Targeting the miR‑342‑5p/Wnt3a Axis Stabilizes Atherosclerotic Plaques and Suppresses Angiogenesis in ApoE−/− Mice

Abstract

MicroRNA‑342‑5p (miR‑342‑5p) has been implicated in atherosclerosis (AS), yet its mechanistic role remains unclear. In this study, we investigated how miR‑342‑5p regulates the Wnt3a signalling pathway and influences plaque vulnerability and neovascularisation in an ApoE‑/‑ mouse model of AS. Twenty‑four‑week‑old male ApoE‑/‑ mice were fed a high‑fat diet for 16 weeks to induce vulnerable plaques. miR‑342‑5p and Wnt3a expression were quantified by RT‑qPCR and Western blotting. Functional effects of miR‑342‑5p inhibition (antagomir) and Wnt3a over‑expression (oe‑Wnt3a vector) were assessed on serum lipids, inflammatory and oxidative‑stress cytokines, plaque morphology, and micro‑vessel density (MVD). Results revealed that miR‑342‑5p was up‑regulated while Wnt3a and β‑catenin were down‑regulated in AS aortae, and miR‑342‑5p directly targeted the 3′‑UTR of Wnt3a. Antagonising miR‑342‑5p or over‑expressing Wnt3a lowered total cholesterol, triglycerides, LDL‑C, and pro‑inflammatory cytokines (IL‑5, IL‑12p70, IFN‑γ, TNF‑α), increased HDL‑C and SOD activity, reduced lipid core area, increased collagen content, decreased macrophage infiltration, and lowered MVD. In contrast, miR‑342‑5p over‑expression exacerbated all these parameters. Thus, miR‑342‑5p promotes plaque instability and angiogenesis by repressing Wnt3a; restoring Wnt3a mitigates these effects, suggesting a therapeutic target for AS.

Introduction

Atherosclerosis (AS) remains the leading cause of cardiovascular morbidity and mortality worldwide. Plaque destabilisation—characterised by lipid‑rich necrotic cores, thin fibrous caps, macrophage infiltration, and reduced collagen—leads to rupture and acute events. While lipid‑lowering therapies have improved outcomes, many patients continue to experience cardiovascular events, underscoring the need for novel molecular interventions.

MicroRNAs (miRNAs) are short non‑coding RNAs that post‑transcriptionally regulate multiple target genes. miR‑342‑5p, located within the 14q32 miRNA cluster, has been reported to influence angiogenesis via TGF‑β and VEGF pathways and to modulate inflammatory responses in vascular disease. However, its specific role in AS and interaction with key signalling cascades remain incompletely defined.

The Wnt/β‑catenin pathway is critical for vascular cell homeostasis, influencing smooth‑muscle cell proliferation, endothelial integrity, and plaque composition. Prior work indicates that miR‑342‑5p negatively regulates Wnt3a in other disease contexts, suggesting a potential regulatory axis in AS.

Here we elucidate the miR‑342‑5p/Wnt3a interaction in AS and evaluate its impact on plaque stability and angiogenesis in ApoE‑/‑ mice.

Materials and Methods

Animals and Ethics

Male ApoE‑/‑ and C57BL/6J mice (8 weeks old) were housed under a 12 h light/12 h dark cycle and fed standard chow or a high‑fat diet (20% fat, 0.25% cholesterol) for 16 weeks. All procedures adhered to NIH guidelines and were approved by the Institutional Animal Care and Use Committee of Qinghai Provincial People’s Hospital (ethical number 201870726).

Experimental Design

ApoE‑/‑ mice were randomly allocated to six groups (n = 12 each): AS, NC (saline), miR‑342‑5p agomir, miR‑342‑5p antagomir, oe‑Wnt3a, and miR‑342‑5p agomir + oe‑Wnt3a. Treatments (40 mg/kg, tail vein) were administered biweekly for 8 weeks. C57BL/6J mice served as normal controls.

Biochemical Analyses

Serum total cholesterol (TC), triglycerides (TG), LDL‑C, HDL‑C, malondialdehyde (MDA), and superoxide dismutase (SOD) were measured using commercial kits (NanJing JianCheng Bioengineering Institute). Cytokines (IL‑5, IL‑12p70, IFN‑γ, TNF‑α) were quantified by ELISA (MultiSciences).

Tissue Processing

After 12‑h fasting, mice were euthanised; aortae were harvested, washed in RNA‑free PBS, and divided for histology (HE, Oil Red O, Sirius Red) or molecular assays (RT‑qPCR, Western blot). Samples for histology were embedded in paraffin, sectioned (4 µm), and stained according to standard protocols.

Gene Expression and Protein Analysis

RT‑qPCR quantified miR‑342‑5p, Wnt3a, β‑catenin, and reference genes (U6, β‑actin) using the 2^−ΔΔCt method. Western blotting assessed Wnt3a, β‑catenin, CD34, α‑SMA, and β‑actin, with ImageJ densitometry.

Immunohistochemistry

Sections were immunostained for MOMA‑2 (macrophages), α‑SMA (smooth‑muscle cells), and CD34 (endothelial cells). Staining intensity was quantified via Image Pro Plus 6.0, and the plaque vulnerability index was calculated as (macrophages + lipid %) / (smooth‑muscle cells + collagen %). MVD was determined by counting CD34⁺ micro‑vessels/mm².

Luciferase Reporter Assay

The Wnt3a 3′‑UTR (wild‑type and mutant) was cloned into GP‑miRGLO. Mouse aortic endothelial cells were co‑transfected with the reporter and varying concentrations of miR‑342‑5p agomir; luciferase activity was measured after 48 h.

Statistical Analysis

Data were presented as mean ± SD. Two‑group comparisons used unpaired t‑tests; multiple groups used one‑way ANOVA with Tukey post‑hoc tests. Significance was set at *p* < 0.05.

Results

miR‑342‑5p Is Up‑regulated and Wnt3a Is Down‑regulated in AS Aortae

RT‑qPCR showed a significant increase in miR‑342‑5p (≈3.5‑fold, *p* < 0.01) and a concurrent decrease in Wnt3a (≈0.4‑fold, *p* < 0.01) in AS mice versus normals. Western blot confirmed reduced Wnt3a and β‑catenin protein levels. Luciferase assays demonstrated that miR‑342‑5p directly binds the Wnt3a 3′‑UTR, reducing reporter activity by up to 64% at 100 pM, an effect abrogated by mutating the binding site.

miR‑342‑5p Antagomir or Wnt3a Over‑expression Ameliorates Lipid Profiles

miR‑342‑5p antagomir and oe‑Wnt3a markedly lowered TC, TG, and LDL‑C (≈30–40% reduction, *p* < 0.01) and raised HDL‑C (≈20% increase, *p* < 0.05) relative to AS and NC groups. miR‑342‑5p agomir exacerbated dyslipidaemia. Over‑expression of Wnt3a rescued the adverse effects of miR‑342‑5p over‑expression.

Inflammatory and Oxidative Stress Markers are Suppressed by miR‑342‑5p Inhibition

Serum IL‑5, IL‑12p70, IFN‑γ, TNF‑α, and MDA were elevated in AS mice (*p* < 0.01). miR‑342‑5p antagomir or oe‑Wnt3a reduced these cytokines and MDA by ~40% and increased SOD activity by ~35% (*p* < 0.01). miR‑342‑5p agomir further intensified inflammatory and oxidative markers.

Plaque Morphology Improves with miR‑342‑5p Inhibition or Wnt3a Over‑expression

HE staining revealed that miR‑342‑5p antagomir and oe‑Wnt3a groups exhibited smaller plaque areas, thicker fibrous caps, reduced lipid cores, and increased collagen content (Sirius Red) compared with AS and NC groups (*p* < 0.01). Oil Red O quantification confirmed a ~30% decrease in lipid content in treated groups. The plaque vulnerability index decreased by 45% in antagomir and oe‑Wnt3a groups, whereas miR‑342‑5p agomir increased it by 60% (*p* < 0.01).

Macrophage Accumulation and Smooth‑Muscle Cell Presence Are Modulated

Immunohistochemistry showed that miR‑342‑5p antagomir and oe‑Wnt3a reduced MOMA‑2⁺ macrophage percentages by ~50% and increased α‑SMA⁺ smooth‑muscle cells by ~30% relative to AS and NC groups (*p* < 0.01). The combined miR‑342‑5p agomir + oe‑Wnt3a treatment restored macrophage and smooth‑muscle cell levels toward baseline.

Angiogenesis is Attenuated by Targeting the miR‑342‑5p/Wnt3a Axis

CD34 immunostaining and Western blot demonstrated that miR‑342‑5p antagomir and oe‑Wnt3a reduced MVD by ~55% compared with AS mice (*p* < 0.01). miR‑342‑5p agomir increased MVD by ~70% (*p* < 0.01). The combined treatment moderated angiogenic responses.

Discussion

Our findings reveal a previously uncharacterised miR‑342‑5p/Wnt3a axis that governs lipid metabolism, inflammation, oxidative stress, plaque composition, and neovascularisation in atherosclerosis. miR‑342‑5p up‑regulation in AS aortae directly suppresses Wnt3a, thereby dampening β‑catenin signalling and promoting a vulnerable plaque phenotype. Conversely, inhibiting miR‑342‑5p or restoring Wnt3a re‑establishes protective lipid profiles, reduces pro‑inflammatory cytokines, enhances antioxidant defenses, stabilises plaque architecture, and curbs pathological angiogenesis.

These results align with reports of miR‑342‑5p involvement in macrophage activation and inflammation, and with the established role of Wnt signalling in vascular biology. The therapeutic potential of modulating this axis is underscored by the robust reversal of AS hallmarks observed in vivo.

Conclusion

This study is the first to demonstrate that miR‑342‑5p promotes plaque vulnerability and angiogenesis by targeting Wnt3a in ApoE‑/‑ mice. Pharmacological inhibition of miR‑342‑5p or up‑regulation of Wnt3a stabilises atherosclerotic plaques and may represent a promising strategy for AS treatment.

Availability of Data and Materials

All data generated during this study are included in the article and its supplementary materials. Further inquiries can be directed to the corresponding author.

Abbreviations

miR‑342‑5p

MicroRNA‑342‑5p

A

Atherosclerosis

α‑SMA

α‑Smooth muscle actin

MVD

Micro‑vessel density

miRNA

MicroRNA

oe

Over‑expression

NC

Negative control

PBS

Phosphate‑buffered saline

TC

Total cholesterol

TG

Triglyceride

LDL‑C

Low‑density lipoprotein cholesterol

HDL‑C

High‑density lipoprotein cholesterol

ELISA

Enzyme‑linked immunosorbent assay

IL

Interleukin

TNF‑α

Tumor necrosis factor alpha

IFN

Interferon

MDA

Malondialdehyde

SOD

Superoxide dismutase

RT‑qPCR

Reverse transcription quantitative polymerase chain reaction