| Stroke is the most common causes of serious, long-term disability in the population and for several decades, most efforts to treat brain stroke were based on neuroprotection. However, it shows very little beneficial activity in ischemic stroke patients. Studies have shown that while neuron death was caused following cerebral ischemia, a regenerative response is also triggered in the ischemic area, including angiogenesis, vascular remodeling, and other pathological activation in the ischemic zone. This discovery has encouraged scientists to explore angiogenesi-based strategies for the treatment of brain ischemia, and recent studies have focused stroke therapy on timely revascularization in ischemic region. In our previous study, we demonstrated that VEGF-Notch signaling is involved in the regulation of ischemia-induced angiogenesis after stroke, but little is known about the complex upstream regulation of this process. While mir-210 is known as the hypoxia-induced miRNA and researches have demonstrated a significant correlation between mir-210, angiogenesis and hypoxia, we proposed mir-210 could have a critical role in the regulation of angogenesis after stroke.In vivo experiments, we investigated the expression changes of mir-210 and ephrinA3 in adult rat ischemic brain cortex after stroke. The experiments were divided into four groups: 1, 3, 7 days post-ischemia groups and sham group. Rat middle cerebral artery occlusion (MACO) models were produced by obstructing MCA using a single strand nylon thread. Rats were killed at 1, 3, 7 days after ischemia, and the impaired cortical areas were extracted for examine. The sham group was not operated as control. MiRNAs were isolated from ischemic cortical areas and real time PCR was used to determine the expression change of mir-210 of different groups. Then RT-PCR and immunofluorescence were used to determine the mRNA or protein expression change of mir-210's target gene ephrinA3 in ischemic cortical areas of different groups. The results showed that mir-210 expression was significantly up-regulated after ischemia, about 7.2±0.56 (1 d), 20.1±0.87 (3 d) and 20.3±0.76 (7 d) fold changes in ischemic cortical areas relative to sham group. RT-PCR showed that mRNA expression of ephrinA3 in ischemic cortical areas was gradually up-regulated after ischemia, achieving the highest expression at 7d post-ischemia. However, the result of immunofluorescence showed a higher percentage of ephrinA3-positive cells of sham group (66%±0.55), compared to 1 d(45%±0.38) ,3 d (36%±0.65) and 7 d (27%±0.28)post-ischemia groups. In vitro experiments, Mir-210 was over-expressed in HUVE-12 by lentivirus transfection and the experiments were divided into two groups: mir-210 overexpression group (LV-mir-210-GFP) and control group (LV-GFP). EphrinA3 protein change was detected by Flow cytometry analysis, and both mRNA and protein expression changes of VEGF-Notch signaling moleculars were detected by real time PCR, immunofluorescence and western blot. At last capillary formation assay was performed to explore the ability of HUVE-12 to form capillary in two groups. The results showed a significant down-regulation of ephrinA3 protein levels after Mir-210 over-expression in HUVE-12 compared to control group, while both mRNA and protein levels of VEGF, VEGFR2 and Notch1 were up-regulated after Mir-210 over-expression in HUVE-12. Moreover, Capillary formation assay results showed increased capillary-like structures after Mir-210 over-expression in HUVE-12 compared to control group. We concluded that the hypoxic conditions in ischemic cortex could up-regulate Mir-210 expression significantly and thus enhance angiogenesis by up-regulating VEGF-Notch signaling moleculars expressions.
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