Inhibition of miR-148a-3p leads to a pro-angiogenic and anti-senescent effect on endothelial cells in the repair and healing process of myocardial infarction

To improve the healing and partial regeneration after myocardial infarction (MI), a well-coordinated angiogenic response is necessary. Since current therapeutic strategies to enhance post-MI angiogenesis are lacking, new approaches are needed. MicroRNAs (miRNAs) have been identified as potential regulators of angiogenesis, however, effective strategies to improve angiogenesis by targeting a specific miRNA are challenging. In this study, we investigate miR-148a-3p as a novel microRNA candidate with therapeutic potential to improve neovascularization following MI, especially in the context of cellular senescence.

We initiated this study by performing an expression analysis of miR-148a-3p in human vascular endothelial cells (EC) and murine tissue. The miR-148a-3p was characterized regarding gene and protein expression for their contribution to EC function, i.e. proliferation, migration, wound healing, and metabolism after either pre-or anti-miR transfection. All experiments were performed in non-senescent and replicative senescent ECs. Subsequently, we identified potential targets by in silico research and examined them at mRNA and protein levels.

Initial screenings revealed an upregulated expression of miR-148a-3p in murine aortic ECs of old mice (24 months old) as well as in a  wire-induced injury model of the femoral artery (p<0.05). Notably, in vitro in replicative senescent ECs, there was a significant upregulation of miR-148a-3p expression (p<0.01), especially in senescent cells under hypoxic conditions (p<0.05). Furthermore, the expression of miR-148a-3p is reduced under growth conditions compared to basal medium conditions, which suggests a negative correlation between this miR and EC-growth. In line with this, downregulation of miR-148a-3p improves the proliferation of senescent ECs (p<0.001), whereas we also observed a significant regulation of the migration capacity of senescent ECs (p<0.05). To investigate the influence of miR-148a-3p on specific molecular pathways, we performed a systematic target research in silico to subsequently focus on Akt1, NFKBIA, HMOX1 and RTN4 as potential targets. Downregulation of miR-148a-3p verified this predictions by significantly upregulating Akt1 (p<0.001), NFKBIA (p<0.01), HMOX1 (p<0.05) and RTN4 (p<0.05). Additionally, overexpression of miR-148a-3p resulted in the shortening of telomeres in ECs, while its knockdown reduced senescence markers, including p16ink4a, p21, p14arf, and CD44 (p<0.05). High-resolution microscopy images of ECs after upregulation of miR-148a-3p preliminary showed larger cell nuclei and cell sizes, indicating a pro-senescent cellular effect of the named miR. In further experiments, we study the dysfunctional as well as the antiangiogenic effect of miR-148a-3p on vascular endothelial cells by targeting all genes indicated.

In summary, our findings demonstrate that varying levels of miR-148a-3p elicit distinct effects on EC function, particularly in replicative senescent cells and under hypoxic conditions. Our experimental data strongly suggest that this miRNA plays a role in the angiogenic response and hold the potential to serve as therapeutic target. Currently in future experiments, we will evaluate the microRNA potential and safety on MI healing in vivo.