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Open AccessArticle

MicroRNA-24-3p Targets Notch and Other Vascular Morphogens to Regulate Post-ischemic Microvascular Responses in Limb Muscles

Bristol Heart Institute, School of Clinical Sciences, University of Bristol, Bristol BS2 8HW, UK
National Heart and Lung Institute, Imperial College London, London SW3 6LY, UK
BHF Centre for Cardiovascular Science, University of Edinburgh, Edinburgh EH164TJ, UK
Author to whom correspondence should be addressed.
Current Affiliation: Evox Therapeutics Ltd., Oxford OX4 4HG, UK.
Current Affiliation: Cardiovascular Research Unit, Sanofi R&D, Chilly-Mazarin 91380, France.
Current Affiliation: College of Health and Life Sciences, Hamad Bin Khalifa University, Education City, Doha P.O. Box: 34110, Qatar.
Current affiliations: Health Economics and Outcomes Research Ltd., Cardiff CF23 8RB, UK.
Int. J. Mol. Sci. 2020, 21(5), 1733;
Received: 28 January 2020 / Revised: 19 February 2020 / Accepted: 21 February 2020 / Published: 3 March 2020
(This article belongs to the Special Issue RNAs in Cardiovascular Diseases-CardioRNA EU COST Action)
MicroRNAs (miRs) regulate complex processes, including angiogenesis, by targeting multiple mRNAs. miR-24-3p-3p directly represses eNOS, GATA2, and PAK4 in endothelial cells (ECs), thus inhibiting angiogenesis during development and in the infarcted heart. miR-24-3p is widely expressed in cardiovascular cells, suggesting that it could additionally regulate angiogenesis by acting on vascular mural cells. Here, we have investigated: (1) new miR-24-3p targets; (2) the expression and the function of miR-24-3p in human vascular ECs; (3) the impact of miR-24-3p inhibition in the angiogenesis reparative response to limb ischemia in mice. Using bioinformatics target prediction platforms and 3′-UTR luciferase assays, we newly identified Notch1 and its Delta-like ligand 1 (Dll1) to be directly targeted by miR-24-3p. miR-24-3p was expressed in human ECs and pericytes cultured under normal conditions. Exposure to hypoxia increased miR-24-3p in ECs but not in pericytes. Transfection with a miR-24-3p precursor (pre-miR-24-3p) increased miR-24-3p expression in ECs, reducing the cell survival, proliferation, and angiogenic capacity. Opposite effects were caused by miR-24-3p inhibition. The anti-angiogenic action of miR-24-3p overexpression could be prevented by simultaneous adenovirus (Ad)-mediated delivery of constitutively active Notch intracellular domain (NICD) into cultured ECs. We next demonstrated that reduced Notch signalling contributes to the anti-angiogenic effect of miR-24-3p in vitro. In a mouse unilateral limb ischemia model, local miR-24-3p inhibition (by adenovirus-mediated miR-24-3p decoy delivery) restored endothelial Notch signalling and increased capillary density. However, the new vessels appeared disorganised and twisted, worsening post-ischemic blood perfusion recovery. To better understand the underpinning mechanisms, we widened the search for miR-24-3p target genes, identifying several contributors to vascular morphogenesis, such as several members of the Wingless (Wnt) signalling pathway, β-catenin signalling components, and VE-cadherin, which synergise to regulate angiogenesis, pericytes recruitment to neoformed capillaries, maturation, and stabilization of newly formed vessels. Among those, we next focussed on β-catenin to demonstrate that miR-24-3p inhibition reduces β-catenin expression in hypoxic ECs, which is accompanied by reduced adhesion of pericytes to ECs. In summary, miR-24-3p differentially targets several angiogenesis modulators and contributes to autonomous and non-autonomous EC crosstalk. In ischemic limbs, miR-24-3p inhibition increases the production of dysfunctional microvessels, impairing perfusion. Caution should be observed in therapeutic targeting of miR-24-3p. View Full-Text
Keywords: miR-24-3p; angiogenesis; endothelial cells; Notch; β-catenin; limb ischemia miR-24-3p; angiogenesis; endothelial cells; Notch; β-catenin; limb ischemia
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Marchetti, M.; Meloni, M.; Anwar, M.; Al-Haj-Zen, A.; Sala-Newby, G.; Slater, S.; Ford, K.; Caporali, A.; Emanueli, C. MicroRNA-24-3p Targets Notch and Other Vascular Morphogens to Regulate Post-ischemic Microvascular Responses in Limb Muscles. Int. J. Mol. Sci. 2020, 21, 1733.

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