Macrophage-derived HB-EGF activates EGFR signaling to impair atherogenic macrophage–vascular smooth muscle cell crosstalk in ASCVD

Background

Atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of death worldwide. Despite advances in lipid-lowering therapies, substantial residual cardiovascular risk persists, highlighting the need to identify non-lipid-mediated mechanisms of disease susceptibility. Inflammation plays a central role in plaque initiation and progression; however, the specific pathways driving this process remain incompletely understood.

Purpose

We sought to identify immune pathways associated with ASCVD in severe hypercholesterolemia and to define their mechanistic contribution to vascular inflammation.

Methods

Fifteen individuals ≥60 years old with severe hypercholesterolemia (LDL-C ≥175 mg/dL or ApoB ≥129 mg/dL) were stratified by the presence or absence of ASCVD (ASCVD group, n=7; non-ASCVD group, n=8). Whole-genome sequencing and single-cell RNA sequencing of peripheral blood mononuclear cells were performed to calculate polygenic risk scores (PRS) and characterise immune transcriptional profiles across groups. Serum samples from the same individuals were analyzed by ELISA to quantify circulating protein levels. Mechanistic studies were conducted in vitro using THP1-derived macrophages and human aortic vascular smooth muscle cells (AoVSMCs).

Results

Whole-genome sequencing revealed no differences in the PRS between ASCVD and non-ASCVD individuals. Single-cell RNA sequencing identified CD14⁺ monocytes as the cell population with the greatest number of differentially expressed genes between ASCVD and non-ASCVD groups (FDR<0.05). Pathway enrichment analysis demonstrated epidermal growth factor receptor (EGFR) signaling as the top-enriched pathway in ASCVD (normalized enrichment score=2.15). Within this pathway, the ligands amphiregulin (AREG, padj=1.45E-7) and heparin-binding-EGF-like growth factor (HB-EGF, padj=0.007) were significantly upregulated in ASCVD subjects.

Circulating HB-EGF levels were significantly higher in ASCVD individuals (t-test, p<0.05), whereas AREG levels did not differ between groups (t-test, p>0.05). Functional studies showed that recombinant HB-EGF reduced oxidized LDL uptake without affecting native LDL uptake. In co-culture experiments, macrophages promoted AoVSMCs proliferation (n=10, 1-way ANOVA, p<0.0001), an effect attenuated by HB-EGF silencing (n=10, 1-way ANOVA, p<0.05).

AoVSMCs treated with HB-EGF demonstrated decreased expression of the contractile marker calponin-1 (CNN1, n=6, t-test, p< 0.001) and increased expression of SRY-box transcription factor 9 (SOX9, n=6, t-test, p< 0.001) and a trend towards increased expression of the proliferation marker Ki-67 (MKI67, n=6, t-test, p=0.054), consistent with a shift towards a synthetic and proliferative phenotype when compared with vehicle-treated cells.

Conclusions

These findings implicate the HB-EGF-EGFR signaling axis in residual inflammatory risk in ASCVD and support HB-EGF as a potential therapeutic target.Proposed mechanism HB-EGF in atherosclerosis