Inhibition of miR-148a-3p as a therapeutic strategy to counteract endothelial senescence and improve vascular repair

Endothelial senescence is a key factor in the development of cardiovascular disease (CVD), particularly in conditions such as myocardial infarction (MI). Following MI, an effective angiogenic response is essential for tissue repair and regeneration, a process inhibited by senescent endothelial cells (ECs). Senescent ECs contribute to endothelial dysfunction by reducing regenerative capacity and impairing angiogenesis. This study investigates miR-148a-3p as a potential therapeutic agent to counteract endothelial senescence, enhance vascular repair, and improve recovery following MI, offering insights into new approaches for CVD management.

Expression levels of miR-148a-3p were assessed in Human Coronary Artery Endothelial Cells (HCAECs) and murine tissue via qRT-PCR analysis. The impact of miR-148a-3p on cellular function (proliferation, migration, tube, and sprout formation) and cellular senescence (senescence marker expression, SA-β-gal activity, morphological analysis) following anti-miR transfection was determined in both replicative and non-replicative senescent HCAECs. Target gene and protein expressions were evaluated by qPCR and Western Blot analysis.

Using an online dataset of miRNA expression across different ages and various organs and tissues, our research group developed a machine learning tool to identify key regulators of aging. This analysis revealed three microRNAs that appear to play a central, organ-independent role in aging. MiR-148a-3p was one of the three microRNAs consistently upregulated across all organs with age. To further elucidate its role in cardiac aging, we first screened murine aortic ECs from C57BL/6J mice (3 vs. 24 months old), showing a significant upregulation of miR-148a-3p with aging (p<0.05). A similar effect was observed in vitro in replicative senescent HCAECs (p<0.01), particularly under hypoxic conditions (p<0.001). Furthermore, we detected significantly enhanced functional properties in anti-miR-148a-3p-transfected ECs (both non-senescent and senescent), including increased proliferation (p<0.01 and p<0.001, respectively), migration (p<0.05), sprouting (p<0.05), and tube formation (p<0.05). MiR-148a-3p knockdown reduced markers of cellular senescence in (non-) senescent ECs, such as p16INK4a (p<0.001), p21 (p<0.05 and p<0.001, respectively), p14ARF (p<0.05 and p<0.0001, respectively), and CD44 (p<0.01). It also decreased senescence-associated SA-β-gal activity (p<0.05) and increased the number of smaller cells and small cell nuclei, as observed in morphological analysis (p<0.05). Downregulation of miR-148a-3p leads to a decreased apoptosis in non-senescent ECs. Systemic target research revealed Akt1 (p<0.001), NFKBIA (p<0.01), and RTN4 (p<0.5) as potential targets, which were significantly upregulated following miR-148a-3p downregulation in non-senescent ECs. Additionally, nRas (p<0.05) and IL6ST (p<0.01) were significantly downregulated in replicative-senescent ECs following miR-148a-3p knockdown. Target regulation findings were further confirmed at the protein level (p<0.05).

In summary, we demonstrate that inhibiting miR-148a-3p improves key EC functions, especially in replicative senescent cells. This strongly suggests that miRNA-148a-3p plays a critical role in the angiogenic response and holds promise as a therapeutic target to combat senescence-induced endothelial dysfunction in CVDs. Future experiments will evaluate its potential in vivo using ultrasound-guided MI induction in mice.