Genetic depletion of smooth muscle cells (SMCs) from atheroprogression fundamentally reshapes murine brachiocephalic artery (BCA) lesions

Background

Smooth muscle cells (SMCs) are key contributors to atherosclerosis, exerting both protective and detrimental effects within plaques. Although their heterogeneity is well described, their overall impact on plaque progression and clinical outcomes remains unclear. Complete SMC ablation would clarify their net function, but systemic depletion is lethal and has precluded such studies.

We developed a model enabling plaque-specific, non-lethal SMC depletion via inhibition of cytoskeletal dynamics (Apr2/3 protein complex). Our data demonstrate that SMCs are indispensable for plaque stability, as their depletion markedly reduces collagen deposition and disrupts fibrous cap formation. Conversely, SMCs promoted plaque expansion and luminal narrowing. These findings suggest that selective modulation of SMC proliferation may limit detrimental effects while preserving stabilizing functions.

Methods

We generated Myh11-CreERT2 × Arpc2fl/fl × ApoE-/- mice (Arpc2-SMCΔ/Δ) enabling SMC-specific Arpc2 deletion after tamoxifen induction prior to plaque development, followed by Western diet. Arpc2 deletion disrupts actin dynamics, inhibiting SMC proliferation and migration. Lesions were analyzed by histology and immunofluorescence.

We analyzed human and murine single-cell RNA-seq datasets identifying high ANXA2 and S100A10 co-expression as a possible driver of proliferation. Moreover, we treated established ApoE plaques with the annexin A2 heterotetramer inhibitor A2ti-1. Brachiocephalic and aortic root lesions were assessed histologically, and Annexin A2 protein quantified by Western blot.

Results

Brachiocephalic artery plaques of Arpc2-SMCΔ/Δ mice exhibited pronounced architectural alterations, characterized by significantly reduced plaque size and cellularity. ECM remodelling was profound, with near-complete loss of collagen fibers. Arpc2-SMCΔ/Δ mice demonstrated severely impaired fibrous cap formation. Despite reduced plaque burden, lesions displayed features consistent with structural instability.

Cross-analysis of scRNA-seq datasets identified Annexin A2/S100A10 as selectively upregulated in proliferating SMCs. We propose that this signaling axis functions as a key regulator of SMC-lineage cell (SLC) proliferation in murine atherosclerotic lesions. Pharmacological inhibition of Annexin A2 heterotetramer formation using A2ti-1 significantly reduced SMC proliferation in advanced lesions. However, this was accompanied by fibrous cap thinning in late-stage atherosclerosis, promoting plaque destabilization.

Conclusions

SMC ablation from atheroprogression produces smaller, hypocellular plaques lacking collagen and fibrous caps. The Arpc2-SMCΔ/Δ model enables in vivo dissection of SMC functions, highlighting their central roles in plaque expansion, extracellular matrix maintenance, and cap formation. Annexin A2/S100A10 signaling drives SMC-lineage proliferation in murine atherosclerosis and may represent a conserved pathway in human disease. Summary of murine model findings