. Statistical analysis Data are expressed as mean ± standard deviation (SD). Analysis of variance (ANOVA) was used to determine significant differences among groups. Comparisons between two groups were conducted with the Student’s t test. Data analysis was performed using SPSS software. P values under 0.05 were considered statistically significant.

Results Establishment of ischemic injury model TTC staining relies on the oxidation of TTC by intact mitochondrial dehydrogenase, which yields the carmine red product formazan. Infarcted tissues lack dehydrogenase activity and therefore do not stain. The infarcted area (white) can be easily distinguished from the surrounding intact tissues (red). As shown in Fig. 1a, the brain tissue was pale near the right MCA and the uninjured tissue was red. Response of microvascular density to ischemic injury Neovascularization is a major physiological phenomenon that occurs in response to transient ischemic cerebral

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Fig. 1 Coronal sections of rat brain after MCA occlusion and ischemia-induced angiogenesis in the brain. a TTC staining of ischemic injury brain tissue after MCA occlusion showed clearly visible red staining of normal brain tissue (right) and unstained part of the infarct areas (white, left) with distinct border. b Factor VIIIrelated antigen was used as a marker for microvessels. The number of positive cells at 3rd and 7th day after ischemia was significantly higher in the experimental group than in the sham control group.

c Changes in Notch1 expression in the microvessels were confirmed by immunofluorescent staining with confocal laser scanning microscopy. Factor VIII-related antigen (red signal) was used as a marker for microvessels. After ischemic brain injury, there were far more endothelial cells positive for Notch1 expression (green signal) in the experimental group than in the sham group. (9200) *P \ 0.05 versus. sham control. (Color figure online)

injury. To assess ischemia-induced angiogenesis in the ischemic cortex area with respect to changes in microvascular density, we used vascular endothelial cell marker Factor VIII-related antigen to identify brain vascular endothelium by immunohistochemical staining. Microvessels were analyzed using Werdner et al.’s method (1991). The capillary density in the ischemic cortex after 3 days ischemic brain injury were significantly higher than in the sham group and remained high until 7 days (4.47 ± 0.44 at 3rd day and 5.63 ± 0.69 at 7th day, respectively, P \ 0.05) (Fig. 1b). This indicates that angiogenesis was present in the brain cortex area after ischemic injury. After 1 day injury, there was no significant difference between the number of Factor VIII-related antigen-positive cells in the endothelial layer in the ischemic group (2.85 ± 0.51) and sham group (2.79 ± 0.21).

Expression of miR-210 in ischemic cortex

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To determine whether miR-210 could be activated by ischemia, we used quantitative real-time PCR to assay changes in miR-210 expression after injury. As shown in Fig. 2a, miR-210 expression was markedly higher in the ischemic group than in the sham control group. Fold changes were about 7.2 ± 0.56 (1 day), 20.1 ± 0.87 (3 days), and 20.3 ± 0.76 (7 days) (P \ 0.05). This indicates that miR210 may be involved in the regulation of the neovascularization in response to transient ischemic cerebral injury. Response of the Notch pathway to ischemic injury in endothelial cells Vascular endothelial cells were found to express the Notch molecule. The current study focuses on the contribution of


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Fig. 2 miR-210 expression in vitro and in vivo. a At 1 day, 3 day, and 7 day after brain ischemia, miR-210 levels were significantly higher in the experimental group than in the sham group. b miR-210

up-modulation by hypoxia. c miR-210 overexpression in HUVE-12 cells, *P \ 0.05 versus control

the Notch pathway to postnatal vascularization. In this study, we found expression of the Notch1 protein in the ischemic cortex area to be up-regulated 1 day after the ischemic insult, and this effect was still observable after 7 days insult (P \ 0.05) (Fig. 3a). To determine whether the Notch pathway participated in the angiogenesis of cerebral vessels, levels of Notch1 in endothelial cells were assessed using immunofluorescence double staining. As shown in Fig. 1c, Factor VIII-related antigen labeled with TRITC and Notch1 labeled with FITC were located in the cytoplasm. A small number of cells expressed both Factor VIII-related antigen and Notch1 in the ischemic cortex 1 day after ischemic brain injury. The number of doublepositive cells had increased by 3 days post-ischemia. After 7 days ischemia, however, endothelial cells were negative for the expression of Notch1 in the ischemic cortex. The sham group showed no Notch1-positive microvessels or endothelial cells in the cerebral cortex. These results suggest that the Notch1 pathway mediates angiogenesis in response to ischemic injury to the cortex and may play a crucial role in regulating the biological processes of the endothelial cells of the blood vessels.

significantly up-regulated relative to the control group (P \ 0.05) (Fig. 3b).

Changes in expression of miR-210 and Notch1 in HUVE-12 under hypoxia in vitro Hypoxic induction of miR-210 and changes in the expression of Notch1 signal molecule were tested in vitro in HUVE-12 cells exposed to low oxygen tension. We observed that HUVE-12 cells exposed to 1 % O2 for 12 h showed strongly activated HIF-1a signal molecules (Fig. 3b). This induced positive miR-210 modulation (4.73 ± 0.55-fold change relative to control, P \ 0.05) (Fig. 2b). We evaluated the levels of Notch1 in HUVEC-12 cell line under hypoxic conditions. The expression of Notch1 protein in hypoxic HUVE-12 cells was found to be

Effects of miR-210 overexpression on angiogenesis in vitro After examining miR-210 expression and the changes in Notch signaling molecule in vivo and in vitro, we hypothesized that miR-210 may regulate the Notch pathway with respect to angiogenesis after ischemic brain injury. To confirm the mechanisms of miR-210 in the regulation of angiogenesis, we transfected HUVE-12 cells with recombinant lentiviral expression vector containing miR-210 to create a stable cell line consistently expressing miR-210. As shown in Fig. 4a, more than 90 % of the cells were expressed miR-210 with GFP. qPCR showed miR210 expression to have been more than 32.1-fold times the level of control cells (P \ 0.05) (Fig. 2c). To determine whether overexpression of miR-210 enhanced angiogenesis in vitro, we studied the ability of endothelial cells to form capillary-like structures on Matrigel. Cells overexpressing miR-210 clearly formed more capillary-like structures (17.33 ± 6.33) than control cells did (6.33 ± 2.33) (P \ 0.05) (Fig. 4b and 4c). These findings demonstrate that miR-210 plays an important role in angiogenesis. Mechanisms of angiogenesis under miR-210 expression To determine the possible mechanisms underlying the angiogenic activity of miR-210 in endothelial cells in vitro, levels of Notch1 expression were evaluated in HUVEC-12 cell lines overexpressing miR-210. As shown in Fig. 4d, overexpression of miR-210 was shown to significantly enhance the expression level of Notch1. These findings indicate that the Notch1 signaling pathway is involved in the formation of capillaries under miR-210 overexpression.

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Fig. 3 Protein expression of Notch1 and HIF-1a. a Protein levels of Notch1 were found to be more pronounced in the ischemic injury group than in the sham group at 1 day. This difference was still clear

at 7th day. b The protein levels of HIF-1a and Notch1, as measured by Western blotting, were significantly higher in hypoxic HUVE-12 cells,*P \ 0.05 versus control

Fig. 4 Angiogenesis under miR-210 overexpression conditions in HUVE-12 cells and Notch1 expression in miR-210 overexpressing cells. a GFP-positive cells were transfected with lentivirus containing miR-210. More than 90 % of the cells expressed miR-210 with GFP. Bar = 100 lm. b, c Up-regulation of miR-210 was found to enhance

neovascularization. More capillary-like structures per field were observed in cells overexpressing miR-210 than in controls. Bar = 20 lm. d The expression of Notch1 was significantly upregulated in cells overexpressing miR-210, *P \ 0.05 versus control

Discussion

restoration of cerebral blood perfusion of ischemic brain tissues is vital to the treatment of ischemic strokes. Angiogenesis involves many growth factors and signaling molecules. It is based on endogenous vascular endothelial cell proliferation, migration, and remodeling. These ultimately lead to the formation of new blood vessels. The Notch pathway is an evolutionarily conserved signaling pathway. Its main components include Notch receptors, ligands, and downstream target genes. It is responsible for cell differentiation and tissue organization [12]. Upon the binding of ligand to receptor, Notch signaling is activated and the intracellular domain is released

After an ischemic stroke, ischemia and anoxia cause the degeneration and necrosis of large numbers of neurons. This can lead to death or severe neurological dysfunction in survivors. Studies have shown that neuronal necrosis takes place in the core of the ischemic penumbra. However, the degeneration of neurons at the perimeter of the penumbra has been found to be reversible. In addition, the survival, proliferation, and differentiation of endogenous and transplanted stem cells require suitable micro-environments. For this reason, the immediate induction of angiogenesis and

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(Notch ICN). This induces the transcription of Notch target genes (Hes1, Hey1, Hey2, etc.). Both in vitro and in vivo experiments suggest that Notch1 and Dll4 expression can simultaneously induce the formation of the vascular endothelial superficial layer as well as VEGF. The Notch signaling pathway is responsible for the precise control of endothelial cell budding, tubular structural formation, and maturation in new vasculature [13]. Because Notch1 exerts a pleiotropic effect in response to cerebral ischemic injury, we performed double immunofluorescent staining analysis to determine whether the Notch pathway is involved in angiogenesis after ischemic brain insult. Our data show that levels of Notch1 protein localized on endothelial cells in the cerebral cortex were more significantly up-regulated in experimental cells than in the cells of the control group. This indicates that the Notch pathway may form the molecular foundation of the regulation of ischemia-induced angiogenesis in ischemic cortexes. MiR-210, a hypoxia-specific miRNA, depends on HIF activation. It is stably upregulated after hypoxia [14, 15]. When miR-210 is overexpressed in human umbilical vein endothelial cells, the ability of those cells to form blood vessels becomes significantly pronounced more than that of cells with normal levels of expression. Further research has indicated that miR-210 may facilitate angiogenesis through the negative regulation of its target gene, ephrin A3, which is an important member of the ephrin angiogenesis regulatory gene family [16, 17]. Brain tissue expresses a variety of miRNAs, but little is known about the involvement of miRNA in the regulation of the angiogenic response to cerebral ischemia injury [18]. Brain tissue is highly sensitive to hypoxia. Focal, widespread brain ischemia can lead to the over expression of HIF-1a miRNA in the ischemic penumbra [19]. We speculate that certain hypoxia-induced miRNAs can regulate post-ischemia angiogenesis through the hypoxia/HIF-Notch signaling pathway. In the current study, we observed increased miR-210 expression levels in postischemia brain tissue using quantitative real-time PCR analysis. To determine the specific mechanism of miR-210 regulation in vitro, we overexpressed miR-210 in human umbilical vein endothelial cells. We observed significant increases in the expression of Notch1 protein, which further enhanced blood vessel formation by endothelial cells. In conclusion, the results of the current study imply that miR-210 may be a regulatory factor targeting the expression of Notch to regulate angiogenesis and the maturation of vasculature in post-ischemic brain tissue. The miR-210Notch signaling pathway may be one of the major signaling pathways involved in the regulation of post-ischemia angiogenesis. Acknowledgments This study was supported by the National Natural Science Foundation of China (Grant No. 30960396), (Grant No.

51 81060324) and the National S&T Major Special Project on Major New Drug Innovation (2011ZX09102-010-01).

References 1. Krupinski J, Kaluza J, Kumar P, Kumar S, Wang JM (1994) Role of angiogenesis in patients with cerebral ischemic stroke. Stroke 25:1794–1798 2. Marti HH, Bernaudin M, Petit E, Bauer C (2000) Neuroprotection and angiogenesis: dual role of erythropoietin in brain ischemia. News Physiol Sci 15:225–229 3. Hamada Y, Gonda K, Takeda M, Sato A, Watanabe M, Yambe T, Satomi S, Ohuchi N (2011) In vivo imaging of the molecular distribution of the VEGF receptor during angiogenesis in a mouse model of ischemia. Blood 118:e93–e100 4. Hedhli N, Dobrucki LW, Kalinowski A, Zhuang ZW, Wu X, Russell RR 3rd, Sinusas AJ, Russell KS (2012) Endothelial-derived neuregulin is an important mediator of ischaemia-induced angiogenesis and arteriogenesis. Cardiovasc Res 93:516–524 5. Gridley T (2007) Vascular biology: vessel guidance. Nature 445: 722–723 6. Hofmann JJ, Luisa Iruela-Arispe M (2007) Notch expression patterns in the retina: an eye on receptor-ligand distribution during angiogenesis. Gene Expr Patterns 7:461–470 7. Liu R, Trindade A, Sun Z, Kumar R, Weaver FA, Krasnoperov V, Naga K, Duarte A, Gill PS (2012) Inhibition of Notch signaling by Dll4-Fc promotes reperfusion of acutely ischemic tissues. Biochem Biophys Res Commun 418:173–179 8. Ambros V (2004) The functions of animal microRNAs. Nature 431:350–355 9. He L, Hannon GJ (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5:522–531 10. Bentwich I, Avniel A, Karov Y, Aharonov R, Gilad S, Barad O, Barzilai A, Einat P, Einav U, Meiri E, Sharon E, Spector Y, Bentwich Z (2005) Identification of hundreds of conserved and nonconserved human microRNAs. Nat Genet 37:766–770 11. Yang WJ, Yang DD, Na S, Sandusky GE, Zhang Q, Zhao G (2005) Dicer is required for embryonic angiogenesis during mouse development. J Biol Chem 280:9330–9335 12. Ehebauer M, Hayward P, Arias AM (2006) Notch, a universal arbiter of cell fate decisions. Science 314:1414–1415 13. Lobov IB, Renard RA, Papadopoulos N, Gale NW, Thurston G, Yancopoulos GD, Wiegand SJ (2007) Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting. Proc Natl Acad Sci USA 104:3219–3224 14. Chan SY, Loscalzo J (2010) MicroRNA-210: a unique and pleiotropic hypoxamir. Cell Cycle 9:1072–1083 15. Kelly TJ, Souza AL, Clish CB, Puigserver P (2011) A hypoxiainduced positive feedback loop promotes hypoxia-inducible factor 1alpha stability through miR-210 suppression of glycerol-3phosphate dehydrogenase 1-like. Mol Cell Biol 31:2696–2706 16. Corn PG (2008) Hypoxic regulation of miR-210: shrinking targets expand HIF-1’s influence. Cancer Biol Ther 7:265–267 17. Fasanaro P, D’alessandra Y, Di Stefano V, Melchionna R, Romani S, Pompilio G, Capogrossi MC, Martelli F (2008) MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem 283:15878–15883 18. Jeyaseelan K, Lim KY, Armugam A (2008) MicroRNA expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion. Stroke 39:959–966 19. Jin KL, Mao XO, Nagayama T, Goldsmith PC, Greenberg DA (2000) Induction of vascular endothelial growth factor and hypoxia-inducible factor-1alpha by global ischemia in rat brain. Neuroscience 99:577–585

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