Abstract Body (Do not enter title and authors here): Atherosclerosis is a primary contributor to cardiovascular mortality. The pathological proliferation, migration to the intima, and phenotypic switching of Smooth Muscle Cells (SMCs) leads to vascular remodeling and plaque development. MicroRNAs (miRs) have emerged as a promising target for modulating SMC activity while maintaining the proper function of Endothelial Cells (ECs).

This study investigates whether modulating miR-331-3p can therapeutically target SMCs in atherosclerosis and how this affects ECs.

MiR-331-3p levels in SMCs, ECs, and murine tissue were measured via qRT-PCR. Pre- and anti-miR transfection was used to assess its upregulation or inhibition on proliferation, migration, metabolism, and apoptosis. Morphological changes were analyzed by immunofluorescence microscopy. In silico predicted targets were validated at the mRNA level, and protein-level validation is ongoing.

Initial investigations revealed a significant increase in miR-331-3p expression during disease progression (p<0.0001) in ApoE--/- mice, a commonly used atherosclerosis model. Further research uncovered that miR-331-3p is highly conserved across species, suggesting its relevance for subsequent in vitro experiments with human SMCs and ECs. Cytokines such as IFNγ, IL-1β, and TNFα were found to influence miR-331-3p expression in SMCs. Following pre- or anti-miRNA transfection, the cellular morphology of SMCs and ECs remained unchanged. Microscopically, the increase in SMC cell area observed 24 hours after miR-331-3p overexpression (p<0.0277) was linked to enhanced proliferation, as validated by BrdU assay results (p<0.05). In contrast, inhibition of miR-331-3p inhibited SMC migration (p<0.0178) and revealed divergent effects on SMC and EC function. Cell death assays showed that miR-331-3p causes anti-apoptotic effects in SMCs 48 hours after regulation. In silico analyses, utilizing literature research, target prediction tools, and public sequencing data, identified several potential targets, including KLF16, BAK1, PHLPP1, SOCS1, TNFα, TGFBR1, and DUSP5. Their upregulation in SMCs was confirmed 24 and 48 hours after miR-331-3p overexpression (p<0.05), while ECs showed remarkably few, if any, target changes.

In conclusion, inhibiting miR-331-3p efficiently modulates vascular remodeling in SMCs without impairing EC function, highlighting its potential as a valuable target for cardiovascular treatment strategies potentially by majorly regulating the identified targets DUSP5 and PHLPP1.