O iginal Con ibu ion
Mi ochond ial dys unc ion p omo ed by Po phy omonas gingi alis lipopolysaccha ide
as a possible link be ween ca dio ascula disease and pe iodon i is
Ped o Bullon
a
, Ma io Da id Co de o
b
, José Luis Quiles
c
, Juan Manuel Mo illo
a
,
Ma ia del Ca men Rami ez-To osa
d
, Mau izio Ba ino
e,
⁎
a
Depa men o Pe iodon ology, Den al School, Uni e si y o Se illa, Se illa, Spain
b
Cen o Andaluz de Biología del Desa ollo, Uni e sidad Pablo de Ola ide–CSIC, and Cen o de In es igación Biomédica en Red de En e medades Ra as, ISCIII, Se illa 41013, Spain
c
Depa men o Physiology, Ins i u e o Nu i ion and Food Technology “José Ma aix,”Biomedical Resea ch Cen e , Uni e si y o G anada, G anada, Spain
d
Depa men o Biochemis y and Molecula Biology II, Ins i u e o Nu i ion and Food Technology “José Ma aix,”Biomedical Resea ch Cen e , Uni e si y o G anada, G anada, Spain
e
Depa men o Biochemis y, Biology, and Gene ics, Facul y o Medicine, Uni e si à Poli ecnica delle Ma che, 60100 Ancona, I aly
abs ac a icle in o
A icle his o y:
Recei ed 11 Oc obe 2010
Re ised 3 Feb ua y 2011
Accep ed 15 Feb ua y 2011
A ailable online 24 Feb ua y 2011
Keywo ds:
Pe iodon i is
CoQ
10
Mi ochond ial memb ane po en ial
Apop osis
Reac i e oxygen species
Ca dio ascula disease
F ee adicals
Oxida i e s ess is one o he ac o s ha could explain he pa hophysiological mechanism o inflamma o y
condi ions ha occu in ca dio ascula disease (CVD) and pe iodon i is. Such inflamma o y esponse is o en
e oked by specific bac e ia, as he lipopolysaccha ide (LPS) o Po phy omonas gingi alis is a key ac o in his
p ocess. The aim o his esea ch was o s udy he ole o mi ochond ial dys unc ion in pe iphe al blood
mononuclea cells (PBMCs) om pe iodon i is pa ien s and o e alua e he influence o LPS on fib oblas s o
be e unde s and he pa hophysiology o pe iodon i is and i s ela ionship wi h CVD. PBMCs om pa ien s
showed lowe CoQ
10
le els and ci a e syn hase ac i i y, oge he wi h high le els o ROS p oduc ion. LPS- ea ed
fib oblas s p o oked inc eased oxida i e s ess and mi ochond ial dys unc ion by a dec ease in mi ochond ial
p o ein exp ession, mi ochond ial mass, and mi ochond ial memb ane po en ial. Ou s udy suppo s he
hypo hesis ha LPS-media ed mi ochond ial dys unc ion could be a he o igin o oxida i e s ess in pe iodon al
pa ien s. Abno mal PBMC pe o mance may p omo e oxida i e s ess and al e cy okine homeos asis. In
conclusion, mi ochond ial dys unc ion could ep esen a possible link o unde s anding he in e ela ionships
be ween wo p ominen inflamma o y diseases: pe iodon i is and CVD.
© 2011 Else ie Inc. All igh s ese ed.
Wi hin mos cells, he mi ochond ion is he main sou ce o
eac i e species, which a e by-p oduc s o cell ene gy p oduc ion.
Inside mi ochond ia he p ima y eac i e oxygen species (ROS)
p oduced is supe oxide, mos o which is con e ed o hyd ogen
pe oxide by he ac ion o supe oxide dismu ase. The mi ochond ial
p oduc ion o supe oxide has been asc ibed o se e al elec on
anspo chain enzymes, including complex I and complex III. These
complexes along wi h coenzyme Q
10
(CoQ
10
) may leak elec ons,
which in u n may in e ac wi h oxygen, hus o ming ROS [1,2]. All
condi ions able o al e mi ochond ial e ficiency can enhance ROS
p oduc ion, wi h a di ec and c i ical e ec on oxida i e s ess. In his
espec , CoQ
10
also has an impo an lipid-soluble an ioxidan ac i i y,
in addi ion o i s well-known edox ole as elec on/p o on ca ie ,
and is syn hesized by he o ganism unde physiological condi ions.
Oxida i e s ess is defined as a pe sis en imbalance be ween he
p oduc ion o highly eac i e molecula species (e.g., ROS and eac i e
ni ogen species (RNS)) and an ioxidan de enses [3]. When he
p oduc ion o ROS/RNS exceeds he capaci y o an ioxidan de enses,
oxida i e s ess may ha e a ha m ul e ec on he unc ional and
s uc u al in eg i y o biological issue. Oxida i e s ess is in ol ed in
a he oscle osis, hype ension, insulin esis ance, hea ailu e, and he
aging p ocess [3]. Specifically, mi ochond ial oxida i e s ess has been
ela ed o myoca dial dys unc ion and hea ailu e [4]. Mos o he
oxida i e s ess diseases a e conside ed ch onic diseases and hei
influence on mo bidi y and mo ali y, in he u u e, will be a c i ical
public heal h opic [5]. Among hem, ca dio ascula diseases (CVDs)
a e conside ed he majo cause o dea h in Wes e n coun ies [6] and
a e ela ed o some isk ac o s such as obesi y, diabe es, insulin
esis ance, and me abolic synd ome. Recen ly, CVDs ha e also been
associa ed wi h an o al disease: pe iodon i is [7–11]. Pe iodon i is is a
gene ally ch onic diso de cha ac e ized by he b eakdown o he
oo h-suppo ing issues p oducing he loss o den i ion. The cause is
due o an ecological imbalance be ween he mic obial biofilm on ee h
and an impai ed hos inflamma o y esponse. One o he main
challenges is he sea ch o ac o s ha may explain hese ela ion-
ships. The e is e idence suppo ing he ole o inflamma ion in all he
F ee Radical Biology & Medicine 50 (2011) 1336–1343
Abb e ia ions: CAL, clinical a achmen le el; CoQ
10
,coenzymeQ
10
;CVD,
ca dio ascula disease; GM, gingi al ma gin; LPS, lipopolysaccha ide; PD, pe iodon al
p obing dep h; PBMC, pe iphe al blood mononuclea cell; RNS, eac i e ni ogen
species; ROS, eac i e oxygen species.
⁎Co esponding au ho . Fax: +39 0712204123.
E-mail add ess: [email p o ec ed] (M. Ba ino).
0891-5849/$ –see on ma e © 2011 Else ie Inc. All igh s ese ed.
doi:10.1016/j. ee adbiomed.2011.02.018
Con en s lis s a ailable a ScienceDi ec
F ee Radical Biology & Medicine
jou nal homepage: www.else ie .com/loca e/ ee adbiomed
p e iously indica ed pa hological e en s as a mee ing poin [11].
Oxida i e s ess is one o he main ac o s s udied ha may be able o
explain he pa hophysiological mechanism o inflamma o y condi ions
ha occu in a he oscle osis, CVD, and pe iodon i is. Se e al s udies
ha e demons a ed an inc ease in p oduc s om oxida i e damage in
plasmaand se um o subjec s wi h pe iodon i is compa ed wi h heal hy
indi iduals [12–14]. Mo eo e , he e is e idence o a dec eased
an ioxidan capaci y in subjec s wi h pe iodon i is, e alua ed by a ious
assays [15–17].
Pe iodon i is, as an in ec ious disease, has been ela ed also o a
specific g oup o bac e ia, h ee o which ha e been conside ed he
main pe iodon al pa hogens: Tanne ella o sy hia, Agg ega ibac e
ac inomyce emcomi ans, and Po phy omonas gingi alis [18]. In he
o al en i onmen he inflamma o y esponse is o en e oked by
specific bac e ia, such as P. gingi alis, able o p oduce an inflamma o y
esponse ha can be co ela ed wi h a he oscle osis and ul ima ely
h ombo ic complica ions [19].P. gingi alis bac e ial DNA has been
ound in co ona y a e y biopsy samples [20]. The lipopolysaccha ide
(LPS) o P. gingi alis is a key ac o in he de elopmen o pe iodon i is.
Gingi al fib oblas s, which a e he majo cons i uen s o gingi al
connec i e issue, may di ec ly in e ac wi h bac e ia and bac e ial
p oduc s, including LPS, in pe iodon al lesions. I has been sugges ed
ha gingi al fib oblas s play an impo an ole in he hos esponses
o LPS in pe iodon al disease [21].
The pu pose o his s udy was o s udy he ole o mi ochond ial
dys unc ion in pe iphe al blood mononuclea cells (PBMCs) om
pe iodon i is pa ien s. The influence o P. gingi alis LPS was also
in es iga ed in cul u ed human p ima y fib oblas s o p o ide new
app oaches ha could con ibu e o he comp ehension o he
pa hophysiology o pe iodon i is and i s ela ionship wi h CVD.
Ma e ials and me hods
Pa ien s
A o al o 58 pa ien s, all o e 35 yea s o age, a ending Se ille
Uni e si y Den al School, we e en olled in he s udy. All pa ien s who
we e accep ed ga e olun a y w i en in o med consen . P o ocol and
consen o ms had been p e iously app o ed by he Commi ee o
E hics and Resea ch o Se illa Uni e si y (16 Decembe 2006). All
pa ien s me he ollowing inclusion c i e ia: hey had mo e han 20
ee h, hey had no aken an ibio ics o an i-inflamma o y d ugs in he
p e ious 6 mon hs, hey we e no a ec ed by immunodeficiency, hey
we e gene ally heal hy, and hey had unde gone no p e ious
pe iodon al ea men . Pa ien s we e ec ui ed o e a pe iod o
10 mon hs and one blood sample was aken om each pa ien as he o
she was ec ui ed.
Rou ine labo a o y es s yielded no mal esul s o glucose 94.7±
8.7 mg/dl (no mal alues (n ) 76–110), u ea 36.3±10.10 mg/dl (n
10–45), u ic acid 5.3±1.2 mg/dl (n 2.5–7.5), o al p o ein 7.1±
0.4 g/dl (n 6.6–8.7), c ea inine 0.5±0.1 mg/dl (n 0.5–1.1), aspa a e
amino ans e ase 24.3±12.5 mU/ml (n 10–40), alanine amino-
ans e ase 25.2±15.1 mU/ml (n 10–40), choles e ol 192.7±
42.3 mg/dl (n b220), and iglyce ides 97.1±44.8 mg/dl (n 70–
170). Also, blood p essu e yielded no mal esul s (sys olic blood
p essu e115±10 mm Hgand dias olic blood p essu e 70±12 mm Hg).
A baseline pe iodon al examina ion was pe o med, and a single
examine collec ed ull medical and den al his o ies. A single ained
den al examine eco ded pe iodon al da a. The pe iodon al p obing
dep h (PD) and he ecession o he gingi al ma gin (GM) ela i e o he
cemen oenamel junc ion a six si es pe oo h we e eco ded. Clinical
a achmen le el (CAL)was calcula edby adding ecession o PD. PD and
CAL we e eco ded o he nea es highes millime e by means o he
No h Ca olina pe iodon al p obe (Hu-F iedy, Chicago, IL, USA), 15 mm
in leng h and 0.35 mm in diame e . Acco ding o he c i e ia es ablished
by Mach ei e al. [22], he clinical en i y o pe iodon i is is based on he
p esence o CAL≥6 mm in wo o mo e ee h and one o mo e si es
wi h PD≥5 mm. Pa ien s we e di ided in o wo g oups: one wi h
pe iodon i is (n=38) and he o he wi hou pe iodon i is (n=20).
Blood mononuclea cell and fib oblas cul u es
Hepa inized and coagula ed blood samples we e collec ed omeach
pa ien and cen i uged a 3800 g o 5 min, and he sepa a ed plasma
and se um we e s o ed a −80 °C. PBMCs we e pu ified by isopycnic
cen i uga ion using His opaque-1119 and His opaque-1077 (Sigma
Chemical Co., S . Louis, MO, USA). Mononuclea cells we e cul u ed a
37 °C in a 5% CO
2
a mosphe e in RPMI 1640 medium supplemen ed wi h
L-glu amine, an an ibio ic/an imyco ic solu ion (Sigma Chemical Co.),
and 10% e al bo ine se um (Gibco, In i ogen, Eugene, OR, USA).
Fib oblas s om he skin o heal hy olun ee s (30 yea s o age)
we e cul u ed in DMEM (4500 mg/L glucose, L-glu amine, py u a e;
Gibco, In i ogen) supplemen ed wi h 20% e al bo ine se um (FBS;
Gibco, In i ogen) and an ibio ics (Sigma Chemical Co.). Cells we e
incuba ed a 37 °C in a 5% CO
2
a mosphe e.
Fib oblas ea men
Fib oblas s we e cul u ed wi h 10 μg/ml LPS o P. gingi alis (Nuclibe
S.A., Spain) in he absence o p esence o a known dose, 30 μM, o CoQ
10
o 24 h [23]. LPS used o ea men o cell cul u es was dilu ed wi h
e al bo ine se um.
Measu emen o CoQ
10
le els
Lipid ex ac ion om PBMCs and skin fib oblas s was pe o med as
desc ibed p e iously [23]. Coenzyme Q
9
was used as an in e nal
s anda d. B iefly, cells we e lysed wi h 1% SDS and o exed o 1 min. A
mix u e o e hanol:isop opanol (95:5) was added and he samples we e
o exed o 1 min. To eco e CoQ
10
, 5 ml o hexane was added and
samples we e cen i uged a 1000 g o 5 min a 4 °C. The uppe phases
om h ee di e en ex ac ions we e eco e ed and d ied by a o a o y
e apo a o (Ro a apo R-210; Büchi Labo echnik AG, Flawil,
Swi ze land). Lipid ex ac was esuspended in 1 ml e hanol, d ied in
a Speed-Vac (Exp ess SC250EXP; The mo Fishe Scien ific. Wal ham,
MA, USA), and kep a −20 °C un il used. Samples we e suspended in
60 μl o e hanol be o e HPLC injec ion. Lipid componen s we e sepa a ed
by a Beckmann 166–126 HPLC sys em equipped wi h a 15-cm K omasil
C-18 column in a column o en se o 40 °C, wi h a flow a e o 1 ml/min
and a mobile phase con aining 65:35 me hanol:n-p opanol and
1.42 mM li hium pe chlo a e. CoQ
10
le els we e analyzed wi h a UV
de ec o (Sys em Gold 168; Beckman Coul e , B ea, CA, USA).
Mi ochond ial memb ane po en ial (ΔΨ
m
)
PBMCs and fib oblas s we e cul u ed in six-well pla es (35-mm-
diame e well) un il confluen . Mi oT acke (100 nM; Mi oT acke
Red CMXRos packing aging; Molecula P obes, Eugene, OR, USA) was
added and incuba ed o 30 min. Once he incuba ion was finished,
he cells we e ha es ed, incuba ed wi h esh medium, washed,
cen i uged (500 g), esuspended in RPMI medium, and analyzed by
flow cy ome y in an Epics XL cy ome e (Beckman Coul e ; exci a ion
wa eleng h 579 nm, emission wa eleng h 599 nm).
Measu emen o ci a e syn hase ac i i y
Ci a e syn hase-specific ac i i y in whole-cell ex ac s p epa ed
om PBMCs and fib oblas s was measu ed a 412 nm minus 360 nm
(13.6 mM
−1
cm
−1
) using 5,5-di hiobis-(2-ni obenzoic acid) o de ec
ee sul hyd yl g oups in coenzyme A as desc ibed p e iously [24].
1337P. Bullon e al. / F ee Radical Biology & Medicine 50 (2011) 1336–1343
Wes e n blo ing o mi ochond ial p o ein
Whole cellula lysa e om fib oblas s was p epa ed by gen le
shaking wi h a bu e con aining 0.9% NaCl, 20 mM T is–HCl, pH 7.6,
0.1% T i on X-100, 1 mM phenylme hylsul onyl fluo ide, and 0.01%
leupep in. Elec opho esis was ca ied ou by 10–15% ac ylamide
SDS–PAGE. P o eins we e ans e ed o Immobilon memb anes
(Ame sham Pha macia, Pisca away, NJ, USA). Mouse an i-complex I
(39-kDa subuni ) and mouse an i-complex III (co e 1 subuni )
an ibodies we e used o de ec p o eins by Wes e n blo ing. P o eins
we e elec opho esed, ans e ed o ni ocellulose memb anes, and,
a e being blocked o e nigh a 4 °C, incuba ed wi h he espec i e
an ibody solu ion dilu ed a 1:1000. Memb anes we e hen p obed
wi h hei espec i e seconda y an ibody (1:2500). Immunolabeled
p o eins we e de ec ed by using a chemiluminescence me hod
(Immun S a HRP subs a e ki ; Bio-Rad Labo a o ies, He cules, CA,
USA). P o ein was de e mined by he B ad o d me hod [25].
Mi ochond ial ROS p oduc ion
Mi ochond ial ROS gene a ion in PBMCs and fib oblas s was assessed
using Mi oSOX ed, a ed mi ochond ial supe oxide indica o . Mi oSOX
ed is a no el fluo ogenic dye ecen ly de eloped and alida ed o highly
selec i e de ec ion o supe oxide in he mi ochond ia o li e cells [26].
Mi oSOX ed eagen is li e-cell pe mea i e and is apidly and selec i ely
a ge ed o he mi ochond ia. Once in he mi ochond ia, Mi oSOX ed
eagen is oxidized by supe oxide and exhibi s ed fluo escence.
Fluo escence mic oscopy
Cells g own on mic oscope slides in six-well pla es o 24 h we e
incuba ed wi h Mi oSOX ed o 30 min a 37 °C, washed wice in PBS,
fixed wi h 4% pa a o maldehyde in PBS o 0.5–1 h a oom empe a u e,
and washed wice wi h PBS. Cells we e hen incuba ed o 10 min a
37 °C wi h an i-cy och ome can ibody (In i ogen, Ba celona, Spain) o
label mi ochond ia. Slides we e analyzed by immunofluo escence
mic oscopy (Mi oSOX ed; exci a ion wa eleng h 555/28; emission
wa eleng h 617/73).
Flow cy ome y
App oxima ely 1×10
6
cells we e incuba ed wi h 1 μM Mi oSOX
ed o 30 min a 37 °C, washed wice wi h PBS, esuspended in 500 μl
o PBS, and analyzed by flow cy ome y in an Epics XL cy ome e
(Beckman Coul e ; exci a ion a 510 nm and fluo escence de ec ion
a 580 nm).
Analysis o apop osis
Apop osis in fib oblas s was assessed by obse ing nuclei
condensa ion by Hoechs 33342 (In i ogen, Molecula P obes)
s aining (0.05 μg/ml) and an i-ac i e caspase-3 (Cell Signaling
Technology, Be e ly, MA, USA) ac i a ion by immunofluo escence
mic oscopy and Wes e n blo . Ten andom fields and mo e han 500
cells we e coun ed in each expe imen o ob ain cell dea h
pe cen age conside ing he numbe o condensed nuclei. Cells we e
g own on 1-mm
2
glass co e slips o 24 h in DMEM cul u e medium
con aining 10% FBS. A e 24 h ea men , cells we e insed once wi h
PBS, fixed in 3.8% pa a o maldehyde o 5 min a oom empe a u e,
and pe meabilized in 0.1% saponin o 5 min. Fo immunos aining,
glass co e slips we e incuba ed wi h an i-ac i e caspase-3 (17 kDa)
an ibodies dilu ed 1:100 in PBS o 1–2 h a 37 °C in a humidified
chambe . Excess an ibody binding was emo ed by washing he
co e slips wi h PBS ( h ee imes, 5 min). The seconda y an ibodies, a
e ame hyl hodamine goa an i- abbi IgG (Molecula P obes)
dilu ed 1:100 in PBS, we e added and incuba ed o 1 h a 37 °C.
Co e slips we e hen insed wi h PBS o 3 min, incuba ed o 1 min
wi h PBS con aining Hoechs 33342 (1 μg/ml), and washed wi h PBS
( h ee imes, 5 min). Finally, he co e slips we e moun ed on o
mic oscope slides using Vec ashield moun ing medium (Vec o
Labo a o ies, Bu lingame, CA, USA) and analyzed using a fluo es-
cence mic oscope (BX 41, Olympus, Ba celona, Spain). Wes e n
blo ing was pe o med using s anda d me hods p e iously
desc ibed.
Table 1
Pe iodon al da a, CoQ
10
le el, and ci a e syn hase ac i i y in pe iodon i is and
nonpe iodon i is pa ien s.
Pe iodon i is (n=38) Nonpe iodon i is (n=20)
Age (yea s) 45±11 44.3±8
BMI (kg/m
2
) 27.7±2.7 24.6±1.6
CoQ
10
(pmol Q /mg p o ein) 60.2±16* 150.4±13.5
Ci a e syn hase (EA) 1.9±0.6* 8.6±0.3
Pe iodon al da a
GM 0.79±0.08* 0.18±0.02
PD 3.5±0.5* 1.9±0.2
CAL 4±0.3* 2.1±0.45
Den al plaque 45.1±4.8* 26.4±2.21
Gingi al bleeding 60.3±5.3* 41.31±5.9
Da a ep esen he means±SD. BMI, body mass index; CoQ
10
, coenzyme Q
10
; EA,
enzyma ic ac i i y; CAL, clinical a achmen le el; GM, ecession o he gingi al ma gin;
PD, pe iodon al p obing dep h.
*Pb0.001; significan ly di e en be ween pe iodon i is and nonpe iodon i is.
Fig. 1. ROS p oduc ion in pe iodon i is pa ien s. (A) ROS p oduc ion was analyzed in
PBMCs om pe iodon i is and nonpe iodon i is pa ien s by flow cy ome y as
desc ibed unde Ma e ials and me hods. Da a ep esen he means±SD o h ee
sepa a e expe imen s. *Pb0.001 be ween pe iodon i is and nonpe iodon i is pa ien s.
(B) E ec o CoQ10 on ROS gene a ion. PBMCs o a ep esen a i e pe iodon i is pa ien
and a nonpe iodon i is pa ien we e ea ed wi h 30 μM CoQ
10
o 24 h. Da a ep esen
he means±SD o h ee sepa a e expe imen s. *Pb0.001 be ween pe iodon i is and
nonpe iodon i is; **Pb0.001 be ween he absence and he p esence o CoQ
10
.
1338 P. Bullon e al. / F ee Radical Biology & Medicine 50 (2011) 1336–1343
S a is ical analysis
All esul s a e exp essed as means±SD unless s a ed o he wise. The
unpai ed S uden es was used o e alua e he significance o
di e ences be ween g oups, accep ing Pb0.05 as he le el o significance.
Resul s
Clinical da a
Thi y-eigh o he 58 pa ien s who me he inclusion c i e ia and
accep ed o pa icipa e in he s udy we e diagnosed wi h pe iodon i is.
Table 1 summa izes he esul s o he pe iodon al examina ion wi h
significan di e ences in all he pa ame e s s udied (Pb0.001 o GM,
PD, CAL, den al plaque, and gingi al bleeding de e mina ions), whe eas
no significan di e ences we e ound o age o body mass index among
he conside ed g oups.
Mi ochond ial dys unc ion in pe iodon i is pa ien s
CoQ
10
le els, de e mined in PBMCs isola ed om he 38 pe iodon al
pa ien s, we e abou 56% lowe han in nonpe iodon i is pa ien s
(Table 1). To u he examine mi ochond ial dys unc ion in PBMCs om
pe iodon i is pa ien s, we de e mined mi ochond ial mass by ci a e
syn hase ac i i y. Table 1 shows a s a is ically significan low le el o
ci a e syn hase ac i i y in PBMC in pe iodon i is pa ien s (1.99±0.59
sp ac ) compa ed o nonpe iodon i is pa ien s (8.61±0.27 sp ac ).
Fig. 2. E ec o LPS on mi ochond ial componen s. (A) E ec o LPS on mi ochond ial memb ane po en ial (ΔΨ
m
). Fib oblas s we e ea ed wi h 10 μg/ml LPS o 30 μMCoQ
10
+10μg/ml LPS o
24 h. (B) Ci a e syn hase specific ac i i y in con ol and LPS- ea ed PBMCs was assayed as desc ibed unde Ma e ials and me hods. (C) Mi ochond ial p o ein exp ession le els in LPS- ea ed
cells. P o eins (50 μg) om con ol and ea ed fib oblas ex ac s we e immunos ained wi h an ibodies agains complexes I (39-kDa subuni ) and III (co e 1 subuni ). P o ein le els we e
de e mined by densi ome ic analysis (IOD, in eg a ed op ical in ensi y) o h ee di e en Wes e n blo s and no malized o GAPDH signal. *Pb0.05, be ween con ol and LPS- ea ed cells;
**Pb0.05, be ween he absence and he p esence o CoQ
10
.(D)CoQ
10
le el a e LPS ea men . Resul s a e exp essed as pmol o CoQ
10
pe mg o p o ein. Da a ep esen he means±SD o h ee
sepa a e expe imen s. *Pb0.01.
1339P. Bullon e al. / F ee Radical Biology & Medicine 50 (2011) 1336–1343
Oxida i e s ess in pe iodon i is pa ien s
Quan ifica ion o ROS p oduc ion by flow cy ome y analysis
showed high le els o mi ochond ial ROS p oduc ion in PBMCs om
pe iodon i is pa ien s compa ed o nonpe iodon i is pa ien s
(Fig. 1A).
To u he examine he ole o ROS gene a ion in pe iodon i is,
PBMCs o one ep esen a i e pa ien we e incuba ed wi h CoQ
10
, and
mi ochond ial ROS p oduc ion (Fig. 1B) was moni o ed, showing a
significan educ ion in mi ochond ial ROS p oduc ion in he p esence
o CoQ
10
.
LPS induces mi ochond ial dys unc ion in fib oblas s
The ΔΨ
m
was significan ly educed by LPS ea men . Flow cy ome y
analysis showed a ΔΨ
m
dec ease o abou 35% compa ed o un ea ed
fib oblas s (Fig. 2A). LPS-induced ΔΨ
m
dec ease was also pa ially
p e en ed by CoQ
10
.Fig. 2B shows a s a is ically significan dec ease in
ci a e syn hase ac i i y a e LPS ea men compa ed o con ols, also
pa ially p e en ed by CoQ
10
. Enzyma ic ac i i y da a we e 293.3±4.3 o
con ol, 261.6±7 o LPS, and 296.4±1.5 o LPS+CoQ
10
. LPS ea men
induced a significan dec ease in complex I (39-kDa subuni ) and
complex III (co e 1 subuni ) exp ession le els wi h a concomi an
dec ease in CoQ
10
le els compa ed o con ol fib oblas s (67.29±0.8
and 117.244±5.2 pmol/mg p o ein, espec i ely; Figs. 2CandD).CoQ
10
supplemen a ion e ficien ly p e en ed LPS-induced down- egula ion o
mi ochond ial p o eins.
LPS induces a high le el o mi ochond ial ROS p oduc ion in fib oblas s
Mi oSOX ed fluo escence colocalized wi h mi ochond ial cy o-
ch ome coxidase (Fig. 3A). Quan ifica ion o ROS p oduc ion by flow
cy ome ic analysis indica ed ha LPS ea men induced a significan
inc emen in mi ochond ial ROS p oduc ion (634.65±9 a bi a y uni s)
compa ed o con ol (579.25±18) (Fig. 3B). The addi ion o a widely
ecognized memb ane an ioxidan , such as CoQ
10
, a enua ed ROS
de ec ion (577.7±7).
LPS ini ia es he in insic pa hway o caspase-3-dependen apop osis in
fib oblas s
LPS caused an inc emen in apop o ic nuclei condensa ion and
caspase-3 ac i a ion. In con as , CoQ
10
highly p e en ed he LPS-
induced caspase-3 ac i a ion and apop osis a e (Figs. 4A, B, and C).
Discussion
Oxida i e s ess and ee adical gene a ion, as p ima y o
seconda y e en s, play impo an oles in he de elopmen o
sys emic diseases such as ype 2 diabe es, a he oscle osis, and
ca dio ascula diseases [27,28], as mi ochond ia a e he majo sou ce
o ROS. CoQ
10
le els ha e been sugges ed o be use ul as a
mi ochond ial dys unc ion ma ke [29] and CoQ
10
deficiency has
been ound in gingi al biopsies and leukocy es om pe iodon al
pa ien s [30,31]. To assess mi ochond ial dys unc ion in pe iodon i is,
Fig. 3. E ec o LPS on mi ochond ial ROS gene a ion. (A) Mi oSOX ed s ain e ealed inc eased supe oxide anion. Mi oSOX ed colocalized wi h cy och ome cin me ged images,
indica ing ha supe oxide anion p oduc ion occu ed mainly in mi ochond ia. (B) Flow cy ome y quan ifica ion o ROS p oduc ion. Da a ep esen he means±SD o h ee sepa a e
expe imen s. *Pb0.001, be ween con ol and LPS- ea ed cells; **Pb0.001, be ween he absence and he p esence o CoQ
10
.
1340 P. Bullon e al. / F ee Radical Biology & Medicine 50 (2011) 1336–1343

PBMCs o pe iodon al pa ien s we e s udied, showing a dec ease in
CoQ
10
le els and ci a e syn hase ac i i y. In e es ingly, a posi i e
co ela ion be ween he con en o CoQ
10
in PBMCs and skele al
muscle and fib oblas s [32,33] was demons a ed, he e o e sugges -
ing ha CoQ
10
deficiency and mi ochond ial dys unc ion in pe iodon-
al pa ien s could also be p esen in o he cells and issues.
Fu he mo e, fib oblas s o some pa ien s wi h CoQ
10
deficiency
show a highe p oduc ion o ROS in mi ochond ia [34]. In his espec ,
high le els o mi ochond ial ROS p oduc ion we e obse ed in PBMCs
o ou pe iodon i is pa ien s.
P e ious wo k has shown ha P. gingi alis, one o he key e iological
ac o s in pe iodon al pa hology, induces oxida i e s ess in i o [35],
wi h bac e ial LPS playing a majo ole in he pa hogenesis o
pe iodon al pa hology [36].
To assess he mi ochond ial damage induced by LPS, we de e mined
mi ochond ial ΔΨ
m
and mi ochond ial mass in bo h con ol and LPS-
ea ed fib oblas s. Ci a e syn hase is a mi ochond ial ma ix p o ein
whose ac i i y has been shown o co ela e well wi h mi ochond ial
mass [24]. We ound ha LPS ea men in cul u ed fib oblas s p o oked
inc eased oxida i e s ess and mi ochond ial dys unc ion, cha ac e ized
by a dec ease in mi ochond ial p o ein exp ession, mi ochond ial mass,
and CoQ
10
le els, as well as a educ ion in he mi ochond ial memb ane
po en ial.
I has been epo ed ha CoQ
10
deficiency de e mined a dec ease
in complex II+III, complex III, and complex IV ac i i ies; a dec ease in
he exp ession o mi ochond ial p o eins in ol ed in oxida i e
phospho yla ion; a dec ease in he mi ochond ial ΔΨ
m
; and an
inc ease in ROS p oduc ion [34]. To s udy he pa hophysiological
mechanisms o LPS-induced mi ochond ial dys unc ion in cul u ed
p ima y fib oblas s, we assessed he exp ession le els o c i ical
componen s o he mi ochond ial espi a o y chain, obse ing a
dec emen in exp ession le els o p o eins o complex I (39-kDa
subuni ) and complex III (co e 1 subuni ), oge he wi h a educ ion in
CoQ
10
le els.
Nex , o in es iga e he possible mi ochond ial o igin o ROS
p oduc ion in ela ion o pe iodon i is, LPS- ea ed and con ol
fib oblas s we e exposed o Mi oSOX ed, a fluo och ome specific
o supe oxide anion, which is p oduced in he inne mi ochond ial
Fig. 4. LPS induced apop osis in fib oblas s ea ed wi h LPS 10 μg/ml. (A and B) LPS-induced apop osis was assessed by caspase-3 ac i a ion by immunofluo escence mic oscopy and
Wes e n blo as desc ibed unde Ma e ials and me hods. (C) LPS-induced apop osis is p e en ed by CoQ
10
. Fib oblas s we e ea ed wi h 10 μg/ml LPS in he absence o p esence o
30 μM CoQ
10
o 24 h. Apop osis was assessed as desc ibed unde Ma e ials and me hods. Da a ep esen he means±SD o h ee sepa a e expe imen s. *Pb0.01, be ween con ol
and LPS- ea ed cells; **Pb0.01, be ween he absence and he p esence o CoQ
10
.
1341P. Bullon e al. / F ee Radical Biology & Medicine 50 (2011) 1336–1343
compa men , showing high le els o mi ochond ial ROS by LPS
ea men . In ag eemen wi h he hypo hesis ha ROS p oduc ion was
caused by a mi ochond ial dys unc ion, we ound ha ROS we e
mainly gene a ed in mi ochond ia o LPS- ea ed fib oblas s.
I should be aken in o accoun ha ROS a e essen ial in cells and
issues o many li e-sus aining p ocesses, bu hey can also induce
cell damage and dea h [37], especially when hei ac i i ies a e
uncon olled. ROS can be eleased in o he cy osol and igge “ROS-
induced ROS elease”in neighbo ing mi ochond ia. This mi ochon-
d ion- o-mi ochond ion ROS signaling cons i u es a posi i e eed-
back mechanism o enhanced ROS p oduc ion po en ially leading o
significan mi ochond ial inju y [38]. In his con ex , cy och ome cis
eleased and p ocaspase-9, caspase-3, and endonuclease G a e
ac i a ed, esul ing in DNA deg ada ion and apop o ic dea h. In
ou in es iga ion, an inc ease in apop osis was obse ed in LPS-
ea ed fib oblas s by caspase-3 ac i a ion, sugges ing ha LPS
ea men induces apop osis by he ac i a ion o a leas he in insic
pa hway.
Finally, i can be conside ed ha oxida i e s ess is one o he key
ac o s explaining some o he pa hophysiological mechanisms
associa ed wi h inflamma o y condi ions such as CVD and pe iod-
on i is [39,40], as lipid pe oxida ion is one o i s mos well known
e ec s. In ac , inc eased lipid pe oxida ion has been obse ed in
pe iodon i is [8], and i is accep ed ha lipid pe oxida ion indi ec ly
eflec s in acellula ROS gene a ion. I is in e es ing o no e ha
supe oxide plays a majo ole in he elease o cy okines ( o
example, TNF-α,IL-1β,andIL-6)[41], which a e in ol ed in he
pa hogenesis o pe iodon al disease and CVD [7] and hisisalso
induced by P. gingi alis LPS ea men . The findings o his s udy
show ha CoQ
10
ea men amelio a ed mi ochond ia dys unc ion
and oxida i e s ess and educed cell dea h. Because CoQ
10
is a
pi o al elemen in he mi ochond ial espi a o y chain and, a he
same ime, is an impo an an ioxidan , hese esul s sugges ha
mi ochond ial dys unc ion is c ucial in he pa hophysiology o
pe iodon al disease.
Ou s udy suppo s he hypo hesis ha LPS-media ed mi ochon-
d ial dys unc ion could be a he o igin o oxida i e s ess in
pe iodon al pa ien s. Abno mal PBMC pe o mance may p omo e
oxida i e s ess and al e cy okine homeos asis. In conclusion,
mi ochond ial dys unc ion could ep esen a possible link o unde -
s anding he in e ela ionships be ween wo p ominen inflamma o y
diseases: pe iodon i is and CVD.
Acknowledgmen
The au ho s a e indeb ed wi h Ms. M. Glebocki o ex ensi e
edi ing o he manusc ip .
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