Forced expression of microRNA-146b reduces TRAF6-dependent inflammation and improves ischemia-induced neovascularization in hypercholesterolemic conditions

Atherosclerosis. 2019 Oct:289:73-84. doi: 10.1016/j.atherosclerosis.2019.08.010. Epub 2019 Aug 23.

Abstract

Background and aims: MicroRNA (miR)-146 is a key regulator of inflammation, endothelial activation and atherosclerosis. This study sought to define its potential role for the modulation of ischemia-induced neovascularization in atherosclerotic conditions.

Methods: Next generation sequencing and qRT-PCR analyses were used to compare microRNA expression in the ischemic muscles of hypercholesterolemic ApoE-deficient (ApoE-/-) mice vs. wild type mice, and in HUVECs exposed or not to oxLDL. Neovascularization was investigated in a mouse model of hindlimb ischemia and the functional activities of HUVECs and pro-angiogenic cells (PACs) were assessed in vitro.

Results: We found that miR-146b (but not miR-146a) is significantly reduced in the ischemic muscles of ApoE-/- mice, and in HUVECs exposed to oxLDL. Inhibition of miR-146b reduces angiogenesis in vitro, whereas forced expression of miR-146b rescues oxLDL-mediated impairment of endothelial cell proliferation and tube formation. Mechanistically, miR146b directly targets tumor necrosis factor-alpha (TNFa) Receptor Associated Factor 6 (TRAF6) to inhibit inflammation. We found that hypercholesterolemia and oxLDL exposure are associated with higher levels of TRAF6, and increased expression of TNFa. However, forced expression of miR-146b in high cholesterol conditions reduces the expression of these inflammatory factors. In vivo, intramuscular injection of miR-146b mimic reduces ischemic damages and restores blood flow recuperation and capillary density in the ischemic muscles of ApoE-/- mice. Treatment with miR-146b also increases the number and functional activities of pro-angiogenic cells (PACs).

Conclusions: Hypercholesterolemia is associated with reduced expression of miR-146b, which increases TRAF6-dependent inflammation and is associated with poor neovascularization in response to ischemia. Forced expression of miR-146b using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in atherosclerotic conditions.

Keywords: Angiogenesis; Hypercholesterolemia; Inflammation; Neovascularization; microRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blood Flow Velocity
  • Cell Movement
  • Cell Proliferation
  • Hindlimb / metabolism
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Hypercholesterolemia / metabolism*
  • Inflammation / metabolism*
  • Intracellular Signaling Peptides and Proteins / genetics
  • Ischemia / physiopathology
  • Lipoproteins, LDL / metabolism
  • Mice
  • Mice, Knockout, ApoE
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism
  • Neovascularization, Pathologic / metabolism*
  • Reactive Oxygen Species / metabolism
  • Sequence Analysis, RNA
  • THP-1 Cells
  • TNF Receptor-Associated Factor 6 / genetics*

Substances

  • Intracellular Signaling Peptides and Proteins
  • Lipoproteins, LDL
  • MIRN146 microRNA, human
  • MicroRNAs
  • Mirn146 microRNA, mouse
  • Reactive Oxygen Species
  • TNF Receptor-Associated Factor 6
  • TRAF6 protein, mouse
  • Tifab protein, human
  • oxidized low density lipoprotein