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Open AccessArticle

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

1
Bristol Heart Institute, School of Clinical Sciences, University of Bristol, Bristol BS2 8HW, UK
2
National Heart and Lung Institute, Imperial College London, London SW3 6LY, UK
3
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; https://doi.org/10.3390/ijms21051733
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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