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

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 cardiovascu...

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Autores principales: Marchetti, Micol, Meloni, Marco, Anwar, Maryam, Al-Haj-Zen, Ayman, Sala-Newby, Graciela, Slater, Sadie, Ford, Kerrie, Caporali, Andrea, Emanueli, Costanza
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084374/
https://www.ncbi.nlm.nih.gov/pubmed/32138369
http://dx.doi.org/10.3390/ijms21051733
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author Marchetti, Micol
Meloni, Marco
Anwar, Maryam
Al-Haj-Zen, Ayman
Sala-Newby, Graciela
Slater, Sadie
Ford, Kerrie
Caporali, Andrea
Emanueli, Costanza
author_facet Marchetti, Micol
Meloni, Marco
Anwar, Maryam
Al-Haj-Zen, Ayman
Sala-Newby, Graciela
Slater, Sadie
Ford, Kerrie
Caporali, Andrea
Emanueli, Costanza
author_sort Marchetti, Micol
collection PubMed
description 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.
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spelling pubmed-70843742020-03-24 MicroRNA-24-3p Targets Notch and Other Vascular Morphogens to Regulate Post-ischemic Microvascular Responses in Limb Muscles Marchetti, Micol Meloni, Marco Anwar, Maryam Al-Haj-Zen, Ayman Sala-Newby, Graciela Slater, Sadie Ford, Kerrie Caporali, Andrea Emanueli, Costanza Int J Mol Sci Article 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. MDPI 2020-03-03 /pmc/articles/PMC7084374/ /pubmed/32138369 http://dx.doi.org/10.3390/ijms21051733 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Marchetti, Micol
Meloni, Marco
Anwar, Maryam
Al-Haj-Zen, Ayman
Sala-Newby, Graciela
Slater, Sadie
Ford, Kerrie
Caporali, Andrea
Emanueli, Costanza
MicroRNA-24-3p Targets Notch and Other Vascular Morphogens to Regulate Post-ischemic Microvascular Responses in Limb Muscles
title MicroRNA-24-3p Targets Notch and Other Vascular Morphogens to Regulate Post-ischemic Microvascular Responses in Limb Muscles
title_full MicroRNA-24-3p Targets Notch and Other Vascular Morphogens to Regulate Post-ischemic Microvascular Responses in Limb Muscles
title_fullStr MicroRNA-24-3p Targets Notch and Other Vascular Morphogens to Regulate Post-ischemic Microvascular Responses in Limb Muscles
title_full_unstemmed MicroRNA-24-3p Targets Notch and Other Vascular Morphogens to Regulate Post-ischemic Microvascular Responses in Limb Muscles
title_short MicroRNA-24-3p Targets Notch and Other Vascular Morphogens to Regulate Post-ischemic Microvascular Responses in Limb Muscles
title_sort microrna-24-3p targets notch and other vascular morphogens to regulate post-ischemic microvascular responses in limb muscles
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084374/
https://www.ncbi.nlm.nih.gov/pubmed/32138369
http://dx.doi.org/10.3390/ijms21051733
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