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. 2019 May 17;17:49–62. doi: 10.1016/j.omtn.2019.05.002

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© 2019 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Inhibition of miR-15a/16 Improves Blood Flow (BF) Recovery, Post-ischemic Neovascularization, and Toe Survival following Limb Ischemia

Limb ischemia was induced in mice by femoral artery ligation, and Ad.Luc or Ad.Luc-Decoy was injected into the ischemic adductor muscle immediately after surgery. (A and B) Line graph shows the time course of post-ischemic BF recovery (calculated as the ratio between the BF of the ischemic foot and the contralateral foot) in mice injected with Ad.Luc or Ad.Luc-Decoy. Representative color laser Doppler images taken at days 0, 7, 14, and 21 post-ischemia are provided. Yellow frames show the ischemic foot analyzed. Data are presented as mean ± SEM (n ≥ 10 per group). *p < 0.05, **p < 0.01 versus time-matched Ad.Luc condition. (C) Immunohistological analysis and representative microphotographs showing the capillary density in mice injected with Ad.Luc or Ad.Luc-Decoy 21 days after LI and in contralateral (control) muscle of mice injected with Ad.Luc (n = 5 per group), assessed by immunolabelling of CD31 as a marker of endothelial cells. Scale bar, 50 μm. (D) Immunohistological analysis and representative microphotographs showing arteriolar density in mice injected with Ad.Luc or Ad.Luc-Decoy (n = 11 or 8 mice per group, respectively) after limb ischemia, assessed by immunolabelling of α-smooth muscle actin (αSMA) as a marker of vascular smooth muscle cells. Arterioles were quantified according to vessel diameter (<20 μm or ≥20 μm). Data are presented as mean + SEM. Scale bar, 20 μm. (E) Cumulative proportion of toe survival over 21 days of follow-up after ischemia in mice injected with Ad.Luc or Ad.Luc-Decoy (n ≥ 10 mice per group). p = 0.0013, log rank test. (F) Tie2 expression in ischemic adductor muscles, 3 days after limb ischemia surgery and injection with either Ad.Luc or Ad.Luc-Decoy virus, was assessed at mRNA and protein levels by RT-PCR and western blot, respectively. Results were normalized to GAPDH for mRNA analysis (n = 6) or to α/β-tubulin for protein analysis (n = 9) and presented as mean ± SEM. *p < 0.05 versus Ad.Luc group.