miR-15a/-16 Inhibit Angiogenesis by Targeting the Tie2 Coding Sequence: Therapeutic Potential of a miR-15a/16 Decoy System in Limb Ischemia

Marie Besnier, Saran Shantikumar, Maryam Anwar, Parul Dixit, Aranzazu Chamorro-Jorganes, Walid Sweaad, Graciela Sala-Newby, Paolo Madeddu, Anita C. Thomas, Lynsey Howard, Sobia Mushtaq, Enrico Petretto, Andrea Caporali, Costanza Emanueli*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)

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Abstract

MicroRNA-15a (miR-15a) and miR-16, which are transcribed from the miR-15a/miR-16-1 cluster, inhibit post-ischemic angiogenesis. MicroRNA (miRNA) binding to mRNA coding sequences (CDSs) is a newly emerging mechanism of gene expression regulation. We aimed to (1) identify new mediators of the anti-angiogenic action of miR-15a and -16, (2) develop an adenovirus (Ad)-based miR-15a/16 decoy system carrying a luciferase reporter (Luc) to both sense and inhibit miR-15a/16 activity, and (3) investigate Ad.Luc-Decoy-15a/16 therapeutic potential in a mouse limb ischemia (LI) model. LI increased miR-15a and -16 expression in mouse muscular endothelial cells (ECs). The miRNAs also increased in cultured human umbilical vein ECs (HUVECs) exposed to serum starvation, but not hypoxia. Using bioinformatic tools and luciferase activity assays, we characterized miR-15a and -16 binding to Tie2 CDS. In HUVECs, miR-15a or -16 overexpression reduced Tie2 at the protein, but not the mRNA, level. Conversely, miR-15a or -16 inhibition improved angiogenesis in a Tie2-dependent manner. Local Ad.Luc-Decoy-15a/16 delivery increased Tie2 levels in ischemic skeletal muscle and improved post-LI angiogenesis and perfusion recovery, with reduced toe necrosis. Bioluminescent imaging (in vivo imaging system [IVIS]) provided evidence that the Ad.Luc-Decoy-15a/16 system responds to miR-15a/16 increases. In conclusion, we have provided novel mechanistic evidence of the therapeutic potential of local miR-15a/16 inhibition in LI.

Original languageEnglish
Pages (from-to)49-62
Number of pages14
JournalMolecular Therapy - Nucleic Acids
Volume17
Early online date17 May 2019
DOIs
Publication statusPublished - 6 Sep 2019

Keywords

  • angiogenesis
  • angiopoietins
  • ischemia
  • IVIS
  • microRNAs
  • miR decoy
  • miR-15a
  • miR-16a
  • mouse ischemia model
  • Tie2

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    Besnier, M., Shantikumar, S., Anwar, M., Dixit, P., Chamorro-Jorganes, A., Sweaad, W., Sala-Newby, G., Madeddu, P., Thomas, A. C., Howard, L., Mushtaq, S., Petretto, E., Caporali, A., & Emanueli, C. (2019). miR-15a/-16 Inhibit Angiogenesis by Targeting the Tie2 Coding Sequence: Therapeutic Potential of a miR-15a/16 Decoy System in Limb Ischemia. Molecular Therapy - Nucleic Acids, 17, 49-62. https://doi.org/10.1016/j.omtn.2019.05.002