miR-331-3p as a modulator of vascular smooth muscle cell function in atherosclerosis

Atherosclerosis progresses with age and underlies many cardiovascular diseases that lead to death. Human Coronary Artery Smooth Muscle Cells (SMCs), which contribute to vascular contraction and extracellular matrix production in the tunica media, play a pivotal role in vascular remodeling. Under pathological conditions, such as pro-inflammatory cytokine release, SMCs proliferate and undergo phenotype switching from a contractile to a synthetic state during migration to the intima, contributing to neointima formation or plaque destabilization. MicroRNAs (miRs) have emerged as promising targets to modulate the functions and behavior of SMCs and prevent atherosclerotic progression. Thereby, it is crucial to maintain the protective barrier and angiogenesis of Human Coronary Artery Endothelial Cells (ECs).

This study aims to elucidate the effects of miR-331-3p by its targeted regulation on cellular functions and properties of SMCs compared to ECs to examine its therapeutic potential in atherosclerosis.

The levels of miR-331-3p in human SMCs and ECs, and murine tissue were measured using qRT-PCR. The targeted modulation of miR-331-3p expression was achieved by pre- and anti-miR transfection. Following this, the impact of miR-331-3p on cellular processes, including proliferation, migration, metabolic activity, and apoptosis, was evaluated. Immunofluorescence microscopy was performed to appraise morphological changes. Subsequent in silico research uncovered possible targets of miRNA-331-3p, which are verified on mRNA and protein levels.

Preliminary studies reveal a strong upregulation of miR-331-3p in vivo in ApoE-knockout mice, an established model for atherosclerosis, during disease progression (p<0.0001). Analysis revealed miR-331-3p to be highly conserved among species, making it suitable for further research in vitro in human SMCs and ECs. In replicative senescence, miR-331-3p levels are divergently regulated in SMCs compared to ECs, which indicates regulatory mechanisms that occur with age and influence atherosclerosis. While cytokines such as IFNγ, IL1-β, and TNFα influence miR-331-3p expression in SMCs, in ECs, hypoxia influences miR-311-3p expression. The morphological cell shape of SMCs and ECs is not affected by transfection with either pre- or anti-miR. The optically quantified increase in cell area (p<0,0277) of SMCs 24 hours after pre-miR transfection can be attributed to increased proliferation and confirms BrdU assay (p<0,05). Whereas anti-miRs hampers SMC migration (p<0,0178). Particularly, anti-miR transfection appears to have an opposing effect on functional parameters in SMCs and ECs. Cell death assay revealed anti-apoptotic tendencies of miR-331-3p in SMCs 48 hours after regulation. Subsequent in silico research based on literature, target prediction databases, and publically available sequencing data sets revealed KLF16, BAK1, PHLPP1, SOCS1, TNFa, TGFBR1, NPR2 and DUSP5 as potential targets. We confirmed their upregulation in SMCs 24 and 48 hours after miR-331-3p overexpression (p<0,05). ECs demonstrate much fewer target regulations as SMCs.

In summary, miR-331-3p may act as an important factor in atherosclerosis. The treatment with anti-miR can be efficient against vascular remodeling. Its impact on functions and characteristics in ECs appears to be less pronounced from its effects in SMCs, highlighting miR-331-3p as a promising target for potential cardiovascular therapies with protected endothelial cell function.