Effect of miR-181b Treatment on Senescent Human Aortic Smooth Muscle Cells
Authors: Moreno Denisse G , Gogulamudi Venkateswara , Lesniewski Lisa , Donato Anthony , Tuday Eric 
Published: May 2024

Large Artery Stiffness (LAS) is independently associated with increased cardiovascular morbidity and mortality and its development is multifactorial and has been associated with the onset of cellular senescence (CS). CS is defined as a stable cell cycle arrest, characterized by an altered cell phenotype with impaired function and detrimental secretory profile. Vascular smooth muscle cells (VSMCs) are present in two main phenotypes: the contractile phenotype seen in young populations, and the synthetic phenotype related to aging and senescent VSMCs. Micro-RNAs (miR) are non-coding RNAs that can alter protein expression. miR-181b has been demonstrated to undergo downregulation with aging with a close to 80% decrease, this correlates with an upregulation of markers of senescence. To test the hypothesis that miR-181b could modulate the senescent phenotype in VSMCs and promote a more contractile phenotype, a cellular model comprising young and old human aortic smooth muscle cells (HASMCs) was employed. These cells were transfected with either a miR-181b mimic or a scrambled miR control (SCR), resulting in an 11-fold increase in the young and a 3.3-fold increase in the old. Cell proliferation was measured using electric cell-substrate impedance sensing (ECIS) technology, where it was found to be lower in older HASMCs (1.05±0.001) compared to the young (1.1±0.001, p<0.05). When miR-181b was introduced, there was a significant increase in proliferation for both age groups by 0.03-fold compared to SCR (p<0.05). Old HASMCs demonstrated a decreased average ionomycin-induced contractile response (3.65%±0.46) when compared to the young (7.17%±0.60, p<0.05). Lipofectamine-mediated transfection of HASMCs with miR-181b resulted in an increased contractility response in the old HASMCs (5.46%±0.91) compared to scrambled control (1.41%±0.39, p<0.05), while there was no significant difference for the young cells. The percentage of Senescence-Associated-β-galactosidase (Saβ-gal) positive cells, a marker of senescence, was higher in old (39.74%±3.04) vs young (4.35%±0.72, p<0.05). The percentage of Saβ-gal positive cells tended to decrease in old VSMCs after miR-181b transfection (SCR 79.65%±4.38 vs Old181b 70.39±4.03, p=0.2.02), with no effect on young cells. Immunofluorescence in situ hybridization (IF-FISH) staining for the double-stranded DNA break marker (53BP1) was lower in the young (4.65%) compared to old HASMCs (32.69%). Likewise, the percentage of cells with one or more 53BP1 foci colocalization in telomeres, referred to as Telomere dysfunction-induced foci (TIF), was higher in old (11.54%) compared to young (2.33%) VSMCs (p=0.002). However, miR-181b transfection did not impact these markers regardless of age (p=0.94). Transfection of HASMCs with miR-181b did not impact senescent cell abundance, but improved contractility and cellular proliferation. Combined with our prior data demonstrating a senomorphic effect of miR-181b transfection to improve cellular stiffness and favorably modulate of senescence-associated secretory factors, our results demonstrate an enhancement in the phenotypic characteristics of the old HASMCs group, suggesting that miR-181b participates in the modulation of the senescent VSMCs phenotype. This translates to a potential improvement in HASMC health that could reduce the development of LAS. National Institutes of Health Awards: K08 AG070281 (ET), T32 HL139451 (DM), R01 AG048366 (LAL), R01 AG060395 (AJD), and Western Institute for Veterans Research (ET). This is the full abstract presented at the American Physiology Summit 2024 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.