Inhibition of microRNA-494-3p activates Wnt signaling and reduces proinflammatory macrophage polarization in atherosclerosis

Eva van Ingen; Amanda C Foks; Tamar Woudenberg; M Leontien van der Bent; Alwin de Jong; Philipp J Hohensinner; Johann Wojta; Ilze Bot; Paul H A Quax; Anne Yaël Nossent

doi:10.1016/j.omtn.2021.10.027

Inhibition of microRNA-494-3p activates Wnt signaling and reduces proinflammatory macrophage polarization in atherosclerosis

Mol Ther Nucleic Acids. 2021 Nov 4:26:1228-1239. doi: 10.1016/j.omtn.2021.10.027. eCollection 2021 Dec 3.

Authors

Eva van Ingen^{1

2

3}, Amanda C Foks³, Tamar Woudenberg^{1

2}, M Leontien van der Bent^{1

2}, Alwin de Jong^{1

2}, Philipp J Hohensinner⁴, Johann Wojta^{4

5}, Ilze Bot³, Paul H A Quax^{1

2}, Anne Yaël Nossent^{1

2

4

6}

Affiliations

¹ Department of Surgery, Leiden University Medical Center, 2300 RC Leiden, the Netherlands.
² Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 2300 RC Leiden, the Netherlands.
³ Division of BioTherapeutics, LACDR, Leiden University, 2333 CC Leiden, The Netherlands.
⁴ Department of Internal Medicine II, Medical, University of Vienna, 1090 Vienna, Austria.
⁵ Ludwig Boltzmann Institute for Cardiovascular Research, 1090 Vienna, Austria.
⁶ Department of Laboratory Medicine, University of Vienna, 1090 Vienna, Austria.

Abstract

We have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in early and established plaques, with reduced macrophage content in established plaques. Within the plaque, different subtypes of macrophages are present. Here, we aimed to investigate whether miR-494-3p directly influences macrophage polarization and activation. Human macrophages were polarized into either proinflammatory M1 or anti-inflammatory M2 macrophages and simultaneously treated with 3GA-494 or a control antisense (3GA-ctrl). We show that 3GA-494 treatment inhibited miR-494-3p in M1 macrophages and dampened M1 polarization, while in M2 macrophages miR-494-3p expression was induced and M2 polarization enhanced. The proinflammatory marker CCR2 was reduced in 3GA-494-treated atherosclerosis-prone mice. Pathway enrichment analysis predicted an overlap between miR-494-3p target genes in macrophage polarization and Wnt signaling. We demonstrate that miR-494-3p regulates expression levels of multiple Wnt signaling components, such as LRP6 and TBL1X. Wnt signaling appears activated upon treatment with 3GA-494, both in cultured M1 macrophages and in plaques of hypercholesterolemic mice. Taken together, 3GA-494 treatment dampened M1 polarization, at least in part via activated Wnt signaling, while M2 polarization was enhanced, which is both favorable in reducing atherosclerotic plaque formation and increasing plaque stability.

Keywords: 14q32 locus; DLK1-DIO3 locus; Wnt signaling; antisense oligonucleotides; atherosclerosis; cardiovascular disease; inflammation; macrophage polarization; microRNA; microRNA-494-3p.