RATIONALE Angiogenesis promotes neurological recovery after stroke and is associated with longer survival of stroke patients. Cerebral angiogenesis is tightly controlled by certain microRNAs (miRs), such as the miR-15a/16-1 cluster, among others. However, the function of the miR-15a/16-1 cluster in endothelium on postischemic cerebral angiogenesis is not known. OBJECTIVE To investigate the functional significance and molecular mechanism of endothelial miR-15a/16-1 cluster on angiogenesis in the ischemic brain. METHODS AND RESULTS Endothelial cell-selective miR-15a/16-1 conditional knockout (EC-miR-15a/16-1 cKO) mice and wild-type littermate controls were subjected to 1 hour middle cerebral artery occlusion followed by 28-day reperfusion. Deletion of miR-15a/16-1 cluster in endothelium attenuates post-stroke brain infarction and atrophy and improves the long-term sensorimotor and cognitive recovery against ischemic stroke. Endothelium-targeted deletion of the miR-15a/16-1 cluster also enhances post-stroke angiogenesis by promoting vascular remodeling and stimulating the generation of newly formed functional vessels, and increases the ipsilateral cerebral blood flow. Endothelial cell-selective deletion of the miR-15a/16-1 cluster up-regulated the protein expression of pro-angiogenic factors VEGFA (vascular endothelial growth factor), FGF2 (fibroblast growth factor 2), and their receptors VEGFR2 (vascular endothelial growth factor receptor 2) and FGFR1 (fibroblast growth factor receptor 1) after ischemic stroke. Consistently, lentiviral knockdown of the miR-15a/16-1 cluster in primary mouse or human brain microvascular endothelial cell cultures enhanced in vitro angiogenesis and up-regulated pro-angiogenic proteins expression after oxygen-glucose deprivation, whereas lentiviral overexpression of the miR-15a/16-1 cluster suppressed in vitro angiogenesis and down-regulated pro-angiogenic proteins expression. Mechanistically, miR-15a/16-1 translationally represses pro-angiogenic factors VEGFA, FGF2, and their receptors VEGFR2 and FGFR1, respectively, by directly binding to the complementary sequences within 3'-untranslated regions of those messenger RNAs. CONCLUSIONS Endothelial miR-15a/16-1 cluster is a negative regulator for postischemic cerebral angiogenesis and long-term neurological recovery. Inhibition of miR-15a/16-1 function in cerebrovascular endothelium may be a legitimate therapeutic approach for stroke recovery.

中文翻译：

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靶向内皮细胞的microRNA-15a / 16-1缺失促进中风后血管生成，并改善长期神经功能。

原理血管生成促进中风后的神经功能恢复，并与中风患者的更长生存期相关。脑血管生成受到某些microRNA（miR）的紧密控制，例如miR-15a / 16-1簇。但是，内皮中的miR-15a / 16-1簇对缺血后脑血管新生的功能尚不清楚。目的探讨内皮miR-15a / 16-1簇在缺血性脑血管生成中的功能意义和分子机制。方法和结果对内皮细胞选择性miR-15a / 16-1条件性基因敲除（EC-miR-15a / 16-1 cKO）小鼠和野生型同窝小鼠进行了1小时的大脑中动脉闭塞治疗，然后进行了28天的再灌注。内皮中miR-15a / 16-1簇的缺失可减轻中风后脑梗塞和萎缩，并改善针对缺血性中风的长期感觉运动和认知恢复。针对内皮细胞的miR-15a / 16-1簇的缺失还通过促进血管重塑和刺激新形成的功能性血管的生成来增强中风后的血管生成，并增加了同侧脑血流量。miR-15a / 16-1簇的内皮细胞选择性缺失上调了促血管生成因子VEGFA（血管内皮生长因子），FGF2（成纤维细胞生长因子2）及其受体VEGFR2（血管内皮生长）的蛋白表达缺血性脑卒中后，T细胞受体2）和FGFR1（成纤维细胞生长因子受体1）。一致地，在原代小鼠或人脑微血管内皮细胞培养物中对miR-15a / 16-1簇进行慢病毒抑制可增强体外血管生成，并在缺氧-葡萄糖剥夺后上调促血管生成蛋白的表达，而miR-15a /的慢病毒过表达16-1簇抑制体外血管生成并下调促血管生成蛋白表达。机械上，miR-15a / 16-1通过直接结合那些信使RNA的3'-非翻译区域内的互补序列，分别翻译抑制促血管生成因子VEGFA，FGF2及其受体VEGFR2和FGFR1。结论内皮miR-15a / 16-1簇是缺血后脑血管新生和长期神经功能恢复的负调节剂。