Abstract 4365714: Inhibition of miRNA-375-3p in vascular cells restores cellular functions in the context of cellular ageing

Abstract

Background: Ageing is a major driving factor for vascular changes. Over time, senescent endothelial and smooth muscle cells accumulate in the vasculature, promoting endothelial dysfunction and cardiovascular disease (CVD). MicroRNAs serve a key function in CVD and cellular senescence. Methods: Expression levels of microRNA-375-3p were analyzed in non-senescent and replicative senescent human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC) by qRT-PCR. To evaluate its effects regarding cellular functions and cellular senescence, miR-375-3p was downregulated by transfection of a LNA-inhibitor. In silico research identified targets of miR-375-3p, which were validated on mRNA and protein level. Results: In a machine-learning based analysis of over 1200 microRNA datasets including 1800 distinct miRNAs from various tissue samples from patients between the age of 20 to 90 years, we showed that miR-375-3p is an important miR upregulated in aged individuals showing an exceptionally strong centrality and targeting more than 80% of the respective age-associated targets . In vitro, expression analyses showed especially a 10-fold upregulation of miR-375-3p in senescent compared to non-senescent HCAEC (p<0.05). In aortic endothelial cells of mice, miR-375-3p is upregulated in old compared to young mice. Transfection with a LNA-inhibitor downregulated the microRNA successfully. By the inhibition of miR-375-3 we observed improved angiogenic capacities in non-senescent and replicative senescent HCAEC by tube formation and sprouting assays (p<0.05). However, cell death in non-senescent cells was elevated by inhibition. In morphology analyses, downregulation of the miR led to an increase in cell nuclei size in non-senescent cells. Inhibition induced the expression of pro-inflammatory cytokines like TNF-alpha (p<0.01) and IL-1beta (p<0.05) in senescent HCAEC. Conclusion: A machine-learning based analysis identified microRNA-375-3p as a significantly upregulated microRNA in senescent endothelial cells, which was verified in vitro and in vivo. Inhibition of miR-375-3p improved angiogenic capacities in non-senescent and replicative senescent HCAEC. However, its role in cellular senescence remains inconclusive and needs further evaluation.