m6A Modification of Profilin-1 in Vascular Smooth Muscle Cells Drives Phenotype Switching and Neointimal Hyperplasia via Activation of the p-ANXA2/STAT3 Pathway.

Abstract

Background: In-stent restenosis is characterized by a significant reduction in lumen diameter within the stented segment, primarily attributed to excessive proliferation of vascular smooth muscle cells (VSMCs) and neointimal hyperplasia. PFN1 (profilin-1), an actin-sequestering protein extensively studied in amyotrophic lateral sclerosis, remains less explored in neointimal hyperplasia.

Methods: Utilizing single-cell RNA sequencing alongside data from in-stent restenosis patients and various experimental in-stent restenosis models (swine, rats, and mice), we investigated the role of PFN1 in promoting VSMC phenotype switching and neointimal hyperplasia.

Results: Single-cell RNA sequencing of stenotic rat carotid arteries revealed a critical role for PFN1 in neointimal hyperplasia, a finding corroborated in stented swine coronary arteries, in-stent restenosis patients, PFN1^SMC-IKO (SMC-specific PFN1 knockout) mice, and PFN1 overexpressed mice. PFN1 deletion was shown to suppress VSMC phenotype switching and neointimal hyperplasia in PFN1^SMC-IKO mice subjected to a wire-injured model. To elucidate the observed discordance in PFN1 mRNA and protein levels, we identified that METTL3 (N⁶-methyladenosine methyltransferase) and YTHDF3 (N⁶-methyladenosine-specific reader) enhance PFN1 translation efficiency in an N⁶-methyladenosine-dependent manner, confirmed through experiments involving METTL3 knockout and YTHDF3 knockout mice. Furthermore, PFN1 was mechanistically found to interact with the phosphorylation of ANXA2 (annexin A2) by recruiting Src, promoting the phosphorylation of STAT3, a typical transcription factor known to induce VSMC phenotype switching.

Conclusions: This study unveils the significance of PFN1 N⁶-methyladenosine modification in VSMCs, demonstrating its role in promoting phenotype switching and neointimal hyperplasia through the activation of the p-ANXA2 (phospho-ANXA2)/STAT3 pathway.