Hyperdynamic circulatory syndrome in a mouse model transgenic for SerpinB3

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ContentslistsavailableatScienceDirect

Annals

of

Hepatology

j o ur na l ho me p ag e:w w w . e l s e v i e r . e s / a n n a l s o f h e p a t o l o g y

Original

article

Hyperdynamic

circulatory

syndrome

in

a

mouse

model

transgenic

for

SerpinB3

Gianmarco

Villano

a

_,

_Alberto

_Verardo

b

_,

_Andrea

_Martini

b,∗

_,

_Silvia

_Brocco

b

_,

_Paola

_Pesce

b

_,

Erica

Novo

c

_,

_Maurizio

_Parola

c

_,

_David

_Sacerdoti

b

_,

_Marco

_Di

_Pascoli

b

_,

_Marny

_Fedrigo

d

_,

Chiara

Castellani

d

_,

_Annalisa

_Angelini

d

_,

_Patrizia

_Pontisso

b

_,

_Massimo

_Bolognesi

b a_Dept._of_Surgery,_Oncology_and_{Gastroenterology,}_University_of_Padova,_Padova,_Italy

b_Dept._of_Medicine,_University_of_Padova,_Padova,_Italy

c_Dept._of_Clinical_and_Biological_Sciences,_Unit_of_Experimental_Medicine_and_{Interuniversity}_Center_for_Liver_{Pathophysiology,}_University_of_Torino,_Torino,

Italy

d_Dept._of_Cardiac,_Thoracic_and_Vascular_Sciences,_University_of_Padova,_Padova,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received2April2019 Accepted28June2019

Availableonline18September2019 Keywords: Serpin Angiotensin Fibrogenesis Cardiacoutput Splanchnicvasodilation

a

b

s

t

r

a

c

t

Introductionandobjectives:SerpinB3isacysteineproteaseinhibitorinvolvedinseveralbiological activ-ities.Itisprogressivelyexpressedinchronicliverdisease,butnotinnormalliver.Theroleinvascular reactivityofthisserpin,belongingtothesamefamilyofAngiotensinII,isstillunknown.Ouraimwasto evaluatetheinvivoandinvitroeffectsofSerpinB3onsystemicandsplanchnichemodynamics. Materialandmethods:Differenthemodynamicparameterswereevaluatedbyultrasonographyintwo coloniesofmice(transgenicforhumanSerpinB3andC57BL/6Jcontrols)atbaselineandafterchronic car-bontetrachloride(CCl4)treatment.InvitroSerpinB3effectonmesentericmicrovesselsof5Wistar-Kyoto ratswasanalyzedmeasuringitsdirectactionon:(a)preconstrictedarteries,(b)dose–responsecurves tophenylephrine,beforeandafterinhibitionofangiotensinIItype1receptorswithirbesartan.Hearts ofSerpinB3transgenicmiceandofthecorrespondingcontrolswerealsoanalyzedbymorphometric assessment.

Results:InSerpinB3transgenicmice,cardiacoutput(51.6±21.5vs30.1±10.8ml/min,p=0.003), hep-aticarterypulsatilityindex(0.85±0.13vs0.65±0.11,p<0.001)andportalveinbloodﬂow(5.3±3.2 vs3.1±1.8ml/min,p=0.03)weresigniﬁcantlyincreased,comparedtocontrols.Invitro,recombinant SerpinB3hadnodirecthemodynamiceffectonmesentericarteries,butitincreasedtheirsensitivityto phenylephrine-mediatedvasoconstriction(p<0.01).Thiseffectwassuppressedbyinhibitingangiotensin IItype-1receptors.

Conclusions:Intransgenicmice,SerpinB3isassociatedwithahyperdynamiccirculatorysyndrome-like pattern,possiblymediatedbyangiotensinreceptors.

1. Introduction

Incirrhosis,portalhypertensionresultsfromanincreaseinthe

intrahepaticresistancetoportalﬂowduetoliverﬁbrosisand

alter-ationinintrahepaticvasculature.Portalhypertensionisalsocaused

byanincreaseinportalbloodinﬂow,duetoincreasedsplenicand

mesentericblood ﬂow causedbythevasodilationof splanchnic

arteries[1].Systemicandsplanchnicvasodilationisthemaincause

∗ Correspondingauthorat:Dept.ofMedicine,UniversityofPadova,viaGiustiniani, 2,35128Padova,Italy.

E-mailaddress:andrea.martini.10@phd.unipd.it(A.Martini).

oftheonsetofthehyperdynamiccirculatorysyndromeofcirrhosis,

characterizedbyincreasedcardiacoutputandheartrate,decreased

systemicvascularresistanceandlowarterialbloodpressure[1].

Accordingtothe“peripheralarterialvasodilationtheory”[1],

splanchnic vasodilationleadstorenal sodiumretention and,as

aconsequence,hypervolemiawhichcontributestothe

hyperdy-namicsyndromebyincreasingcardiacpreload.Thehyperdynamic

syndrome,inturn,maintainsandenhancesportalhypertension.

Thehyperdynamiccirculatorysyndromeispivotalforthe

devel-opment of severalcomplications of cirrhosis [1,2]. The leading

mechanismforthedevelopmentofthishemodynamicsyndrome

seemsto be thealtered sensitivity to vasoconstrictorsand the

increasedeffectofvasodilators[3].

https://doi.org/10.1016/j.aohep.2019.06.021

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SerpinB3(SB3) is a member of the ov-serpins/cladeB

fam-ily,protease inhibitorsinvolvedin multiplebiological functions

and cell homeostasis control [4]. This serpin is physiologically

expressedinsquamousepithelium[4],insweatglands,in

endothe-lialcellsoftheveinsandinarterieswalls[5].SB3isnotdetectable

in normal hepatocytes [6], while its expression progressively

increases in chronic liver disease, cirrhosis and hepatocellular

carcinoma[7].Adirectcorrelationofthismoleculewith

Transform-ingGrowthFactor-_␤1(TGF-_␤1)expressionhasbeenreportedin

chronichepatitisandhepatocellularcarcinoma[8]andin

exper-imentalmodelsoflung ﬁbrogenesisin SB3transgenicmice[9].

Moreover,ithasbeenrecentlyobservedthatSB3over-expression

signiﬁcantlyincreasestheexpressionofpro-ﬁbrogenicgenes,and

activateshepaticstellatecells,favoringcollagendeposition,intwo

murine modelsof liver ﬁbrosis[10].SB3 has ananti-apoptotic

effect[11]and,inaddition,itinducesepithelialtomesenchymal

transitionandinterleukin-6(IL-6)expressionasaconsequenceof

unfoldedproteinresponse[11].Theinvolvementofthisserpinin

thecontrolofproteolyticprocesseshasimportantimplicationsin

ﬁbrogenesisandneoplastictransformation,whereasthebalance

betweenproteasesandtheirinhibitorscanaffecttheextracellular

matrixcomponents,cellmotility,invasiveness,proliferationand

apoptosis[11].

Takingintoaccountthatmembersoftheserpinfamily,suchas

AngiotensinII,areknowntobeinvolvedintheregulationof

arte-rialresistance[12]andareabletostimulatecellproliferation[13]

andTGF-␤1andﬁbronectingeneexpression[14],wehypothesized

thatSB3mighteithercontributeortobeinvolvedinthegenesis

and/ormaintenanceofthehyperdynamiccirculatorysyndromeof

cirrhosis.

The aim of the study was to investigate the relationships

between SB3 and systemic and splanchnic hemodynamics. For

thispurpose,weanalyzedthehemodynamicdifferencesbetween

transgenicmiceexpressinghumanSB3andcontrolanimals,and

howsuchdifferencesweremodiﬁedbytheinductionofliver

ﬁbro-sisbychroniccarbontetrachloride(CCl4)treatment.Inthisstudy,

wealsoevaluatedliverandhearthistopathologyandinvestigated

invitroeffectsofhumanSB3onmesentericregulation.

2. Materialsandmethods

2.1. Experimentalanimals

C57BL/6Jmicetransgenic(TG)forhumanSB3(kindlyprovided

byProfessorG.Cassani,TechnogenS.c.p.A,PianadiMonteVerna,

CE,Italy)andC57BL/6Jwildtype(WT)controlmice(CharlesRiver

ItaliaS.p.A,Calco,Lecco,Italy)wereusedasexperimentalmodel.

TransgenicmiceshowedhighSerpinB3expressionintheliver,

eval-uatedbymRNAquantiﬁcationandimmunohistochemistry,while

thisserpinwasbarelydetectableintheliverofcontrolmice(Suppl.

Figure1andRefs.[9,15]).

Moreover,5WTWistar-Kyotorats(male,16weeksold,body

weight,200–250g;CharlesRiverLaboratories,Calco,Lecco,Italy)

wereusedtoanalyzetheeffectsofrecombinantSB3on

mesen-tericvascularreactivity.Miceandratswerekeptunderspeciﬁc

pathogen-freeconditionsandmaintainedwithfreeaccesstopellet

foodandwaterattheAnimalCareFacilityoftheCenterof

Experi-mentalSurgery,UniversityofPadua,Italy.

Allinstitutionaland nationalguidelinesforthecareand use

oflaboratoryanimalswerefollowed.Theexperimentswere

car-riedoutinaccordancewiththeItalianlegislation(D.L.27/01/1992

116),whichcomplieswiththeEuropeanCommunityguidelines

(CEEDirective86/609)forthecare/useofexperimentalanimals.

Allanimalsreceivedhumanecareaccordingtothecriteria

out-linedinthe“GuidefortheCareandUseofLaboratoryAnimals”

preparedbytheNationalAcademyofSciencesandpublishedby

theNationalInstitutesofHealth(NIHpublication86-23revised

1985).Theexperimentalprotocolswereapprovedbythelocal

Eth-icalcommitteeandbytheItalianMinistryofHealth(ethicsnumber:

202/2011-B).Allmeasuresweretakentominimizeanypainor

discomforttotheanimals.

2.2. Inductionofliverﬁbrosis

LiverﬁbrosiswasinducedbyCCl4(Sigma-Aldrich,Milan,Italy)

inbothWTandTGmice(males,aged12–14weeksold).CCl4

intox-icationisawidelyusedmodelofexperimentalliverﬁbrosis[16],

thatinducesnecrosisandhepatocyteapoptosiswithactivationof

hepaticstellatecellandtissueﬁbrosis[17].

Sixteenmice(8TGand8WT)wereinoculatedintraperitoneally

withCCl4atadoseof50␮l/100gbodyweight,dissolvedinoliveoil

(20%),twiceaweekfor10weeks[18].Thecontrolgroup(6TGand

6WT)receivedinjectionsofoliveoilvehicletwiceaweekfor10

weeks.Allmiceweresacriﬁced24hafterthelastdoseoftreatment.

Aftersacriﬁce,bloodwasobtainedbycardiacpunctureandtheliver

andspleenexcised,weighedanddividedintosamples.Atleasttwo

tissuesamplesweretakenfromtheliverofeachanimal:theﬁrst

onewasformalinﬁxedandparafﬁnembeddedforhistological

anal-ysis(hematoxylin–eosinandSiriusredstaining),whiletheother

wasimmediatelyfrozeninliquidnitrogenandstoredat−80◦_C_for

molecularstudies.Fortheevaluationofheartremodeling,3TGand

3WTmicewithoutCCl4treatmentwereconsidered.

2.3. Mouseliverhistology

Hematoxylin–eosinandSiriusred stainingswerecarriedout

accordingtostandardprotocols. ImmunohistochemistryforSB3

and␣-smoothmuscleactin(␣-SMA)wasperformedon

parafﬁn-embeddedsectionsofallliverspecimens,obtainedfromtreated

and control mice using peroxidase block to eliminate

endoge-nous peroxidase activity. After washing, sections (2␮m thick)

wereincubatedwithanti-␣-SMA(1:200dilution;Sigma-Aldrich,

Milan,Italy)andwithmonoclonalantibodyagainstSerpinB3(Santa

Cruz Biotechnology,CA,USA; dilution 1:50)respectively.These

antibodieswereuseddilutedinphosphate-bufferedsaline

supple-mentedwithbovineserumalbumin,overnightat4◦Cinahumid

environment,followedbyincubationwithperoxidaselabeled

poly-mer conjugated tosecondary antibodies(HRP-labelled System;

DAKO,Milan,Italy).Theimmunoreactionproductwasvisualized

byaddingsubstrate-chromogendiaminobenzidine(DAB)solution,

resultingwithbrownishcolorationatantigensites.Negative

con-trolswereperformedbyreplacingtherespectiveprimaryantibody

byisotypeandconcentrationsmatchedirrelevantantibody.

2.4. Quantitativereal-timePCR(SYBRgreenassay)

The expression of SerpinB3 and Collagen 1 genes in the

liverwasassessedby real-timePCR usingthefollowing setsof

primers:forSB3senseprimer5-GCAAATGCTCCAGAAGAAAG-3,

reverse primer 5-CGAGGCAAAATGAAAAAGATG-3. For Collagen

1 senseprimer: 5-GGGCAAGACAGTCATCGAAT-3,antisense: 5

-GGTGGAGGGAGTTTACACGA-3. The housekeeping gene mouse

␤-actin was analyzed in all ampliﬁcation sets to assess the

integrity of total RNA extracts and to deﬁne therelative

tran-script quantiﬁcationofeachgene ofinterest.Thesetof␤-actin

primersis:sense:5-AGAGCTACGAGCTGCCTGAC-3andantisense:

5-GGATGCCACAGGACTCCA-3.Therelativeexpressionwas

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2.5. Mousehearthistologyandtissueanalysis

Macroscopicandmicroscopicanalyseswereperformedonthe

WTandTGspecimens.Allsampleswereﬁxedin10%formalinin

0.1mol/L(pH=7.4)phosphatebuffer,dehydratedinaseriesof

ris-ingalcoholconcentrations,andthenembeddedinparafﬁnforlight

microscopy. Four micrometer-thick sectionswere stained with

hematoxylinand eosin and Heidenheimmodiﬁed Azan-Mallory

[20].

2.6. Morphometricassessmentoftheheart

The following indices were calculated by computerized

planimeter(ImageProplus)onformalin-ﬁxedtransversesectionof

thehearttakeninthemiddleportionofinterventricularseptum:

-Leftventricularmass/rightventricularmass(LVM/RVM),indexof

hypertrophy.

-Leftventricularmass/leftventricularvolume(LVM/LVV),indexof

dilatationofleftventricle.

-Right ventricular mass/right ventricular volume (RVM/RVV),

indexofdilatationofrightventricle.

Theseindiceswerecalculatedwithavalidatedprocedure

cur-rentlyadoptedinourlaboratory[20–22].

2.7. SilvernitratestainingforAgNOR

To assess cellularproliferation, Nucleolar Organizer Regions

(AgNOR)were evaluated in sectionsof formalin-ﬁxed

parafﬁn-embeddedhearth tissue samples, as previously described [23].

Brieﬂy,Gelatinwasdissolvedin1%aqueousformicacidata

con-centrationof2%.Thissolutionwasmixedwith50%aqueoussilver

nitrate solution (1:2), and the ﬁnal solution was poured over

thetissuesections,whichwereincubatedfor30minunderdark

roomconditionsat roomtemperature.Theywerethen washed

withdeionizeddistilledwaterbeforecounterstainingwithMayer’s

hematoxylinand dehydrationbyalcoholsand xylene.Synthetic

medium wasused tomount thesections. Thetotal number of

AgNORdots/nucleusata1000×magniﬁcation(oilimmersion)in

200representativenucleiwerecounted.

2.8. Hemodynamicsevaluation

Afteranesthesia inducedwith3% isoﬂurane andmaintained

with1.5%isoﬂuraneduringconstantmonitoringoftemperature,

respirationrate,andECG,allTGandWTmice,treatedanduntreated

withCCl4,underwenttocardiac,hepatic,renalandsplenic

hemo-dynamicsevaluationusingtheVevo2100apparatus(Visualsonics,

Toronto, Canada), a high-resolution in vivo micro-imaging

sys-temforsmallanimals.Thefollowingparametersweremeasured:

cardiacoutput(CO),portalvein(PV)diameter,portalveinblood

velocity(PBV)andﬂowvolume(PBF),hepatic,splenicandrenal

arterial resistanceindices: resistance index (RI)and pulsatility

index(PI).Alltheseparameterswerecalculatedasdescribed

else-where[24–26].Miceunderwentanesthesia,inducedinachamber

for inhalation of isoﬂurane,before undergoingthe sonographic

examination.Micewerethenputsupineona speciﬁctable,on

whichtheyweremaintainedanesthetizedwithisoﬂurane,while

body temperature,heart andrespiratory rates weremonitored.

Thorax and abdomen of mice were then shaved to allow an

adequateultrasonographicwindow. Toeliminate inter-observer

variabilityofDopplerresults,allthemeasurementswererecorded

byasingleoperator,whowasunawareofthetreatmentandthe

characteristicsoftheinvestigatedmice.

Fig.1.Relativeweightofliver(panelA)andspleen(panelB)inTGandWTanimals afteroliveoil(control)orCCl4treatment.Resultsreferto16treatedmice(8TGand

8WT)andto12untreatedcontrolmice(6TGand6WT).Theboxindicatesthelower andtheupperquartileandthemiddlelineindicatesthemedian;barsindicatethe rangeofvaluesdistribution(Mann–Whitneytest).

2.9. Isolatedmicrovesselpreparation

SmallresistancemesentericarteriesofWistar-Kyotorats

(diam-eter <500␮m) were studied as previously described [26]. The

no-ﬂowmodelwaschosentoavoidinterferencefromshearstress.

2.10. EvaluationofthedirecteffectofSB3onmesentericarteries

preconstrictedwithphenylephrine

ThepreparationofisolatedmesentericarteriesofWistar-Kyoto

rats[26]wasusedtoevaluatetheinvitroeffectofrecombinantSB3

onvasculardiameter.TherecombinantproteinSB3wasprepared

aspreviouslydescribed [27].ThedirecteffectofSB3on

mesen-tericarterieswasevaluatedafterpreconstrictionof thearteries

withphenylephrine(PHE)(1␮M).RecombinantSB3wasdissolved

into physiological salt solution(PSS) toobtain a concentration

of10−7M.Thisconcentrationwasempiricallychosen,

consider-ingthevasoactiveeffectofanequimolardoseofanotherserpin,

AngiotensinII[28].SB3wasadministeredintheperfusion

cham-ber(extraluminalapplication)anditseffectwasevaluatedforat

least5min,measuringthechangeinarterialdiameterwithavideo

system[26].

2.11. Evaluationofdose–responsecurvestophenylephrineand

SB3onmesentericarteries,beforeandafterinhibitionof

AngiotensinIItype1-receptorswithirbesartan

ThepreparationofisolatedmesentericarteriesofWistar-Kyoto

ratsdescribedelsewhere[26]wasusedtoevaluatetheeffectof

(4)

Table1

HemodynamicparametersofTGandWTmicebeforeandaftertreatmentwithCCl4.

BeforeCCl4treatment AfterCCl4treatment TGvsWTbeforeCCl4 TGvsWTafterCCl4

TGmice WTmice TGmice WTmice

CO(ml/min) 51.6±21.5 30.1±10.8 59.2±12.6 59.8±29.2 p=0.003 p=NS PVdiameter(mm) 1.1±0.2 1.1±0.2 1.3±0.2 1.2±0.2 p=NS p=NS PBV(mm/s) 151±60 97±54 131±25 118±101 p=0.010 p=NS PBF(ml/min) 5.3±3.2 3.1±1.8 5.6±3.6 5.7±6.5 p=0.030 p=NS HepaticRI 0.58±0.06 0.49±0.11 0.60±0.11 0.55±0.05 p=0.001 p=NS HepaticPI 0.85±0.13 0.65±0.11 0.89±0.22 0.81±0.13 p=0.001 p=NS SplenicRI 0.45±0.08 0.50±0.09 0.41±0.06 0.42±0.07 p=NS p=NS SplenicPI 0.62±0.16 0.72±0.21 0.53±0.10 0.54±0.15 p=NS p=NS RenalPI 0.58±0.08 0.55±0.09 0.60±0.05 0.59±0.13 p=NS p=NS RenalRI 0.87±0.17 0.81±0.18 0.93±0.11 0.89±0.27 p=NS p=NS Heartrate(bpm) 451±49 478±50 504±19 446±54 p=NS p=NS

TG:transgenicforhumanSB3;WT:wild-type;CO:cardiacoutput;PV:portalvein;PBV:portalbloodﬂowvelocity;PBF:portalbloodﬂowvolume;RI:arterialresistance index;PI:arterialpulsatilityindex;bpm:beatsperminute.

Boldvaluescorrespondtosigniﬁcantpvalues(p<0.05)

Fig.2. HemodynamicparametersinTGandWTmice,beforeandafterchronicadministrationofCCl4.Resultsreferto16treatedmice(8TGand8WT)andto12untreated controlmice(6TGand6WT).Estimatedofvarianceare±SD.

theequilibrationperiod,concentration–responsecurvestoPHEat

cumulativedoses(10−8to10−4M)wereevaluated.Afteraproper

washoutperiodofatleast20min,mesentericarterieswere

chal-lengedagainwithincreasingdosesofPHE(10−8to10−4M)and

SB3(10−8to10−4M)simultaneouslyadministeredbyextraluminal

application,andconcentration–responsecurveswerere-evaluated.

The effect of SB3 after inhibition of Angiotensin II type

1-receptors with irbesartan was also analyzed. Irbesartan at the

concentrationof10−5Mwasaddedtothesolution,aspreviously

described[29].Aftera20-minperiodofequilibration,mesenteric

arterieswereﬁrstchallengedwithPHEaloneatincreasing

concen-trations(10−8to10−4M)andconcentration–responsecurveswere

recorded.Then,afterawashoutwithPSSandirbesartanforatleast

20min,thearterieswerere-challengedwithincreasingdosesof

PHE(10−8 to10−4M)and SB3(10−8 to10−4M)simultaneously

administeredbyextraluminalapplication.

2.12. Dataanalysis

Parametersof ﬁbrosis and morphometric analysis onhearts

were expressed as mean_±SD or median_±SEM and analyzed

using theunpaired t-test, unpairedt-test withWelch’s

correc-tionandnon-parametricMann–WhitneyUtestwhenappropriate.

Hemodynamicdatawereexpressedas mean±SD andanalyzed

withunpairedt-test.TheeffectsofPHEandSB3wereevaluated

asvariations intheinternal diameterofthevessels. Data were

expressedasmean_±SE.Two-wayANOVAwasusedtocompare

concentration–responsecurvesfromcontrolsandtreatedgroups.

Thenullhypothesiswasrejectedatp<0.05.

3. Results

3.1. Pathologicalﬁndingsoftheliver

AfterCCl4 treatment, TG miceshoweda signiﬁcantincrease

of the relative volume of the liver and of the spleen,

com-paredtountreatedmice(liver/bodyweightratio:6.01±0.50%vs

5.55±0.63%,p=0.016;spleen/bodyweightratio;0.77±0.51%vs

0.28_±0.06%,p=0.008),whileWTmicedidnotshowsigniﬁcant

differences(Fig.1AandBrespectively).Accordingly,liversofTG

vsWTmicetreatedwithCCl4showedanincreasedcollagen

syn-thesis/accumulationandanexacerbatedmyoﬁbroblastactivation

(Suppl.Figure2aand2b),asresultedbydatarelatedtocollagen1

mRNAexpression(Suppl.Figure2c),conﬁrmingthat

overexpres-sionofSB3inTGmiceispro-ﬁbrogenicinthismodelofchronic

(5)

Transgenic mice

Hematoxylin and eosin

Azan-mallory

AgNOR staining

Wild type mice

A

B

C

D

E

F

Fig.3.Hearthistologyandtissueanalysis.Hematoxylinandeosin(A,B)andAzan-Mallorystain(C,D)oftransgenicmiceshowedthenormalorientation,diameter,nuclear shapeandnoincreaseofinterstitialfibrosisinrespectofwildtypemice.PanelsA–D:originalmagnification40×.AgNORstainingwasthesameintransgenicandwildtype mice(E,F).NoteintranuclearblackdotsinthenucleusofcardiomyocytesthatindicatesthepresenceofAgNORsites.PanelsE,F:originalmagnification63×.Resultsreferto 6untreatedmice(3TGand3WT).

Ratio 0 1 2 3 4 5 6 LVM/RVM RVM/RVV LVM/LVV p=0.01 p=0.90 p=0.14 TG WT

Fig.4.MorphometricassessmentoftheheartinWTandTGmice.TheindexLVM/RVMratioissigniﬁcantlyhigherinTGmicecomparedtothecontrols.Resultsreferto 6untreatedmice(3TGand3WT).Columnsaremeans±SD.LVM:leftventricularmass;RVM:rightventricularmass;LVV:leftventricularvolume;RVV:rightventricular volume.

3.2. HemodynamicschangesinrelationtoCCl4treatment

BeforeCCl4treatment,CO,PBV,PBF,hepaticRIvaluesand

hep-aticPIvalues were signiﬁcantlyhigher in TGmice thanin WT

animals(Table1andFig.2).AfterCCl4 treatment,CO,PBV,PBF,

hepaticRIandPIvalueswerenotmodiﬁedinTGmice,whilethey

increasedinWTmice.TheseparametersweresimilarinallCCl4

treatedanimals.Asfarasheartrate,splenicandrenalresistance

indicesareconcerned,nosigniﬁcantdifferencewasfoundamong

theinvestigatedgroups.

3.3. Histologicalevaluationoftheheart

Heart histological evaluation of WT mice and TG mice did

not show signiﬁcant differences in terms of morphometric

(6)

516.2±49.5n/mm2 _{respectively),} _myocyte _diameter _(12.1_±_1.5

and11.2±1.2␮m),asreportedintherepresentativeFig.3Aand

B.SilvernitratestainforAgNORproteinsitesdidnotreveala

dif-ferentnumberofproteinssitesinwildtypemicevstransgenicmice

(2.74±0.71and3.37±0.74,respectively)(Fig.3EandF).

Intransgenicmicetheindexofhypertrophy(LVM/RVMratio)

was signiﬁcantly higher compared to wild type mice (p=0.01)

(Fig.4).

3.4. EffectofSB3onpreconstrictedratmesentericarteries

Mesenteric arteries vasoconstriction obtained after PHE

(10−6M)challengewasnotfurtherinﬂuencedbytheextraluminal

administrationofrecombinantSB3(Fig.5A).

This molecule alone had no acute hemodynamic effect on

mesentericarteriesinvitro,howeverit increasedthesensitivity

toPHEofsmallresistancemesentericarteries(two-wayANOVA:

p<0.01)(Fig.5B).

ArterialsensitivitytoPHEwasdecreasedbypre-treatmentwith

irbesartan(two-wayANOVA:p<0.01)butwasnotfurthermodiﬁed

bytheadministrationofSB3(two-wayANOVA:p=NS)(Fig.5C).

4. Discussion

ThepresentstudydemonstratesthatSB3hasavasoactiveeffect

onmesentericcirculation andmayparticipatein thegenesisof

thehyperdynamiccirculatorysyndromeoflivercirrhosis,besides

beinginvolvedintheprogressionofhepaticﬁbrogenesis[10].

Thenoveltyofourresultsresidesonthefactthatinour

experi-mentalmodel,despiteCCl4treatedTGmiceshowedmoreadvanced

morphologicalandmolecularfeaturesofhepaticﬁbrosis,compared

tocontrols,SB3hasdemonstratedavasoactiveeffectonsplanchnic

circulation.Adirectparticipationofthis moleculeinthe

patho-physiologyofthehyperdynamiccirculatorysyndromeofcirrhosis,

whichmaintainsandworsensportalhypertension,maytherefore

behypothesized.

Ourresultsonuntreatedmicehaveshownthat,comparedtoWT

animals,TGmicehavesigniﬁcantlyhighervaluesnotonlyofportal

veinbloodvelocityandﬂowvolume,butalsoofcardiacoutput.In

addition,inuntreatedTGmice,anincreaseinhepaticartery

resis-tanceindexwasdetectedandthisﬁndingisinagreementwiththe

socalled“hepaticarterialbufferresponsetheory”.Accordingtothis

theory,hepaticarterialresistancephysiologicallyincreaseswhen

PBFincreases,tomaintaintotalhepaticbloodﬂowconstant[30].

Ontheotherhand,adirecteffectofSB3onintrahepaticarterial

resistancecannotberuledout.Onelimitationofthisworkisthe

lackofmurineurineanalysistoconﬁrmthepresenceofanhigher

sodiumretentioninTGmicecomparedtoWTmice.However

differ-enthemodynamicparametershavebeenevaluatedinthesemiceby

anhigh-resolutioninvivomicro-imagingsystem,andnoconﬂicting

resultshavebeenfound.

Both cardiac and splanchnic hemodynamic modiﬁcations

describedabovearecommonlyfoundinlivercirrhosis,supporting

thefactthatourmurineTGmodelforSB3presentshemodynamic

alterationtypicalofcirrhosisintheabsenceofliverstructural

sub-version.HemodynamicdifferencesbetweenTGandWTmicewere

abolishedafterCCl4treatment,whenalsoWTmicedevelopedthe

hyperdynamiccirculatorysyndromeinacontestofadvanced

ﬁbro-sisdeposition.

Inlinewiththepresentedﬁndings,morphometricanalysison

hearttissuesamplesshowedanincreasedcardiachypertrophyin

TGmice,possiblylinkedtotheirhyperdynamiccirculation,while

0.0 0.2 0.4 0.6 0.8 1.0 baseline -8 -7 -6 -5 -4 Contraction Phe (log[M]) Baseline SerpinB3 0.00 0.20 0.40 0.60 0.80 1.00 baseline -8 -7 -6 -5 -4 Contraction Phe (log[M])

Irbesartan Irbesartan + SerpinB3 0 50 100 150 200 250 300 350 400 baseline 1 mi n 2 mi n 3 mi n 4 mi n 5 mi n Internal diameter (µm) p<0.01 p: ns p: ns

A

B

C

Fig.5.PanelA:effectofSB3(10−7_M)_on_small_resistance_mesenteric_arteries

(isolatedfromto 5WTWistar-Kyotorats)preconstrictedwithphenylephrine (10−6_M)._No _signiﬁcant_variation_in_arterial _diameter_was_observed. _Panel_B:

concentration–responsecurvetophenylephrine(PHE)obtainedinsmallresistance mesentericarteries.TheadministrationofSB3increasedthesensitivitytoPHEofthe arteries(two-wayANOVA:p<0.01).PanelC:concentration–responsecurvetoPHE obtainedinsmallresistancemesentericarteriesincubatedwithirbesartan(10−5_M)

toinhibitAngiotensinIItype1-receptors.Afterpre-incubationwithirbesartan,the administrationofSB3didnotmodifythesensitivitytoPHEofthearteries(two-way ANOVA:p:NS).Estimatedofvarianceare±SD.

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capillarydensity,numbersofmyocytesorproliferationindexwere

notsigniﬁcantlyincreased.

ThestudyofvascularreactivityonWistar-Kyotoratsshowed

thatinvitrorecombinantSB3hasnodirecthemodynamiceffect

onmesentericarterieswhereasitsacuteadministrationmodiﬁes

thesensitivity ofthearterytoalpha-adrenergicstimuli.

Consis-tentlywiththedata abovediscussed,we cansupposethat this

effectmightbemediatedby therenin–angiotensin–aldosterone

system(RAAS),particularlybyAngiotensinIItype1-receptors.This

hypothesis,that needstobefurtherexploredinfuture studies,

hasbeenconﬁrmedandsupportedbydatafromexperimentsin

whichSB3-mediatedincreaseinmesentericvasoconstriction,was

blockedbytheadministrationofirbesartan,aspeciﬁcinhibitorof

AngiotensinIItype1-receptors.

In conclusion, SB3 seems to participate to the genesis of

the hyperdynamic circulatory syndrome, possibly mediated by

angiotensinreceptors,besideitsinvolvementintheprogression

ofliverﬁbrosis,leadingtothemechanicalincreaseinintrahepatic

portalresistanceofcirrhosis.

Abbreviations

CCl4 carbontetrachloride

SB3 SerpinB3

TGF-␤1 TransformingGrowthFactor-␤1

IL-6 interleukin-6

TG transgenic

WT wildtype

CO cardiacoutput

PV portalveindiameter

PBV portalveinbloodvelocity

PBF portalbloodﬂow

␣-SMA ␣-smoothmuscleactin

RI resistanceindex

PI pulsatilityindex

PHE phenylephrine

LVM leftventricularmass

RVM rightventricularmass

LVV leftventricularvolume

RVV rightventricularvolume

PSS physiologicalsaltsolution

RAAS renin–angiotensin–aldosteronesystem

NORs NucleolarOrganizerRegions

AgNOR silver-stainedNucleolarOrganizerRegions

Financialsupport

ThisworkwassupportedinpartbyNationalMinistryof

Educa-tion,UniversityandResearch[FIRBProjectProt.RBLA03S4SP005]

andbytheUniversityofPadova[ProjectNo.CPDA110795].

Conﬂictofinterest

None.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,atdoi:10.1016/j.aohep.2019.06.021.

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