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Anti-inflammatory effect of ubiquinol-10 onyoung and senescent endothelial cells viamiR-146a modulation
Fabiola Olivieri, Raffaella Lazzarini, Lucia Babi-ni, Francesco Prattichizzo, Maria Rita Rippo,Luca Tiano, Silvia Di Nuzzo, Laura Graciotti,Roberto Festa, Francesca Brugè, Patrick Orlan-do, Sonia Silvestri, Miriam Capri, Linda Palma,Mauro Magnani, Claudio Franceschi, Gian PaoloLittarru, Antonio Domenico Procopio
PII: S0891-5849(13)00243-8DOI: http://dx.doi.org/10.1016/j.freeradbiomed.2013.05.033Reference: FRB11579
To appear in: Free Radical Biology and Medicine
Received date: 8 December 2012Revised date: 12 April 2013Accepted date: 22 May 2013
Cite this article as: Fabiola Olivieri, Raffaella Lazzarini, Lucia Babini, FrancescoPrattichizzo, Maria Rita Rippo, Luca Tiano, Silvia Di Nuzzo, Laura Graciotti,Roberto Festa, Francesca Brugè, Patrick Orlando, Sonia Silvestri, Miriam Capri,Linda Palma, Mauro Magnani, Claudio Franceschi, Gian Paolo Littarru, AntonioDomenico Procopio, Anti-inflammatory effect of ubiquinol-10 on young andsenescent endothelial cells via miR-146a modulation, Free Radical Biology andMedicine, http://dx.doi.org/10.1016/j.freeradbiomed.2013.05.033
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1
Anti-inflammatory effect of ubiquinol-10 on young and senescent endothelial cells via miR-
146a modulation
Fabiola Olivieri1,2�, Raffaella Lazzarini1�, Lucia Babini1, Francesco Prattichizzo1, Maria Rita
Rippo1, Luca Tiano3, Silvia Di Nuzzo1, Laura Graciotti1, Roberto Festa1, Francesca Brugè3, Patrick
Orlando3, Sonia Silvestri3, Miriam Capri4, Linda Palma5, Mauro Magnani5, Claudio Franceschi4,
Gian Paolo Littarru3 and Antonio Domenico Procopio1,2
� These authors contributed equally to the manuscript
1-Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona,
Italy; 2-Center of Clinical Pathology and Innovative Therapies, I.N.R.C.A. National Institute,
Ancona, Italy; 3-Department of Dentistry and Clinical Sciences, Università Politecnica delle
Marche, Ancona, Italy; 4-Department of Experimental Diagnostic and Specialty Medicine, “Alma
Mater Studiorum” Università di Bologna and Centro Interdipartimentale Galvani “CIG”, Alma
Mater Studiorum Università di Bologna, Bologna, Italy; 5-Department of Biomolecular Sciences,
Università degli Studi di Urbino “Carlo Bo”, Urbino, Italy.
Running title: Anti-inflammatory effect of ubiquinol-10 on endothelial cells
Corresponding author
Fabiola Olivieri, Ph.D,
Dept. of Clinical and Molecular Sciences (DISCLIMO),
Università Politecnica delle Marche, Ancona
Via Tronto 10/A - 60020 Ancona (Italy),
Ph: +39 071 220 6242, Fx: +39 071 220 6240
e-mail: f.olivieri@univpm.it
2
Abstract
Clinical evidence demonstrates that ubiquinol-10, the reduced active form of Coenzyme Q10
(CoQ10H2), improves endothelial function through its antioxidant and likely anti-inflammatory
properties. We previously reported that a biomarker combination including miR-146a, its target
protein IL-1 receptor-associated kinase (IRAK-1), and released interleukin (IL)-6, here collectively
designated as MIRAKIL, indicates senescence-associated secretory phenotype (SASP) acquisition
by primary human umbilical vein endothelial cells (HUVEC). We explore the ability of short- and
long-term CoQ10H2 supplementation to affect MIRAKIL in HUVEC, used as a model of vascular
aging, during replicative senescence in absence/presence of lipopolysaccharide (LPS), a
proinflammatory stimulus.
Senescent HUVEC had the same ability as young cells to internalize CoQ10 and exhibit an
improved oxidative status. LPS-induced NF-�B activation diminished after CoQ10H2 pretreatment
both in young and senescent cells. However, short-term CoQ10H2 supplementation attenuated LPS-
induced MIRAKIL changes in young cells; in senescent cells CoQ10H2 supplementation
significantly attenuated LPS-induced miR-146a and IRAK-1 modulation but failed to curb IL-6
release. Similar results were obtained with long-term CoQ10H2 incubation. These findings provide
new insights into the molecular mechanisms by which CoQ10H2 stems endothelial cell
inflammatory responses and delays SASP acquisition. These phenomena may play a role in
preventing the endothelial dysfunction associated with major age-related diseases.
Keywords: miRs-146a, replicative senescence, HUVEC, Coenzyme Q10, SASP, IL-6
3
Introduction
The biological actions of Coenzyme Q10 (CoQ10) include a bioenergetic role as a proton and
electron carrier in the mitochondrial respiratory chain, and an action as a ubiquitous lipid soluble
antioxidant protecting against lipid peroxidation, extensively described both in vitro and in vivo,
and probably also contributing to its anti-inflammatory and anti-apoptotic properties [1]. CoQ10 is
synthesized by the mevalonate pathway and is subsequently converted to its active reduced form,
ubiquinol-10 (CoQ10H2). In plasma CoQ10 is transported by lipoproteins, where it is found mainly
in the reduced form [2]. Oxidative stress and low-grade inflammation are the hallmarks of the aging
process and are enhanced in many age-related degenerative diseases, such as cardiovascular disease
(CVD), type 2 diabetes mellitus (T2DM), Alzheimer disease (AD) and cancer [3, 4]. Recent studies
suggest that inflammation and oxidative stress can modulate endothelial progenitor cell (EPC)
bioactivity [5]. EPCs move toward injured endothelium or inflamed tissue and are incorporated into
foci of neovascularization, thus enhancing blood flow and the tissue repair process [5].
During aging oxidative stress increases, contributing to the formation of multi-protein inflammatory
complexes called inflammasomes, whereas antioxidant defences decline [4, 6]. Quinone reductase
activity is reduced during aging, and CoQ10 declines in aged animals and humans [6, 7]. CoQ10
supplementation is therefore widely prescribed to aging subjects and to patients with age-related
diseases, like CVD [8, 9]. In CVD patients CoQ10 supplementation improves endothelial function
as measured by flow-mediated dilation (FMD) [8]. CoQ10 supplementation has also been
associated with significant beneficial effects on vascular elasticity, measured as pulse-wave velocity
(PWV), and endothelial function, measured by digital thermal monitoring (DTM) [9].
A recent meta-analysis of human randomized controlled trials has disclosed that CoQ10
supplementation improves endothelial function in patients both with and without overt CVD [10].
However, its administration to patients receiving conventional therapies for T2DM and
hypertension has failed to yield conclusive results [11, 12].
4
The antioxidant effect on endothelial function is probably mediated by a reversal of mitochondrial
dysfunction, even though other molecular mechanisms have also been proposed [8, 13, 14].
Antioxidants can inhibit the nuclear export of telomerase reverse transcriptase, delaying replicative
senescence of endothelial cells [13]. CoQ10 can also induce endothelium-bound extracellular
superoxide dismutase activity, concomitantly improving flow-dependent endothelium-mediated
dilation [14]. Recently, a crucial role for non-mitochondrial CoQ10 has been documented in nitric
oxide (NO) signaling, highlighting a new molecular link between CoQ10 supplementation and its
effect on endothelial function [15].
Transcriptomic approaches have recently shown that CoQ10 affects gene expression by acting on
inflammatory pathways [16]. These findings have been strengthened by data showing that CoQ10
supplementation can modify the expression of proinflammatory and stress-related genes, such as
lipopolysaccharide-sensitive genes and chemokine ligand genes [17, 18]. CoQ10 has also been
suggested to be a modulator of the expression of microRNA (miR), a new class of gene expression
regulators acting at the post-transcriptional level via an interference mechanism [16, 19]. A rapid
increase in miR-146a expression has been reported during inflammatory and immune responses
[20]. IRAK-1 and TNF receptor-associated factor-6 (TRAF-6), established as molecular targets of
miR-146a, are known to participate in the common signaling pathway derived from toll-like
receptor (TLR-1, -2, -4, -6, -7 and -9) and affecting NF-kappaB (NF-�B)-controlled gene
expression. Interestingly, miR-146a induction is NF-�B-dependent [21]. Thus, increased miR-146a
expression during inflammatory responses might be involved in a negative feedback loop aimed to
curb production of proinflammatory cytokines [22, 23]. NF-�B is found in nearly all animal cell
types and is involved in cellular responses to stimuli such as stress, cytokines, ultraviolet
irradiation, bacterial or viral antigens, and also free radicals [24]. Accordingly, supplementation of
CoQ10, acting as a free-radical scavenger, was reported to modulate monocyte miR-146a
expression, probably via NF-�B activation [25]. Raised miR-146a expression has also been
described during replicative senescence in association with acquisition of the senescence-associated
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secretory phenotype (SASP) in fibroblasts [26]. However, little information is currently available on
human vascular endothelial cells, which could be used as a model for vascular aging and endothelial
dysfunction.
In a recent study we described a biomarker combination, including miR-146a expression, that
indicates acquisition of SASP by HUVEC, and demonstrated the functional relationships between
these components [22]. In this study we aimed to explore the anti-inflammatory effect exerted by
CoQ10 on HUVEC via NF-�B activation and miR-146a expression modulation, hypothesizing that
such modulation could help understand the molecular basis of the role played by CoQ10 on
inflammation-related and senescence-associated endothelial dysfunction.
6
Materials and methods
Cell model
HUVEC were purchased from Clonetics and cultured in endothelial growth medium (EGM-2; both
from Lonza, Basel, Switzerland). Replicative senescence was studied in endothelial cells passaged
until the 13th passage (P13) as described previously [22].
Human monocytic THP-1 cells were purchased from ATCC (Rockville, MD, USA) and maintained
in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1%
penicillin/streptomycin and 1% L-Glutamine (all from Euroclone, Milano, Italy).
Senescence-associated �-galactosidase staining
The senescent status of cells was verified by staining for senescence-associated �-galactosidase (SA
�-gal) as previously described [27]. Non-confluent HUVEC cultured in 6-well plates were washed
twice in phosphate-buffered saline (PBS), pH 7.4, and fixed for 10 min in 2% formaldehyde and
0.2% glutaraldehyde in PBS. After two washes in PBS, they were incubated for 18 h at 37 °C with
freshly prepared �-gal staining solution containing 1 mg/ml 5-bromo-4-chloro-3indolyl- �-D-
galactopyranoside, 5 mM potassium ferrocyanide, 150 mM NaCl, 2 mM MgCl2, and 40 mM citric
acid titrated with NaH2PO4 to pH 6.0. �-gal was microscopically revealed by the presence of a blue,
insoluble intracellular precipitate. The percentage of �-gal-positive cells was determined by
counting at least 500 cells per sample.
Lipopolysaccharide treatment
THP-1 cells and HUVEC were cultured in 6-well plates at a concentration of 80,000 cells/well
(70% confluence) in RPMI-1640 and EGM-2, respectively. Then 1 μg/ml lipopolysaccharide (LPS)
(Sigma-Aldrich, Taufkirchen, Germany) was added to the medium for 30 min or 5, 24 or 48 h.
7
Ubiquinol-10 supplementation and high-performance liquid chromatography
Ubiquinol-10 (CoQ10H2), kindly donated by Kaneka (Kaneka Corporation, Japan), was solubilized
in water using a mixture of glycerol and the emulsifying agent PEG 60-hydrogenated castor oil
(Cremophor®, BASF SE Chemical Company, Germany) (CoQ10:glycerol:HCO60 0.4:0.6:1). A 1
mM stock solution was kept at –80 °C until use. Oxidation in these conditions was minimal for
several months.
Short-term CoQ10H2 supplementation involved HUVEC treatment with 10 μM CoQ10H2 or
vehicle (Cremophor) for 24 h followed by treatment with LPS or medium for 30 min, 5, 24 or 48 h.
Long-term CoQ10H2 supplementation consisted of treatment with 10 μM CoQ10H2 or vehicle
(Cremophor®) for about 60 days, i.e. until acquisition of the senescent phenotype.
CoQ10H2 concentration in the medium, stability of the reduced form in solution, and incorporation
by cells were verified by high-performance liquid chromatography (HPLC) using a single dilution
step after extraction and vigorous vortexing of 50 �l of culture medium or cell suspension in PBS.
The extraction mixture was centrifuged for 1 min at 13,000 x g and 40 �l of supernatant was
injected into a dedicated HPLC system with electrochemical detector (ECD; Shiseido, Tokyo,
Japan). Two mobile phases were used: mobile phase 1 for loading and concentrating the sample (50
mM sodium perchlorate in methanol/water 95/5 v/v) and mobile phase 2 for the analytical
procedure (50 mM sodium perchlorate in methanol/isopropanol 95/5 v/v). A peculiarity of the
system was a post-separation reducing column (Shiseido CQR; 20 x 2.0 mm) capable of fully
reducing the oxidized CoQ10 peak, thus enabling simultaneous detection of both the reduced and
the oxidized form (ECD oxidation potential, 650 mV). CoQ10H2 content in culture medium was
expressed as μg/ml; cell content was expressed as μg CoQ10H2/1x10^6 cells.
Cell viability assay
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used as an indicator of
cell viability. Cells were grown in 96-well plates at a density of 2×103 cells/well. After 18 h cells
8
were washed with fresh medium then treated with 10 μM CoQ10H2. After 24 h pretreatment, 100 �l
MTT (1 mg/ml) was added and incubated for 4 h. Finally, the formed formazan salt was solubilized
by adding 200 �l dimethyl sulfoxide and its amount was determined by measuring optical density
(OD) at 540 nm using a microplate reader (MPT Reader, Invitrogen, Milano, Italy). Data are
expressed as mean of at least three independent experiments.
Cytokine production
Culture supernatants were collected at the end of each incubation, centrifuged and stored at –20 °C
until use in the assays. IL-6 concentration was measured using a commercially available high-
sensitivity enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen).
RNA isolation
Total RNA from HUVEC and THP-1 cells was isolated using an RNA purification kit (Norgen
Biotek, Thorold, ON, Canada) which allows isolation of both miR and larger RNA species. RNA
was stored at -80 °C until use. �Quantitative RT-PCR of mature microRNAs
MiR expression values obtained with quantitative real-time (RT-q)PCR were normalized using
nucleolar RNA (RNU44). Human miR-146a and human RNU44 expression was quantified using
TaqMan MicroRNA Assay (Applied Biosystems, Foster City, CA, USA), with some modifications.
Briefly, total RNA was reverse-transcribed with the TaqMan MicroRNA reverse transcription kit; 5
�l of RT mix contained 1 �l of each miR specific stem-loop primers, 1.67 �l input RNA, 0.4 �l of
10 mM dNTPs, 0.3 �l reverse transcriptase, 0.5 �l of 10x buffer, 0.6 �l RNAse inhibitor diluted
1:10, and 0.5 �l H2O2. The mixture was incubated at 16 °C for 30 min, at 42 °C for 30 min, and at
85 °C for 5 min. Subsequently RT-qPCR was performed in 20 �l of PCR reaction mix containing 1
�l 20x Taqman MicroRNA Assay—which in turn contained PCR primers and probes (5’-FAM)—
9
10 �l of 2x TaqMan Universal PCR Master mix No AmpErase® UNG (Applied Biosystems), and 5
�l of reverse-transcribed product. The reaction was first incubated at 95 °C for 2 min followed by
40 cycles at 95 °C for 15 sec and at 60 °C for 1 min. Data were analyzed with an Opticon Monitor 2
RT-PCR machine (MJR, Bio-Rad Laboratories, Hercules, CA, USA), with the automatic
comparative threshold (Ct) setting for adapting baseline. Detection thresholds were set at 35 Ct. The
relative amount of miR-146a was calculated using the Ct method with �Ct = Ct(MiR146a)-
Ct(RNU44).
Protein extraction and immunoblotting
Total proteins were extracted using RIPA buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1 % SDS,
1.0 % Triton X-100, 5 mM EDTA, pH 8.0) containing protease inhibitor cocktail (Roche Applied
Science, Indianapolis, IN, USA). Protein concentration was determined using Bradford Reagent
(Sigma-Aldrich, St. Louis, MO, USA). Total protein extracts (40 �g) were separated by 10 % SDS-
PAGE and transferred to nitrocellulose membranes (Whatman, Dassel, Germany). Membranes were
blocked in TBS with 0.1% Tween20 (TBS-T) containing 5 % fat-free dry milk for 60 min and then
incubated overnight at 4 °C with primary rabbit polyclonal antibodies against IRAK-1 (MBL
International, Woburn, MA, USA), TRAF-6 and �-actin (both from Santa Cruz Biotechnology,
Santa Cruz, CA, USA) all diluted 1:10,000; �-actin was used to check the uniformity of blotting.
Membranes were washed in TBS-T and incubated for 60 min with the secondary antibody diluted
1:10,000 (Sigma-Aldrich, St. Louis) followed by washing in TBS-T. Proteins were visualized by
ECL according to the instructions of the manufacturer (Amersham, Piscataway, NJ, USA) and
quantified using Quantity One software (Bio-Rad).
�
Electrophoretic mobility shift assay (EMSA)
Native whole cell extracts were prepared in ice using a lysis buffer containing 50 mM Tris-HCl, pH
7.5, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1% TRITON X-100, 0.5% NP-40, 10% glycerol, 2
mM DTT, 4 mM AEBSF, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, 10 μg/ml leupeptin, 10 μg/ml
10
pepstatin, 10 mM �-glycerolphosphate, and 2 mM p-nitrophenyl phosphate. Protein concentration
was determined according to Bradford [28]. The NF-�B-speci�c double-stranded oligonucleotide
probe (5’-TCA ACA GAG GGG ACT TTC CGA GAG GCC-3’) was end-labeled with [�-32P]ATP,
by T4 polynucleotide kinase (New England Biolabs) and puri�ed through Sephadex G-25. Cell
extracts (10 μg) were incubated for 30 min at room temperature with labeled oligonucleotide in a
reaction containing 0.2 mg/ml of poly(dI-dC). DNA–protein complexes were resolved by
electrophoresis on 5% non-denaturating polyacrylamide gels in a TBE buffer system at a constant
voltage of 170 V, for 3 h at 4 °C. The gels were dried and subjected to autoradiography in a GS250
Molecular Imager system and to densitometric analysis with Molecular Analyst software (Bio-Rad).
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Statistical analysis
Data are the means ± standard deviation (S.D.) of independent experiments. Independent samples t
test was used to determine whether differences between samples were significant. P values < 0.05
were considered significant.
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Results
MiR-146a expression, IRAK-1 protein levels and amount of IL-6 released during HUVEC
replicative senescence
The senescent status of HUVEC was verified by SA-�-gal staining; accordingly cells were defined
as “young” at the second passage (P2) and as “senescent” at P13 (P2 vs. P13: 5 % ± 3 % vs. 87 % ±
7 %) (Fig. 1a). HUVEC SASP acquisition was evaluated by a combination of three biomarkers:
miR-146a expression, the level of its target protein IRAK-1, and the amount of IL-6 released into
the culture medium. For the sake of simplicity we designated this combination as MIRAKIL
(MicroRNA, IRAK, IL-6).
Significant MIRAKIL modulation was observed in senescent vs. young cells. In particular,
senescent cells were characterized by a 5-fold greater expression of miR-146a (Fig. 1b, A) and by a
significant 12-fold decrease in IRAK-1 protein levels, a miR-146a validated target (P2 vs. P13, %
vs. �-actin; 76.46 ± 7.10 vs. 6.2 ± 1.05) (Fig. 1b, C). The amount of IL-6 released into the medium
by senescent HUVEC was about 5-fold greater than that released by P2 cells (IL-6 pg/ml, P2 vs.
P13; 31 ± 09 vs. 145 ± 30) (Fig. 1b, B).
.
Effect of lipopolysaccharide on MIRAKIL and NF-�B activation
In this study we first tested the MIRAKIL modulation induced by a proinflammatory stimulus.
Since it has been reported that LPS can modulate MIRAKIL in THP-1 cells [23], we tested the
effect of 1 �g/ml LPS on MIRAKIL in young and senescent HUVEC (Fig. 2) using THP-1 cells as
a positive control. Thirty-minute incubation with LPS significantly enhanced miR-146a expression
in young but not in senescent cells (Tab. 1), whereas 5 h incubation was needed to achieve a
significant increase in senescent cells (Tab. 1 and Fig. 2a, A). Similar effects were seen in the
amount of IL-6 released into the medium (Tab. 1 and Fig. 2a, B). LPS stimulation for 5 h also
affected MIRAKIL in THP-1 cells, where miR-146a expression increased about 140-fold (Fig. 2a,
C), whereas in young and senescent HUVEC the increase was 1.8- and 2.5-fold, respectively (Fig.
13
2a, A). In contrast, LPS induced a similar increase in IL-6 release both in THP-1 cells (about 2-
fold) and in HUVEC, both young (about 5-fold) and senescent (about 2-fold) (Fig. 2a, B and D).
MiR-146a modulation thus seemed to affect IL-6 release to a similar extent in both cell types, even
though it was weaker in HUVEC. Interestingly, LPS proinflammatory stimulation mainly induced
TNF-alpha release in monocytic cells and IL-6 release in endothelial cells (Fig. 2b).
Interestingly, miR-146a expression and IL-6 levels reverted to baseline faster in young HUVEC
(about 24 h) than in senescent HUVEC (about 48 h) (Fig. 2a, A and B).
After 5 h LPS exposure IRAK-1 protein levels were significantly reduced both in HUVEC (Fig. 2c,
A and B) and in THP-1 cells (Fig. 2c, C), whereas the level of TRAF-6 protein was not
significantly changed (Fig. 2c, A, B and C).
Figure 2d shows NF-�B activation in young and senescent HUVEC at baseline and after 30 min
and 5 h stimulation with 1 �g/ml LPS. NF-�B activation was significantly higher in senescent than
in younger HUVEC. Moreover, 30 min incubation with 1 �g/ml LPS induced a significant increase
in NF-�B activation both in young and senescent cells, whereas NF-�B activation reverted to basal
levels after 5 h LPS stimulation
Effects of short-term CoQ10H2 supplementation on MIRAKIL and NF-�B activation
Young and senescent HUVEC were treated with LPS and CoQ10H2 to explore the anti-
inflammatory effects of CoQ10. We first tested the stability of the reduced form of CoQ10H2 in the
culture medium, showing that CoQ10H2 was stable for at least 48 h (data not shown). Then we
tested the ability of young and senescent HUVEC to internalize CoQ10H2 and its effects on cell
viability. To do so HUVEC were incubated with rising CoQ10H2 concentrations (from 0.1 to 100
μM) for 24 h; this treatment had no toxic effects (Supplementary Fig. 1). The 10 �M concentration
was selected for the in vitro studies because it is in the same order of magnitude as the plasma
concentrations achieved in humans receiving standard CoQ10 oral supplements [29]. Figure 3
illustrates the ability of young and senescent HUVEC to internalize CoQ10. Its intracellular
14
concentration was increased about 7-fold both in young and senescent cells (Fig. 3a). LPS treatment
induced no appreciable effect on CoQ10 intracellular levels. Elevation in CoQ10 content
significantly improved the cell oxidative status in all conditions, producing a significant decrease in
the percentage of oxidized CoQ10 (Fig. 3b).
The anti-inflammatory effect of CoQ10H2 was assessed by pre-treating young and senescent
HUVEC with 10 �M CoQ10H2 prior to LPS exposure. As shown in Fig. 4a, CoQ10H2 exerted a
stronger anti-inflammatory effect on young cells, probably via miR-146a modulation. In fact, LPS-
induced miR-146a expression was significantly reduced in CoQ10H2-treated young cells (Fig. 4a,
fold-change after 30 min LPS treatment in P2 HUVEC without and with CoQ10H2 pretreatment:
2.3 ± 0.31 vs. 0.8 ± 0.25), whereas the same effect in senescent cells required 5 h LPS treatment
(Fig. 4a, miR-146a fold-change after 5 h LPS treatment in P13 HUVEC without and with CoQ10H2
pretreatment: 2.01 ± 0.25 vs. 0.95 ± 0.21).
CoQ10H2 pretreatment significantly reduced NF-�B activation induced by 30 min LPS treatment
both in young and in senescent cells (Fig. 4b); 5 h LPS exposure had no significant effect on NF-
�B activation (Fig. 2d), therefore CoQ10H2 pretreatment did not affect 5 h LPS treatment (Fig. 4b).
Similarly, CoQ10H2 pretreatment limited the LPS-induced decrease in IRAK-1 protein after 5 h
LPS treatment in young and senescent cells (Fig. 4c).
In addition, CoQ10H2 pretreatment significantly reduced LPS-induced IL-6 release by young cells
at both 30 min and 5 h, whereas a slight, non-significant reduction was observed in senescent cells
at 5 h (Fig. 5).
Interestingly, CoQ10H2 pretreatment significantly reduced IL-6 release by young cells also in the
absence of LPS stimulation, whereas it did not curb IL-6 release by senescent cells not exposed to
the LPS stimulation (Fig. 5, Table 1). A similar effect was observed on miR-146a expression
(Table 1).
15
Effects of long-term CoQ10H2 supplementation on MIRAKIL
The effect of prolonged CoQ10H2 incubation on MIRAKIL was evaluated by supplementing
HUVEC with CoQ10H2 until acquisition of the senescent phenotype (which took about 60 days).
The percentage of �-gal-stained cells and the levels of miR-146a expression in the absence /
presence of CoQ10H2 supplementation during replicative passages are reported in Fig. 6. The
proportion of �-gal-positive cells was significantly reduced at P9 and P10 in presence of CoQ10H2
supplementation compared with cells without CoQ10H2 supplementation (% of �-gal-positive cells,
- CoQ10H2 vs. + CoQ10H2; P9: 20 ± 3 vs. 10 ± 2; P10: 23 ± 3 vs. 12 ± 2) (Fig. 6 A). A
concomitant, significant decline in miR-146a expression was seen in CoQ10H2-supplemented cells
at P9 (miR-146a expression in arbitrary units, - CoQ10H2 vs. + CoQ10H2, P9: 0.14 ± 0.3 vs. 0.06 ±
0.02) (Fig. 6 B). However, CoQ10H2 supplementation was unable to curb IL-6 released by HUVEC
undergoing replicative senescence (data not shown).
16
Discussion
The main findings of our study are that CoQ10H2 exerts an anti-inflammatory effect on endothelial
cells both in basal condition and after LPS stimulation, and that the strength of this effect depends
on the senescent status of supplemented cells.
In a previous study we described a combination of biomarkers associated with SASP acquisition by
HUVEC that included miR-146a expression, the level of its target protein IRAK-1, and the amount
of IL-6 released into the culture medium [20]. In this study we called this marker combination
“MIRAKIL”. In the previous investigation we demonstrated a direct correlation between the
MIRAKIL components by transfecting young and senescent HUVEC with miR-146a mimic and
antagomir and documenting the consequent decrease and increase in the levels of its target protein
IRAK-1 [20].
The MIRAKIL biomarkers belong to the NF-�B proinflammatory pathway, which is activated by
toll like receptor family (TLR) signaling. Interestingly, intracellular ROS are capable of
influencing, or being influenced by, NF-�B activity [22]. Therefore it is not surprising that ROS
could modulate an NF-�B response. Accordingly CoQ10, acting as a free-radical scavenger, could
modulate, directly or via NF-�B, the expression of both coding and non-coding (miR-146a) genes.
To highlight the molecular mechanisms involved in the anti-inflammatory action of CoQ10 we
therefore explored its ability to modulate MIRAKIL and NF-kB activation. Our findings clearly
show that both young and senescent HUVEC incorporate CoQ10, and that after LPS stimulation
NF-kB activation and miR-146a expression are reduced both in young and senescent HUVEC.
However, CoQ10 had a more pronounced anti-inflammatory effect on young than senescent
endothelial cells. In fact, CoQ10H2 pretreatment significantly affected MIRAKIL, reducing miR-
146a expression and IL-6 release and raising IRAK-1 protein levels in young cells both at baseline
and after LPS stimulation. In senescent cells CoQ10H2 pretreatment significantly curbed the
increase of miR-146a expression and the decrease of IRAK-1 protein levels, and did so only after
17
LPS treatment, not at baseline; however, and most importantly, it failed to reduce IL-6 release. In
fact baseline IL-6 levels and NF-�B activation were both significantly higher in senescent than in
younger cells. Consequently, the effect of LPS in further enhancing IL-6 release is greater in young
than in senescent HUVEC, as is the anti-inflammatory effect of CoQ10H2. These data suggest that
antioxidants can participate in the negative feedback loop that curbs IL-6 release via miR-146a in
HUVEC. However, the anti-inflammatory action of CoQ10 is efficient in younger cells living in a
milieu devoid of proinflammatory stimuli, but less so in a milieu rich in proinflammatory cytokines,
like the culture medium of senescent cells. We previously documented that senescent HUVEC
release large amounts of a number of proinflammatory cytokines in surrounding medium, including
tumor necrosis factor (TNF)-alpha and IL-6 [22]. TNF-alpha and IL-6, can induce continuous NF-
�B signaling activation, acting by autocrine and paracrine mechanisms. In fact, human endothelial
cells respond both to LPS and to inflammatory cytokines, which enhance NF-�B activity [30]. LPS
acts mainly through TLR-4, IRAK-1 and TRAF-6 signaling, whereas TNF-alpha acts through
specific TNF-R1 and TNF-R2 receptors that converge on TRAF-2 protein, which in turn can
activate NF-�B and JNK signaling pathways [30]. Thus, LPS and interleukins (TNF-alpha and IL-
6) may induce NF-�B activation through pathways that are differentially regulated by miR-146a.
Interestingly, LPS-induced inflammatory signals are known to mediate activation of NF-�B through
ROS [31]. It has recently been shown that various antioxidants, such as vitamin C and vitamin E,
inhibit ROS-induced inflammatory responses by preventing activation of NF-�B [32]. The present
study indicates that CoQ10 prevents LPS-induced miR-146a expression, which in turn is induced
directly by NF-�B. Further, CoQ10 was able to reduce the consequent release of inflammatory
mediators, like IL-6. Interestingly, this ability of CoQ10 seems to be impaired in senescent cells,
which are characterized by a distinctive inflammatory noise (inflammaging) and by a delayed
response to LPS. In fact, our data show that young cells respond faster than senescent ones to LPS
stimulation, suggesting a more efficient stimulus-induced response by young cells. Similar to short-
term CoQ10 supplementation, prolonged HUVEC treatment with CoQ10H2 did affect MIRAKIL,
18
but did not curb IL-6 release. These data confirm our previous finding that SASP acquisition during
replicative senescence is not entirely controlled by the IRAK-1 pathway [20]. Indeed they suggest
an IRAK-1-dependent IL-6 transcription process in young cells, with the transcription likely being
induced predominantly by LPS, whereas the increased IL-6 transcription seen in senescent cells
would be IRAK-1-independent, probably via TNF-alpha binding and NF-�B-independent
signalling.
Notably MIRAKIL levels showed greater variability in senescent than in young cells, and may thus
have prevented achievement of significant differences in the former cells. Indeed earlier work by
our group showed a high variability of the systemic inflammatory status in elderly-old humans in
vivo [33] and a higher degree of variability in IL-6 released by cells from elderly individuals
compared with cells from younger subjects [34].
The anti-inflammatory effect of CoQ10H2 on endothelial cells demonstrated here could have
clinical relevance. Mounting evidence has been showing that endothelial inflammation and/or
senescence status contribute greatly to the development of multiple vascular diseases [35, 36].
Early work on miR-146a expression indicated that it is involved in innate immunity against
bacterial pathogens. Further analysis of its biological role disclosed that its expression regulates
production of many cytokines both in cells responding to an immunological stimulus and in cells
undergoing replicative senescence [20]. Interestingly, these miR-146a effects were documented not
only in macrophages [21, 37, 38], but also in lung epithelial [39], intestinal epithelial [40],
fibroblast [41, 18], astrocyte [42], dental pulp [43] and endothelial cells [22, 44]. These findings
suggest that several cell types can contribute to the maintenance of a systemic inflammatory status.
Here we showed that, probably as a consequence of SASP acquisition, endothelial senescent cells
release an amount of IL-6 that is similar to the amount released by LPS-stimulated young
endothelial cells. These data suggest that senescent endothelial cells may contribute to activate and
maintain a systemic inflammatory response similar in effectiveness to the one generated by young
cells exposed to microbial infections. Thus, SASP acquisition by endothelial cells during aging
19
could greatly contribute to the age-related low-grade systemic inflammatory status that has been
called “inflammaging” [45, 46]. It is reasonable to hypothesize that young endothelial cells receive
more effective CoQ10H2 supplementation compared with senescent cells, whose milieus are
characterized by large amounts of proinflammatory molecules that may impair the antioxidant
effect. Indeed, prolonged CoQ10H2 supplementation seems to delay acquisition of the senescent
phenotype, even though SASP acquisition cannot be delayed indefinitely. Thus, despite the
unchanged ability of older cells to incorporate CoQ10, ubiquinol supplementation in older humans
may be insufficient to affect the age-associated constitutive inflammatory status [47, 48], even
though in our experimental conditions it was nonetheless capable to sustain an acute response to
LPS. It is also conceivable that greater benefit would accrue from CoQ10 supplementation provided
at an earlier age.
20
Supporting Information list
The viability of young (P2) and senescent (P13) HUVEC cultured with rising CoQ10H2
concentrations (from 0.1 to 100 μM) for 24 h was evaluated by (3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT). This treatment had no toxic effects (Supplementary Fig. 1).
Limitations
This study was performed in vitro, therefore further work is needed to establish optimal CoQ10H2
supplementation dosages in vivo. Moreover, even though our data demonstrate that CoQ10H2 exerts
an anti-inflammatory action on endothelial cells and that such action is mediated by miR-146a
modulation, we did not establish whether this effect is a cause or a result of miR-146a modulation.
Research Funding:� The present study was supported by the following Italian Health Ministry
grants: Università Politecnica delle Marche RFPS-2007-6-654027 and INRCA - Programma
Strategico RFPS-2007-6-654027. The work was also supported by grants from the Università
Politecnica delle Marche to ADP and FO.
Acknowledgement. The authors are grateful to Dr S. Modena for the language revision.
Author Contributions: All of the authors contributed significantly to the work: (1) conception and
design of the study: FO, RL, RF; (2) data collection, analysis and interpretation: LG, LB, FP, SD,
PO, SS, FB, MC, LP, MM; and (3) drafting and revising the manuscript for intellectual content: FO,
RL, LT, MRR, GPL, CF, ADP.
Conflict of Interest Statement
None of the authors have any conflict of interest.
21
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27
Table 1. MiR-146a expression and levels of IL-6 released by young (P2) and senescent (P13)
HUVEC in different experimental conditions
Fig. 1a. Percentage of �-Gal-positive cells during HUVEC replicative senescence
The percentage of �-gal-stained HUVEC during replicative senescence (from P1 to P13). P =
number of culture passages; * T-test, p<0.01, senescent vs. young (P2) cells. Data are the means ±
S.D. of 3 independent experiments.
Fig. 1b. MiR-146a expression (A), amount of IL-6 released into the culture medium (B), and
IRAK-1 protein levels (C) during HUVEC replicative senescence
MiR-146a expression (fold-increase vs. P2 considered as 1) (A) and amount of IL-6 released into
the culture medium (B) by HUVEC during replicative senescence (from P1 to P13). IRAK-1
protein levels in young (P2) and senescent (P13) HUVEC (C). P = number of culture passages;
*p<0.01 senescent vs. young cells. Data are the means ± S.D. of 3 independent experiments.
Fig. 2a. MiR-146a expression levels and IL-6 release induced in young (P2) and senescent
(P13) HUVEC and in THP-1 cells by treatment with 1 �g/ml LPS
Relative MiR-146a expression (A) and IL-6 release into the culture medium (B) measured in young
(P2) and senescent (P13) HUVEC without LPS treatment and treated with 1 �g/ml LPS for 30 min
28
and 5 h. Relative MiR-146a expression (C) and IL-6 release into the culture medium (D) measured
in THP-1 cells without LPS treatment and treated with 1 �g/ml LPS for 30 min.
A.u. = arbitrary units; NT= LPS-untreated cells; *p<0.01 vs. NT; P = number of culture passages.
Data are the means ± S.D. of 3 independent experiments.
Fig. 2b. IL-6 and TNF-a (pg/ml) released by HUVEC and THP-1 LPS-untreated and treated
with 1 �g/ml LPS
IL-6 and TNF-alpha concentration was measured using an high-sensitivity enzyme-linked
immunosorbent assay (ELISA) kit. *p<0.01 vs. LPS-untreated cells; P = number of culture
passages. Data are the means ± S.D. of 3 independent experiments.
Fig. 2c. IRAK-1 and TRAF-6 protein levels induced by treatment with 1 �g/ml LPS in young
(P2) and senescent (P13) HUVEC and in THP-1
IRAK-1 and TRAF-6 protein levels in young (P2) and senescent (P13) HUVEC without LPS
treatment and treated with 1 �g/ml LPS for 30 min and 5 h, and in THP-1 cells without LPS
treatment and treated with 1 �g/ml LPS for 5 h. Western blot and densitometric analysis are shown.
Densitometric analysis was performed with Quantity-One software (Bio-Rad). Protein expression
values are reported as percentage vs. actin. NT = LPS-untreated cells; * T-test, p<0.01 vs. NT; P =
number of culture passages. Data are the means ± S.D. of 3 independent experiments.
Fig. 2d. NF-�B activation in young (P2) and senescent (P13) HUVEC by treatment with 1
�g/ml LPS
29
NF-�B activation was evaluated by electrophoretic mobility shift assay (EMSA) and reported as
volume-adjusted counts/mm2. Densitometric analysis was performed by the Molecular Analyst
software (Bio-Rad). Autoradiographic analysis of dried gels was performed by the GS250
Molecular Imager system (Bio-Rad). Data are the means ± S.D. of 3 independent experiments.
Fig. 3a. Intracellular CoQ10H2 levels in young (P2) and senescent (P13) HUVEC in the
absence / presence of 10 �M CoQ10H2 supplementation and 1 �g/ml LPS stimulation
Intracellular CoQ10H2 levels in young (P2) and senescent (P13) HUVEC are reported as mg/106
cells. NT = LPS-untreated cells; *p<0.01 vs. CoQ10H2-unsupplemented cells; P = number of
culture passages. Data are the means ± S.D. of 3 independent experiments.
Fig. 3b. Percentage of oxidized CoQ10 in young (P2) and senescent (P13) HUVEC in absence /
presence of 10 μM CoQ10H2 supplementation and 1 �g/ml LPS stimulation
Percentage of oxidized intracellular CoQ10 in HUVEC without LPS treatment and treated with 1
�g/ml LPS for 30 min and 5 h. Ox = oxidized; NT= LPS-untreated cells; *p<0.01 vs. CoQ10H2-
unsupplemented cells; # p<0.01 vs. NT; P = number of culture passages. Data are the means ± S.D.
of 3 independent experiments.
Fig. 4a. Effect of CoQ10H2 incubation on miR-146a expression in LPS-treated young (P2) and
senescent (P13) HUVEC
30
MiR-146a relative expression in young (P2) and senescent (P13) HUVEC without LPS treatment
(NT) and treated with 1 �g/ml LPS for 30 min and 5 h. *p<0.01 vs. CoQ10H2-unsupplemented
cells. P = number of culture passages. Data are the means ± S.D. of 3 independent experiments.
Fig. 4b. Effect of CoQ10H2 incubation on NF-�B activation in LPS-treated young (P2) and
senescent (P13) HUVEC
NF-�B activation in young (P2) and senescent (P13) HUVEC cells treated with 1 �g/ml LPS for 30
min and 5 h. *T test, p<0.05 vs. LPS-untreated cells. P = number of culture passages.
NF-�B activation was evaluated by electrophoretic mobility shift assay (EMSA) and reported as
volume-adjusted counts/mm2. Densitometric analysis was performed by the Molecular Analyst
software (Bio-Rad). Autoradiographic analysis of dried gels was performed by the GS250
Molecular Imager system (Bio-Rad).
Fig. 4c. Effect of CoQ10H2 incubation on IRAK-1 expression in LPS-treated young (P2) and
senescent (P13) HUVEC
IRAK-1 protein levels in young (P2) and senescent (P13) HUVEC without LPS treatment and
treated with 1 �g/ml LPS for 5 h. Western blot and densitometric analysis are shown. Densitometric
analysis was performed with Quantity-One software (Bio-Rad). Protein expression values were
reported as percentage vs. actin. NT = LPS-untreated cells; * t-test, p<0.01 vs. NT; P = number of
culture passages. Data are the means ± S.D. of 3 independent experiments.
31
Fig. 5. IL-6 release in young (P2) and senescent (P13) HUVEC stimulated with 1 �g/ml LPS in
the absence / presence of CoQ10H2 supplementation
IL-6 pg/ml released into the culture medium by young (P2) and senescent (P13) HUVEC without
LPS treatment and treated with 1 �g/ml LPS for 30 min and 5 h. Dotted lines indicate the amount of
IL-6 released by LPS-untreated and CoQ10H2-unsupplemented senescent (P13) cells. NT = LPS-
untreated cells; *p<0.01 vs. CoQ10H2-unsupplemented cells; # p<0.01 vs. NT cells; P = number of
culture passages. Data are the means ± S.D. of 3 independent experiments.
Fig. 6. Percentage of �-Gal-positive cells (A) and miR-146a expression levels (B) in HUVEC
during replicative senescence with and without CoQ10H2 supplementation
Percentage of �-gal-stained cells and levels of miR-146a relative expression in the absence /
presence of CoQ10H2 supplementation in HUVEC from P7 to P12. Data are the means of 3
independent experiments. *t-test: p<0.05; a.u. = arbitrary units; P = number of culture passages;
SA-�-Gal = Senescence Associated �-Galactosidase.
32
NT CoQ10H2 LPS 30 min CoQ10H2+
LPS 30 min
LPS 5 h CoQ10H2 +
LPS 5 h
P2 HUVEC
MiR-146a
(a.u.)
0.13±0.05 0.05±0.03#
# p = 0.024
0.32±0.04°
° p = 0.003
0.16±0.06#
# p = 0.004
0.27±0.03°
° p = 0.006
0.20±0.03#
# p = 0.048
IL-6
(pg/ml)
31±09 13±5#
# p = 0.003
89±34°
° p = 0.003
24±5#
# p = 0.003
100±14°
° p = 0.001
23±6#
# p = 0.001
P13 HUVEC
MiR-146a
(a.u.)
0.53±0.10*
* p = 0.001
0.50±0.09*
* p = 0.001
0.72±0.18*
* p = 0.001
0.70±0.10*
* p = 0.001
1.27±0.09*°
* p = 0.001
° p = 0.003
1.01±0.08*°#
* p = 0.001
° p = 0.013
# p = 0.045
IL-6
(pg/ml)
145±30*
* p = 0.001
150±45*
* p = 0.001
120±45*
* p = 0.001
129±19*
* p = 0.001
250±44*°
* p = 0.001
° p = 0.001
189±75*
* p = 0.001
a.u. = arbitrary units; NT= LPS-untreated cells; * P13 vs. P2, p<0.05; # CoQ10H2 supplementation
vs. non-supplementation, p<0.05 ;° LPS-treated vs. NT. LPS = 1�g/ml. CoQ10H2 = 10 �M.
33
Highlights 1. We explored the anti-inflammatory effect of CoQ10 on endothelial cells (HUVECs)
2. CoQ10H2 pretreatment reduces IL-6 release via miR-146a modulation in young cells
3. CoQ10 has a more pronounced anti-inflammatory effect on young than on senescent cells
4. The strength of CoQ10 effects depends on the senescence status of supplemented cells