Author's Accepted Manuscript

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

5

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).

11

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.

12

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

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