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

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.

Highlights

Atherosclerosis is a chronic inflammatory disease characterized by formation of lipid-rich plaques in the arterial wall
We have shown previously that expression of miR-494-3p upregulates in specific cell types and even whole tissues after treatment with 3GA-494,26 likely via an autoregulatory mechanism. 3GA-494treated M2 macrophages increased miR-494-3p secretion via extracellular vesicles (EVs) (p = 0.01), whereas in EVs from M0 and M1 macrophages no differences were observed in miR-494-3p secretion between 3GA-ctrl and 3GA-494, except for a general increased secretion by M1 macrophages treated with 3GA-494 for both miR-494-3p and U6 (p = 0.1 and p = 0.03, respectively; Figures S1A–S1C)
transducing b-like 1 X-linked (TBL1X) and transcription factor 7-like 2 (TCF7L2) were upregulated, significantly or on trend (p = 0.05 [Figure 4G] and p = 0.1 [Figure 4H], respectively), in 3GA-494 M1 macrophages compared with 3GA-ctrl

Summary

Introduction

Atherosclerosis is a chronic inflammatory disease characterized by formation of lipid-rich plaques in the arterial wall. Vulnerable plaques may eventually rupture and result in a cardiovascular event, such as myocardial infarction or ischemic stroke.[1] Macrophages are cells of the innate immune system that play a central role in atherosclerosis. Macrophages can polarize in response to signals from cytokines and chemokines, and from bioactive lipids such as cholesterol and oxidized lowdensity lipoproteins (LDLs).[2,3,4,5,6]. M1 macrophages polarize in response to interferon-g (IFNg) and lipopolysaccharide (LPS). Activated M2 macrophages polarize in response to interleukin-4 (IL-4) and IL-13. M2 macrophages induce an anti-inflammatory response whereby they counteract activation of the immune system.[7,8,9,10] The in vitro M1/M2 classification, is an oversimplification compared with the in vivo situation. Macrophage plasticity is highly dynamic, and macrophages continuously adapt to the signals they receive from their environment.[3,9,10,11]

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