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Vascular Endothelial Cell-specific MicroRNA-15a Inhibits Angiogenesis in Hindlimb Ischemia*

  • Ke-Jie Yin
    Correspondence
    To whom correspondence may be addressed. Tel.: 734-763-7838; Fax: 734-936-2641
    Affiliations
    Cardiovascular Center, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • Karl Olsen
    Affiliations
    Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan 48109
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  • Milton Hamblin
    Affiliations
    Cardiovascular Center, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • Jifeng Zhang
    Affiliations
    Cardiovascular Center, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • Steven P. Schwendeman
    Affiliations
    Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan 48109
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  • Y. Eugene Chen
    Correspondence
    To whom correspondence may be addressed. Tel.: 734-763-7838; Fax: 734-936-2641
    Affiliations
    Cardiovascular Center, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants NS066652, HL089544 (to Y. E. C.), and HL68345 (to S. P. S.) and T32 HL007853 (to M. H.). This work was also supported by American Heart Association Grants 0630209N (to K.-J. Y.), 0835237N (to J. Z.), and 0840025N (to Y. E. C.).
    This article contains supplemental Figs. S1–S4.
    ♦ This article was selected as a Paper of the Week.
Open AccessPublished:June 12, 2012DOI:https://doi.org/10.1074/jbc.M112.364414
      The effects and potential mechanisms of the vascular endothelial cell (EC)-enriched microRNA-15a (miR-15a) on angiogenesis remain unclear. Here, we show a novel finding that EC-selective miR-15a transgenic overexpression leads to reduced blood vessel formation and local blood flow perfusion in mouse hindlimbs at 1–3 weeks after hindlimb ischemia. Mechanistically, gain- or loss-of-miR-15a function by lentiviral infection in ECs significantly reduces or increases tube formation, cell migration, and cell differentiation, respectively. By FGF2 and VEGF 3′-UTR luciferase reporter assays, Real-time PCR, and immunoassays, we further identified that the miR-15a directly targets FGF2 and VEGF to facilitate its anti-angiogenic effects. Our data suggest that the miR-15a in ECs can significantly suppress cell-autonomous angiogenesis through direct inhibition of endogenous endothelial FGF2 and VEGF activities. Pharmacological modulation of miR-15a function may provide a new therapeutic strategy to intervene against angiogenesis in a variety of pathological conditions.

      Introduction

      Angiogenesis is a biological process that generates new blood vessels from existing vascular endothelial cells (ECs) to deliver nutrients and oxygen to various organs and tissue (
      • Folkman J.
      Angiogenesis in cancer, vascular, rheumatoid and other disease.
      ,
      • Carmeliet P.
      Angiogenesis in life, disease and medicine.
      ). In physiological conditions, angiogenesis plays a critical role in embryonic development, wound healing, and in response to ovulation. However, pathological angiogenesis may give rise to abnormally rapid proliferation of blood vessels, thus contributing to the pathogenesis or tissue repair processes of many human diseases (
      • Folkman J.
      Angiogenesis in cancer, vascular, rheumatoid and other disease.
      ,
      • Carmeliet P.
      Angiogenesis in life, disease and medicine.
      ). It has been well established that stimulation of angiogenesis can be therapeutic in ischemic heart disease, cerebrovascular disease, peripheral arterial disease, and wound healing (
      • Yanagisawa-Miwa A.
      • Uchida Y.
      • Nakamura F.
      • Tomaru T.
      • Kido H.
      • Kamijo T.
      • Sugimoto T.
      • Kaji K.
      • Utsuyama M.
      • Kurashima C.
      Salvage of infarcted myocardium by angiogenic action of basic fibroblast growth factor.
      ,
      • Baumgartner I.
      • Pieczek A.
      • Manor O.
      • Blair R.
      • Kearney M.
      • Walsh K.
      • Isner J.M.
      Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia.
      ,
      • Zhang Z.G.
      • Zhang L.
      • Jiang Q.
      • Zhang R.
      • Davies K.
      • Powers C.
      • Bruggen N.
      • Chopp M.
      VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain.
      ). However, inhibition of angiogenesis can also be therapeutic in cases of cancer, ophthalmic conditions, rheumatoid arthritis, and other diseases (
      • Carmeliet P.
      Angiogenesis in life, disease and medicine.
      ,
      • Huang D.
      • Ding Y.
      • Li Y.
      • Luo W.M.
      • Zhang Z.F.
      • Snider J.
      • Vandenbeldt K.
      • Qian C.N.
      • Teh B.T.
      Sunitinib acts primarily on tumor endothelium rather than tumor cells to inhibit the growth of renal cell carcinoma.
      ).
      Normally, angiogenesis is strictly controlled by a balance between pro-angiogenic and anti-angiogenic factors. Disruption of this balance favors pathological angiogenesis, and there is a localized accumulation of endogenous pro-angiogenic molecules, including growth factors, matrix metalloproteinases, cytokines, and integrins. More specifically, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), and epidermal growth factor (EGF) can induce the division of cultured endothelial cells, thus indicating a direct action on these cells (
      • Olsson A.K.
      • Dimberg A.
      • Kreuger J.
      • Claesson-Welsh L.
      VEGF receptor signalling: in control of vascular function.
      ,
      • Cross M.J.
      • Claesson-Welsh L.
      FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition.
      ).
      MicroRNAs (miRs)
      The abbreviations used are: miR
      microRNA
      HUVEC
      human umbilical vein endothelial cell
      LDPI
      laser Doppler perfusion imaging
      TG
      transgenic.
      have been recently discovered as a novel family of noncoding small RNAs that negatively modulate protein expression in various organisms (
      • Bartel D.P.
      MicroRNAs: genomics, biogenesis, mechanism, and function.
      ,
      • Bartel D.P.
      MicroRNAs: target recognition and regulatory functions.
      ). It is now evident that miRs are able to regulate expression of at least one third of the human genome and play a critical role in various biological processes, including cell differentiation, apoptosis, development, angiogenesis, and metabolism (
      • Bartel D.P.
      MicroRNAs: genomics, biogenesis, mechanism, and function.
      ,
      • Wang S.
      • Olson E.N.
      AngiomiRs: key regulators of angiogenesis.
      ,
      • Kuehbacher A.
      • Urbich C.
      • Dimmeler S.
      Targeting microRNA expression to regulate angiogenesis.
      ,
      • Suárez Y.
      • Sessa W.C.
      MicroRNAs as novel regulators of angiogenesis.
      ). Recent studies have also revealed important roles for miRs in regulating angiogenesis (
      • Wang S.
      • Olson E.N.
      AngiomiRs: key regulators of angiogenesis.
      ,
      • Kuehbacher A.
      • Urbich C.
      • Dimmeler S.
      Targeting microRNA expression to regulate angiogenesis.
      ,
      • Suárez Y.
      • Sessa W.C.
      MicroRNAs as novel regulators of angiogenesis.
      ). For example, mice with EC-specific deletion of Dicer display defective postnatal angiogenesis (
      • Kuehbacher A.
      • Urbich C.
      • Zeiher A.M.
      • Dimmeler S.
      Role of Dicer and Drosha for endothelial microRNA expression and angiogenesis.
      ,
      • Suárez Y.
      • Fernández-Hernando C.
      • Yu J.
      • Gerber S.A.
      • Harrison K.D.
      • Pober J.S.
      • Iruela-Arispe M.L.
      • Merkenschlager M.
      • Sessa W.C.
      Dicer-dependent endothelial microRNAs are necessary for postnatal angiogenesis.
      ). Discovering the function of individual miRs in angiogenesis is just beginning to be elucidated.
      MiR-15a and miR-16-1 are located on chromosome 13 in humans and are clustered at the 13q14 region. The miR-15a/16-1 cluster is the first group of identified miR genes associated with mammalian carcinogenesis (
      • Calin G.A.
      • Dumitru C.D.
      • Shimizu M.
      • Bichi R.
      • Zupo S.
      • Noch E.
      • Aldler H.
      • Rattan S.
      • Keating M.
      • Rai K.
      • Rassenti L.
      • Kipps T.
      • Negrini M.
      • Bullrich F.
      • Croce C.M.
      Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia.
      ,
      • Calin G.A.
      • Cimmino A.
      • Fabbri M.
      • Ferracin M.
      • Wojcik S.E.
      • Shimizu M.
      • Taccioli C.
      • Zanesi N.
      • Garzon R.
      • Aqeilan R.I.
      • Alder H.
      • Volinia S.
      • Rassenti L.
      • Liu X.
      • Liu C.G.
      • Kipps T.J.
      • Negrini M.
      • Croce C.M.
      MiR-15a and miR-16-1 cluster functions in human leukemia.
      ). In our previous publication (
      • Yin K.J.
      • Deng Z.
      • Hamblin M.
      • Xiang Y.
      • Huang H.
      • Zhang J.
      • Jiang X.
      • Wang Y.
      • Chen Y.E.
      Peroxisome proliferator-activated receptor δ regulation of miR-15a in ischemia-induced cerebral vascular endothelial injury.
      ), we demonstrated that miR-15a, by negatively regulating the Bcl-2 protein, has an anti-survival role in oxygen-glucose deprivation-induced cerebral vascular endothelial cell death. Additionally, recent studies have shown that miR-16-1 displays anti-angiogenic characteristics by directly inhibiting VEGF protein expression in carcinoma cell lines (

      Deleted in proof

      ,
      • Hua Z.
      • Lv Q.
      • Ye W.
      • Wong C.K.
      • Cai G.
      • Gu D.
      • Ji Y.
      • Zhao C.
      • Wang J.
      • Yang B.B.
      • Zhang Y.
      MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia.
      ,
      • Karaa Z.S.
      • Iacovoni J.S.
      • Bastide A.
      • Lacazette E.
      • Touriol C.
      • Prats H.
      The VEGF IRESes are differentially susceptible to translation inhibition by miR-16.
      ) as well as in endothelial cells (
      • Chamorro-Jorganes A.
      • Araldi E.
      • Penalva L.O.
      • Sandhu D.
      • Fernández-Hernando C.
      • Suárez Y.
      MicroRNA-16 and microRNA-424 regulate cell-autonomous angiogenic functions in endothelial cells via targeting vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1.
      ). However, whether the miR-15a is able to synergistically regulate cell autonomous angiogenesis in vascular endothelial cells, especially during in vivo settings, is still unexplored.
      In the present study, we utilized EC-selective miR-15a transgenic mice to explore the effects and molecular mechanisms of the vascular miR-15a on hindlimb ischemia-induced angiogenesis. We have identified the miR-15a as a novel anti-angiogenic microRNA. Moreover, we further demonstrated that FGF2 and VEGF are direct downstream targets of miR-15a translational repression, and this inhibition contributes to miR-15a-mediated anti-angiogenic activity against ischemic insults.

      DISCUSSION

      In this study, we addressed the potential role and molecular mechanisms of the vascular miR-15a in regulating cell-autonomous angiogenesis. We demonstrated for the first time that EC-selective transgenic overexpression of the miR-15a led to a significantly lower recovery of local blood flow and decreased capillary density in mice following hindlimb ischemia. Moreover, lentivirus-mediated gain- or loss-of-miR-15a function in vascular endothelial cells effectively reduces or increases EC tube formation, migration, and differentiation. We further identified FGF2 and VEGF as two direct downstream targets of miR-15a-mediated repression at the post-transcriptional level. These inhibitory pathways are responsible for the anti-angiogenic effects of the miR-15a after ischemic insults.
      The discovery of miRs that mediate post-transcriptional silencing of specific target genes has shed light on how noncoding RNAs can play critical roles in angiogenesis (
      • Wang S.
      • Olson E.N.
      AngiomiRs: key regulators of angiogenesis.
      ,
      • Kuehbacher A.
      • Urbich C.
      • Dimmeler S.
      Targeting microRNA expression to regulate angiogenesis.
      ,
      • Suárez Y.
      • Sessa W.C.
      MicroRNAs as novel regulators of angiogenesis.
      ). The initial evidence showing the importance of miRs in the regulation of angiogenesis arose from several experiments using mice with a genetically manipulated Dicer gene (
      • Kuehbacher A.
      • Urbich C.
      • Zeiher A.M.
      • Dimmeler S.
      Role of Dicer and Drosha for endothelial microRNA expression and angiogenesis.
      ,
      • Suárez Y.
      • Fernández-Hernando C.
      • Yu J.
      • Gerber S.A.
      • Harrison K.D.
      • Pober J.S.
      • Iruela-Arispe M.L.
      • Merkenschlager M.
      • Sessa W.C.
      Dicer-dependent endothelial microRNAs are necessary for postnatal angiogenesis.
      ). Dicer knock-out mice exhibit embryonic lethality because of abnormal vascular wall structure and disarrangement. Mice with vascular-selective Dicer knock-out have been reported to demonstrate a pathological phenotype showing impaired angiogenic ability, such as reduced endothelial tube formation and slowed EC migration, which may have resulted from functional alteration of some key angiogenesis-related genes. Thereafter, an increasing number of individual miRs have recently been shown to regulate angiogenesis signaling pathways, thus modulating endothelial migration, proliferation, and vascular-forming patterns (
      • Wang S.
      • Olson E.N.
      AngiomiRs: key regulators of angiogenesis.
      ,
      • Kuehbacher A.
      • Urbich C.
      • Dimmeler S.
      Targeting microRNA expression to regulate angiogenesis.
      ,
      • Suárez Y.
      • Sessa W.C.
      MicroRNAs as novel regulators of angiogenesis.
      ). In general, angiogenesis-related miRs can be classified into two groups with often opposing effects. Among them, let-7, the miR-17–92 cluster, miR-27b, miR-126, miR-130a, miR-210, miR-296, and miR-378 are found to have pro-angiogenic effects; whereas miR-221/222, miR-328, miR-92a, and miR-214 have been thought to be anti-angiogenic (
      • Suárez Y.
      • Sessa W.C.
      MicroRNAs as novel regulators of angiogenesis.
      ,
      • Zhang C.
      MicroRNAs in vascular biology and vascular disease.
      ). Of note, the effects of these miRs on endothelial cell biology and angiogenesis have been identified in both cultured endothelial cells and in ischemia-induced angiogenesis (
      • Bonauer A.
      • Carmona G.
      • Iwasaki M.
      • Mione M.
      • Koyanagi M.
      • Fischer A.
      • Burchfield J.
      • Fox H.
      • Doebele C.
      • Ohtani K.
      • Chavakis E.
      • Potente M.
      • Tjwa M.
      • Urbich C.
      • Zeiher A.M.
      • Dimmeler S.
      MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice.
      ,
      • Wang S.
      • Aurora A.B.
      • Johnson B.A.
      • Qi X.
      • McAnally J.
      • Hill J.A.
      • Richardson J.A.
      • Bassel-Duby R.
      • Olson E.N.
      The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis.
      ).
      MiR-15a and miR-16-1 are the first identified miR genes related to human cancer. Deletion of the miR-15a at chromosome 13q14 has been frequently shown in patients with chronic lymphocytic leukemia (
      • Calin G.A.
      • Dumitru C.D.
      • Shimizu M.
      • Bichi R.
      • Zupo S.
      • Noch E.
      • Aldler H.
      • Rattan S.
      • Keating M.
      • Rai K.
      • Rassenti L.
      • Kipps T.
      • Negrini M.
      • Bullrich F.
      • Croce C.M.
      Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia.
      ,
      • Calin G.A.
      • Cimmino A.
      • Fabbri M.
      • Ferracin M.
      • Wojcik S.E.
      • Shimizu M.
      • Taccioli C.
      • Zanesi N.
      • Garzon R.
      • Aqeilan R.I.
      • Alder H.
      • Volinia S.
      • Rassenti L.
      • Liu X.
      • Liu C.G.
      • Kipps T.J.
      • Negrini M.
      • Croce C.M.
      MiR-15a and miR-16-1 cluster functions in human leukemia.
      ). Classically, both miRs can directly bind to and inhibit the anti-apoptotic protein, Bcl-2, to block cell cycle progression and thus induce apoptosis of leukemic cells (
      • Calin G.A.
      • Cimmino A.
      • Fabbri M.
      • Ferracin M.
      • Wojcik S.E.
      • Shimizu M.
      • Taccioli C.
      • Zanesi N.
      • Garzon R.
      • Aqeilan R.I.
      • Alder H.
      • Volinia S.
      • Rassenti L.
      • Liu X.
      • Liu C.G.
      • Kipps T.J.
      • Negrini M.
      • Croce C.M.
      MiR-15a and miR-16-1 cluster functions in human leukemia.
      ,
      • Cimmino A.
      • Calin G.A.
      • Fabbri M.
      • Iorio M.V.
      • Ferracin M.
      • Shimizu M.
      • Wojcik S.E.
      • Aqeilan R.I.
      • Zupo S.
      • Dono M.
      • Rassenti L.
      • Alder H.
      • Volinia S.
      • Liu C.G.
      • Kipps T.J.
      • Negrini M.
      • Croce C.M.
      MiR-15 and miR-16 induce apoptosis by targeting Bcl2.
      ) and prostate cancer cells (
      • Bonci D.
      • Coppola V.
      • Musumeci M.
      • Addario A.
      • Giuffrida R.
      • Memeo L.
      • D'Urso L.
      • Pagliuca A.
      • Biffoni M.
      • Labbaye C.
      • Bartucci M.
      • Muto G.
      • Peschle C.
      • De Maria R.
      The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities.
      ). In addition to the regulation of cell death, the effects of miR-16-1 on angiogenesis and angiogenesis-related genes have been currently reported in several carcinoma cell lines. For example, Hua et al. have found that miR-16 can inhibit VEGF mRNA and protein levels in CNE cells, a human nasopharyngeal carcinoma cell line, by directly binding to the 3′-UTR of human VEGF (
      • Hua Z.
      • Lv Q.
      • Ye W.
      • Wong C.K.
      • Cai G.
      • Gu D.
      • Ji Y.
      • Zhao C.
      • Wang J.
      • Yang B.B.
      • Zhang Y.
      MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia.
      ). This finding was later confirmed in HeLa cells (
      • Karaa Z.S.
      • Iacovoni J.S.
      • Bastide A.
      • Lacazette E.
      • Touriol C.
      • Prats H.
      The VEGF IRESes are differentially susceptible to translation inhibition by miR-16.
      ). A miR-16 anti-angiogenic effect was similarly documented in multiple myeloma cells (

      Deleted in proof

      ) showing that inhibition of miR-16 results in a significant increase in cell proliferation, invasive capacity, tumor load, and angiogenesis. MiR-16 inhibition of its downstream targets, VEGF and FGFR1, is responsible for this anti-angiogenic activity. In addition, Bonci's group has also documented that miR-15 and miR-16 are down-regulated in prostate cancer-associated fibroblasts and therefore enhances tumor cell growth and progression through the lower translational suppression of FGF-1 and FGFR1 (
      • Musumeci M.
      • Coppola V.
      • Addario A.
      • Patrizii M.
      • Maugeri-Saccà M.
      • Memeo L.
      • Colarossi C.
      • Francescangeli F.
      • Biffoni M.
      • Collura D.
      • Giacobbe A.
      • D'Urso L.
      • Falchi M.
      • Venneri M.A.
      • Muto G.
      • De Maria R.
      • Bonci D.
      Control of tumor and microenvironment cross-talk by miR-15a and miR-16 in prostate cancer.
      ). However, the direct role of miR-15a on vascular endothelial cells in the setting of angiogenesis (cell-autonomous angiogenesis) has not been determined although it is extensively expressed in the vasculature.
      In this study, we found that in vitro lentivirus-mediated gain-of-miR-15a function results in significantly reduced EC tube formation, cell migration, and differentiation, whereas loss-of-miR-15a function has the opposite effect. We also found that fusion of the FGF2 or VEGF 3′-UTR fragment to the luciferase reporter vector results in a functional expression in vitro, and this activity is significantly suppressed or enhanced by lentivirus-derived exogenous miR-15a or a miR-15a inhibitor, respectively. Furthermore, up-regulation of miR-15a leads to an even stronger reduction of FGF2 and VEGF secretion and mRNA expression in endothelial cell cultures, whereas down-regulation of miR-15a increases FGF2 and VEGF translation. Of significance, to further confirm our in vitro finding of a miR-15a anti-angiogenic role, we developed an EC-selective miR-15a transgenic mouse line and demonstrated that genetic overexpression miR-15a in vivo significantly hindered the recovery of local blood flow, reduced capillary density, and inhibited FGF2 and VEGF activities in mice after hindlimb ischemia. Taken together, our experimental data extend previous findings in carcinoma cell lines and further identify that miR-15a can repress FGF2 and VEGF translation in vascular endothelial cells by directly binding to the 3′-UTRs of FGF2 and VEGF and subsequently suppress angiogenesis in vivo and in vitro following ischemic insults. To our knowledge, we are the first to document that the miR-15a has a causative role in the regulation of EC-autonomous angiogenesis.
      A most recent study from Suarez's group (
      • Chamorro-Jorganes A.
      • Araldi E.
      • Penalva L.O.
      • Sandhu D.
      • Fernández-Hernando C.
      • Suárez Y.
      MicroRNA-16 and microRNA-424 regulate cell-autonomous angiogenic functions in endothelial cells via targeting vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1.
      ) has reported that miR-16, together with miR-424, inhibits cell-autonomous angiogenesis in ECs by targeting VEGFR2, FGFR1, and VEGF. They showed that overexpression of miR-16 or miR-424 reduced proliferation, migration, and cord formation of ECs in vitro, and lentiviral overexpression of miR-16 reduced the ability of ECs to form blood vessels in vivo. In our current study, we demonstrate that miR-15a, another miR-15/16 family member, regulates cell-autonomous angiogenic activity in vascular endothelial cells in vitro and in vivo. Indeed, we have found VEGF as one of direct targets of miR-15a-mediated anti-angiogenic function, which is consistent with previous findings. However, we further identified FGF2 as a novel downstream target of miR-15a to mediate miR-15a anti-angiogenic activity in vasculature. MiR-15a and miR-16-1 appear as a cluster and share most functions because they have identical miR-binding sites and may post-transcriptionally repress the same mRNA targets. However, it is unclear whether these miRs complement each other or whether one of these miRs takes on the actions of the other completely. In addition, these particular miRs may have different biological functions or molecular mechanisms, in part by recruiting different coregulators to the miRISC (miRNA-containing RNA-induced silencing complex). Our study employed an EC-selective miR-15a transgenic mouse model; thus, we generated direct and convincing data defining the role of miR-15a in the regulation of EC-intrinsic angiogenesis in the in vivo setting. Furthermore, the utilization of LDPI to examine local blood flow recovery provides a clinically relevant approach for detecting functional recovery after ischemic insults. Certainly, we realize that other angiogenic growth factors may also be important mediators responsible for miR-15a anti-angiogenic activity. Further studies using EC-specific miR-15a transgenic and knock-out animal models, combined with proteomics approaches, are necessary to further validate targets of the miR-15a.
      Angiogenesis is a normal physiological process in tissue growth and development that may also occur as a natural defense response against ischemic cerebrovascular and cardiovascular diseases, such as ischemic stroke and myocardial infarction (
      • Yanagisawa-Miwa A.
      • Uchida Y.
      • Nakamura F.
      • Tomaru T.
      • Kido H.
      • Kamijo T.
      • Sugimoto T.
      • Kaji K.
      • Utsuyama M.
      • Kurashima C.
      Salvage of infarcted myocardium by angiogenic action of basic fibroblast growth factor.
      ,
      • Baumgartner I.
      • Pieczek A.
      • Manor O.
      • Blair R.
      • Kearney M.
      • Walsh K.
      • Isner J.M.
      Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia.
      ,
      • Zhang Z.G.
      • Zhang L.
      • Jiang Q.
      • Zhang R.
      • Davies K.
      • Powers C.
      • Bruggen N.
      • Chopp M.
      VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain.
      ). Extensive studies have shown that post-ischemic angiogenesis plays a crucial role in the recovery of blood flow in affected tissue (
      • Hayashi T.
      • Deguchi K.
      • Nagotani S.
      • Zhang H.
      • Sehara Y.
      • Tsuchiya A.
      • Abe K.
      Cerebral ischemia and angiogenesis.
      ,
      • Arai K.
      • Jin G.
      • Navaratna D.
      • Lo E.H.
      Brain angiogenesis in developmental and pathological processes: neurovascular injury and angiogenic recovery after stroke.
      ,
      • Zhang Z.G.
      • Chopp M.
      Neurorestorative therapies for stroke: underlying mechanisms and translation to the clinic.
      ). Thus, angiogenic vessels in the ischemic boundary zone may contribute to recovery of tissue at risk by restoring metabolism in surviving neurons or cardiomyocytes as well as provide the essential trophic support to newly generated neuronal or cardiac cells. Indeed, increased microvessel density has been observed in the penumbral areas, and the number of new angiogenic vessels is correlated with longer survival in both stroke and heart attack patients (
      • Krupiński J.
      • Kaluza J.
      • Kumar P.
      • Kumar S.
      • Wang J.M.
      Some remarks on the growth-rate and angiogenesis of microvessels in ischemic stroke: morphometric and immunocytochemical studies.
      ), suggesting that active angiogenesis may be beneficial for neurological or cardiac functional recovery. Using state-of-the-art molecular techniques and genetically manipulated animal models, our present data show miR-15a regulation of post-ischemic angiogenesis via inhibition of FGF2 and VEGF signaling pathways. These findings will lead us to better understand the mechanisms of miR-mediated regulation of angiogenesis and establish a human translational basis for the development of novel restorative therapy to enhance functional recovery following stroke and myocardial infarction.

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