Senomorphic properties of miR-181b in aged human aortic smooth muscle cells

Abstract

Introduction: Age associated large artery stiffness (LAS) is an independent predictor of adverse cardiovascular health outcomes in humans and has also been shown to be associated with the development of heart failure, kidney disease, and even dementia. Furthermore, the development of cellular senescence in the aorta, the permanent arrest of the cell cycle and development of pro-inflammatory, pro-fibrotic senescence associated secretory phenotype (SASP), has also been shown to contribute to age associated LAS. MicroRNAs (miR) are small, non-coding RNAs that down-regulate protein expression in a semi-specific manner. miR-181b has been shown to be downregulated with aging, targets many of the secreted factors associated with the aortic SASP and has previously been shown to have a beneficial effect on vascular health. With smooth muscle cells being the predominant cell type in the large arteries, we wanted to address the hypothesis that miR-181b has the potential to act as a senomorphic in human aortic smooth muscle cells (HASMCs) and promote beneficial changes in individual cells that influence LAS. Methods: To test our hypothesis, we obtained young and old HASMCs and transfected them with miR-181b or a scrambled miR control utilizing a lipofectamine transfection protocol. Total RNA was isolated from the HASMCs and quantitative PCR was performed to assess changes of the SASP profile. Cell proliferation was measured using electric cell-substrate impedance sensing (ECIS) technology. Lastly, individual HASMC stiffness measurements were made using twisting magnetic microscopy. Results: Transfection of HASMCs with miR-181b resulted in reduced expression of markers of cell cycle arrest (p16 and p21) and the SASP profile (IL-6, TNF-alpha, MCP1, TGF-b1, IL-1alpha, and MMP2) when compared to scrambled control (p<0.05). HASMC proliferation was decreased in old HASMCs compared to young (p<0.05) and was increased with miR-181b transfection when compared to scramble control (p<0.05). Lastly, individual HASMC stiffness was lower in young cells compared to old (0.4382 ± 0.29 vs. 0.9959 ± 0.72 Pa/nm, n=120, p<0.001). Cell stiffness was decreased in old HASMCs when treated with miR-181b when compared with control (0.7856 ± 0.144 vs. 1.18 ± 0.288 Pa/nm, n=76, p<0.001), but there was no difference in young HASMCs. Conclusions: miR-181b appears to act as a senomorphic agent when transfected in old HASMCs. This is evidenced by noticeable changes in the expression of markers of cellular senescence which corresponded with positive changes in cellular characteristics that are associated with LAS, namely the improvement in cellular proliferation and reduction of individual cell stiffness in the old HASMC groups. Together, these results suggest the modulation of miR-181b may be a potential therapeutic target to improve age associated LAS through an effect on cellular senescence. K08 AG070281 (ET), Western Institute for Veterans Research (ET), U-CARES (ET), R01 AG048366 (LAL), R01 AG050238 (AJD), R01 AG060395 (AJD) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.