Liver-specific deletion of the Plpp3 gene alters plasma lipid composition and worsens atherosclerosis in apoE−/− mice
Full text
1
SCIENTIFIC REPORTSȁͽǣͺͺͻͶȁǣͷͶǤͷͶ;ȀͺͺͻͶ
ǤǤȀ
ǦƤ
−/−
ͷ,*ǡͷ,*ǡǡiͷǡǤͷǡ
ͷǡǡ¡ǡǦͺǡǤͷǡ
Ƭͷ
Ǥ
ǦƤ
Ǥơ
Ǥǡ
ǦƤƪ
ǤǦ
−/−Ȁ−/−Ǧ+Ȁ−/−Ǧ−ơǡ
Ǥͷǡ
ǤǡǦ+Ǧ−ǡ
ǤƤ
Ƥǡǡ
ǡǤǡ
Ǥ
ǦǦǡ
Ǥ
Lipid phospha e phospha ases (LPPs) a e in eg al memb ane p o eins wi h six ansmemb ane domains, which
display b oad subs a e speci ici y and ca alyse he dephospho yla ion o lipid subs a es including phospha idic
acid, lysophospha idic acid, ce amide 1-phospha e, sphingosine 1-phospha e and diacylglyce ol py ophospha e1.
They belong o a la ge phospha ase/phospho ans e ase amily ha includes bo h memb ane and soluble amily
membe s2.
The LPP amily is composed o h ee enzymes in mammals: LPP1, LPP2 and LPP3, which a e encoded by
h ee independen genes named PLPP1, PLPP2 and PLPP3, espec i ely3,4. The exp ession o LPP2 mRNA is
ound mainly in he b ain, panc eas and placen a, whe eas LPP1 and LPP3 mRNA exp ession is ubiqui ous5.
Al hough he h ee enzymes demons a e o e lapping ca aly ic ac i i ies and subs a e p e e ences, selec i e
a ge ed inac i a ion o he Plpp genes in mice indica es ha he enzymes ha e non- edundan unc ions6. In ac ,
whe eas gene inac i a ion o Plpp1/LPP1 o Plpp2/LPP2 does no esul in any obse able pheno ype7,8, Plpp3/
LPP3 a ge ed inac i a ion causes p o ound de elopmen al de ec s, hus indica ing he essen ial ole o LPP3 in
mouse emb yonic de elopmen 9.
In e es in PLPP3/LPP3 has ecen ly been aised by he esul s o genome-wide associa ion s udies (GWAS)10,11
ha ha e iden i ied he i able single nucleo ide polymo phisms in he PLPP3 gene and ha e sugges ed PLPP3 as
a no el locus associa ed wi h co ona y a e y disease (CAD) suscep ibili y. In e es ingly, he PLPP3 isk allele
ͷDepa men o Pha macological and Biomolecula Sciences, Uni e si à degli S udi di Milano, Milano, I aly. Zo a
Biosciences Oy, Espoo, Finland. Depa men o Physiology, Ins i u e o Biomedicine, Uni e si y o Tu ku, Tu ku,
Finland. ͺIns i u o de Fisiología Celula , Di isión de Neu ociencias Uni e sidad Nacional Au ónoma de México, Cd.
ǤͶͺͻͷͶǡ±Ǥ*These au ho s con ibu ed equally o his wo k. Co espondence and eques s o ma e ials
should be add essed o G.C. (email: [email p o ec ed])
RǣͶͿͶͷͼ
AǣͶ;Ͷͷͽ
PǣͷͺͶͷͽ
www.na u e.com/scien i ic epo s/
2
SCIENTIFIC REPORTSȁͽǣͺͺͻͶȁǣͷͶǤͷͶ;ȀͺͺͻͶ
independen ly p edic s CAD and lacks associa ions wi h adi ional isk ac o s such as hype ension, choles e ol,
diabe es melli us, obesi y o smoking.
The gene a ion o a condi ional Plpp3-null allele12 has allowed o he in es iga ion o issue-speci ic dele ions
o he Plpp3 gene and hei e ec s on ascula heal h. In p e ious s udies, a lack o Plpp3 exp ession in smoo h
muscle cells has been epo ed o enhance in imal hype plasia and ascula in lamma ion13, and mo e ecen ly,
he a ge ed inac i a ion o Plpp3 in endo helial and haema opoie ic cells has indica ed ha LPP3 se es as a
nega i e egula o o endo helial pe meabili y and ascula in lamma ion14.
In he p esen s udy, a possible ole o Plpp3 in a he oscle osis de elopmen was in es iga ed. The a ge o gan
chosen o he selec i e inac i a ion o Plpp3/LPP3 was he li e , because i is one o he main o gans in ol ed
in lipid me abolism. Hepa ic Plpp3/LPP3-de icien mice we e hen c ossed wi h a he o-p one apolipop o ein E
knock-ou mice apoE−/−.
The esul s indica ed ha he lack o hepa ic Plpp3 exp ession inc eased he le els o se e al p o-a he ogenic
plasma lipid species and led o accele a ed a he oscle osis p og ession.
Ǥ To es he C e ecombinase exp ession speci ici y
and ac i i y, he Plpp3 locus was p elimina ily examined in he genomic DNA ex ac ed om li e and ail sam-
ples o which he la e se ed as a e e ence non- a ge issue. Genomic DNA ex ac ed om he ails o bo h
Plpp3 / apoE−/− Alb-C e− and Plpp3 / apoE−/− Alb-C e+ animals showed only a PCR band co esponding o he
loxed, unp ocessed locus (Fig.1A, lanes A-B, E-F). In con as , he Alb-C e ansgene was ac i e in he li e o
Plpp3 / apoE−/− Alb-C e+ mice, whe e he p ocessed allele was de ec ed oge he wi h he loxed, unp ocessed
allele (Fig.1A, lanes G-H). As expec ed, he Plpp3 locus was unp ocessed in he li e o Plpp3 / apoE−/− Alb-C e−
animals (Fig.1A, lanes C-D).
Ȁ−/−Ǧ+ǡ
Ǥ To in es iga e he e ec o C e−media ed Plpp3 locus ecombina ion
on Plpp3 ansc ip ion, wo quan i a i e PCR p ime se s we e designed, a ge ing he 5′ (ups eam o he C e
ecombinase media ed excision o c ucial Plpp3 exons) and 3′ end (loca ed wi hin he DNA sequences excised
by he C e ecombinase) o Plpp3 mRNA. This app oach was able o e alua e Plpp3 p omo e ac i i y, by p obing
Plpp3 mRNA exp ession ups eam o he si e o ac ion o C e ecombinase in he genome (5′ end p ime pai ) and
he unc ional e ec o Alb-C e−media ed ecombina ion, which esul s in unca ed and non- unc ional Plpp3
mRNA (3′ end p ime pai ).
The C e ecombinase had no e ec on he 5′ end o Plpp3 mRNA in bo h mouse lines; bo h Plpp3 /
apoE−/− Alb-C e− and Plpp3 / apoE−/− Alb-C e+ issues showed compa able exp ession le els (see
Supplemen a yFigu eS1). In con as , quan i a i e PCR using he 3′ end p ime pai showed a ma ked dec ease
o Plpp3 mRNA exp ession in he li e s o Plpp3 / apoE−/− Alb-C e+ mice compa ed o he li e s o Plpp3 /
apoE−/− Alb-C e− mice (Fig.1B). No ele an di e ences we e obse ed be ween he wo geno ypes in all he
o he issues analysed (Fig.1B). A majo li e -speci ic dec ease o LPP3 p o ein exp ession in Plpp3 / apoE−/−
Alb-C e+ mice was also con i med by Wes e n blo ing analysis (Fig.1C).
ơǤ The
hepa ic pa enchyma was una ec ed by li e -speci ic Plpp3 dele ion; haema oxylin and eosin (H&E) s aining
showed no di e ences in he li e s om Plpp3 / apoE−/− Alb-C e− and Plpp3 / apoE−/− Alb-C e+ mice ed chow
die o 32 weeks s a ing a e weaning (8 weeks o age) (Supplemen a yFigu eS2). The Wes e n die , which was
adminis e ed o 12 weeks s a ing a e weaning, inc eased lipid accumula ion in bo h geno ypes bu esul ed in
no signi ican di e ences be ween he wo mouse lines. Speci ically, he pe cen age o Oil Red O posi i e a ea o e
he o al a ea was 77.4 ± 2.1% in Plpp3 / apoE−/− Alb-C e− and 80.3 ± 5.2% in Plpp3 / apoE−/− Alb-C e+ mice,
n = 10, p > 0.05 (Supplemen a yFigu eS2). In addi ion, an Gieson and Pe iodic acid-Schi (PAS) s aining did
no e eal any di e ences in collagen and glycogen deposi ion be ween Plpp3 / apoE−/− Alb-C e+ and Plpp3 /
apoE−/− Alb-C e− mice (Supplemen a yFigu eS2).
Ȁ−/−Ǧ+
Ǥ To in es iga e he ole played by hepa ic LPP3 du ing a h-
e oscle osis de elopmen , Plpp3 / apoE−/− Alb-C e− and Plpp3 / apoE−/− Alb-C e+ mice we e ed, a e weaning,
a chow die o 32 weeks o a Wes e n die o 12 weeks.
No di e ences in bo h plaque size and composi ion we e obse ed a he ao ic sinuses in he wo mouse
lines ed he chow die (Fig.2A,C,E and Table1). In con as , when challenged wi h a Wes e n die , Plpp3 /
apoE−/− Alb-C e+ mice showed a 35% inc ease in a he oscle osis de elopmen , as compa ed wi h Plpp3 /
apoE−/− Alb-C e- mice (p = 0.031) (Fig.2B,D,F and Table1). This inc ease appea ed o be p ima ily a ibu able
o a la ge nec o ic co e a ea (Table1 and Fig.3). A end owa ds inc eased accumula ion o ex acellula ma ix
(ECM) was obse ed in Plpp3 / apoE−/− Alb-C e+ mice (p = 0.063). No signi ican a ia ions we e ound in neu-
al lipid deposi ion, mac ophages and smoo h muscle cell accumula ion (de ec ed wi h Oil Red O, Mac-2 and
α SMA s aining, espec i ely). These a ia ions in he plaque componen s esul ed in a highe pe cen age o he
nec o ic co e and a concomi an ly dec eased pe cen age o neu al lipids and mac ophages (Table1 and Fig.3).
En- ace analysis o he en i e ao a o he Wes e n die - ed mice showed a d ama ic inc ease in ao ic a he o-
scle osis in Plpp3 / apoE−/− Alb-C e+ mice, speci ically in he ao ic a ch and he ho acic segmen , as compa ed
wi h Plpp3 / apoE−/− Alb-C e− mice (Fig.4A,B). No di e ences in a he oscle osis bu den we e obse ed in he
abdominal ao as o ei he geno ype (Fig.4C).
www.na u e.com/scien i ic epo s/
3
SCIENTIFIC REPORTSȁͽǣͺͺͻͶȁǣͷͶǤͷͶ;ȀͺͺͻͶ
Figu e 1. (A) Geno yping o he loxed and p ocessed Plpp3 locus. A ep esen a i e PCR sc eening is
shown. Tail ips (A-B) and li e issue (C-D) o Plpp3 / apoE−/− Alb-C e− mice show only he 235 bp band
co esponding o he loxed, unp ocessed Plpp3 locus. Plpp3 / apoE−/− Alb-C e+ mice also display he
unp ocessed Plpp3 locus band in he ail ips (E-F), bu in he li e , bo h he unp ocessed and p ocessed allele
(162 bp) bands a e p esen (G-H). Li e PCR amplicons om a wild ype Plpp3w /w mouse (I), Plpp3 /w
he e ozygous mouse (L) and Plpp3 / pa en mouse (M) a e shown as con ols. Molecula weigh ma ke ,
100 bp New England Biolabs (N); (B) Quan i a i e exp ession o he Plpp3 mRNA 3′ end. Exp ession le els o
Plpp3 mRNA a e shown, quan i ied by qPCR analysis de ec ing a egion downs eam o he C e ecombinase-
media ed loxed exons excision si e. The alues we e no malized o he exp ession o he ansc ip in each
co esponding issue o he Plpp3 / apoE−/− Alb-C e− mice (n = 6 o li e , n = 3 o all he o he issues;
*p = 0.0022 in Plpp3 / apoE−/− Alb-C e+ s Plpp3 / apoE−/− Alb-C e− mouse li e by Wilcoxon ank-sum
es ). Wa = whi e adipose issue; (C) Wes e n blo ing analysis o LPP3 exp ession. A ep esen a i e image o
he immunoblo expe imen s. A owheads indica e he p esence o unglycosyla ed (lowe band, 29 KDa) and
glycosyla ed (uppe bands, 33–38 KDa) o ms o LPP3. The esul s we e con i med in h ee biological eplica es.
Wes e n blo ing indica ed a subs an ial dec ease in LPP3 in he li e s o Plpp3 / apoE−/− Alb-C e+ mice
compa ed wi h Plpp3 / apoE−/− Alb-C e− mice. In con as , LPP3 le els emained unchanged in he kidney and
b ain.
www.na u e.com/scien i ic epo s/
4
SCIENTIFIC REPORTSȁͽǣͺͺͻͶȁǣͷͶǤͷͶ;ȀͺͺͻͶ
Ȁ−/−Ǧ+-
Ǥ On a chow die , Plpp3 / apoE−/− Alb-C e+ and Plpp3 / apoE−/− Alb-C e− mice did no show signi i-
can di e ences in ci cula ing o al-choles e ol (TC) and HDL-choles e ol (HDL-C) le els, as well as in plasma
iglyce ides (TAG) and phospholipids (PL) (TC: 296.4 ± 47.3 s 271.5 ± 53.1 mg/dl, espec i ely; HDL-C:
Figu e 2. Rep esen a i e H&E pho omic og aphs and quan i ica ion o he maximum plaque a ea a
he ao ic sinuses in Plpp3 / apoE−/− Alb-C e+ and Plpp3 / apoE−/− Alb-C e− mice. The a he oscle osis
de elopmen in he chow ed mice (n = 7–8) was compa able be ween he wo geno ypes (A,C,E). Wes e n die
adminis a ion signi ican ly wo sened a he oscle osis de elopmen in Plpp3 / apoE−/− Alb-C e+ compa ed
wi h Plpp3 / apoE−/− Alb-C e− mice (n = 16–17) (B,D,F). The da a a e shown as he mean ± SEM; *p = 0.031 s
Plpp3 / apoE−/− Alb-C e− by Wilcoxon ank-sum es . Ba leng h = 500 μ m.
www.na u e.com/scien i ic epo s/
5
SCIENTIFIC REPORTSȁͽǣͺͺͻͶȁǣͷͶǤͷͶ;ȀͺͺͻͶ
14.3 ± 2.7 s 15.0 ± 4.8 mg/dl, espec i ely; TAG: 47.7 ± 9.6 s 53.0 ± 13.8 mg/dl, espec i ely; PL: 231.1 ± 34.3
s 228.2 ± 46.1 mg/dl, espec i ely; n = 10, p > 0.05). In con as , when he mice we e adminis e ed a Wes e n
die , a signi ican inc ease o TAG was obse ed in Plpp3 / apoE−/− Alb-C e+ s Plpp3 / apoE−/− Alb-C e− mice
(TAG: 301.1 ± 103.0 s 162.4 ± 48.3 mg/dl, espec i ely, p = 0.0012). A end owa ds an inc ease in plasma PL
le els was also obse ed in Plpp3 / apoE−/− Alb-C e+ (703.9 ± 106.6 s 588.6 ± 103.7 mg/dl, espec i ely, n = 10,
p = 0.054). No signi ican di e ences we e obse ed o TC le els (939.2 ± 190.5 s 899.5 ± 218.8 mg/dl, espec-
i ely, p > 0.05).
ǡ-
Ǥ To gain insigh s in o changes caused by Plpp3
dele ion in plasma lipid le els, lipidomic analysis was pe o med using global lipidomics me hods as well as a -
ge ed ce amide and lysophospholipid pla o ms. The s a is ical esul s o all lipids and compa isons oge he wi h
q- alues con olling he alse disco e y a e a e p esen ed in Supplemen a yDa aS1. The Wes e n die , compa ed
wi h chow die , inc eased he le els o mos lipid classes in bo h mouse lines (see Supplemen a yTableS1). The
e ec s o hepa ic Plpp3 de iciency on he plasma lipidome in mice ed chow o Wes e n die s is shown in Tables2
and 3 as well as Supplemen a yFigu eS3. The hepa ic Plpp3 dele ion inc eased he le els o lac osylce amides
(LacCe ) in mice on bo h he chow and Wes e n die s. In addi ion, on Wes e n die , he le els o lysophospha idic
acids (LPA) and lysophospha idylinosi ols (LPI) we e signi ican ly inc eased in Plpp3 / apoE−/− Alb-C e+ s
Plpp3 / apoE−/− Alb-C e− mice (Table2). Globo iaosylce amides (Gb3) we e also inc eased, al hough his a i-
a ion did no each s a is ical signi icance (p = 0.055).
Table3 and Supplemen a yFigu eS3 show he molecula lipids ha we e signi ican ly al e ed by hepa ic
Plpp3 dele ion in mice ed chow o a Wes e n die . Se e al LacCe lipids we e signi ican ly inc eased in Plpp3 /
apoE−/− Alb-C e+ mice on bo h die s. LPAs, lysophospha idyle hanolamines (LPEs) and LPIs we e also inc eased
wi h bo h die a y ea men s, bu eached s a is ical signi icance only wi h he Wes e n die . In hese lipid classes,
he a y acids a ied om sho -sa u a ed o long-polyunsa u a ed a y acids, hus indica ing ha he al e a ions
we e due o b oad changes o hese lipid classes a he han o al e a ions in indi idual a y acids.
On he basis o he esul s o GWAS ha associa ed he PLPP3 gene polymo phisms wi h CAD suscep ibili y10,11,
a possible ole o Plpp3/LPP3 in a he oscle osis de elopmen was in es iga ed in a gene ically modi ied mouse
model. The unc ion o LPP3, he enzyme encoded by Plpp3, is ha o dephospho yla ing lipid subs a es, and
he e o e, he li e was chosen as he a ge o gan o Plpp3 dele ion, because he li e is among he main sou ces
o ci cula ing plasma lipids and lipop o eins, which signi ican ly con ibu e o he a he oscle osis p ocess. I is
well known ha , du ing a he ogenesis, LDLs en e he a e ial wall, ei he oxidized o enzyma ically deg aded,
and ollow he a he ogenic pa hway ha , h ough he in ol emen o mac ophages and smoo h muscle cells, leads
o choles e ol deposi ion and a he oscle o ic plaque o ma ion15. Impo an ly, he lipop o eins esponsible o
choles e ol anspo , mainly LDLs and HDLs, also con ain hund eds o o he associa ed lipid species ha may
exhibi a ious bioac i e p ope ies ha a ec he cou se o he disease16. Among hose, LPP3 lipid subs a es o
hei p ecu so s ha e been iden i ied as componen s o he ci cula ing lipop o eins17.
Chow die Wes e n die
Plpp3 / apoE−/−
Alb-C e−
Plpp3 / apoE−/−
Alb-C e+
Plpp3 / apoE−/−
Alb-C e−
Plpp3 / apoE−/−
Alb-C e+
Lesion size (μ m 2) 494,884 ± 142,501 547,335 ± 150,002 355,308 ± 138,729 479,720 ± 150,857
(p = 0.031)
Nec o ic co e a ea (μ m 2) 54,237 ± 23,029 51,201 ± 17,107 40,104 ± 23,571 92,879 ± 35,903
(p = 0.0001)
% o al plaque a ea 11.3 ± 4.3 9.5 ± 2.6 11.0 ± 4.5 19.5 ± 5.1
(p = 0.0001)
Mac-2 posi i e a ea (μ m 2) 92,274 ± 53,696 78,784 ± 24,882 147,094 ± 70,170 144,314 ± 49,215
% o al plaque a ea 19.1 ± 8.0 15.8 ± 8.7 41.0 ± 10.0 31.0 ± 8.1
(p = 0.0077)
ORO-posi i e a ea (μ m 2) 216,346 ± 73075 226,550 ± 59293 195,505 ± 75,335 232,692 ± 70,157
% o al plaque a ea 44.4 ± 2.8 42.4 ± 9.1 55.9 ± 7.3 49.6 ± 9.8
(p = 0.045)
ECM-posi i e a ea (μ m 2) 341,054 ± 96,641 420,028 ± 164,825 180,185 ± 71,971 254,436 ± 104,916
% o al plaque a ea 70.3 ± 14.1 74.5 ± 15.2 50.6 ± 7.4 52.0 ± 10.4
α SMA-posi i e a ea (μ m 2) 12,208 ± 4,412 9,569 ± 5,184 12,738 ± 8,922 16,419 ± 6,668
% o al plaque a ea 2.4 ± 0.6 1.9 ± 1.3 3.4 ± 1.2 3.5 ± 1.1
CD3-posi i e cells (cells/100000 μ m 2) 1.1 ± 0.8 0.6 ± 0.8 3.7 ± 3.4 3.4 ± 3.0
Table 1. Plaque size and composi ion a he ao ic sinuses o Ppap2b / apoE−/− Alb-C e− and Ppap2b /
apoE−/− Alb-C e+ mice. Da a a e exp essed as he mean ± SD; n = 7–8 in Chow die , n = 16–17 in WD. Mac-2
(mac ophages), ORO (Oil Red O, neu al lipids), ECM (ex acellula ma ix), α SMA (smoo h muscle cells),
CD3 (T lymphocy es).
www.na u e.com/scien i ic epo s/
6
SCIENTIFIC REPORTSȁͽǣͺͺͻͶȁǣͷͶǤͷͶ;ȀͺͺͻͶ
Figu e 3. His ological and immunohis ochemical cha ac e iza ion o plaques a he ao ic sinuses
in Plpp3 / apoE−/− Alb-C e− and Plpp3 / apoE−/− Alb-C e+ mice ed a Wes e n die . Rep esen a i e
pho omic og aphs o he nec o ic co e (A,B), mac ophages (Mac-2 + cells; C,D), neu al lipids (Oil Red O;
E,F), ex acellula ma ix (ECM; G,H), smoo h muscle cells (α SMA + celIs; I,J) and CD3 + cells (K,L). Ba
leng h = 200 μ m. As e isks ma k he localiza ion o he nec o ic co e (A,B).
www.na u e.com/scien i ic epo s/
7
SCIENTIFIC REPORTSȁͽǣͺͺͻͶȁǣͷͶǤͷͶ;ȀͺͺͻͶ
The li e -speci ic dele ion o Plpp3 was achie ed by c ossing Plpp3- loxed mice wi h animals exp essing C e
ecombinase unde he con ol o he hepa ocy e-speci ic albumin p omo e . In he li e s o Plpp3 / apoE−/−
Alb-C e+ mice, compa ed wi h C e ecombinase nega i e mice, he Plpp3 exp ession le els dec eased o e
i e- old. The p esence o a esidual Plpp3 unc ional gene in li e , also demons a ed ia PCR analysis o li e
genomic DNA, may be explained by he p esence o non-pa enchymal, i.e. non-hepa ocy e, cell ypes, such as
sinusoidal endo helial cells, Kup e cells, hepa ic s ella e cells and, o en, in ahepa ic lymphocy es18,19. I should
be no ed ha hese cells, which do no exp ess albumin20, con ibu e o only 6.5% o he li e olume, bu accoun
o app oxima ely 40% o he o al numbe o li e cells19. Compa able exp ession le els we e obse ed in all
he o he o gans/ issues assayed in he Plpp3 / apoE−/− Alb-C e+ and Plpp3 / apoE−/− Alb-C e− animals, hus
demons a ing he high speci ici y o he albumin p omo e exp ession and C e ecombinase ac i i y.
Figu e 4. Rep esen a i e pho omic og aphs and quan i ica ion o plaque a ea de ec ed by en- ace analysis
o he ao as o mice ed a Wes e n die . A e 12 weeks o die a y ea men , whole ao as we e collec ed and
en- ace analysis was pe o med o quan i y he pe cen age o ao ic su ace co e ed by a he oscle o ic plaques.
A he oscle o ic plaque de elopmen in he a ches (A) and in he ho acic segmen s (B) was signi ican ly
inc eased in he ao as om Plpp3 / apoE−/− Alb-C e+ mice (whi e do s) compa ed wi h Plpp3 / apoE−/− Alb-
C e− mice (black do s). A he oscle osis de elopmen a he abdominal segmen (C) was no di e en be ween
he wo geno ypes. Da a a e shown as he mean ± SEM; n = 7–8 mice pe g oup, p = 0.0016 in he a ch and
p = 0.0016 in he ho acic segmen s Plpp3 / apoE−/− Alb-C e− mice by Wilcoxon ank-sum es .
www.na u e.com/scien i ic epo s/
8
SCIENTIFIC REPORTSȁͽǣͺͺͻͶȁǣͷͶǤͷͶ;ȀͺͺͻͶ
The main esul o he p esen manusc ip is he demons a ion ha hepa ocy e-speci ic Plpp3 dele ion is
associa ed wi h inc eased a he oscle osis p og ession. This esul was obse ed when mice we e ed Wes e n die
(+ 35% plaque a ea a he ao ic sinus and + 86% plaque ex en a he ao ic a ch o Alb-C e+ s Alb-C e− mice),
whe eas no di e ences we e ound in a he oscle osis de elopmen be ween he wo geno ypes, when mice we e
ed a chow die . The plaque composi ion was no di e en be ween he wo mouse lines on a chow die ; howe e ,
unde a Wes e n die , a la ge nec o ic co e was obse ed in Plpp3 / apoE−/− Alb-C e+ mice. In e es ingly, no
di e ences in he main ci cula ing plasma lipid le els we e ound in chow ed animals, whe eas Wes e n die - ed
Plpp3 / apoE−/− Alb-C e+ mice displayed inc eased TAG plasma le els s Plpp3 / apoE−/− Alb-C e− mice.
Because Lpp3 is a lipid phospha ase wi h a la ge numbe o subs a es, speci ic in es iga ions in o all he indi-
idual Lpp3 a ge s would no be possible. Thus, wi h he aim o elucida ing he e ec s o hepa ic Plpp3 dele ion
on ci cula ing lipid le els, which migh a ec a he oscle osis de elopmen , a lipidomic analysis was conduc ed
on Plpp3 / apoE−/− Alb-C e− and Plpp3 / apoE−/− Alb-C e+ mouse plasma. S a is ical analyses o he lipidomics
da a e ealed ha none o he lipids we e signi ican ly di e en a e mul iple hypo hesis co ec ion. Howe e ,
signi ican non-adjus ed p- alues o se e al lipids in he same o ela ed lipid classes we e obse ed, pa icula ly
in mice on he Wes e n die . The e o e, we conside hese esul s in mice on he Wes e n die o be biologically
ele an .
Compa ison o Plpp3 / apoE−/− Alb-C e− and Plpp3 / apoE−/− Alb-C e+ mice ed ei he a chow die o a
Wes e n die indica ed a signi ican inc ease in LacCe in Plpp3 / apoE−/− Alb-C e+ mice. A di ec link be ween
LacCe accumula ion and Plpp3 dele ion is di icul o es ablish, gi en he complexi y o he biosyn he ic ou es
ha con e ce amide o o he bioac i e sphingolipids in mammalian cells21. Howe e , a ecen s udy wi h
apoE−/− mice, al hough in a di e en expe imen al se ing, has epo ed an inc ease in he neu al glycosphin-
golipid LacCe when Plpp3 exp ession is dec eased22. In animal models, inc eased le els o LacCe and o he
sphingolipids ha e been associa ed wi h he de elopmen o a he oscle osis23,24, and se e al enzymes in he gly-
cosphingolipid syn hesis pa hway ha e been es ed as po en ial an i-a he oscle o ic d ug a ge s25,26. Ele a ed
sphingolipid le els in human plasma ha e also been indica ed as a isk ac o o a he oscle osis de elopmen 27.
In e es ingly, in a ecen clinical s udy, di e en sphingolipids and glycosphingolipids, especially LacCe , ha e
been associa ed wi h CAD ou come and plaque ulne abili y, which was iden i ied on he basis o an enla ged
nec o ic co e28. This obse a ion suppo s ou esul s, because in Plpp3 / apoE−/− Alb-C e+ mice ed a Wes e n
die , he wo sening o a he oscle osis was associa ed wi h a la ge nec o ic co e. In Plpp3 / apoE−/− Alb-C e+
mice ed a chow die , howe e , he inc ease in LacCe le els was no associa ed wi h inc eased a he oscle osis
de elopmen . Toge he , hese obse a ions do no indica e a s aigh o wa d link be ween highe plasma LacCe
concen a ions and a he oscle osis. Howe e , i canno be excluded ha his plasma lipid pe u ba ion none he-
less con ibu ed o he accele a ed a he oscle o ic p ocess obse ed in mice ed he Wes e n die .
To al lipids
Chow Wes e n die
Mean ± SEM (μmol/L) Mean ela i e
di e ence (%) P- alue
Mean ± SEM (μmol/L) Mean ela i e
di e ence (%) P- alueAlb-C e+Alb-C e−Alb-C e+Alb-C e−
CE 8491 ± 486 8156 ± 509 n.s. 0.863 32812 ± 2757 28131 ± 2238 n.s 0.460
Ce d18:0 0.34 ± 0.02 0.33 ± 0.02 n.s. 0.605 2.2 ± 0.2 1.8 ± 0.1 n.s 0.173
Ce d18:1 9.9 ± 1.1 11.8 ± 1.4 n.s. 0.340 50.1 ± 3.0 50.2 ± 6.9 n.s 0.696
DAG 5.7 ± 0.4 5.9 ± 0.5 n.s. 0.779 22.4 ± 1.2 22.4 ± 2.1 n.s 1.000
Gb3 1.3 ± 0.1 1.1 ± 0.1 n.s. 0.387 4.6 ± 0.2 3.8 ± 0.3 n.s 0.055
Glc/GalCe 28.4 ± 2.3 28.3 ± 2.2 n.s. 1.000 125.3 ± 5.8 113.9 ± 4.8 n.s 0.315
LPA 0.20 ± 0.01 0.19 ± 0.01 n.s. 1.000 0.25 ± 0.02 0.20 ± 0.01 27% 0.034
LPC 582 ± 30 584 ± 18 n.s. 0.796 1113 ± 29 1059 ± 46 n.s 0.408
LPE 23.0 ± 2.1 21.1 ± 2.0 n.s. 0.436 57.2 ± 4.7 46.9 ± 3.5 n.s 0.146
LPG 9.5 ± 2.8 12.2 ± 4.1 n.s. 0.863 15.8 ± 4.4 12.9 ± 3.6 n.s 0.829
LPI 1.3 ± 0.1 1.2 ± 0.1 n.s. 0.387 3.8 ± 0.2 3.1 ± 0.2 23% 0.016
LSM 0.006 ± 0.0004 0.006 ± 0.0004 n.s. 0.436 0.014 ± 0.0015 0.012 ± 0.0012 n.s 0.829
LacCe 4.0 ± 0.3 3.0 ± 0.3 34% 0.040 21.5 ± 1.5 15.9 ± 1.6 35% 0.021
PC 1328 ± 48 1399 ± 105 n.s. 0.730 3742 ± 160 3573 ± 216 n.s 1.000
PC O 17.2 ± 1.4 14.5 ± 1.3 n.s. 0.161 61.9 ± 2.3 56.9 ± 5.2 n.s 0.360
PE O 2.3 ± 0.2 2.3 ± 0.3 n.s. 0.536 1.4 ± 0.2 1.9 ± 0.3 n.s 0.202
PI 29.0 ± 2.7 29.8 ± 2.3 n.s. 0.796 55.3 ± 2.5 52.2 ± 3.2 n.s 0.829
SM 401 ± 24 378 ± 25 n.s. 0.730 1134 ± 47 981 ± 86 n.s 0.146
Table 2. Pe cen age changes in he o e all amoun s o lipids in classes caused by hepa ic Plpp3 dele ion in
mice ed a chow o Wes e n die . The da a a e exp essed as he mean ± SEM; n = 8–10, Wilcoxon ank-sum
es . CE = Choles e yl es e ; Ce d18:0 = Ce amide d18:0; Ce d18:1 = Ce amide d18:1; DAG = Diacylglyce ol;
Gb3 = Globo iaosylce amide; Glc/GalCe = Glucosylce amide/Galac osylce amide; LPA = Lysophospha idic
acid; LPC = Lysophospha idylcholine; LPE = Lysophospha idyle hanolamine; LPG = Lysophospha idylglyce ol;
LPI = Lysophospha idylinosi ol; LSM = Lysosphingomyelin; LacCe = Lac osylce amide; PC/PC
O = Phospha idylcholine; PE O = Phospha idyle hanolamine; PI = Phospha idylinosi ol; SM = Sphingomyelin.
www.na u e.com/scien i ic epo s/
9
SCIENTIFIC REPORTSȁͽǣͺͺͻͶȁǣͷͶǤͷͶ;ȀͺͺͻͶ
The Wes e n die , compa ed wi h he chow die , in bo h mouse lines, led o highe plasma concen a ions o
choles e yl es e s, sphingolipids and mos glyce ophospholipids. In e es ingly, Plpp3 / apoE−/− Alb-C e+ mice
showed a s a is ically signi ican inc ease in TAG and LPA le els s Plpp3 / apoE−/− Alb-C e− mice only when
ed he Wes e n die .
LPA is a well-known subs a e o LPP3, which dephospho yla es and hus e mina es LPA’s ecep o -media ed
signalling ac ions29. P e ious s udies ha e demons a ed ha he li e plays a majo ole in LPA ca abolism30.
The inc ease in LPA obse ed in Plpp3 / apoE−/− Alb-C e+ mice ed a Wes e n die suppo s hese obse a ions
and indica es he in ol emen o hepa ic LPP3 in LPA deg ada ion. Mo eo e , because LPA is an obliga e in e -
media e in TAG syn hesis31, he inc eased LPA le els in Plpp3 / apoE−/− Alb-C e+ mice a e consis en wi h he
ele a ed plasma TAG le els obse ed. In humans, i has been obse ed ha hype lipidemia p edisposes o he
gene a ion o LPA in he ci cula ion32, and highe concen a ions o se um LPA ha e been associa ed wi h he
occu ence o acu e co ona y synd omes33. Mo eo e , in apoE−/− mice, sys emic ea men wi h unsa u a ed LPA
has been shown o ha e p o-in lamma o y and p o-a he oscle o ic e ec s34. In ou s udy, we obse ed inc eased
ci cula ing amoun s o LPA 18:1 in he sn-2 posi ion and 20:4 in he sn-1 posi ion. These LPA molecula species
ha e p e iously been ecognized o ha e high a he ogenic and h ombogenic po ency in he lipid- ich co es
o human a he oscle o ic plaques35 as well as o p omo e dyslipidemia36. In e es ingly, an associa ion be ween
nec o ic co e o ma ion and LPA accumula ion has been highligh ed in p e ious s udies35. These indings suppo
a link be ween he ele a ed plasma LPA concen a ion and he inc eased nec o ic co e a eas obse ed in he ao ic
sinus o Plpp3 / apoE−/− Alb-C e+ mice ed a Wes e n die .
The Plpp3 / apoE−/− Alb-C e+ mice ed a Wes e n die also displayed a signi ican inc ease o LPI plasma con-
cen a ion compa ed wi h ha in Wes e n- ed Plpp3 / apoE−/− Alb-C e− mice. LPI is syn hesized by se e al cell
ypes37–39 and is gene a ed by phospholipase A2, which ca alyses he hyd olysis o phospha idylinosi ol and gen-
e a es LPI and ee a achidonic acid40. Mos o he biological e ec s o LPI a e media ed by he ecep o GPR5541.
LPI may play a p o-a he ogenic ole by p omo ing endo helial dys unc ion. Indeed, se e al s udies ha e indica ed
ha LPI nega i ely egula es endo helial cells mig a ion and induces VCAM-1 and ICAM-1 exp ession42,43.
Finally, Gb3 showed a end owa ds inc eased le els in Plpp3 / apoE−/− Alb-C e+ mice ed a Wes e n die .
This inc ease is consis en wi h he obse ed ele a ion o LacCe , because Gb3 is syn hesized om LacCe by α
1,4-galac osyl ans e ase44. Ci cula ing Gb3 is anspo ed in LDL and HDL pa icles45,46. In he a e ial wall,
Gb3 accumula ion leads o ex acellula ma ix deposi ion and calci ica ion in he media47, whe eas, wi hin he
endo helium, i p omo es he p oduc ion o eac i e oxygen species, up egula es he exp ession o adhesion mol-
ecules48, and causes endo helial dys unc ion49.
In conclusion, he p esen wo k p o ides he i s demons a ion o he ole o Plpp3/LPP3 in a he oscle o-
sis de elopmen in an animal model and u he p o ides expe imen al e idence suppo ing clinical obse a-
ions ela ing PLPP3 polymo phisms o CAD suscep ibili y. Addi ionally, plasma lipidomic analysis sugges ed a
Lipid
Chow Wes e n die
Mean ± SEM (μmol/L) Mean ela i e
di e ence (%) P- alue
Mean ± SEM (μmol/L) Mean ela i e
di e ence (%) P- alueclass name Alb-C e+Alb-C e−Alb-C e+Alb-C e−
LPA LPA 0:0/18:1 0.006 ± 0.0005 0.005 ± 0.0007 n.s 0.505 0.012 ± 0.0012 0.007 ± 0.0006 66% 0.009
LPA LPA 20:4/0:0 0.019 ± 0.002 0.015 ± 0.002 n.s 0.190 0.035 ± 0.003 0.026 ± 0.002 31% 0.016
LPC LPC 0:0/18:2 10.0 ± 0.8 11.0 ± 0.3 −9% 0.040 5.1 ± 0.3 4.7 ± 0.3 n.s. 0.408
LPE LPE 18:0 4.3 ± 0.3 3.2 ± 0.3 36% 0.027 9.5 ± 0.9 8.6 ± 1.0 n.s. 0.633
LPE LPE 20:3/0:0 0.12 ± 0.02 0.16 ± 0.03 n.s 0.605 0.77 ± 0.14 0.47 ± 0.05 64% 0.043
LPE LPE 20:4/0:0 0.59 ± 0.07 0.50 ± 0.09 n.s 0.387 2.19 ± 0.35 1.32 ± 0.14 66% 0.009
LPE LPE 22:6/0:0 0.73 ± 0.10 0.79 ± 0.18 n.s 0.863 1.95 ± 0.18 1.43 ± 0.07 36% 0.016
LPG LPG 18:1/0:0 0.008 ± 0.001 0.009 ± 0.001 n.s 1.000 0.065 ± 0.004 0.052 ± 0.002 24% 0.012
LPI LPI 18:1/0:0 0.025 ± 0.002 0.021 ± 0.002 n.s 0.436 0.400 ± 0.032 0.278 ± 0.012 44% 0.001
LPI LPI 18:2/0:0 0.064 ± 0.006 0.049 ± 0.002 30% 0.024 0.093 ± 0.009 0.072 ± 0.003 n.s. 0.068
LPI LPI 22:5/0:0 0.007 ± 0.001 0.006 ± 0.001 n.s 0.200 0.033 ± 0.003 0.025 ± 0.002 32% 0.034
LPI LPI 22:6/0:0 0.019 ± 0.002 0.017 ± 0.001 n.s 0.666 0.071 ± 0.006 0.054 ± 0.005 31% 0.043
LacCe LacCe d18:1/16:0 1.11 ± 0.11 0.80 ± 0.07 38% 0.031 6.01 ± 0.39 4.6 ± 0.4 31% 0.043
LacCe LacCe d18:1/22:0 0.62 ± 0.04 0.52 ± 0.05 n.s 0.113 2.96 ± 0.18 2.28 ± 0.25 29% 0.027
LacCe LacCe d18:1/24:0 0.43 ± 0.04 0.35 ± 0.03 n.s 0.222 3.22 ± 0.25 2.31 ± 0.30 39% 0.034
LacCe LacCe d18:1/24:1 1.37 ± 0.12 0.97 ± 0.10 42% 0.031 6.65 ± 0.57 4.64 ± 0.57 44% 0.034
PC O PC P-18:0/20:4
(PC O-18:1/20:4) 2.26 ± 0.16 1.57 ± 0.12 44% 0.002 6.21 ± 0.35 5.38 ± 0.56 n.s. 0.173
SM SM 42:2 95.4 ± 4.7 91.5 ± 4.3 n.s 0.863 231.9 ± 9 195.4 ± 12.5 19% 0.027
Table 3. Pe cen age changes in he amoun s o lipid species caused by hepa ic Plpp3 dele ion in mice
ed a chow o Wes e n die . Da a a e exp essed as mean ± SEM; n = 8–10, Wilcoxon ank-sum es .
LPA = Lysophospha idic acid; LPC = Lysophospha idylcholine; LPE = Lysophospha idyle hanolamine;
LPG = Lysophospha idylglyce ol; LPI = Lysophospha idylinosi ol; LacCe = Lac osylce amide; PC
O = Phospha idylcholine; SM = Sphingomyelin.