A senescent phenotype in endothelial cells is associated with increased apoptosis, reduced endothelial nitric oxide synthase (eNOS) and inflammation, which are implicated in arterial dysfunction and disease in humans. We tested the hypothesis that changes in microRNAs are associated with a senescent phenotype in human aortic endothelial cells (HAEC). Compared with early-passage HAEC, late-passage HAEC had a reduced proliferation rate and increased staining for senescence-associated beta-galactosidase and the tumor suppressor p16INK4a. Late-passage senescent HAEC had reduced expression of proliferation-stimulating/apoptosis-suppressing miR-21, miR-214 and miR-92 and increased expression of tumor suppressors and apoptotic markers. eNOS-suppressing miR-221 and miR-222 were increased and eNOS protein and eNOS activation (phosphorylation at serine1177) were lower in senescent HAEC. Caveolin-1 inhibiting miR-133a was reduced and caveolin-1, a negative regulator of eNOS activity, was elevated in senescent HAEC. Inflammation-repressing miR-126 was reduced and inflammation–stimulating miR-125b was increased, whereas inflammatory proteins were greater in senescent HAEC. Development of a senescent arterial endothelial cell phenotype featuring reduced cell proliferation, enhanced apoptosis and inflammation and reduced eNOS is associated with changes in miRNAs linked to the regulation of these processes. Our results support the hypothesis that miRNAs could play a critical role in arterial endothelial cell senescence.
SummaryInhibition of mammalian target of rapamycin, mTOR, extends lifespan and reduces age‐related disease. It is not known what role mTOR plays in the arterial aging phenotype or if mTOR inhibition by dietary rapamycin ameliorates age‐related arterial dysfunction. To explore this, young (3.8 ± 0.6 months) and old (30.3 ± 0.2 months) male B6D2F1 mice were fed a rapamycin supplemented or control diet for 6–8 weeks. Although there were few other notable changes in animal characteristics after rapamycin treatment, we found that glucose tolerance improved in old mice, but was impaired in young mice, after rapamycin supplementation (both P < 0.05). Aging increased mTOR activation in arteries evidenced by elevated S6K phosphorylation (P < 0.01), and this was reversed after rapamycin treatment in old mice (P < 0.05). Aging was also associated with impaired endothelium‐dependent dilation (EDD) in the carotid artery (P < 0.05). Rapamycin improved EDD in old mice (P < 0.05). Superoxide production and NADPH oxidase expression were higher in arteries from old compared to young mice (P < 0.05), and rapamycin normalized these (P < 0.05) to levels not different from young mice. Scavenging superoxide improved carotid artery EDD in untreated (P < 0.05), but not rapamycin‐treated, old mice. While aging increased large artery stiffness evidenced by increased aortic pulse‐wave velocity (PWV) (P < 0.01), rapamycin treatment reduced aortic PWV (P < 0.05) and collagen content (P < 0.05) in old mice. Aortic adenosine monophosphate‐activated protein kinase (AMPK) phosphorylation and expression of the cell cycle‐related proteins PTEN and p27kip were increased with rapamycin treatment in old mice (all P < 0.05). Lastly, aging resulted in augmentation of the arterial senescence marker, p19 (P < 0.05), and this was ameliorated by rapamycin treatment (P < 0.05). These results demonstrate beneficial effects of rapamycin treatment on arterial function in old mice and suggest these improvements are associated with reduced oxidative stress, AMPK activation and increased expression of proteins involved in the control of the cell cycle.
Oxidative stress and inflammation develop in the vascular endothelium with aging in rodents and humans and cause dysfunction. In vitro culture of endothelial cells to senescence may be a useful model of endothelial aging in humans, but this has not been established. Human aortic endothelial cells (HAEC) were analyzed by western blotting at early (passage 7–10) and senescent (passage 22–24) stages and compared with endothelial cells obtained in vivo from antecubital veins of healthy young (18–35 yr, n=26) and older (55–71 yr, n=26) adult males and analyzed using quantitative immunofluorescent staining. Nitrotyrosine, a marker of oxidative stress, was increased (P<0.05) with age both in vivo (1.6±0.2‐fold) and in vitro (1.9±0.1‐fold). The oxidant enzyme NADPH oxidase increased with age in vivo (1.6±0.1‐fold, P<0.05) and tended to increase in vitro (1.5±0.1‐fold, P=0.14). The antioxidant enzymes catalase and copper zinc superoxide dismutase (SOD) did not differ with age either in vivo or in vitro, whereas manganese SOD was selectively increased in senescent HAECs (1.6‐fold, P<0.05). The inflammatory proteins tumor necrosis factor α, nuclear factor κ B and monocyte chemoattractant protein‐1 increased (all P<0.05) with age both in vivo (1.6±0.2‐, 1.6±0.2‐, 1.4±0.2‐fold) and in vitro (2.4±0.5‐, 1.7±0.3‐, 1.6±0.1‐fold). In vitro culture of vascular EC to senescence may be a useful model of in vivo EC aging in humans for assessing oxidative stress and protein expression of oxidant and antioxidant enzymes and inflammatory molecules.NIH AG013038 and the Swedish Medical Research Council
Aging leads to vascular endothelial dysfunction, but the mechanisms involved are incompletely understood. We assessed the effects of aging on endothelial cell (EC) senescence in vivo and the possible role of the anti‐senescence and anti‐inflammatory deacetylase, SIRT1, in vitro. Compared with young adult (5 mo) mice, old (30 mo) mice had reduced endothelium‐dependent dilation and a higher % of aortic EC that stained for the senescence marker p16INK4a (31 vs. 6). "Aged" cultures of human aortic EC (HAEC) (35±2 population doublings, PDL) had a higher % of cells stained for p16INK4a (46 vs. 7) and (beta)‐galactosidase, a 2nd marker of senescence (57 vs. 6), than "young" cultures (20±1 PDL). SIRT1 expression was 55% lower in senescent HAEC (0.45±0.08 vs. 1.00 ± 0.10 AUs, P<0.002). Senescent HAEC demonstrated reduced expression of the anti‐inflammatory proteins interleukin‐10 (0.6±0.05 vs. 1.0±0.2 AUs, P<0.05) and interleukin‐1 receptor‐α (0.7±0.05 vs. 1.0±0.2 AUs, P<0.05), and increased expression of the inflammatory protein monocyte chemoattractant protein‐1 (1.6±0.1 vs. 1.0±0.1 AUs, P<0.005). In conclusion, the % of senescent arterial EC in vivo is greater in old age. A decrease in SIRT1 may play an important role in EC senescence and contribute to development of vascular endothelial inflammation and dysfunction.Swedish Research Council; NIH AG006537, AG013038, AG022241, RR00051
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.