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MicroRNA changes in human arterial endothelial cells with senescence: relation to apoptosis, eNOS and inflammation.

ABSTRACT: 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 p16(INK4a). 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.

SUBMITTER: Rippe C

PROVIDER: S-EPMC3245334 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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MicroRNA changes in human arterial endothelial cells with senescence: relation to apoptosis, eNOS and inflammation.

Rippe Catarina C Blimline Mark M Magerko Katherine A KA Lawson Brooke R BR LaRocca Thomas J TJ Donato Anthony J AJ Seals Douglas R DR

Experimental gerontology 20111015 1

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-galactos ...[more]

PMID: 22037549

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Project description:Targeting microRNAs (miRs) using small chemical molecules has become a promising strategy for disease treatment. miR‑216a has been reported to be a potential therapeutic target in endothelial senescence and atherosclerosis via the Smad3/NF‑κB signaling pathway. Ginsenoside Rb2 (Rb2) is the main bioactive component extracted from the plant Panax ginseng, and is a widely used traditional Chinese medicine. In the present study, Rb2 was identified to have a high score for miR‑216a via bioinformatics analysis based on its sequence and structural features. The microscale thermophoresis experiment further demonstrated that Rb2 had a specific binding affinity for miR‑216a and the dissociation constant was 17.6 µM. In both young and senescent human umbilical vein endothelial cells (HUVECs), as well as human aortic endothelial cells, Rb2 decreased the expression of endogenous miR‑216a. Next, a replicative endothelial senescence model of HUVECs was established by infection with pre‑miR‑216a recombinant lentiviruses (Lv‑miR‑216a) and the number of population‑doubling level (PDL) was calculated. Stable overexpression of miR‑216a induced a premature senescent‑like phenotype, whereas the senescent features and increased activity of senescence‑associated β‑galactosidase (SA‑β‑gal) were reversed after Rb2 treatment. The percentage of SA‑β‑gal‑positive cells in senescent PDL25 cells transfected with Lv‑miR‑216a was decreased 76% by Rb2 treatment compared with the Lv‑miR‑216a group without Rb2 treatment (P=0.01). Mechanistically, miR‑216a inhibited Smad3 protein expression, promoted IκBα degradation and activated NF‑κB‑responsive genes, such as vascular cell adhesion molecule 1 (VCAM1), which promoted the adhesiveness of endothelial cells to monocytes. These pro‑inflammatory effects of miR‑216a were significantly suppressed by Rb2 treatment. When Smad3 was suppressed by small interfering RNA, the elevated expression levels of intercellular adhesion molecule 1 and VCAM1 induced by miR‑216a were significantly reversed. Collectively, to the best of our knowledge, the present study demonstrated for the first time that Rb2 exerted an anti‑inflammation effect on the process of endothelial cell senescence and could be a potential therapeutic drug by targeting miR‑216a.

Project description:BackgroundCirculating endothelial progenitor cells (EPCs) play important roles in vascular repair. However, the mechanisms of high-glucose- (HG-) induced cord blood EPC senescence and the role of B2 receptor (B2R) remain unknown.MethodsCord blood samples from 26 patients with gestational diabetes mellitus (GDM) and samples from 26 healthy controls were collected. B2R expression on circulating CD34+ cells of cord blood mononuclear cells (CBMCs) was detected using flow cytometry. The plasma concentrations of 8-isoprostaglandin F2α (8-iso-PGF2α) and nitric oxide (NO) were measured. EPCs were treated with HG (40 mM) alone or with bradykinin (BK) (1 nM). The B2R and eNOS small interfering RNAs (siRNAs) and the PI3K antagonist LY294002 were added to block B2R, eNOS, and PI3K separately. To determine the number of senescent cells, senescence-associated β-galactosidase (SA-β-gal) staining was performed. The level of mitochondrial reactive oxygen species (ROS) in EPCs was assessed by Mito-Sox staining. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assays. Mitochondrial DNA (mtDNA) copy number and the relative length of telomeres were detected by real time-PCR. The distribution of human telomerase reverse transcriptase (hTERT) in the nucleus, cytosol, and mitochondria of EPCs was detected by immunofluorescence. The expression of B2R, p16, p21, p53, P-Ser473AKT, T-AKT, eNOS, and hTERT was demonstrated by Western blot.ResultsB2R expression on circulating CD34+ cells of CBMCs was significantly reduced in patients with GDM compared to healthy controls. Furthermore, B2R expression on circulating CD34+ cells of CBMCs was inversely correlated with plasma 8-iso-PGF2α concentrations and positively correlated with plasma NO levels. BK treatment decreased EPC senescence and ROS generation. Furthermore, BK treatment of HG-exposed cells led to elevated P-Ser473AKT and eNOS protein expression compared with HG treatment alone. BK reduced hTERT translocation in HG-induced senescent EPCs. B2R siRNA, eNOS siRNA, and antagonist of the PI3K signalling pathway blocked the protective effects of BK.ConclusionBK, acting through PI3K-AKT-eNOS signalling pathways, reduced hTERT translocation, increased the relative length of telomeres while reducing mtDNA copy number, and finally protected against EPC senescence induced by HG.

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MicroRNA changes in human arterial endothelial cells with senescence: relation to apoptosis, eNOS and inflammation.

Publications

MicroRNA changes in human arterial endothelial cells with senescence: relation to apoptosis, eNOS and inflammation.

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