MicroRNA-210 overexpression induces angiogenesis and ... - Nature

Gene Therapy (2014) 21, 37–43 & 2014 Macmillan Publishers Limited All rights reserved 0969-7128/14 www.nature.com/gt

ORIGINAL ARTICLE

MicroRNA-210 overexpression induces angiogenesis and neurogenesis in the normal adult mouse brain L Zeng1,2,4, X He2,4, Y Wang1,2, Y Tang2, C Zheng1, H Cai1,2, J Liu1, Y Wang2,3, Y Fu1 and G-Y Yang1,2,3 Angiogenesis and neurogenesis are crucial processes for brain tissue repair and remodeling after brain injury. Current study shows that microRNA-210 (miR-210) promotes vascular endothelial cell migration and tube formation under hypoxia in vitro. Whether miR-210 overexpression promotes focal angiogenesis and neurogenesis in the normal adult brain is unknown. Adult male C57BL/6 mice (n ¼ 54) underwent stereotactic injection of a lentiviral vector carrying miR-210 (LV-miR-210). Following 28 days of miR-210 gene transfer, endothelial cell and neural precursor cell proliferation, microvessel density and downstream angiogenic factor were genotyped. miR-210 was highly expressed in neurons, astrocytes and endothelial cells of the LV-miR-210-injected brain hemisphere. The endothelial cell proliferation and the number of newly formed microvessels were greatly increased in the LV-miR-210-treated mice compared with the controls (Po0.05). Neural progenitor cells in the subventricular zone were greatly increased compared with the controls (Po0.05). The data indicate that miR-210 is a key factor at the microRNA level in promoting angiogenesis and neurogenesis, which was associated with local increased vascular endothelial growth factor (VEGF) levels, suggesting that miR-210 may be a potential target for ischemic stroke therapy. Gene Therapy (2014) 21, 37–43; doi:10.1038/gt.2013.55; published online 24 October 2013 Keywords: angiogenesis; brain; microRNA; microvessel; neurogenesis

INTRODUCTION Many brain proteins with angiogenic and neurogenic characteristics including vascular endothelial growth factor (VEGF),1,2 brain-derived neurotrophic factor (BDNF),3,4 insulin-like growth factor (IGF-1),5,6 angiopoietins, netrin-17,8 and so on are known. Numerous studies have demonstrated that overexpression of these angiogenic factors promotes focal angiogenesis and neurogenesis for damaged brain repair and remodeling and consequently improves neurological function. The combination of several factors could enhance the efficiency of angiogenesis.9 However, most angiogenic factors are relatively large molecules, which find it difficult to cross the blood brain barrier (BBB). In addition, the half-lives of these large protein molecules are short. Multiple injections of these proteins into the brain are impracticable for the induction of angiogenesis and neurogenesis. MicroRNA (miRNA), a novel group of small noncoding RNAs, is recently found to have critical roles in regulating network of gene expression. Current studies demonstrated that miRNA inhibited mRNA transcription or degraded mRNA, subsequently regulate downstream protein expression. miRNAs are involved in many pathophysiological procedures including cell apoptosis and survival, cell proliferation, differentiation, migration and functioning.10,11 The characteristics of miRNAs include their small size, their capability of regulating multiple targeting genes and the detection of their levels in the circulating blood. Twentytwo oligonucleids of small size were able to cross the BBB with the aid of an appropriate vector such as nanoparticles.12 One miRNA can regulate multiple protein functions, which produces important effects.13,14 Circulating miRNA can be associated with the brain miRNA.15 Monitoring the change of circulating miRNA may

provide useful information on miRNA in other organs, especially in the brain. The study of the function of miRNA in inducing angiogenesis and neurogenesis is important as a potential therapeutic target. miR-210, a unique and pleiotropic hypoxamir, is upregulated in many kinds of cells and tissues under hypoxic conditions in vitro.16 MiR-210 affects directly or indirectly many cellular processes such as cell cycle, development, membrane traffic, differentiation, migration/adhesion, DNA binding, amino-acid catabolism and RNA processing.17 Furthermore, miR-210-stimulated VEGF-driven cell migration and capillary-like structure formation occur through the inhibition of receptor tyrosine kinase ligand ephrin-A3 in vitro.18,19 miR-210 also promoted angiogenesis mediated by the VEGF signaling pathway in a mouse renal ischemia/perfusion model in vivo.20 Furthermore, miR-210 can rescue cardiac function after myocardial infarction by upregulating focal angiogenesis and inhibiting apoptosis in a mouse myocardial injury model.21 A study of miRNA profiles shows that miR-210 in human atherosclerotic plaques is fourfold higher than that in control arteries.22 Our previous study demonstrated that circulating blood miR-210 level was associated with brain miR-210 level in a mouse model of ischemia.23 Acute ischemic stroke patients with higher circulating blood miR-210 show better clinical outcomes.23 All these data indicate that miR-210 is a protective factor during hypoxia, including cerebral ischemia. However, miR-210 function in the normal brain is unclear. In this study, it was hypothesized that miR-210 can reach higher expression via LV-miR-210 gene transfer. If so, we will further determine the effect of miR-210 on the induction of brain angiogenesis and neurogenesis in adult mice.

1 Department of Neurology, Ruijin Hospital, Shanghai, China; 2Neuroscience and Neuroengineering Center, Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China and 3School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China. Correspondence: Professor Y Fu or Professor G-Y Yang, Neuroscience and Neuroengineering Center, Med-X Research Institute, Shanghai Jiao Tong University, 1954 Hua Shan Road, Shanghai 200030, China. E-mail: gyyang0626@gmail.com 4 These authors contributed equally to this work. Received 24 April 2013; revised 22 August 2013; accepted 9 September 2013; published online 24 October 2013

MicroRNA-210 induces angiogenesis and neurogenesis L Zeng et al

38 RESULTS Lentiviral vector-mediated miR-210 overexpression in adult mouse brain To determine the transfer efficiency of lentiviral vector in the adult mouse brain, copGFP expression was examined from 3 to 28 days (Figure 1). The green fluorescence marker was observed in the left basal ganglia around the needle tract as early as 3 days after gene

transfer. The intensity and extent of green staining increased over time and reached the plateau after 14 days of gene transfer, which persisted at a higher level for at least 28 days. The double staining showed that copGFP þ cells colocalized with NeuN, GFAP and CD31, indicating that the lentiviral vector could be overexpressed in neurons, astrocytes and endothelial cells (Figure 1e). Real-time PCR measurement confirmed that miR-210 was highly

Figure 1. Lentivirus-mediated miR-210 expression in adult mouse brain. (a) Cresyl violet staining of coronal sections in the mouse brain. Arrow indicates the lentivirus injection hemisphere in the left basal ganglia of the mouse brain. The box shows the distribution of green fluorescence after lentiviral gene transfection. (b) Photomicrographs show the full view of the intense boundary of copGFP signal (green) around the injecting site at 14 days after lentiviral gene transfer. Bar ¼ 100 mm. (c) Bar graph shows the quantitative PCR analysis of miR-210 expression around the injection site at 14 days after LV-miR-210, LV-GFP or NS administration (data are presented as mean±s.d. *Po0.05 versus NS-treated group, #Po0.05 versus LV-GFP-treated group, n ¼ 6). (d) Photomicrographs show copGFP signal distribution after lentivirus injection at 3, 7, 14 and 28 days. Bar ¼ 50 mm. (e) Photomicrographs show double immunofluorescent staining of copGFP/NeuN, copGFP/GFAP and copGFP/CD31 after lentivirus injection. Bar ¼ 50 mm. Gene Therapy (2014) 37 – 43

& 2014 Macmillan Publishers Limited


39 upregulated in the left basal ganglia in LV-miR-210-treated mice compared with the LV-GFP-treated and saline-treated mice (LVmiR-210 ¼ 9.2±0.3, LV-GFP ¼ 1.9±0.6 and NS ¼ 0.9±1.0; Po0.05), indicating that miR-210 can be overexpressed through gene transfer in the adult mouse brain. miR-210 overexpression induced focal angiogenesis in the brain Microvessel density and proliferating endothelial cells were examined to determine whether miR-210 overexpression induced angiogenesis in the brain. The number of CD31 þ vessels greatly increased in ipsilateral basal ganglia in the LV-miR-210treated mice compared with the control mice after 28 days of gene transfer (Figure 2, microvessel density pixel/field, LV-miR-210 ¼ 217109±24465, LV-GFP ¼ 147061±21182 and NS ¼ 172688±5668, Po0.05). However, the number of SMA þ vessels was not different between the three groups (Figure 3, LV-miR-210 ¼ 3.5±1.1, LV-GFP ¼ 3.0±1.3 and NS ¼ 3.2±1.6, P40.05). Double immunostaining indicated that the number of PCNA þ /CD31 þ double-labeled cells increased in the LV-miR-210treated mice compared with the controls (Figure 4, LV-miR210 ¼ 4.4±1.1, LV-GFP ¼ 2.0±0.7 and NS ¼ 1.4±0.6, Po0.05), suggesting that miR-210 could induce focal angiogenesis in the brain.

miR-210 overexpression increased VEGF expression VEGF expression in the left basal ganglia around the injection region was measured after LV-miR-210 transduction. VEGF þ cells were greatly increased in the LV-miR-210-treated mice after 28 days compared with the LV-GFP-treated and the saline-treated mice (Figures 6a–d, LV-miR-210 ¼ 22.4±4.8, LV-GFP ¼ 7.8±1.3 and NS ¼ 6.8±1.3, Po0.05). Similar results were obtained using western blot analysis. The VEGF protein was significantly higher in the LV-miR-210-treated mice at 28 days after transduction compared with that in LV-GFP-treated and saline-treated mice (Figures 6e and f, LV-miR-210 ¼ 5.9±3.4, LV-GFP ¼ 1.1±0.7, NS ¼ 1.0±0.5; Po0.05). To determine whether VEGF-derived newly formed vessels were leaky in the LV-miR-210-treated mice, BBB integrity was determined using IgG extravasation staining at 28 days after gene transfer. IgG staining in the LV-miR-210-, LVGFP- and saline-treated mice were similar (Figure 7, LV-miR210 ¼ 81.0±22.9, LV-GFP ¼ 82.6±0.9, NS ¼ 80.6±11.7; P40.05), indicating intact blood vessels. DISCUSSION In the current study, we demonstrated that brain miR-210 could be overexpressed for at least 28 days after gene transfer. Neurons, astrocytes and endothelial vascular cells can express miR-210.

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miR-210 overexpression induced neurogenesis in SVZ of adult mouse brain To determine whether miR-210 overexpression induced neurogenesis, neural precursor cells were measured. The DCX þ cells significantly increased in the SVZ in LV-miR-210-treated mice compared with the controls (Figure 5c, LV-miR-210 ¼ 60.3±5.1, LV-GFP ¼ 36.3±1.5 and NS ¼ 30.3±3.5; Po0.05). Double immunostaining showed that the number of BrdU þ /DCX þ double-

labeled cells increased in LV-miR-210-treated mice compared with the controls (Figure 5d, LV-miR-210 ¼ 4.4±1.1, LV-GFP ¼ 2.0±0.7 and NS ¼ 1.4±0.6; Po0.05), indicating that miR-210 could induce focal neurogenesis in the brain.

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Figure 2. miR-210 overexpression increased focal microvessels in mouse brain. (a) Photomicrographs show the CD31 þ microvessel staining at 7, 14 and 28 days around the needle tract after LV-miR210, LV-GFP or NS injection. Bar ¼ 100 mm. (b) Bar graph shows the quantification of microvessel density from (a). Data are presented as mean±s.d. *Po0.05 versus NS-treated group, #Po0.05 versus LV-GFP-treated group, n ¼ 6 in each group. & 2014 Macmillan Publishers Limited

Figure 3. miR-210 overexpression does not induce the increased smooth muscle cell numbers in mouse brain. (a) Photomicrographs show the double staining of CD31 (green) and SMA (red) around the injected site at 28 days after NS, LV-GFP or LV-miR-210 injection. Bar ¼ 100 mm. Arrows indicate the small arteries. (b) Bar graph shows the number of small arteries in NS-, LV-GFP- and LV-miR-210-treated mice. Data are presented as mean±s.d., Po0.05 versus NS-treated group, Po0.05 versus LV-GFP-treated group, n ¼ 6 in each group. Gene Therapy (2014) 37 – 43


40

Figure 4. miR-210 overexpression increased endothelial cell proliferation. (a) Double staining of PCNA (green) and CD31 (red) after NS, LV-GFP or LV-miR-210 injection in normal mouse brain. Yellow shows that PCNA and CD31 are well merged, indicating that the microvessel was newly formed. Bar ¼ 100 mm. Arrows indicate PCNA/CD31-positive cells. (b) Bar graph shows the quantification of PCNA and CD31 double-positive cells. Data are presented as mean±s.d. *Po0.05 versus NS, #Po0.05 versus LV-GFP, n ¼ 6 in each group.

Furthermore, the microvessel density and the number of neuronal progenitor cells in the brain were increased. miR-210 could promote vascular endothelial cell proliferation and neuronal cell proliferation and maturation. miR-210 can then upregulate downstream VEGF expression. We provided strong evidence that miR-210 is associated with focal angiogenesis and neurogenesis via upregulation of VEGF. MiR-210 is a potential promising factor for stimulating focal angiogenesis. Lentiviral vector delivery miR-210 gene has several advantages. First, lentiviral vector-mediated pre-miRNA can induce mature miRNA and be sustained for a longer-term expression. The green fluorescence could persist for at least 28 days after lentivirus GFP gene transfer, suggesting that lentivirus-delivered gene expression could be successfully upregulated in the brain. It is feasible for monitoring therapeutic efficiency without multiple injections, especially the repair procedure including angiogenesis and neurogenesis after cerebral ischemia. Second, the efficacy of lentiviral vector transduction in vivo showed that it had a higher adaptability in experimental brain tissue. We demonstrated that lentivirus could transduce to neurons, astrocytes and endothelial cells. Third, lentivirus has low undesirable side effects. This means low immune response after virus transduction compared with an adenovirus vector.24 Mice receiving lentivirus show no epilepsy or other neurological disorders. Although, studies show that lentiviral vectors integrate into the host genome, which might alter host gene expression and cause cancer.25 However, in clinical trials using lentivirus for the HIV therapy; or in animal model using lentivirus for metabolic disease, both do not produce detectable insertion mutagenesis.26–27 Gene Therapy (2014) 37 – 43

Angiogenesis and neurogenesis are two major responses to cerebral ischemia, as part of the endogenous adaptive procedure for restoring brain function.28–29 In the ischemic core, influx was interrupted by thrombosis, and neuronal death occurred. The newly formed microvessels could revascularize and increase blood flow in the penumbra. The increase in microvessel density showed better recovery after ischemic stroke.30–31 In the mammalian brain, neurogenesis was observed in the subvericular zone (SVZ) of the lateral ventricle and subgranular zone (SGZ) of the dentate gyrus in the hippocampus. After cerebral ischemia, neurogenesis enhanced in the SVZ, which not only increased the number of neural stem cells but also induced new matured neurons migrating to the ischemic region.28 Notably, the migration path is along the vessels.28 However, endogenous self-repair is not sufficient for repairing ischemia-induced brain injury. Regenerative therapy is considered to be a potential approach for the late repair process. Promoting focal angiogenesis and neurogenesis provides a novel approach for ischemic stroke therapy. The use of miR-210 has proven its angiogenic properties in vitro. The present study provides evidence that miR-210 can promote focal angiogenesis in vivo in the normal mouse brain. The number of microvessels and the endothelial cells around the injected site increased up to 28 days after LV-miR-210 gene transfer. In our study, we found that the number of CD31 þ vessels increased, but the number of SMA þ vessels did not increase in the mir-210-injected mice. As we know, the increase in CD31 þ vessels but not SMA vessels indicated that mir-210 promotes angiogenesis but not arteriogenesis. This result may be because of the following: (1) miR-210 only promotes angiogenesis, and (2) we used normal mice under the normal condition, which was different from the ischemic condition. Many angiogenic factors, chemokines and inflammatory mediators could be released locally during ischemic condition; as a result, angiogenesis and arteriogenesis could occur at the same time. We revealed that another novel function of miR-210 was to promote neurogenesis. The number of neural precursor cells and their proliferation in the SVZ increased in the ipsilateral hemisphere after miR-210 gene transfer. MiR-210, the linking bridge between angiogenesis and neurogenesis, could aid to form the neurovascular niche. The neurovascular niche was the key unit in the regenerative therapy following ischemic brain injury.32,33 VEGF is an important angiogenic34 and neurogenic factor35 with therapeutic potential in ischemic disorders, including ischemic stroke.1 Numerous studies demonstrate that VEGF could combine with the VEGF-2 receptor and promote angiogenesis and neurogenesis in vivo and in vitro.36 AAV-mediated VEGF attenuated infarct volume and improved neurological outcomes in the mouse model of brain ischemia.37 We demonstrated that miR-210 overexpression increased VEGF expression in the adult mouse brain. MiR-210 upregulation augments VEGF mRNA and protein expression in the renal ischemia–reperfusion mouse model.20 miR-210 was one of the miRNAs that regulated VEGF under hypoxic conditions in vitro.38 These data suggested that VEGF is a downstream target of miR-210. However, past studies have revealed that VEGF-derived newly formed vessels could be leaky. We performed BBB integrity measurement with IgG staining 28 days after gene transfer. We did not find IgG leaky in the LV-210-treated mice compared with the controls. It is possible that normal mice are capable of forming more matured microvessels. The demonstration of the effect of miR-210 on BBB in the brain ischemic mouse model is needed in the future. Several studies indicate that miR-210 had multiple biological functions targeting other downstream factors.39 For example, miR-210 controlled mitochondrial metabolism by inhibiting the iron– sulfur cluster assembly proteins ISCU1/2 and COX10 expression.40,41 In addition, miR-210 augmented the survival of marrow-derived mesenchymal stem cells (MSCs) in ischemic preconditioning by repressing caspase-8-associated protein 2.42 In cardiomyocytes, & 2014 Macmillan Publishers Limited


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Figure 5. miR-210 overexpression enhanced the number of neural progenitor cells in the mouse brain. (a) Double immunostaining of BrdU (red) and DCX (green) in the SVZ at 28 days after NS, LV-GFP or LV-miR-210 injection. Bar ¼ 50 mm. Bar graphs show the number of BrdU þ cells (b), DCX þ cells (c) and BrdU þ /DCX þ cells (d). Images showed BrdU (red) and DCX (green) double staining in the SVZ at 28 days after LV-miR210 injection under confocal microscopy (e) Data are presented as mean±s.d. *Po0.05 versus NS, #Po0.05 versus LV-GFP, n ¼ 6 in each group.

miR-210 exerts cytoprotective effects by reducing mitochondrial reactive oxygen species.43 The wide multifunctions of miR-210 make it a novel potential target for the treatment of cerebral ischemia. It may affect multiple pathways such as apoptosis, oxidation, mitochondrial function, angiogenesis and neurogenesis in the ischemic brain. In conclusion, the present study demonstrates that miR-210 can be transduced into brain tissue via lentiviral vector. Overexpression of miR-210 can induce focal angiogenesis and neurogenesis in the adult brain, which is associated with VEGF upregulation. The data suggest that miR-210 is a potential target for the treatment of cerebral ischemia.

was harvested and viral particles were further concentrated as described previously.23 Pseudoviral titer was finally determined by real-time PCR.

Experimental groups The procedures for the use of laboratory animals were approved by the Shanghai Jiao Tong University Institutional Animal Care and Use Committee (IACUC), Shanghai, China. Adult male C57BL/6 mice (n ¼ 54) weighing 25–30 grams were divided into three groups: LV-miR-210 (LV-pre-miR-210-copGFP), LV-GFP (LV-scramble-miR-210-copGFP) and NS (normal saline) groups. All mice were injected stereotactically in the left brain hemisphere and killed at 7, 14 and 28 days after gene transduction. Angiogenesis and neurogenesis were further studied in these animals.

Lentiviral vector transduction in mouse brain MATERIALS AND METHODS Lentiviral vector construction and virus packaging The pre-miR-210 hairpin structure and scramble control hairpin were inserted in the lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP (System Biosciences SBI, CA, USA) located downstream of the cytomegalovirus promoter. Packaging of the pCDH expression constructs into pseudoviral particles was performed with the pPACKH1TM Packaging Plasmid mix (SBI) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in a pseudoviral particle producer cell line (293 TN cells). The supernatant & 2014 Macmillan Publishers Limited

Mice were anesthetized intraperitoneally with ketamine/xylazine (100 mg/ 10 mg/kg). Animals were then placed in a stereotaxic apparatus. Administration of LV-miR-210 was described previously.7 A burr hole was drilled at 2.0 mm left lateral to the sagittal suture and 0.5 mm posterior to the bragma. A 10-ml Hamilton syringe was gently inserted 3.0 mm deep into the striatum under the cortex. LV-miR-210 (2.2 ml) containing 2 109 TU/ml was injected into the caudate putamen at a rate of 0.2 ml/minute. The needle was withdrawn after 15 min of injection. The bone hole was sealed by bone wax and animals were returned to their home cages after they woke up. Mice underwent LV-GFP or saline injections as controls. Gene Therapy (2014) 37 – 43


42 expression level detection, as described in our previous paper.23 In brief, reverse transcription and real-time PCR were performed according to the manufacturer’s protocol of the TaqMan miRNA RT Kit and TaqMan miRNA assay Kit (Applied Biosystems, Foster City, CA, USA) by a fast real-time PCR system (7900HT, ABI). The relative miR-210 level was normalized to the endogenous control U6 in triplicate and calculated.23

Immunohistochemistry

Figure 6. miR-210 overexpression increased VEGF expression in mouse brain. Immunostaining of VEGF at 28 days around the needle tract after LV-miR-210 (a), LV-GFP (b) or NS (c) injection. Bar ¼ 100 mm. (d) Bar graph shows the quantification of VEGF expression staining. Data are presented as mean±s.d. *Po0.05 versus NS, # Po0.05 versus LV-GFP, n ¼ 6 in each group. (e) Western blot analysis of VEGF at 28 days around the needle tract after LV-miR-210, LV-GFP or NS injection. (f ) Bar graph shows the quantification of the VEGF protein level. Data are presented as mean±s.d. *Po0.05 versus NS, # Po0.05 versus LV-GFP, n ¼ 4 in each group.

Mice were killed at 7, 14 and 28 days after transduction. They were perfused transcardically with 0.9% NS followed by 4% paraformaldehyde (PFA) solution. Each brain was immersed in 4% PFA overnight and then dehydrated in 20% sucrose solution until the brain sank. The brain was then embedded by Tissue-Tek OCT and cut into 30-mm coronal sections using a cryostat (Leica, Solms, Germany). For immunofluorescent staining, free-floating sections were incubated for 10 min in 0.5% Triton-100 for membrane rupture, blocked for 1 hour in 10% BSA and then incubated with the primary antibodies overnight at 4 1C. Primary antibodies included anti-NeuN (1:100 dilution, Millipore, Billerica, MA, USA), anti-GFAP (1:300 dilution, R&D systems, Tustin, CA, USA), anti-CD31 (1:200 dilution, R&D systems), anti-copGFP (1:200dilution, Evrogen, Moscow, Russia), anti-PCNA (1:200 dilution, Abcam, Cambridge, England), anti-SMA (1:200 dilution, Abcam), anti-DCX (1:200 dilution, Santa Cruz Inc., Santa Cruz, CA, USA), anti-BrdU (1:200 dilution, Santa Cruz Inc.) and anti-VEGF (1:300 dilution, R&D systems). Each section was washed with PBS for 10 min three times, and incubated with Alexa-Fluor-594- or Alexa-Fluor-488-conjugated secondary antibodies (1:500 dilution, Invitrogen) for 1 hour. Each experiment had appropriate positive and negative controls. Four serial sections around the needle track, spaced 200 mm apart, were selected from each animal. There were six animals in each group. Six brain regions were randomly selected from the area of interest for each section at 20 objective (Leica, DM2500) and analyzed by the Image Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA) for quantitative analysis. The IgG extravasation was detected to measure the integrity of the BBB. Brain sections were incubated with biotinylated goat anti-mouse IgG antibody (1:200 dilution, Vector) and visualized as described above.

5-bromo-2-deoxyuridine-5-monophosphate (BrdU) labeling BrdU powder (Sigma, St Louis, MO, USA) was dissolved in normal saline at a concentration of 20 mg/ml. BrdU solution was injected intraperitoneally at a concentration of 50 mg/kg twice a day for 3 days before sample collection. For BrdU double staining, sections were first treated with 2 M HCL for 30 min at room temperature and then with sodium borate for 10 min twice. Sections were then treated with 0.3% Triton/PBS for 30 min, blocked with 5% normal donkey serum and incubated with anti-BrdU (1:200 dilution, Santa Cruz Inc.) at 4 1C overnight. The sections were incubated with secondary antibodies for 60 min at room temperature. After rinsing with PBS, sections were mounted. Photomicrographs were taken with a confocal microscope (Leica). All staining intensity was computed as mean integrated optical density (IOD). Six fields were randomly selected from the area of interest for each section at 20 objective (Leica, DM2500) and analyzed by the Image Pro Plus 6.0 software (Media Cybernetics) for quantitative analysis, where IOD was calculated for arbitrary areas. Four serial sections, spaced 200 mm apart, were selected from each animal, and there are six animals in each group.

Vascular density counting Figure 7. miR-210 overexpression does not influence the BBB integrity. Immunostaining of IgG extravasation at 28 days around the needle tract after LV-miR-210 (a), LV-GFP (b) or NS (c) injection. Bar ¼ 50 mm. (d) Bar graph shows the quantification of IgG expression staining. Data are presented as mean±s.d. Po0.05 versus NS-treated group, Po0.05 versus LV-GFP-treated group, n ¼ 6 in each group.

The number of blood vessels was calculated as the mean of the blood vessel counts obtained from the six brain regions as previously described.34,44 Three brain coronal sections, 1.0 mm anterior, 1.0 mm posterior and the section of the needle track, were chosen. Three areas of the left, right and bottom of the needle track of every section were photographed using a 20 objective. The microvessel density pixel was quantified by the NIH Image J software.44 The mean of the vascular counting was considered as vascular density.

RNA extraction and real-time PCR

Western blot

The mice were killed at 7, 14 and 28 days after LV-miR-210 injection. The brain sample in the left basal ganglia was obtained and transferred into a tube containing TRIzol reagent (Invitrogen) on ice. Total RNA was isolated according to the manufacturer’s protocol. The integrity of RNA was quantified by a NanoDrop 1000 spectrophotometer (Thermo Scientific, UT, USA). Samples with OD260/280 (1.7–2.0) were used for further miR-210

Mice were killed 28 days after lentiviral vector transduction. The brain tissue of the left basic ganglion was dissected. It was mixed with RIPA, PMSF, cocktail and centrifuged for 30 min at 12000 g 4 1C. The protein concentration was calculated by using the BCA Protein Assay Kit (Thermo Pierce, Rockford, USA). The SDS-PAGE was performed and transferred to a nitrocellulose membrane (Whatman Inc, Florham Park, NJ, USA).

Gene Therapy (2014) 37 – 43



43 The membranes were incubated with VEGF primary antibodies (1:500 dilution, Santa Cruz Inc.) overnight at 4 1C and then with a secondary antibody for 1 hour at room temperature. The enhanced ECL substrate (Pierce) was used to visualize each band and recorded by an imaging system (Bio-Rad, Hercules, CA, USA). Finally, the quantify analysis was performed with Quantity-one software.

Statistical analysis Data were presented as mean±s.d. Comparison of the two groups was analyzed by an unpaired Student’s t test. Three group comparison data were analyzed by one-tailed ANOVA and one-tailed ANOVA with Dunnett’s multiple comparison. A probability value of less than 5% was accepted as statistically significant.

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This study is supported by 973 Program of NBRP, China (2011CB504405, GYY, YW), NSFC (30973097, GYY, 81200943, LZ), the Shanghai medical association (SHNR-003, LZ), Shanghai healthy bureau (20124217, LZ) and by the Science and Technology Commission of Shanghai Municipality (12ZR1418600, FY).

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