Eu opean Jou nal o Pha macology 962 (2024) 176216
A ailable online 29 No embe 2023
0014-2999/© 2023 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-
nc-nd/4.0/).
Edoxaban ea men in a pos -in a c ion expe imen al model
Ja ie Ma ínez-Fe n´
andez
a
,
b
,
1
, C is ina Almengl´
o
b
,
1
, Bo ja Baba o
b
, Ram´
on Iglesias-Rey
c
,
Tom´
as Ga cía-Caballe o
b
,
d
, ´
Angel L. Fe n´
andez
e
,
g
, Miguel Sou o-Baya i
a
,
b
, Jos´
e R. Gonz´
alez-
Juana ey
b
,
,
g
,
i
, Ezequiel ´
Al a ez
b
,
g
,
h
,
*
a
Se icio de Radiología, Complexo Hospi ala io Uni e si a io de San iago de Compos ela, San iago de Compos ela, Spain
b
Ins i u o de In es igaci´
on Sani a ia de San iago de Compos ela (IDIS), Complexo Hospi ala io Uni e si a io de San iago de Compos ela (CHUS). SERGAS, T a esía da
Choupana s/n, A Co u˜
na, San iago de Compos ela, 15706, Spain
c
Neu oimaging and Bio echnology Labo a o y (NOBEL), Clinical Neu osciences Resea ch Labo a o y (LINC), Ins i u o de In es igaci´
on Sani a ia de San iago de
Compos ela (IDIS), Complexo Hospi ala io Uni e si a io de San iago de Compos ela (CHUS), SERGAS, T a esía da Choupana s/n, A Co u˜
na, San iago de Compos ela,
15706, Spain
d
Depa men o Mo phological Sciences, School o Medicine, Uni e si y o San iago de Compos ela and Uni e si y Clinical Hospi al, 15782, San iago de Compos ela,
Spain
e
Hea Su ge y Depa men , Uni e si y Hospi al o San iago de Compos ela, San iago de Compos ela, Spain
Depa amen o de Medicina, Uni e sidad de San iago de Compos ela, 15782, A Co u˜
na, Spain
g
CIBERCV, Mad id, Spain
h
Depa amen o de Fa macología, Fa macia y Tecnología Fa mac´
eu ica, Uni e sidad de San iago de Compos ela, 15782, San iago de Compos ela, A Co u˜
na, Spain
i
Se icio de Ca diología y Unidad de Hemodin´
amica. Complexo Hospi ala io Uni e si a io de San iago de Compos ela (CHUS). SERGAS, T a esía da Choupana s/n, A
Co u˜
na, San iago de Compos ela, 15706, Spain
ARTICLE INFO
Keywo ds:
Edoxaban
Acu e myoca dial in a c ion expe imen al
model
Ca diac emodelling a e in a c ion
Ca diac magne ic esonance imaging
Pos -in a c ion an icoagulan ea men
ABSTRACT
Backg ound: The sequelae o myoca dial in a c ion (MI) equi e speci ic pha macological he apy o minimise he
pos -MI emodelling, which in many cases e ol es in o ca dio ascula complica ions. The aim o his s udy was
o analyse he e ec o edoxaban, an o al an icoagulan , on ca diac eco e y in a a model o pe manen co -
ona y a e y liga ion.
Me hods: An expe imen al me hod o assess he pos -MI emodelling in a s o 4 weeks, based on ca diac
magne ic esonance imaging (MRI) and inal his ological analysis o he hea s was pe o med. The in luence o
daily o al ea men wi h edoxaban (20 mg/kg/day) o 28 days pos -MI was analysed in compa ison o ehicle.
Resul s: In ou model, edoxaban was shown o be sa e and bleeding was obse ed in 1 o 10 animals. Gene al
physical eco e y o he ea ed animals was shown by highe body weigh eco e y compa ed wi h non- ea ed
animals (38.6 ±2.9 s. 29.9 ±3.1 g, espec i ely, a e 28 days). The e was no a p onounced e ec o edoxaban
in pos -MI ca diac emodelling, bu mi iga ed ib osis was obse ed by he educed exp ession o ascula
endo helial g ow h ac o and umou g ow h ac o β1 in he pe i-in a c zone.
Conclusions: Ou analysis p o ided he expe imen al basis o suppo he easibili y o MRI o s udy ca diac
unc ion and cha ac e ise myoca dial sca ing in a a model. O e all da a sugges ed he sa e y o edoxaban in
he model, and compa ed o placebo, i showed a be e pos -MI eco e y, p obably by educing ib osis o he
hea . Fu he esea ch on mid- e m ca diac eco e y wi h edoxaban a e MI is jus i ied.
1. In oduc ion
Ca dio ascula disease is he leading cause o dea h globally and he
leading cause o p ema u e dea h in Eu ope, and ischaemic hea disease
is i s mos impo an subg oup (Wo ld Heal h O ganiza ion, 2020).
Acu e myoca dial in a c ion (AMI) su i al has imp o ed because o
diagnos ic and he apeu ical ad ances (Keeley e al., 2003; Kim e al.,
2008); wi h a subsequen inc emen o mo bidi y and heal h
* Co esponding au ho . Ins i u o de In es igaci´
on Sani a ia de San iago de Compos ela (IDIS), Complexo Hospi ala io Uni e si a io de San iago de Compos ela
(CHUS). SERGAS, T a esía da Choupana s/n, A Co u˜
na, San iago de Compos ela, 15706, Spain.
E-mail add ess: [email p o ec ed] (E. ´
Al a ez).
1
These au ho s con ibu ed equally o his wo k and sha e i s au ho ship.
Con en s lis s a ailable a ScienceDi ec
Eu opean Jou nal o Pha macology
jou nal homepage: www.else ie .com/loca e/ejpha
h ps://doi.o g/10.1016/j.ejpha .2023.176216
Recei ed 28 July 2023; Recei ed in e ised o m 4 No embe 2023; Accep ed 16 No embe 2023
Eu opean Jou nal o Pha macology 962 (2024) 176216
2
expendi u e (Ahmed e al., 2012; Klocke e al., 2007). Le en icula
dys unc ion due o ad e se emodelling is he mos impo an p edic o
o poo ou comes (including dea h, ecu en myoca dial in a c ion,
hea ailu e, a hy hmias, angina, and s oke), and i is c ucial o p e-
en i in an ea ly s age (Ahmed e al., 2012; Li e al., 2019). In his ield,
ou challenges a e o unde s and he unde lying pa hophysiological
mechanisms o his p ocess, iden i y and es no el he apeu ic ap-
p oaches, and ansla e his knowledge o clinical p ac ice (Li e al.,
2019; Mallia as e al., 2013).
Bea ing in mind ha i has been sugges ed ha a e he acu e phase
o co ona y synd omes, he hype coagulable s a e is main ained o a
ime (A dissino e al., 2003; O be e al., 2008), an icoagula ion a e
AMI o seconda y p e en ion is a he apeu ic op ion, and his includes
he di ec o al an icoagulan s (DOACs) (Sha ma e al., 2014). DOACs a e
conside ed an in e es ing, sa e, and e icien al e na i e o old i amin K
an agonis s in se e al clinical se ings (Capodanno e al., 2020; Fawzy
e al., 2019; Makam e al., 2018). In pa icula , ac o X-ac i a ed (FXa)
inhibi o s ha e been shown o educe s oke o sys emic embolism, as
well as all-cause mo ali y in pa ien s wi h a ial ib illa ion (Dei elz-
weig e al., 2022). Howe e , hei e ec on acu e co ona y synd ome
p e en ion is less clea (Pop e al., 2019). Ne e heless, in a sub-analysis
o he ENGAGE AF-TIMI 48 ial, he educ ion in ischaemic e en s wi h
edoxaban e sus wa a in was g ea e in pa ien s wi h es ablished co -
ona y a e y disease, while bleeding was signi ican ly educed wi h
edoxaban ega dless o co ona y a e y disease s a us (Zelnike e al.,
2019). Apa om seconda y p e en ion, DOACs could also modula e
he pa hophysiological mechanisms a e AMI su i al, pa icula ly in
he ca diac emodelling p ocess ha always s a s a e an in a c ion. In
his sense, edoxaban has shown pleio opic e ec s on ascula cells ha
could imp o e myoca dial eco e y a e ischaemia (Almenglo e al.,
2020). This is a new hypo hesis, bu conside ing ha he myoca dial
in a c ion e en a es con inue o inc ease as he popula ion ages (Khan
e al., 2020), i is impe a i e o main ain policies o educe ca dio as-
cula isk ac o s and o explo e new app oaches o myoca dial in a c-
ion ea men . One o hese app oaches is o educe ad e se ca diac
emodelling a e in a c ion, and we will es he possible ole o edox-
aban in his se ing.
P eclinical da a abou he e ec s o DOACs in his pa icula pos -
in a c ion se ing should be p o ided. The e o e, c ea ing sui able ani-
mal expe imen a ion models plays an essen ial ole in hose ad ances
(Do e al., 2018; Klocke e al., 2007; Shudo e al., 2011). Howe e , one o
he limi a ions o hese models, especially in small animals, is he use o
ad anced ca diac imaging echniques. In his s udy, we ha e chosen he
a because o i s ease o managemen and i s ela i ely ew e hical
p oblems (Klocke e al., 2007). We chose ca diac magne ic esonance
imaging (MRI) because i is he Gold S anda d echnique in clinical
p ac ice o comp ehensi e e alua ion o pa ien s wi h ca diac ischae-
mic diseases (Ahmed e al., 2012; Kim e al., 2008; O do as and Higgins,
2011). This echnique is cons an ly e ol ing wi h new sequences o
explo e a ious aspec s o he disease (Do e al., 2018; Ga g e al., 2018;
Yla-He uala e al., 2018). In summa y, he objec i e o he s udy was o
analyse he possible in luence o edoxaban in he pos -in a c ion
myoca dial emodelling in a a expe imen al model o pe manen co -
ona y liga ion, unc ionally cha ac e ised by ca diac MRI du ing he
p ocess, and his ologically analysed as he end-poin measu emen . The
p og ess o his objec i e could open new he apeu ic al e na i es o
myoca dial in a c ion and he p o ec ion o ca diac issue, wi h d ugs
and molecula mechanisms no ye explo ed in his ega d. This, ul i-
ma ely, would con ibu e o educing he socio-sani a y bu den o
ischaemic hea disease.
2. Ma e ials and me hods
2.1. Animals, in asi e p ocedu es and pos ope a i e ca e
The en i e s udy and p o ocols we e app o ed by he Bioe hics
Commi ee o he Uni e si y o San iago de Compos ela and he egional
Galician Go e nmen (code 15,005/16/004).
Twen y- wo male Wis a -Kyo o a s (weigh 350 ±50 g; om he
cen al animal acili y, San iago de Compos ela) we e di ided in o an
in e en ion g oup (IG; n =18) and a con ol g oup (CG, sham; n =4). In
he IG, a su gical liga ion o he le an e io descending (LAD) a e y o
he hea was pe o med o c ea e an AMI, ollowing he p ocedu e
desc ibed by P e e e al. wi h sligh modi ica ions (P e e e al., 1979).
In b ie , animals we e anes he ised wi h 60 mg/kg o ke amine and 5
mg/kg o xylazine. A oe and ail pinch con i med seda ion. Then, a
ca he e was inse ed in o he achea and connec ed o a en ila o ha
in oduced anaes hesia om an induc ion chambe wi h 3% iso lu ane
and oxygen o 5–7 min. Anaes hesia was sus ained a e wa ds wi h
2.5% iso lu ane and oxygen a a en ila ion a e o 2.5 l/min. Animals
we e placed in he igh decubi us posi ion on a wa ming pad a 37 ◦C,
and he le la e al su ace o he ches was sha ed and disin ec ed. The
dep h o anaes hesia was con inually moni o ed by assessmen o he
ail-pinch e lex and espi a o y hy hm. Following a le ho aco omy in
he ou h in e cos al space and he applica ion o a ches e ac o , he
pe ica dium was dissec ed and he LAD a e y was liga ed 1–2 mm
below he le au icula appendage wi h a 6/0 polyp opylene h ead
(P olene 6/0; E hicon Inc., Johnson & Johnson, New B unswick, NJ, US)
in animals o he IG, whe eas animals o he CG we e closed as ollows,
wi hou liga ion. The ches e ac o was e i ed, and he in e cos al
space and ches skin we e su u ed wi h 4/0 Vic yl h ead (Vic yl 496 h,
4/0 E hicon Inc.). A cannula was le be ween he su u es ha allowed
in a ho acic ai o be aspi a ed once he wound was closed, and i was
emo ed immedia ely a e his manoeu e. Finally, he en ila o was
u ned o and emo ed when he animal was able o b ea he on i s own.
The esea che s ook all possible measu es o minimise animals’ pain
and su e ing. Bup eno phine (0.1−2.5 mg/kg) was injec ed s. c. 20–30
min be o e he beginning o he anaes hesia and immedia ely be o e he
end o he su ge y as pos ope a i e analgesia.
A e su ge y, animals we e kep sepa a ely in a cage wa med wi h a
hea lamp un il hey had ully eco e ed om anaes hesia. Wel a e
moni o ing o animals ia beha iou al obse a ion was pe o med e e y
day. Food (pelle s) and esh wa e we e p o ided ad libi um. A 12 h: 12
h ligh : da k cycle and a cons an empe a u e o 24 ◦C we e main ained
du ing he en i e expe imen a ion ime. A he end o he s udy, animals
we e humanely killed. Exsanguina ion was pe o med unde gene al
anaes hesia (iso lu ane 2.5% in oxygen a 2.5 l/min), he ho ax was
opened, blood was wi hd awn om he ena ca a, and he hea was
ha es ed.
2.2. Magne ic esonance imaging
MRI p ocedu es we e pe o med unde se o lu ane anaes hesia (5%
induc ion and 3.5% main enance in a gas mix u e o 70% NO
2
and 30%
O
2
) and b ea h moni o ing. Du ing MRI s udies, each animal was ixed
in a Plexiglas holde using a oo h ba , ea ba s and adhesi e ape o
minimise spon aneous mo emen du ing imaging acquisi ion. Each an-
imal unde wen 3 MRI s udies: basal (MRI0), 4 days (MRI1) and 4 weeks
(MRI2) a e he AMI onse . All ca diac MRI s udies (CMR) we e pe -
o med on a 9.4 T ho izon al bo e magne (B uke BioSpin, E lingen,
Ge many) wi h 440 mT/m g adien s and a quad a u e olume coil (7 cm
in diame e ).
The s udy p o ocol consis ed o co e ing he en i e hea wi h
G adien echo (GE) cine MR images wi h elec oca diog am igge ing in
he p one posi ion in he sho axis (SA, o hea unc ion quan i ica ion
pu poses). A single long-axis slice was ob ained in o de o place he
co esponding sho -axis slices co e ing he apex o he base (8–10 sli-
ces). GE sequence was used o acqui e cine ca diac images wi h he
ollowing pa ame e s: echo ime (TE) =3.3 ms, epe i ion ime (TR) =8
ms, slice hickness 1.5 mm, no slice sepa a ion, ield o iew (FOV) =60
×60 mm
2
, ma ix size 256 ×256 (iso opic in-plane esolu ion o 0.234
mm/pixel). Six een cine- ames we e eco ded o co e he ca diac
J. Ma ínez-Fe n´
andez e al.
Eu opean Jou nal o Pha macology 962 (2024) 176216
3
cycle. The p esence o delayed myoca dial enhancemen was assessed
wi h la e gadolinium enhancemen (LGE) sequence a e in a enous
injec ion o a dose o 0.5 mmol/kg body weigh Gd-DTPA. Fo his
pu pose, a as low-angle sho pulse (IR-FLASH) was used. Sho -axis
iews we e ob ained using he ollowing imaging pa ame e s: epe i-
ion ime, 430 ms; echo ime, 2.1 ms; in e pola ed in-plane esolu ion,
0.313 ×0.313 mm; slice hickness, 1.5 mm wi h con iguous 6 slices;
in e sion ime, 350–450 ms. In e sion ime was op imised o he null
poin o no mal myoca dium manually in each indi idual, and a ca diac
ga ing sys em was used in IR-FLASH sequences. The o al acquisi ion
ime was 90 min.
Da a we e collec ed by a adiologis wi h mo e han 15 yea s o
expe ience in CMR. The ollowing pa ame e s we e quan i ied o he
le en icle (LV): end-dias olic olume (EDV), end-sys olic olume
(ESV), and ejec ion ac ion (EF). Calcula ions we e comple ed om he
cine MR sequences on SA planes. The olumes o all LV slices in
maximum elaxa ion and end-con ac ion s ages we e calcula ed o
ob ain EDV and ESV, espec i ely. EF was calcula ed as a a io (%) ac-
co ding o he exp ession: EF =(EDV-ESV)/EDVx100.
The c i e ia ollowed o LV measu emen s a e de ailed as ollows.
Baseline slices, nea he al e plane, we e e ised ca e ully o no
include he le a ium. Papilla y muscles and abeculae we e included
in olume and EF calcula ions as pa o he ca i y because i assu es
lowe in e ope a o a iabili y. Global LV unc ion was quan i ied using
Segmen 2.0 R5165 (h p://segmen .heibe g.se) (Heibe g e al., 2010).
Expe manual d awing o en icula images was pe o med since i was
conside ed he e e ence s anda d in p ac ice.
Abno mali ies (la e enhancemen ) in he LGE sequence o assess
myoca dial sca ing we e analysed in a semi-quan i a i e isual
assessmen scale, using he ollowing ca ego ies: ‘No’, ‘Possible’,
‘P obable’ and ‘Yes’, in inc easing o de o suspicion o pa hology. The
ca ego isa ion was made by consensus be ween wo adiologis s.
2.3. Edoxaban ea men
A e he MRI1, AMI was con i med, and he animals in he IG we e
di ided andomly in o ea ed (TG) and non- ea ed (NTG) g oups. The
day a e MRI1, ea men s a ed in he TG by o al adminis a ion o
edoxaban 20 mg/kg by he in agas ic ga age echnique om a 3 mg/
mL solu ion o edoxaban in wa e ( his co esponds wi h 0.66 ml/100 g
o body weigh ). The NTG was subjec ed o he same in agas ic ga age
echnique, bu wa e , a he same olume as in TG, was adminis a ed
ins ead o edoxaban. Adminis a ions we e done daily, always a he
same ime o he day (9–11 a.m.) un il he day o he las MRI (MRI2) and
sac i ice o he animal (comple ing 28 days o ea men and ollow-up).
A en ion o ex e nal signs o bleeding (in he nose, eyes, nails, blood in
he animal bedding, e c.) was paid o de ec any inc ease in bleeding isk
as soon as possible.
2.4. His ological analysis
Ra s we e sac i iced 28 days pos -su ge y and hei hea s we e
ha es ed o his ological s udies. Animal and hea weigh s we e
eco ded. Hea s we e ixed in 4% pa a o maldehyde-bu e ed solu ion,
pH =7 (Al a Aesa , A an o , Radno , PA, US), o 24 h a oom em-
pe a u e. Hea s we e hen bisec ed and dehyd a ed, h ough a se ies o
e hanol and xylene and embedded in pa a in. Fou blocks o pa a in
we e done o each hea , and he blocks we e done by ou equal sho -
axis slides o 0.5 cm each. Sec ions o 4
μ
m hick we e moun ed on
silanised coa ed slides (Dako-Agilen , San a Cla a, CA). Epi ope e ie al
was pe o med in a PT-Link (Dako-Agilen ) a high pH o 20 min and
hen au oma ically immunos ained in an Au os aine -Link 48 (Dako-
Agilen ), employing he monoclonal an ibodies and p o ocols lis ed in
Table 1. As a de ec ion sys em, we used EnVision FLEX/HRP (Dako-
Agilen ), o 30 min, and 3,3
′
-diaminobenzidine e a-hyd ochlo ide
(DAB) as he ch omogen o 10 min. The ollowing p o eins we e
immunohis ochemically de ec ed: desmin, imen in,
α
-smoo h muscle
ac in (
α
-SMA), ascula endo helial g ow h ac o (VEGF), me al-
lop o ease 9 (MMP9), issue inhibi o o me allop o einases 1 (TIMP1),
supe oxide dismu ase 2 (SOD2), and umou g ow h ac o be a1
(TGFβ1). Fo haema oxylin–eosin (HE) and Masson ich ome s aining
s anda d p o ocols we e used.
Images o ans e sal sec ions o hea s we e cap u ed wi h a loupe
Leica DMD 108 (Leica). We used he slice whe e he sca was la ges o
assess he AMI as sca ed/ o al LV a ea, exp essed in a pe cen age.
Immunohis ochemis y quan i ica ion was pe o med using he ‘Spli
channels’ unc ion (g een channel) om Fiji-ImageJ® so wa e (h p
s://imagej.ne /so wa e/ iji/). Fo each measu emen , a leas n =3
samples we e used o he his ologic expe imen s.
2.5. Da a analysis
The s a is ical analyses we e pe o med wi h SPSS (S a is ical Pack-
age o he Social Sciences), e sion 17.0. The ca ego ical o dicho o-
mous a iables we e exp essed as absolu e alues and pe cen ages and
we e compa ed wi h he Pea son Ҳ2 es . In he case o con inuous
a iables, no mali y was checked wi h he Kolmogo o –Smi no es .
When no mally dis ibu ed, a iables we e desc ibed as he mean ±
s anda d e o o he mean (s.e.m), and when no , as he median and
in e -qua ile ange. The S uden ’s - es was used o he compa isons o
con inuous a iables be ween g oups o pa ien s ( wo- ail dis ibu ion
and equal a iances be ween samples) when a iables ul illed he
condi ion o no mali y, whe eas he Mann–Whi ney U es was used
when a iables did no ul il he condi ion o no mali y. Non-no mal
dis ibu ed a iables we e compa ed wi h he Wilcoxon es . A p- alue
o <0.05 was conside ed s a is ically signi ican .
3. Resul s
3.1. In e en ional p ocedu e and ea men
Fo y-se en a s we e included in he s udy. Two o hem we e
excluded be o e s a ing he in e en ional p og amme a e hey did
no pass he ou ine heal h es s in he animal acili y, nine een died
du ing he su ge y (in aope a i e mo ali y o 42.2%), h ee mo e did
no su i e o he i s 6 h a e su ge y (pe iope a i e mo ali y o
48.9%), and one mo e died du ing he 4-week ollow-up pe iod by in-
e nal bleeding (day 5 o edoxaban’s adminis a ion, o e all mo ali y
Table 1
An ibodies and incuba ion p o ocols.
An igen An ibody’s
Sou ce
Clone Ca alogue No.
&
Manu ac u e
Conc. Incuba ion
ime
SOD2/
Mn
Mouse 349,810 MAB3419,
R&D Sys ems
1:500 20 min
MMP-9 Mouse 4H3 MAB911, R&D
Sys ems
1:20 20 min
TIMP-1 Mouse 63,515 MAB970, R&D
Sys ems
1:100 20 min
VEGF Mouse VG1 MA1-16629,
The moFishe
1:20 20 min
TGF-β1 Mouse 1,018,746 MAB10502,
R&D Sys ems
1:5000 20 min
Vimen in Mouse V9 M0725, Dako-
Agilen
RTU 20 min
α
-SMA Mouse 1A4 M0851, Dako-
Agilen
RTU 20 min
Desmin Mouse D33 M0760, Dako-
Agilen
RTU 20 min
Abb e ia ions:
α
-SMA:
α
-smoo h muscle ac in; Conc.: concen a ion; MMP-9:
me allop o ease 9; RTU: eady o use; SOD2: supe oxide dismu ase 2; TGF-β1:
umou g ow h ac o be a1; TIMP1: issue inhibi o o me allop o einases 1;
VEGF: ascula endo helial g ow h ac o .
J. Ma ínez-Fe n´
andez e al.
Eu opean Jou nal o Pha macology 962 (2024) 176216
4
51.1%). The e o e, 22 animals su i ed un il his ological analysis, om
which ou we e sham-ope a ed (Fig. 1). In he IG, nine a s o he TG
we e ea ed wi h o al edoxaban (20 mg/kg daily) o 28 days. In pa -
allel, nine a s we e included in he NTG and adminis e ed wa e in he
same quan i y as he TG (6.66 mL/kg daily) by he in agas ic ga age
echnique. One ex a-animal was included in he TG ha died o in e nal
bleeding a e day 5 o edoxaban adminis a ion. Ana omic analysis
a e dea h sugges ed ha an inju y o he oesophagus o s omach du ing
he in agas ic ga age echnique caused gas oin es inal bleeding ha
he an icoagulan e ec o edoxaban did no s op. No o he signs o
bleeding we e obse ed in he es o he animals used in he s udy, so i
was no jus i ied o conduc blood sampling du ing he pe iod o edox-
aban ea men o moni o clo ing imes.
3.2. Pos -in a c ion pe iod
A e he su ge y o p o oke he AMI, he animals showed a gene al
eco e y obse ed by hei physical ac i i y and body weigh eco e y.
Animals om he NTG weigh ing 362.4 ±14.2 g on he day o he
su ge y inc eased a mean o 29.9 ±3.1 g a e 28 days o wa e
adminis a ion ( ehicle o edoxaban). On he o he hand, he animals
om he TG (358.1 ±15.0 g on he day o he su ge y) gained a mean o
38.6 ±2.9 g, being s a is ically signi ican in compa ison wi h he NTG
(p =0.047, Mann–Whi ney U). This weigh eco e y ended o be highe
in he TG han in he NTG om he day 11 o ea men , being s a is i-
cally signi ican in he las days o ea men (Fig. 2). The sham g oup
g adually inc eased hei body weigh a e he ho acic su ge y, bu wo
di e ences we e app ecia ed in compa ison wi h he NTG and TG. Fi s ,
a s did no su e a myoca dial in a c ion, so he impac o he su ge y
was less p onounced han in he o he g oups in e ms o body weigh
loss and beha iou in he days a e he in e en ion and be o e s a ing
he ea men (da a no shown). A e eco e y om he anaes hesia, he
animals showed no mal beha iou wi hou signs o pain and no mal
access o ood and wa e . Second, he body weigh gain con inued du ing
he ollowing 28 days, bu i was mo e g adual han in he case o he
o he wo g oups (21.7 ±3.1 g, p =0.024 and p =0.001 wi h espec o
he NTG and TG, espec i ely; Fig. 2).
3.3. MRI, in a c ion con i ma ion and ca diac emodelling ollow-up
Table 2 summa izes he da a om he MRI a he di e en imes and
g oups. No signi ican di e ences we e ound be ween he NTG and TG
ega ding he EDV, ESV, SV, EF o es ima ed LV mass o e he s udy.
AMI induced a educ ion o he EDV, which was eco e ed du ing he
pos -in a c ion ollow-up in bo h he NTG and TG, wi h a mean o e all
inc ease o 0.07 and 0.13 mL ( o NTG and TG, espec i ely). The same
happened wi h he SV, bu he eco e y o his pa ame e in he NTG was
mo e p onounced a e he AMI han in he TG, wi h a mean o e all
inc ease o 0.03 and 0.06 mL ( o NTG and TG, espec i ely). The es i-
ma ed LV mass seemed no o be a ec ed by he AMI, bu an inc ease in
his pa ame e was obse ed o e he s udy, wi h an o e all inc ease o
Fig. 1. Expe imen al design and wo k low. G aphical ep esen a ion o he expe imen al design wi h he linea wo k low o he in e en ions, analysis, and he
animals included and d opou s. Abb e ia ions: AMI: acu e myoca dial in a c ion; MRI: ca diac magne ic esonance imaging; NTG: non- ea ed g oup; Sham: sham
ope a ed animals; TG: ea ed g oup.
a
No passing heal h check;
b
in aope a i e mo ali y;
c
No suppo he in a c ion;
d
gas oin es inal bleeding. *Two animals o
each g oup could no unde go MRI2 because COVID pandemic lockdown.
Fig. 2. Weigh eco e y a e in a c ion. Weigh a ia ion du ing he ea -
men ime o he non- ea ed g oup (NTG, placebo), he ea ed g oup (TG)
wi h edoxaban (20 mg/kg/day), and sham g oup ( ho acic su ge y wi hou
in a c ion induc ion). Poin s ep esen he mean alues o he weigh a ia ion
wi h espec o he i s day o ea men o all he animals in he same g oup.
Ve ical ba s show he s anda d e o o he mean o each alue. *p <0.05
be ween NTG and TG; #p <0.05 be ween Sham and NTG.
J. Ma ínez-Fe n´
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Eu opean Jou nal o Pha macology 962 (2024) 176216
5
0.05 and 0.06 g ( o NTG and TG, espec i ely).
AMI was con i med in he MRI s udies by LGE. We conside ed
‘P obable’ and ‘Yes’ as posi i e diagnos ic ca ego ies in LGE o
myoca dial in a c , and ‘No’ and ‘Possible’ as nega i e diagnos ic ca e-
go ies. We ook as in a c s hose myoca dial sca s which measu ed mo e
han 5% o he LV su ace in his ology and no in a c s hose myoca dial
sca s which measu ed less han 5%. MRI1 had a 33% sensi i i y, an 80%
speci ici y, a 75% posi i e p edic i e alue and a 40% nega i e p e-
dic i e alue, wi h an o e all accu acy o 50%. MRI2 had a 22% sensi-
i i y, a 100% speci ici y, a 100% posi i e p edic i e alue and a 42%
nega i e p edic i e alue, wi h an o e all accu acy o 50%.
No s a is ical di e ence in he de ec ion o he AMI by LGE was
obse ed be ween he NTG and TG a MRI1 (p =0.383). No di e ence
was obse ed be ween hese g oups a MRI2 (p =0.318; Fig. 3A and B).
The e o e, AMI sequelae in MRI we e compa able o bo h g oups.
Finally, al hough he adiologis obse e de ec ed an imp o emen in
LGE be ween MRI1 and MRI2 in h ee o se en a s in he NTG and i e
o se en a s o he TG, his di e ence was no s a is ically signi ican (p
=0.280; Fig. 3C). Fou animals ( wo pe g oup) could no comple e he
MRI2 because o he COVID-19 pandemic lockdown.
3.4. His ologic analysis a e 28 days o ea men pos -in a c ion
The we weigh o he hea s a he end o he s udy was 1.82 ±0.08
g o he NTG and 2.02 ±0.12 g o he TG, wi h no s a is ical di e ence
be ween hem (p =0.169). In he same way, he a io o weigh s be-
ween he hea s and animals was no di e en o bo h g oups (0.49 ±
0.03 and 0.54 ±0.03% o NTG and TG, espec i ely; p =0.215).
In he his ological analysis, we ound small ib o ic a eas loca ed on
he la e al LV wall in all indi iduals, ha a e compa ible wi h well-
healed small myoca dial in a c s. The a ea o in a c ion was measu ed
in he ep esen a i e his ological slides o he AMI, a e Masson’s
s aining, o op imise he image di e en ia ion be ween heal hy issue
and sca issue (Fig. 4A). The a ea o in a c ion was au oma ically
calcula ed by he ‘Spli channels’ unc ion (g een channel) om Fiji-
ImageJ® so wa e (h ps://imagej.ne /so wa e/ iji/).
Assuming a h eshold o 5% o sca ed myoca dial su ace in his o-
logical slices o conside he in a c as signi ica i e, we ob ained ha
72.2% o animals su passed ha h eshold and p esen ed signi ica i e
AMI (77.7 and 66.6 % o TG and NTG, espec i ely, p >0.05).
Sca s we e loca ed in he la e al wall o he LV, as expec ed a e he
liga u e o he LAD a e y in a s. Mean in a c ed myoca dial su aces
we e 6.3 ±3.2 and 14.6 ±6.5 mm
2
, o NTG and TG, espec i ely (p =
0.536). The pe cen age o he in a c ed a ea o e he o al a ea o he
hea sec ion was 9.1 ±2.7 and 11.4 ±3.4% o NTG and TG, espec-
i ely (p =0.514; Fig. 4B). A good co ela ion was obse ed be ween he
in a c ed a ea deno ed by LGE and he sca obse ed in he his ological
analysis (Fig. 4C), which alida ed he da a ob ained by ca diac MRI.
Haemo hagic le el in he en icle wall, leukocy e in il a ion,
en icle wall hinning, dep h o he in a c in he en icle wall, and
nec osis we e e alua ed and ca ego ised in he haema oxylin–eosin
s aining o all he hea s. Leukocy e in il a ion was only alued as
p esen o no , dep h o in a c was ca ego ised as ansmu al o no , and
he es o he a iables we e alued on a h ee-g ade scale (mild,
mode a e, and se e e). The esul s o his analysis a e summa ised in
Supplemen a y Table S1. Speci ic s aining and immunohis ological
analysis we e done o e he slides wi h in a c ion. Masson’s s aining
se ed o e alua e he dimension o he in a c ion-induced sca . The
exp ession o di e en bioma ke s was e ealed by immunohis ological
analysis o cha ac e ise he ca diac issue. The ollowing p o eins we e
analysed in h ee egions (co e, pe iphe y, and su oundings) wi h
espec o he in a c lesions: desmin (ma ke o myo ilamen s),
imen in (ma ke o ib oblas s),
α
-SMA (ma ke o myo ib oblas and/
o smoo h muscle cells), VEGF (angiogenic and p o- emodelling ac o ),
MMP9 ( emodelling p o ease), TIMP1 (me allop o eases inhibi o ),
SOD2 (an ioxidan enzyme), and TGFβ1 (p o ib o ic ma ke ). Co e was
de ined as he a ea clea ly a ec ed by he in a c , and whe e i was
possible o demons a e he o ma ion o collagen his ologically (Mas-
son’s s aining), he loss o desmin o he o ma ion o imen in. Pe-
iphe y was conside ed he hin laye bo de ing he co e, whe e changes
in he no mal his ology o he hea we e e iden . Finally, he su -
oundings we e de ined as he a ea bo de ing he pe iphe y zone and
whe e he his ologic disposi ion o he issue was a ansi ion be ween
he in a c zone and he no mal issue.
S aining pa e ns we e e alua ed using he in ac myoca dial issue
p esen in all slides as an in e nal con ol. S aining o immunohis o-
logical pa e ns we e g aded as G ade 0: no s aining, G ade 1: much less
de ec able, G ade 2: sligh ly less de ec able, G ade 3: de ec able a he
same le el as in ac myoca dium, G ade 4: sligh ly mo e de ec able, and
G ade 5: much mo e de ec able. The esul s o he immunohis ological
analysis a e summa ised in Supplemen a y Table S1.
As a esul , a endency o a highe nec osis le el was obse ed (p =
0.067) in he pe i-in a c ed a ea o he TG wi h espec o he NTG, bu
no changes we e de ec ed o SOD2 (Fig. 5). VEGF exp ession in he
pe iphe al a ea o he in a c ion was signi ican ly less in he TG han in
he NTG (p =0.027). Howe e , his was no associa ed wi h signi ican
changes in
α
-SMA exp ession. Desmin exp ession was sligh ly highe in
Table 2
Resul s om he MRI analysis du ing he s udy.
Va iable MRI* NTG (n =9) TG (n =9) p
a
EDV (mL) MRI0 0.70 ±0.05 0.64 ±0.05 0.805
MRI1 0.67 ±0.03 0.67 ±0.05 0.945
MRI2 0.77 ±0.03 0.77 ±0.06 0.805
p
b
0.734 0.196
p
c
0.042 0.028
p
d
0.027 0.027
ESV (mL) MRI0 0.35 ±0.05 0.30 ±0.03 0.456
MRI1 0.36 ±0.04 0.32 ±0.03 0.295
MRI2 0.39 ±0.02 0.38 ±0.05 0.710
p
b
0.600 0.463
p
c
0.236 0.108
p
d
0.399 0.072
SV (mL) MRI0 0.35 ±0.01 0.34 ±0.02 1.000
MRI1 0.31 ±0.02 0.35 ±0.04 0.731
MRI2 0.38 ±0.02 0.40 ±0.02 0.535
p
b
0.233 0.292
p
c
0.027 0.017
p
d
0.027 0.172
EF (%) MRI0 50.47 ±2.57 53.20 ±1.25 0.456
MRI1 46.74 ±3.42 52.53 ±3.02 0.534
MRI2 49.84 ±1.57 52.30 ±3.20 0.710
p
b
0.028 0.917
p
c
0.499 0.499
p
d
0.735 0.345
e-LVM (g) MRI0 0.56 ±0.02 0.54 ±0.02 0.620
MRI1 0.59 ±0.03 0.59 ±0.03 0.731
MRI2 0.61 ±0.03 0.60 ±0.03 0.805
p
b
0.395 0.068
p
c
0.034 0.034
p
d
0.352 0.891
Da a om sham g oup a e no p esen ed since only MRI0 was done in his g oup
and hei alues we e like he o he g oups (no su gical in e en ions we e done
a his poin ).
* Two animals o each g oup could no unde go MRI2 because COVID pandemic
lockdown.
a
p alue o he U Mann-Whi ney es o he compa ison be ween NTG and TG.
b
p alue o he Wilcoxon es o he compa ison be ween MRI0 and MRI1.
c
p alue o he Wilcoxon es o he compa ison be ween MRI0 and MRI2.
d
p alue o he Wilcoxon es o he compa ison be ween MRI1 and MRI2.
Bold numbe s indica e s a is ical signi icance.
Abb e ia ions: EDV: end-dias olic olume, EF: ejec ion ac ion, e-LVM: es i-
ma ed le en icle mass, ESV: end-sys olic olume, MRI: magne ic esonance
imaging, NTG: non- ea ed g oup, SV: sys olic olume, TG: ea ed g oup.
J. Ma ínez-Fe n´
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Eu opean Jou nal o Pha macology 962 (2024) 176216
6
he su ounding egions o he TG, and he e could be a possible
educ ion o TGFβ1 and MMP9 exp ession in he co e and pe iphe y o
he TG, espec i ely (Fig. 5). Howe e , no di e en ial exp ession was
obse ed o imen in o TIMP1 be ween he NTG and TG. These da a
me i a discussion abou he possibili y o a modi ica ion in he ib osis
p ocess in he TG.
4. Discussion
In his wo k, an expe imen al me hod has been success ully de el-
oped o assess he pos -MI emodelling in a s o e 4 weeks. The me hod
was based on ca diac MRI in li ing animals and a inal his ological
analysis o he hea s. The in luence o a ea men wi h daily o al
edoxaban (20 mg/kg/day) in pos -in a c ed a s du ing he ollow-up
pe iod was analysed and compa ed wi h non- ea ed animals. To ou
knowledge, his was done o he i s ime. In ou expe imen al model,
his ea men was no shown o be dele e ious. The isk o bleeding was
in a easonable ange, as shown by no ex e nal signs o bleeding du ing
he ea men . Only one animal died o in e nal damage induced by he
in agas ic ga age, bu no di ec ly by he ea men . Thus, good da a
on sa e y was obse ed. The gene al physical eco e y o ea ed animals
was be e han non- ea ed animals du ing he ollow-up pe iod.
Al hough he e was no a ma ked e ec o edoxaban in he pos -
in a c ion hea emodelling, a possible weak educ ion o ib osis,
shown by a signi ican educ ion o VEGF, and a endency o less TGFβ1
and MMP9 exp ession, was obse ed. O e all da a sugges ed non-
in e io i y o edoxaban compa ed o placebo, and in some pa ame e s,
edoxaban could be bene icial o hea eco e y du ing he mid- e m
pos -MI pe iod.
The in aope a i e and pe iope a i e mo ali y a es o ou s udy
we e simila o p e ious epo s in his expe imen al model (An onio
e al., 2009; Lin e al., 2008; P e e e al., 1979). EDV, ESV, SV and EF
we e mos ly una ec ed by AMI onse , indings ha a e in ag eemen
wi h p e ious epo s (P e e e al., 1979). On he con a y, EDV and SV
inc eased du ing he pos -in a c ion ollow-up pe iod, main aining he
global alues o EF in bo h he NTG and TG. Only EF was educed a e
in a c ion in some a s, and his pa ame e was no o ally eco e ed
du ing he ollow-up ime, al hough he mean alues did no wo sen in
he TG o NTG. E olu ion o es ima ed LV mass in MRIs and hea mass
a nec opsy showed no signi ican myoca dial mass los du ing he
o e all p ocess. To al weigh gain du ing he ollow-up was always
con inuous, showing he eco e y o he gene al s a e o he animals.
These indings a e in acco dance wi h p e ious wo ks (P e e e al.,
1979), concluding ha he a model o co ona y a e y liga ion does no
Fig. 3. Ca diac MRI s udy. Rep esen a i e images o la e gadolinium enhancemen (LGE) o (A), an animal o he ea ed g oup, and (B) an animal om he non-
ea ed g oup, in he momen o he MRI a he end o he s udy. (C) An example o imp o emen o myoca dial sca obse ed in he MRI s udy o a ea ed animal.
Le image shows a ep esen a i e LGE a e in a c ion induc ion (MRI1), and igh image a 28 days pos - ea men (MRI2). Whi e a ows poin o myoca dial in a c
egions. Scale ba s =1 mm.
J. Ma ínez-Fe n´
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Eu opean Jou nal o Pha macology 962 (2024) 176216
7
p oduce g oss hea ailu e. In his sense, al hough he pe manen liga-
ion o he co ona y a e y is no he mos mime ical model o he
clinical si ua ion in humans, whe e he opening o he co ona y low is a
manda o y objec i e, he pa icula i y o he a s o suppo he in a c s
makes hem sui able o s udy myoca dial eco e y and emodelling a e
AMI.
The e olu ion o ca diac unc ion a e LAD co ona y a e y liga ion
was una ec ed by he ea men wi h edoxaban. The e we e no s a is-
ical di e ences in any pa ame e o he ca diac MRI analysis (EDV, SV,
es ima ed LV mass, and EF) a e he pe iod o ea men be ween he
NTG and he TG. I dese es commen ha we de ec ed i e cases o
imp o emen in he LGE image a e he ea men wi h edoxaban and
only h ee cases in he NTG. Howe e , we could no pe o m he inal
MRI in ou animals, because o he pandemic lockdown, and his
d as ically educed ou s a is ical powe , and as a esul , no signi icance
was eached. The e o e, ca diac unc ion, analysed by MRI, was una -
ec ed by he ea men o edoxaban in ou expe imen al condi ions.
This means ha he e was no wo sening o his unc ion: ca diac oxici y
o damage was no p oduced by edoxaban. Howe e , i would be o
in e es o analyse a possible imp o emen in ca diac unc ion by pos -
MI edoxaban ea men in a la ge s udy.
His ology con i med ha he su gical p ocedu e was e ec i e in
c ea ing a myoca dial sca in all animals, wi h 72.2% o hem o sig-
ni ican size, hese esul s being simila o hose ob ained by P e e e al.
(1979). The mean in a c ed myoca dial su ace was 10.2 ±3.5%, lowe
han da a epo ed by Kainuma e al. (2017), which ob ained 29%, bu
also wi h a g ea a ia ion ( om 4 o 65%) and using emale Lewis a s,
no male Wis a –Kyo o as in ou case. Fu he mo e, we ha e calcula ed
he pe cen age o he in a c ed a ea, in compa ison o all he hea
su aces in he sec ion, no only he LV a ea. All lesions we e loca ed in
he la e al wall o he LV, con i ming he liga u e o he LAD a e y,
which is desc ibed o i iga e ha a ea a leas in some o he s ains
(Kainuma e al., 2017). LGE indings we e consis en wi h he
his ological loca ion o AMI sca s, wi h a 50% global diagnos ic accu-
acy, s ill lesse han obse ed in human diagnos ic MRI (Kim e al.,
2008; O do as and Higgins, 2011), p obably because o he small size o
he hea and in a c .
Edoxaban (20 mg/kg/day) was adminis e ed daily by an in agas ic
ga age in an aqueous solu ion. Wa e was adminis e ed in con ol ani-
mals by he same p ocedu e, in he same olume as in he ea ed g oup.
Ra s suppo ed he manoeu e, bu a en ion should be aken o a oid
he unexpec ed mo emen s o he animal du ing adminis a ion, espe-
cially in ea ed a s. The eason is ha any iolen mo emen could
damage he oesophagus o s omach o he animal and induce in e nal
bleeding. This happened wi h one o he animals ea ed wi h edoxaban,
and i died less han 24 h a e he damage. Because he bleeding was
in e nal, i was unde ec able un il au opsy. Howe e , i was he only
case o bleeding, which allows us o conclude ha he p ocedu e o
in agas ic ga age and he dosage o edoxaban is, wi h a con ollable
isk, sa e o a s. As obse ed in clinical ials, edoxaban is no
exemp ed om bleeding isk, bu he o e all a e o bleeding- ela ed
p oblems is educed compa ing o adi ional an icoagulan s like
i amin K an agonis s (Vilain e al., 2020; Zelnike e al., 2019). The
long- e m e ec s o edoxaban he apy in pa ien s wi h a ial ib illa ion
and s able co ona y a e y disease a e cu en ly unde s udy (Cho e al.,
2022).
An icoagulan ea men was main ained o 28 days in pa allel wi h
placebo. Du ing his ime, animals eco e ed om he AMI and gene al
heal h condi ions we e ollowed by daily weighing. This allowed us o
con i m he eco e y o all he animals bu wi h a signi ican imp o e-
men in he TG wi h espec o he NTG and sham g oup. In e es ingly,
su i al om myoca dial in a c seemed o induce a gene al eac ion in
he animals obse ed by a apid body weigh gain om day 4 a e he
e en and du ing he ollowing 10 days. A e ha , body weigh inc ease
was pa allel o he NTG and sham g oups. Weigh gain in he TG seemed
o be as e om day 11 o ea men in compa ison wi h he NTG and
Fig. 4. In a c cha ac e iza ion. (A) Rep esen a i e images o Masson’s s aining in an in a c ed sec ion, showing he s eps o he calculus o he in a c ed a ea:
om le o igh , he images show 1) he o al a ea o he hea sec ion bounded by a yellow line, 2) he compu a ional con as di e en ia ion o in a c ion
di e en ia ion, and 3) he a ea o hea ee o in a c ion bounded by a yellow line. (B) Mean o he in a c ed a ea on he non- ea ed g oup (NTG, placebo) and
ea ed g oup (TG). Columns ep esen he mean alue o he g oup and symbols show he indi idual alues o each animal. (C) Le image shows an example o he
MRI o la e gadolinium enhancemen , and igh image shows he his ological image a e Masson’s ich ome s aining o he same egion o he hea a he end o he
s udy. A ows ma k he si e o co ela ion in bo h images. Scale ba s =1 mm.
J. Ma ínez-Fe n´
andez e al.
Eu opean Jou nal o Pha macology 962 (2024) 176216
8
sham, and his heal hy s a e could be due o edoxaban ea men . La ge
s udies in he elde ly ha e been done wi h edoxaban, and i has become
he Fi - OR-The-Aged (FORTA) classi ica ion o bene icial (B le el).
Howe e , i s o e was closes o e y bene icial (A le el) (Wehling e al.,
2017). This unde lines he o e all posi i e assessmen o he isk/bene i
a io o his d ug agains a ailable e idence.
Pos -in a c ion ea men wi h edoxaban o 28 days did no ha e a
ema kable e ec on ca diac emodelling in ou expe imen s. This
pe iod o 4 weeks was p e iously used in a models o he e alua ion o
pos -MI ca diac emodelling (Lin e al., 2008; Liu e al., 2009; Saleh
e al., 2012), and his was he eason o main ain he ea men du ing
ha ime. We could also con i m ha animals we e able o eco e hei
gene al physical s a e in ha pe iod. The his ological analysis o he
biopsies allowed a de ailed s udy o he emodelling p ocess. As s a ed,
he e was no change in he size o he in a c ion a e ea men . How-
e e , his was some hing expec ed, conside ing he na u e o his
end-poin pa ame e . Analysing by bioma ke s, he absence o di e -
ences in
α
-SMA and imen in exp ession be ween he NTG and TG
sugges s ha a e 28 days o ea men , edoxaban was no able o
educe ib oblas mig a ion o he sca . The con e sion o ib oblas s o
myo ib oblas s can be ollowed by he exp ession o
α
-SMA (Venugopal
e al., 2022), and i s p esence sugges s he ac i a ion o he sec e ion o
ex acellula ma ix componen s o he sca o he damaged issue. A
p ocess ha seems o be s ill ac i e a e 28 days pos -in a c ion, inde-
penden ly o he edoxaban ea men .
Ca diac emodelling also seems o be ac i e a e edoxaban ea -
men . The immunohis ochemis y o imen in e eals all he non-
myoca dial cell ypes, including endo helial cells, ascula smoo h
muscle, ib oblas s, e c. The e o e, i s posi i i y sugges s he loca ion o
ac i e emodelling (Kondo e al., 2022). Howe e , his could also sug-
ges ha his emodelling could be a mechanism o a oid he o ma ion
o a pe manen sca wi h educed con ac ile p ope ies, since myoca -
dial ib osis a eas did no e eal myo ib oblas s in p e ious wo ks
(Is a oaie e al., 2015). In ac , in ou samples, he e was a sligh ly
highe exp ession o desmin in he su oundings o he sca in he TG.
This elemen o he myo ilamen s could indica e be e p o ec ion o
ca diomyocy es by he ac ion o edoxaban. This p o ein has been ound
in he la e pe iod a e in a c ion du ing he emodelling p ocess (Da a
e al., 2017).
Al hough he le els o TIMP1 we e no a ec ed compa ing TG and
NTG, he possible endency o he educ ion o TGFβ1 and MMP9 shown
in he TG could also sugges educed ib osis a e 28 days o ea men ,
h ough he TGFβ1-Smad-MMP9 signalling pa hway (Wang e al., 2022).
The ac i e pa icipa ion o MMPs in he emodelling p ocess o a hea s
has been p e iously demons a ed (Pa hasa a hy e al., 2014). This
could be suppo ed by he ac o he educ ion obse ed in VEGF
exp ession in he pe iphe y o he in a c ion o he TG wi h espec o
NTG, which could sugges a educed ac i e p ocess o emodelling a e
28 days o ea men (Cheng e al., 2016). Resul s ag ee wi h he possible
an i-in lamma o y and p o ec i e e ec s showed by edoxaban in endo-
helial cells, which could ha e a ole in he p o ec ion and eco e y o
ca diac issue (Almenglo e al., 2020). Mo e ecen ly, i has been
demons a ed ha DOAC, apa om hei an icoagulan e ec s, also
ha e cellula e ec s a ec ing gene exp ession, in lamma o y esponse,
and ib osis (Gadi e al., 2021). These e ec s a e mo e p onounced o
FXa inhibi o s han o h ombin inhibi o s and depend, a leas in pa ,
on he di e en ac i a ion o p o ein C, a signal componen and cy o-
p o ec i e p o ease.
Finally, oxida i e s ess has been sugges ed o pa icipa e in ca diac
emodelling a e AMI in mu ine models (Hou e al., 2018). A educ ion
in supe oxide anions by SOD o hyd ogen pe oxide by ca alase could
educe he issue damage caused by ischaemia. Howe e , he e we e no
changes in SOD2 le els be ween he NTG and TG, in ag eemen wi h
p e ious s udies (Almeida e al., 2014).
4.1. Limi a ions
The main limi a ion o ou s udy is he numbe o animals included in
he s udy. This educes he po ency o he s a is ical analysis. The
a ia ion o he in a c size induced by he expe imen al model con-
ibu es o his limi a ion. Fu he esea ch in la ge popula ions is
needed o con i m ou indings. Second, ou s udy was made only in
males o a pa icula a s ain; hus, he esul s can be di e en
conside ing sex and s ain di e ences. Thi d, he las MRI (MRI2) was
no possible in some o he animal, because he i up ion o he SARS-
CoV-2 pandemic si ua ion closed some acili ies in ou ins i u e.
Fig. 5. His ological analysis. Rep esen a i e his ological images (all acqui ed
a 4×) o in a c egions in he hea s om he non- ea ed g oup (NTG, le
column o images) o om he ea ed g oup (TG, igh column) o animals,
a e s aining wi h Masson’s ich ome and immunos ainings wi h di e en
an ibodies. (A) Masson’s ich ome s aining (COL), which show highe nec osis
in he pe iin a c ed a ea o he TG. Collagen is s ained in blue, muscle in ed
and nec osis in ligh ed/pink (B) Desmin (DES) exp ession was sligh ly highe
in he su ounding egions o TG. Posi i e s aining in b own. (C) VEGF
exp ession in he pe iphe al a ea o he in a c ion was signi ican ly less in TG
han in NTG. Posi i e s aining in b own. (D) Possibly a educ ion o TGFβ1
exp ession in he in a c co e o TG. Posi i e s aining in b own. (E) Reduc ion o
MMP9 exp ession in he pe iphe y a ea o TG. Posi i e s aining in b own.
Do ed lines showing he co e o he in a c egions. Scale ba s =250
μ
m.
J. Ma ínez-Fe n´
andez e al.
Eu opean Jou nal o Pha macology 962 (2024) 176216
9
4.2. Conclusions
In conclusion, ou su gical p o ocol showed e ec i eness in c ea ing
an AMI and he adiological and his ological esul s p o ide some
expe imen al basis o suppo he easibili y o MRI o s udy ca diac
unc ion and cha ac e ise myoca dial sca s in a a model. Howe e ,
u he in i o s udies should be de eloped especially o imp o e he
homogenei y o in a c s and, hus, imp o e he LGE diagnos ic sequence
pe o mance.
Fo he i s ime in ou model, daily o al edoxaban ea men a e
in a c ion was shown o be sa e, wi h a con ollable isk o bleeding. In
compa ison o he placebo, edoxaban showed a be e gene al eco e y
o animals, non-in e io i y hea emodelling, and p omising da a in
pa hophysiological modula ion o ca diac ib osis and angiogenesis.
The e o e, he p esen esul s jus i y u he esea ch on pos -in a c ion
ca diac eco e y by edoxaban ea men . Especially ying o exploi he
possible bene i o pha macological modula ion o ad e se ca diac
emodelling.
Financial suppo
This wo k was suppo ed by Daiichi-Sankyo Spain.
Funding o open access cha ge: Uni e sidade de San iago de Com-
pos ela/CISUG
CRediT au ho ship con ibu ion s a emen
Ja ie Ma ínez-Fe n´
andez: In es iga ion, Fo mal analysis,
W i ing – o iginal d a , W i ing – e iew & edi ing. C is ina Almengl´
o:
In es iga ion, Fo mal analysis, W i ing – o iginal d a , W i ing – e iew
& edi ing. Bo ja Baba o: In es iga ion. Ram´
on Iglesias-Rey: In es-
iga ion, Fo mal analysis, W i ing – e iew & edi ing. Tom´
as Ga cía-
Caballe o: In es iga ion, Fo mal analysis. ´
Angel L. Fe n´
andez: In es-
iga ion. Miguel Sou o-Baya i: Concep ualiza ion, Supe ision,
Funding acquisi ion, In es iga ion, Fo mal analysis, W i ing – e iew &
edi ing. Jos´
e R. Gonz´
alez-Juana ey: Concep ualiza ion, Supe ision,
Funding acquisi ion, In es iga ion, Fo mal analysis. Ezequiel ´
Al a ez:
Concep ualiza ion, Supe ision, Funding acquisi ion, In es iga ion,
Fo mal analysis, W i ing – e iew & edi ing.
Decla a ion o compe ing in e es
The au ho s decla e he ollowing inancial in e es s/pe sonal e-
la ionships which may be conside ed as po en ial compe ing in e es s:
Au ho s decla e ha his wo k was suppo ed by Daiichi-Sankyo
Espa˜
na, bu his did no in luence he esul s o in e p e a ion o his
manusc ip o any o he au ho s.
Da a a ailabili y
Da a will be made a ailable on eques .
Acknowledgemen s
Thanks o Ma ía O e o Alen, by he p ocessing o his ological sam-
ples in he Immunohis ochemis y Lab o he Heal h Resea ch Ins i u e
o San iago de Compos ela.
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j.ejpha .2023.176216.
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