MicroRNA-126 Regulates Angiogenesis and Neurogenesis in a Mouse Model of Focal Cerebral Ischemia

Studies demonstrate that microRNA-126 plays a critical role in promoting angiogenesis. However, its effects on angiogenesis following ischemic stroke are unclear. Here, we explored the effect of microRNA-126-3p and microRNA-126-5p on angiogenesis and neurogenesis after brain ischemia. We demonstrated that both microRNA (miRNA)-126-3p and microRNA-126-5p increased the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) compared with the scrambled miRNA control (p < 0.05). Transferring microRNA-126 into a mouse middle cerebral artery occlusion model via lentivirus, we found that microRNA-126 overexpression increased the number of CD31+/BrdU+ (5-bromo-2′-deoxyuridine-positive) proliferating endothelial cells and DCX+/BrdU+ neuroblasts in the ischemic mouse brain, improved neurobehavioral outcomes (p < 0.05), and reduced brain atrophy volume (p < 0.05) compared with control mice. Western blot results showed that AKT and ERK signaling pathways were activated in the lentiviral-microRNA-126-treated group (p < 0.05). Both PCR and western blot results demonstrated that tyrosine-protein phosphatase non-receptor type 9 (PTPN9) was decreased in the lentiviral-microRNA-126-treated group (p < 0.05). Dual-luciferase gene reporter assay also showed that PTPN9 was the direct target of microRNA-126-3p and microRNA-126-5p in the ischemic brain. We demonstrated that microRNA-126-3p and microRNA-126-5p promoted angiogenesis and neurogenesis in ischemic mouse brain, and further improved neurobehavioral outcomes. Our mechanistic study further showed that microRNA-126 mediated angiogenesis through directly inhibiting its target PTPN9 and activating AKT and ERK signaling pathways. Studies demonstrate that microRNA-126 plays a critical role in promoting angiogenesis. However, its effects on angiogenesis following ischemic stroke are unclear. Here, we explored the effect of microRNA-126-3p and microRNA-126-5p on angiogenesis and neurogenesis after brain ischemia. We demonstrated that both microRNA (miRNA)-126-3p and microRNA-126-5p increased the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) compared with the scrambled miRNA control (p < 0.05). Transferring microRNA-126 into a mouse middle cerebral artery occlusion model via lentivirus, we found that microRNA-126 overexpression increased the number of CD31+/BrdU+ (5-bromo-2′-deoxyuridine-positive) proliferating endothelial cells and DCX+/BrdU+ neuroblasts in the ischemic mouse brain, improved neurobehavioral outcomes (p < 0.05), and reduced brain atrophy volume (p < 0.05) compared with control mice. Western blot results showed that AKT and ERK signaling pathways were activated in the lentiviral-microRNA-126-treated group (p < 0.05). Both PCR and western blot results demonstrated that tyrosine-protein phosphatase non-receptor type 9 (PTPN9) was decreased in the lentiviral-microRNA-126-treated group (p < 0.05). Dual-luciferase gene reporter assay also showed that PTPN9 was the direct target of microRNA-126-3p and microRNA-126-5p in the ischemic brain. We demonstrated that microRNA-126-3p and microRNA-126-5p promoted angiogenesis and neurogenesis in ischemic mouse brain, and further improved neurobehavioral outcomes. Our mechanistic study further showed that microRNA-126 mediated angiogenesis through directly inhibiting its target PTPN9 and activating AKT and ERK signaling pathways. Stroke is one of the leading causes of mortality and morbidity worldwide.1Cai M. Zhang W. Weng Z. Stetler R.A. Jiang X. Shi Y. Gao Y. Chen J. Promoting neurovascular recovery in aged mice after ischemic stroke—prophylactic effect of omega-3 polyunsaturated fatty acids.Aging Dis. 2017; 8: 531-545Crossref PubMed Scopus (31) Google Scholar Currently, only tissue plasminogen activator (tPA) is proven as an effective drug treatment clinically.2Prabhakaran S. Ruff I. Bernstein R.A. Acute stroke intervention: a systematic review.JAMA. 2015; 313: 1451-1462Crossref PubMed Scopus (446) Google Scholar However, due to a limited time window and high hemorrhagic risk, only 1%–2% of patients have benefitted from this treatment.3Ren C. Wang B. Li N. Jin K. Ji X. Herbal formula Danggui-Shaoyao-San promotes neurogenesis and angiogenesis in rat following middle cerebral artery occlusion.Aging Dis. 2015; 6: 245-253Crossref PubMed Scopus (29) Google Scholar There is a critical need to develop new strategies for stroke therapy improvement. Poor blood flow-induced cell death is the main pathology of stroke.4Fan Y. Yang G.Y. Therapeutic angiogenesis for brain ischemia: a brief review.J. Neuroimmune Pharmacol. 2007; 2: 284-289Crossref PubMed Scopus (72) Google Scholar Blood flow restoration could be a potential therapeutic strategy for treating ischemic stroke.5Tang Y. Wang L. Wang J. Lin X. Wang Y. Jin K. Yang G.Y. Ischemia-induced angiogenesis is attenuated in aged rats.Aging Dis. 2015; 7: 326-335Crossref PubMed Scopus (23) Google Scholar Angiogenesis has been demonstrated as a significant event that resulted in brain microvasculature changes following cerebral ischemia. Previous reports have shown that angiogenesis occurred 7 and 14 days after ischemia in rodents and occurred in human brain as early as 3 days following ischemic insult.6Tang Y. Wang J. Lin X. Wang L. Shao B. Jin K. Wang Y. Yang G.Y. Neural stem cell protects aged rat brain from ischemia-reperfusion injury through neurogenesis and angiogenesis.J. Cereb. Blood Flow Metab. 2014; 34: 1138-1147Crossref PubMed Scopus (84) Google Scholar, 7Shen F. Su H. Fan Y. Chen Y. Zhu Y. Liu W. Young W.L. Yang G.Y. Adeno-associated viral-vector-mediated hypoxia-inducible vascular endothelial growth factor gene expression attenuates ischemic brain injury after focal cerebral ischemia in mice.Stroke. 2006; 37: 2601-2606Crossref PubMed Scopus (82) Google Scholar, 8Sbarbati A. Pietra C. Baldassarri A.M. Guerrini U. Ziviani L. Reggiani A. Boicelli A. Osculati F. The microvascular system in ischemic cortical lesions.Acta Neuropathol. 1996; 92: 56-63Crossref PubMed Scopus (41) Google Scholar In addition, clinical observation demonstrated a strong correlation between neovascularization and functional recovery, suggesting that newly formed vessels contributed to behavioral outcomes after ischemic stroke.9Gunsilius E. Petzer A.L. Stockhammer G. Kähler C.M. Gastl G. Serial measurement of vascular endothelial growth factor and transforming growth factor-beta1 in serum of patients with acute ischemic stroke.Stroke. 2001; 32: 275-278Crossref PubMed Scopus (26) Google Scholar Gene-based or stem cell-based therapy could improve neurobehavioral outcomes and reduce ischemic brain injury by promoting angiogenesis.6Tang Y. Wang J. Lin X. Wang L. Shao B. Jin K. Wang Y. Yang G.Y. Neural stem cell protects aged rat brain from ischemia-reperfusion injury through neurogenesis and angiogenesis.J. Cereb. Blood Flow Metab. 2014; 34: 1138-1147Crossref PubMed Scopus (84) Google Scholar, 10Li J. Tang Y. Wang Y. Tang R. Jiang W. Yang G.Y. Gao W.Q. Neurovascular recovery via co-transplanted neural and vascular progenitors leads to improved functional restoration after ischemic stroke in rats.Stem Cell Reports. 2014; 3: 101-114Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 11Lu H. Wang Y. He X. Yuan F. Lin X. Xie B. Tang G. Huang J. Tang Y. Jin K. et al.Netrin-1 hyperexpression in mouse brain promotes angiogenesis and long-term neurological recovery after transient focal ischemia.Stroke. 2012; 43: 838-843Crossref PubMed Scopus (89) Google Scholar Our previous studies demonstrated that netrin-1 (NT-1) and stromal cell-derived factor-1 (SDF-1) overexpression increased angiogenesis in ischemic mouse brains.11Lu H. Wang Y. He X. Yuan F. Lin X. Xie B. Tang G. Huang J. Tang Y. Jin K. et al.Netrin-1 hyperexpression in mouse brain promotes angiogenesis and long-term neurological recovery after transient focal ischemia.Stroke. 2012; 43: 838-843Crossref PubMed Scopus (89) Google Scholar, 12Li Y. Huang J. He X. Tang G. Tang Y.H. Liu Y. Lin X. Lu Y. Yang G.Y. Wang Y. Postacute stromal cell-derived factor-1α expression promotes neurovascular recovery in ischemic mice.Stroke. 2014; 45: 1822-1829Crossref PubMed Scopus (65) Google Scholar, 13Sun H. Le T. Chang T.T. Habib A. Wu S. Shen F. Young W.L. Su H. Liu J. AAV-mediated netrin-1 overexpression increases peri-infarct blood vessel density and improves motor function recovery after experimental stroke.Neurobiol. Dis. 2011; 44: 73-83Crossref PubMed Scopus (56) Google Scholar, 14Fan Y. Shen F. Chen Y. Hao Q. Liu W. Su H. Young W.L. Yang G.Y. Overexpression of netrin-1 induces neovascularization in the adult mouse brain.J. Cereb. Blood Flow Metab. 2008; 28: 1543-1551Crossref PubMed Scopus (54) Google Scholar Although exogenous angiogenic genes increased angiogenesis in experimental cerebral ischemia models, several disadvantages limited its clinical translation. First, gene was transferred to animals before ischemic insult, which was not acceptable for clinical application.7Shen F. Su H. Fan Y. Chen Y. Zhu Y. Liu W. Young W.L. Yang G.Y. Adeno-associated viral-vector-mediated hypoxia-inducible vascular endothelial growth factor gene expression attenuates ischemic brain injury after focal cerebral ischemia in mice.Stroke. 2006; 37: 2601-2606Crossref PubMed Scopus (82) Google Scholar, 11Lu H. Wang Y. He X. Yuan F. Lin X. Xie B. Tang G. Huang J. Tang Y. Jin K. et al.Netrin-1 hyperexpression in mouse brain promotes angiogenesis and long-term neurological recovery after transient focal ischemia.Stroke. 2012; 43: 838-843Crossref PubMed Scopus (89) Google Scholar, 15Zhu W. Fan Y. Frenzel T. Gasmi M. Bartus R.T. Young W.L. Yang G.Y. Chen Y. Insulin growth factor-1 gene transfer enhances neurovascular remodeling and improves long-term stroke outcome in mice.Stroke. 2008; 39: 1254-1261Crossref PubMed Scopus (110) Google Scholar Second, many angiogenic factors were large molecules and difficult to cross the blood-brain barrier (BBB).16Zeng L. He X. Wang Y. Tang Y. Zheng C. Cai H. Liu J. Wang Y. Fu Y. Yang G.Y. MicroRNA-210 overexpression induces angiogenesis and neurogenesis in the normal adult mouse brain.Gene Ther. 2014; 21: 37-43Crossref PubMed Scopus (146) Google Scholar Stem cell-based therapy also showed great potential for promoting angiogenesis. Mesenchymal stem cells (MSCs) enhanced focal angiogenesis by increasing the levels of endogenous vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) in the ischemic perifocal region.17Chen J. Zhang Z.G. Li Y. Wang L. Xu Y.X. Gautam S.C. Lu M. Zhu Z. Chopp M. Intravenous administration of human bone marrow stromal cells induces angiogenesis in the ischemic boundary zone after stroke in rats.Circ. Res. 2003; 92: 692-699Crossref PubMed Scopus (612) Google Scholar Endothelial progenitor cells (EPCs) promoted neurovascular repair and improved long-term neurobehavioral outcomes by the SDF-1/C-X-C chemokine receptor type 4 (CXCR4) signaling pathway.18Fan Y. Shen F. Frenzel T. Zhu W. Ye J. Liu J. Chen Y. Su H. Young W.L. Yang G.Y. Endothelial progenitor cell transplantation improves long-term stroke outcome in mice.Ann. Neurol. 2010; 67: 488-497Crossref PubMed Scopus (260) Google Scholar Despite these promising initial results, its therapeutic efficiency is still low due to poor cell survival.19Tang Y. Cai B. Yuan F. He X. Lin X. Wang J. Wang Y. Yang G.Y. Melatonin pretreatment improves the survival and function of transplanted mesenchymal stem cells after focal cerebral ischemia.Cell Transplant. 2014; 23: 1279-1291Crossref PubMed Scopus (109) Google Scholar Thus, there is a critical need to develop novel strategies to improve angiogenesis after cerebral ischemia. MicroRNAs (miRNAs) were important biological factors because they could cross the BBB and target multiple angiogenesis-associated genes.16Zeng L. He X. Wang Y. Tang Y. Zheng C. Cai H. Liu J. Wang Y. Fu Y. Yang G.Y. MicroRNA-210 overexpression induces angiogenesis and neurogenesis in the normal adult mouse brain.Gene Ther. 2014; 21: 37-43Crossref PubMed Scopus (146) Google Scholar Various miRNAs’ angiogenic function has been tested. Our previous studies showed that miRNA-210 overexpression promoted angiogenesis by upregulating brain-derived neurotrophic factor (BDGF).20Zeng L.L. He X.S. Liu J.R. Zheng C.B. Wang Y.T. Yang G.Y. Lentivirus-mediated overexpression of microRNA-210 improves long-term outcomes after focal cerebral ischemia in mice.CNS Neurosci. Ther. 2016; 22: 961-969Crossref PubMed Scopus (63) Google Scholar miRNA-126, the most abundant miRNA in endothelial cells, was reported to play a vital role in angiogenesis. miRNA-126 possesses two mature strands: miRNA-126-3p and miRNA-126-5p.21Schober A. Nazari-Jahantigh M. Wei Y. Bidzhekov K. Gremse F. Grommes J. Megens R.T. Heyll K. Noels H. Hristov M. et al.MicroRNA-126-5p promotes endothelial proliferation and limits atherosclerosis by suppressing Dlk1.Nat. Med. 2014; 20: 368-376Crossref PubMed Scopus (473) Google Scholar Studies showed miRNA-126-3p promoted angiogenesis in rat heart by activating mitogen-activated protein kinase (MAPK) and AKT signaling pathways.22DA Silva Jr., N.D. Fernandes T. Soci U.P. Monteiro A.W. Phillips M.I. DE Oliveira E.M. Swimming training in rats increases cardiac MicroRNA-126 expression and angiogenesis.Med. Sci. Sports Exerc. 2012; 44: 1453-1462Crossref PubMed Scopus (117) Google Scholar Injection of exosomes that derived from miRNA-126-3p-overexpressing adipose-derived stem cells prevented myocardial injury by protecting myocardial cells from apoptosis, inflammation, fibrosis, and increased angiogenesis.23Luo Q. Guo D. Liu G. Chen G. Hang M. Jin M. Exosomes from miR-126-overexpressing ADSCs are therapeutic in relieving acute myocardial ischaemic injury.Cell. Physiol. Biochem. 2017; 44: 2105-2116Crossref PubMed Scopus (160) Google Scholar However, whether miRNA-126 could promote angiogenesis after stroke remains largely unexplored. In this study, we explored the effects of miRNA-126 in angiogenesis after ischemic stroke and its downstream target. In addition, we also tested the function of miRNA-126 in neurogenesis, and whether such increased angiogenesis and neurogenesis could further improve behavioral outcomes in adult ischemic mice. To determine the effects of miRNA-126-3p and miRNA-126-5p on the function of human umbilical vein endothelial cells (HUVECs), HUVECs were transduced with lentiviral vector carrying miRNA-126-3p, miRNA-126-5p, miRNA-126-3p sponge, or miRNA-126-5p sponge (Figure S1A). Flow cytometry analysis showed that about 70% of HUVECs were transduced with lentivirus (Figure S1B). Real-time PCR analysis showed that miRNA-126-3p and miRNA-126-5p expression were upregulated in HUVECs transduced with lentiviral-miRNA-126-3p (LV-miRNA-126-3p) and miRNA-126-5p (Figure S1C). Interestingly, the expression of miRNA-126-3p and miRNA-126-5p were comparable in HUVECs transduced with LV-miRNA-126-3p sponge, miRNA-126-5p sponge, and LV-GFP sponge (Figure S1C), suggesting transduction of miRNA-126-3p and miRNA-126-5p sponge blocked the binding between miRNA-126-3p and miRNA-126-5p and their target genes, rather than degrading miRNAs. To detect the effects of miRNA-126-3p and miRNA-126-5p on the function of HUVECs, we examined the proliferation and migration of HUVECs transduced with LV-miRNA-126-3p and LV-miRNA-126-5p. We found that both miRNA-126-3p and miRNA-126-5p overexpression promoted HUVEC proliferation and migration; inhibiting miR-126-3p and miRNA-126-5p reversed these functions (Figures S2A and S2B; p < 0.05). We further examined tube formation in the miRNA-126-3p or miRNA-126-5p transduced HUVECs. Results showed that both miRNA-126-3p and miRNA-126-5p overexpression promoted HUVEC tube formation, and inhibiting miR-126-3p and miRNA-126-5p reversed these cells’ function (Figure S2C; p < 0.05). It was noted that miRNA-126-3p transduction significantly increased tube formation of HUVECs compared with the miRNA-126-5p transduced group (Figure S2C; p < 0.05). Meanwhile, miR-126-3p sponge transduction significantly decreased HUVEC tube formation compared with the miR-126-5p sponge transduced group (Figure S2C; p < 0.05). We first examined miRNA-126-3p and miRNA-126-5p expression in an infarct border zone of ischemic mouse brain at 1, 3, 7, and 14 days after middle cerebral artery occlusion (MCAO). miRNA was isolated from the ipsilateral hemisphere of ischemic mice (infarct hemisphere), including cortex and striatum. miRNA-126-3p and miRNA-126-5p expression were significantly decreased from days 3 to 14 after MCAO (Figure 1A). To determine the effects of miRNA-126 on the ischemic brain injury, we injected lentiviral vector carrying miRNA-126 into the mouse brain, and animals were sacrificed 2 and 3 weeks after lentiviral vector injection for histological and biological analysis (Figure 1B). Fluorescent imaging showed GFP successfully expressed in ischemic mouse brain 2 and 3 weeks after lentiviral vector injection, and lentivirus-carrying miRNA-126 transfected both neurons and endothelial cells (Figure S4). Our PCR results demonstrated that miRNA-126 expression was upregulated in both cortex and striatum of ischemic mice (Figures 1C and 1D). We also found that a cavity was detected in mouse brain at 3 and 4 weeks after permanent middle cerebral artery occlusion (pMCAO) even without any virus treatment (Figure S3), suggesting an MCAO injury-induced cavity was formed in the mouse brain. We further evaluated whether miRNA-126 overexpression could reduce infarct volume and improve neurobehavioral recovery. Cresyl violet staining showed that atrophy volume was significantly decreased in the LV-miRNA-126 transduced group compared with the control group (Figure 1E; p < 0.05), and miRNA-126 overexpression significantly reduced neurological deficits and improved behavioral recovery, as shown by modified neurological severity scores (mNSSs) and rotarod test (Figure 1F; p < 0.05). To explore the effects of miRNA-126 on angiogenesis, we measured the length and diameter of blood vessels in ischemic mice brain transduced by lentiviral vector. Our results demonstrated that overexpression of miRNA-126 significantly increased the length and diameter of blood vessels, compared with the GFP group at 2 and 3 weeks after lentiviral vector injection (Figure 2A). In addition, overexpression of miRNA-126 substantially increased the number of 5-bromo-2′-deoxyuridine-positive (BrdU+)/CD31+ cells in the peri-focal region at 2 and 3 weeks after lentiviral vector injection, compared with the GFP group (Figure 2B). These results indicated that miRNA-126 promoted vessel remodeling and angiogenesis after cerebral ischemia. To explore whether miRNA-126 promoted neurogenesis, we performed doublecortin (DCX)/BrdU double staining. As shown in Figure 3, miRNA-126 overexpression significantly increased the number of DCX+/BrdU+ cells in the subventricular zone (SVZ) at 2 and 3 weeks and in the perifocal region at 3 weeks after lentiviral vector injection, suggesting that miRNA-126 promoted neurogenesis. To explore the underlying mechanism of miRNA-126 in angiogenesis and neurogenesis, we examined phosphorylation of AKT and ERK. Protein was isolated from the ipsilateral hemisphere of the brain including cortex and striatum. Western blot results showed miRNA-126 overexpression significantly elevated the expression of p-AKT and p-ERK in the ischemic mouse brain, compared with the control group (Figure 4). We then examined the expression of downstream genes of miRNA-126-3p and miRNA-126-5p, including DUSP10, SPRED1, PTPN9, PTPN7, and PIK3R2, which were all related to AKT and ERK signaling pathways. RNA was isolated from the ipsilateral hemisphere of the brain, including cortex and striatum. Our real-time PCR results demonstrated that PTPN9, SPRED1, and PIK3R2 were significantly decreased 2 and 3 weeks after LV-miRNA-126 treatment, and β-actin was used as a housekeeper (Figures 5A–5E). By searching miRNA-126-3p and miRNA-126-5p seed sequence and mice PTPN9 3′ UTR, we found that PTPN9 3′ UTR was complementary to nucleotides 2–7 of the miRNA-126-5p sequence and nucleotides 2–8 of the miRNA-126-3p sequence (Figure 5F). Our western blot results further demonstrated that miRNA-126-3p and miRNA-126-5p inhibited PTPN9 expression (Figures 5G and 5H). The experimental and matching results illustrated PTPN9 might be a potential target of both miRNA-126-3p and miRNA-126-5p in mice. Further studies showed that overexpression of miR-126 reduced PTPN9 in neurons (Figures S6A–S6C). To confirm whether PTPN9 was the direct target of miRNA-126-3p and miRNA-126-5p, we cloned PTPN9 mRNA 3′ UTR fragment to a luciferase reporter plasmid containing the putative miRNA-126-3p and miRNA-126-5p binding sites. Luciferase reporter plasmid and miRNA mimic were co-transfected in 293T cells. Luciferase activity level was reduced in the cells co-transfected with miRNA-126-3p/miRNA-126-5p mimic and PTPN9 mRNA 3′ UTR fragment group, compared with the mimic control and the PTPN9 3′ UTR fragment group (Figure 5I). Angiogenesis plays an important role in improving neurobehavioral recovery after stroke.24Liu J. Wang Y. Akamatsu Y. Lee C.C. Stetler R.A. Lawton M.T. Yang G.Y. Vascular remodeling after ischemic stroke: mechanisms and therapeutic potentials.Prog. Neurobiol. 2014; 115: 138-156Crossref PubMed Scopus (227) Google Scholar In the present study, we explored the function of miRNA-126-3p and miRNA-126-5p in angiogenesis using in vitro and in vivo models. We found that overexpression of both miRNA-126-3p and miRNA-126-5p promoted the proliferation, migration, and tube formation of HUVECs; contributed to angiogenesis and neurogenesis in the ischemic mice brain; and further improved behavioral recovery by downregulating PTPN9 and activating AKT and ERK signaling pathways. It has been well documented that miRNA-126 was critically involved in regulating angiogenesis. However, the effects of miRNA-126-3p and miRNA-126-5p on angiogenesis are still controversial. Zhou et al.25Zhou Q. Anderson C. Hanus J. Zhao F. Ma J. Yoshimura A. Wang S. Strand and cell type-specific function of microRNA-126 in angiogenesis.Mol. Ther. 2016; 24: 1823-1835Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar demonstrated different effects of miRNA-126-5p and miRNA-126-3p on angiogenesis using different models. They found that inhibition of miRNA-126-3p in the laser injury-induced choroidal neovascularization (CNV) model repressed neovascularization. In the study, anti-miR-126-3p, anti-miR-126-5p, or a scramble control was subretinally injected into the eye immediately following laser injury in three locations and regressed neovascularization at postinjury days 3 and 7. However, silencing of miR-126-5p did not significantly impact neovascularization. In addition, they found that injection of miR-126-3p mimic subretinally after laser injury led to ∼60% decrease in neovascularization, whereas miR-126-5p mimic significantly but mildly enhanced laser-induced neovascularization. They further used HUVEC as an in vitro model to dissect the roles of miR-126-3p and miR-126-5p on angiogenesis. Under starvation condition, miR-126-5p, but not miR-126-3p, significantly increased proliferation and migration of HUVECs in vitro, which is different from the in vivo data.25Zhou Q. Anderson C. Hanus J. Zhao F. Ma J. Yoshimura A. Wang S. Strand and cell type-specific function of microRNA-126 in angiogenesis.Mol. Ther. 2016; 24: 1823-1835Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Conversely, Cao et al.26Cao W.J. Rosenblat J.D. Roth N.C. Kuliszewski M.A. Matkar P.N. Rudenko D. Liao C. Lee P.J. Leong-Poi H. Therapeutic angiogenesis by ultrasound-mediated microRNA-126-3p delivery.Arterioscler. Thromb. Vasc. Biol. 2015; 35: 2401-2411Crossref PubMed Scopus (65) Google Scholar illustrated miRNA-126-3p overexpression promoted HUVEC migration and tube formation, and injection of 2.5 μg of miRNA-126-3p into hindlimb 2 weeks after femoral artery ligation increased microvascular perfusion and vascular density of the ischemic hindlimb, but they did not detect the function of miRNA-126-5p. In our study, we tested and compared the function of both miRNA-126-3p and miRNA-126-5p on angiogenesis by evaluating proliferation, migration, and tube formation of HUVECs. We found overexpression of both miRNA-126-3p and miRNA-126-5p significantly increased the proliferation, migration, and tube formation of HUVECs. Interestingly, miRNA-126-3p showed better effects on tube formation, which is different from Zhou et al.’s25Zhou Q. Anderson C. Hanus J. Zhao F. Ma J. Yoshimura A. Wang S. Strand and cell type-specific function of microRNA-126 in angiogenesis.Mol. Ther. 2016; 24: 1823-1835Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar study. Taken together, all of the studies suggested that the role of miR-126 in angiogenesis depends on cell type, animal model, and strand of miR-126. The majority of patients still suffer from various long-term neurological deficits, even though they survive after timely treatment in the acute phase of ischemic stroke.27Nichols-Larsen D.S. Clark P.C. Zeringue A. Greenspan A. Blanton S. Factors influencing stroke survivors’ quality of life during subacute recovery.Stroke. 2005; 36: 1480-1484Crossref PubMed Scopus (442) Google Scholar One of the major issues is limited angiogenesis and neurogenesis in the post-acute phase.12Li Y. Huang J. He X. Tang G. Tang Y.H. Liu Y. Lin X. Lu Y. Yang G.Y. Wang Y. Postacute stromal cell-derived factor-1α expression promotes neurovascular recovery in ischemic mice.Stroke. 2014; 45: 1822-1829Crossref PubMed Scopus (65) Google Scholar Therapeutic strategies that focus on improving angiogenesis and neurogenesis in the post-acute phase would be beneficial for improving functional recovery after stroke.10Li J. Tang Y. Wang Y. Tang R. Jiang W. Yang G.Y. Gao W.Q. Neurovascular recovery via co-transplanted neural and vascular progenitors leads to improved functional restoration after ischemic stroke in rats.Stem Cell Reports. 2014; 3: 101-114Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Previous studies showed that miRNA-126-3p promoted cardiac angiogenesis by directly downregulating SPRED1 and PIK3R2 and indirectly regulating the VEGF pathway.22DA Silva Jr., N.D. Fernandes T. Soci U.P. Monteiro A.W. Phillips M.I. DE Oliveira E.M. Swimming training in rats increases cardiac MicroRNA-126 expression and angiogenesis.Med. Sci. Sports Exerc. 2012; 44: 1453-1462Crossref PubMed Scopus (117) Google Scholar Mathiyalagan et al.’s28Mathiyalagan P. Liang Y. Kim D. Misener S. Thorne T. Kamide C.E. Klyachko E. Losordo D.W. Hajjar R.J. Sahoo S. Angiogenic mechanisms of human CD34+ stem cell exosomes in the repair of ischemic hindlimb.Circ. Res. 2017; 120: 1466-1476Crossref PubMed Scopus (168) Google Scholar study demonstrated that exosomes derived from CD34+ cells repaired ischemic hindlimb, but knocking down miRNA-126-3p abolished their angiogenic activity and beneficial function both in vitro and in vivo. MSCs modified with miRNA-126-3p released angiogenic factors and activated Notch ligand Delta-like-4, enhancing ischemic angiogenesis in cardiac reparation.29Huang F. Zhu X. Hu X.Q. Fang Z.F. Tang L. Lu X.L. Zhou S.H. Mesenchymal stem cells modified with miR-126 release angiogenic factors and activate Notch ligand Delta-like-4, enhancing ischemic angiogenesis and cell survival.Int. J. Mol. Med. 2013; 31: 484-492Crossref PubMed Scopus (101) Google Scholar However, the angiogenic and neurogenic effects of miRNA-126 in stroke have never been explored. In this study, we overexpressed miRNA-126 in the peri-infarct area of ischemic mice 1 week after stroke. Our results showed that expression of miRNA-126-3p and miRNA-126-5p was increased 2 and 3 weeks after lentiviral vector injection, and such upregulated miRNA-126 promoted angiogenesis and neurogenesis. In our study, miRNA-126 treatment promoted the proliferation of few endothelial cells; however, we also found that injection of miRNA-126 increased the vessel length and vessel diameter, suggesting miRNA-126 is beneficial for promoting vascular remodeling, rather than only promoting angiogenesis. And such miRNA-126-mediated vascular remodeling contributed to behavioral recovery of mice subjected to stroke, suggesting miR-126 treatment holds biological relevance to the overall recovery of the animals. It was noted that higher expression of miRNA-126 was achieved at 3 weeks after lentiviral vector injection than 2 weeks. However, no significant difference of angiogenesis and neurogenesis was observed between 2 and 3 weeks following lentiviral vector injection, suggesting the effects of miRNA-126 in angiogenesis was already saturated at the second week, and increasing the level of miRNA-126 at week 3 could not further promote its angiogenic and neurogenic effects. miRNA-126-mediated neurovascular remodeling was beneficial for reducing brain atrophy volume and improving neurological outcomes. Recent findings suggested that strategies to enhance angiogenesis and neurogenesis for brain injuries may provide promising opportunities to improve clinical outcomes during brain functional recovery.30Xiong Y. Mahmood A. Chopp M. Angiogenesis, neurogenesis and brain recovery of function following injury.Curr. Opin. Investig. Drugs. 2010; 11: 298-308PubMed Google Scholar Newly formed blood vessels removed necrotic tissue and provided trophic factors.31Font M.A. Arboix A. Krupinski J. Angiogenesis, neurogenesis and neuroplasticity in ischemic stroke.Curr. Cardiol. Rev. 2010; 6: 238-244Crossref PubMed Scopus (151) Google Scholar Neurogenesis increased the neuroblasts proliferation, neural stem cells recruitment, and their neuronal differentia