Extracellular Matrix Defects in Aneurysmal Fibulin-4 Mice Predispose to Lung Emphysema

15 Pages·2014·1.45 MB·English

Extracellular Matrix Defects in Aneurysmal Fibulin-4 Mice
Predispose to Lung Emphysema
Natasja W. M. Ramnath1,7, Koen M. van de Luijtgaarden7, Ingrid van der Pluijm1,7, Menno van
Nimwegen2, Paula M. van Heijningen1, Sigrid M. A. Swagemakers1,3, Bibi S. van Thiel1,7,8,
Ruziedi Y. Ridwan1,7,8, Nicole van Vliet1, Marcel Vermeij4, Luuk J. A. C. Hawinkels9, Anne de Munck10,
Oleh Dzyubachyk5,11, Erik Meijering5, Peter van der Spek3, Robbert Rottier10, Hiromi Yanagisawa12,
Rudi W. Hendriks2, Roland Kanaar1,6, Ellen V. Rouwet7, Alex Kleinjan2*, Jeroen Essers1,6,7*
1Department ofGenetics, Cancer Genomics Centre, ErasmusMC,Rotterdam,The Netherlands, 2Department ofPulmonary Diseases, ErasmusMC,Rotterdam,The
Netherlands, 3Department of Bioinformatics, Erasmus MC, Rotterdam, The Netherlands, 4Department of Pathology, Erasmus MC, Rotterdam, The Netherlands,
5Department of Medical Informatics and Department of Radiology, Erasmus MC, Rotterdam, The Netherlands, 6Department of Radiation Oncology, Erasmus MC,
Rotterdam,TheNetherlands,7DepartmentofVascularSurgery,ErasmusMC,Rotterdam,TheNetherlands,8DepartmentofPharmacology,ErasmusMC,Rotterdam,The
Netherlands,9DepartmentofMolecularCellBiologyandCentreforBiomedicalGenetics,Leiden,TheNetherlands,10DepartmentofPediatricSurgery,ErasmusMC,
Rotterdam,TheNetherlands,11DepartmentofRadiology,LeidenUniversityMedicalCentre,Leiden,TheNetherlands,12DepartmentofMolecularBiology,Universityof
TexasSouthwesternMedicalCenter,Dallas,Texas,UnitedStatesofAmerica
Abstract
Background:In this study we set out to investigate the clinically observed relationship between chronic obstructive
pulmonarydisease(COPD)andaorticaneurysms.Wetestedthehypothesisthataninheriteddeficiencyofconnectivetissue
might playa roleinthe combined developmentofpulmonary emphysemaand vascular disease.
Methods:We first determined the prevalence of chronic obstructive pulmonary disease in a clinical cohort of aortic
aneurysms patients and arterial occlusive disease patients. Subsequently, we used a combined approach comprising
pathological,functional,molecularimaging,immunologicalandgeneexpressionanalysistorevealthesequenceofevents
thatculminates inpulmonary emphysema inaneurysmal Fibulin-4deficient(Fibulin-4R)mice.
Results:Here we show that COPD is significantly more prevalent in aneurysm patients compared to arterial occlusive
disease patients, independent of smoking, other clinical risk factors and inflammation. In addition, we demonstrate that
aneurysmal Fibulin-4R/R mice display severe developmental lung emphysema, whereas Fibulin-4+/R mice acquire alveolar
breakdown with age and upon infectious stress. This vicious circle is further exacerbated by the diminished antiprotease
capacity ofthelungs andultimately resultsin thedevelopment ofpulmonary emphysema.
Conclusions:Our experimental data identify genetic susceptibility to extracellular matrix degradation and secondary
inflammationasthe commonmechanisms inboth COPDand aneurysmformation.
Citation:RamnathNWM,vandeLuijtgaardenKM,vanderPluijmI,vanNimwegenM,vanHeijningenPM,etal.(2014)ExtracellularMatrixDefectsinAneurysmal
Fibulin-4MicePredisposetoLungEmphysema.PLoSONE9(9):e106054.doi:10.1371/journal.pone.0106054
Editor:VincenzoLionetti,ScuolaSuperioreSant’Anna,Italy
ReceivedApril7,2014;AcceptedJuly28,2014;PublishedSeptember25,2014
Thisisanopen-accessarticle,freeofallcopyright,andmaybefreelyreproduced,distributed,transmitted,modified,builtupon,orotherwiseusedbyanyonefor
anylawfulpurpose.TheworkismadeavailableundertheCreativeCommonsCC0publicdomaindedication.
DataAvailability:Theauthorsconfirmthatalldataunderlyingthefindingsarefullyavailablewithoutrestriction.Allrelevantdataarewithinthepaperandits
SupportingInformationfiles.
Funding:Thisworkwassupportedbythe‘LijfenLeven’grant(2008):‘Earlydetectionanddiagnosisofaneurysmsandheartvalveabnormalities’(toJEandPvH).
LHissupportedbytheAlpd’HuZes/BasMulderaward2011(UL2011-5051).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*Email:[email protected](JE);[email protected](AK)
Introduction Aortic aneurysm (AA) is one of the cardiovascular diseases
relatedtoCOPD[6,7].Thenatureofthisrelationshipiscurrently
Chronic obstructive pulmonary disease (COPD) is worldwide unknown.PatientswithCOPD,AA,and/oratherosclerosisshare
oneofthemajorcausesofmorbidityandmortality[1].Inaddition anumberofriskfactors,includingage,hypertension,andtobacco
to chronic airflow obstruction due to airway inflammation and smoking [8,9]. Resemblances in risk profiles between these
alveolar destruction, COPD is associated with extrapulmonary conditions, most notably smoking, may account for the relation
manifestations, including cardiovascular diseases [2–5]. These between AA and COPD. Furthermore, a systemic inflammatory
comorbidconditionscontributetotheoveralldisabilityofpatients response hasbeen suggestedas a commondenominator[10].
and complicatethemanagement ofCOPD.
The association between COPD and AA prompted us to
investigate the prevalence of COPD in a large cohort of patients
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withaneurysmalorarterialocclusivedisease(AOD)inrelationto expressionbytranscriptionalinterferencethroughplacementofa
their clinical risk profiles. Here, we found that COPD is much TKneo targeting construct in a downstream gene (Mus81)[14].
moreprevalentinpatientswithAAcomparedtothosewithAOD, While Mus81 knockout mice, from which the selectable marker
irrespectiveofcommonclinicalriskfactors.SinceAA[11,12]and was removed, were born at expected Mendelian frequencies and
COPD [13] are associated with destruction of the extracellular were indistinguishable from wild type littermates in terms of
matrix (ECM),wehypothesized that a primaryECM defectmay development, growth, immune function and fertility [17], our
provide a common ground for the combined development of Fibulin-4hypomorphicmicedisplayeda2-foldlowerexpressionof
COPD and aneurysm formation. We previously demonstrated Fibulin-4 in heterozygous Fibulin-4+/R aortas and a 4-fold
that mice with reduced expression of the ECM glycoprotein downregulation in homozygous Fibulin-4R/R aortas, resulting in
Fibulin-4 exhibit ECM degradation in the aortic wall and AA ECM defects and vascular abnormalities, including aortic
formation [14,15]. We here investigated the role of Fibulin-4 aneurysms in the aorta ascendens [14]. Indeed, comparison of
deficiency inthedevelopment oflung emphysema. haematoxylin-eosin(HE)andelastinstainedaortasof3-monthold
Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R mice showed severe
Results thickening and decellularization of the medial layer of the
ascending aortic wall in Fibulin-4R/R mice and fragmented and
Clinical study disorganizedelastinlaminaeresultingin2–3folddilatedascending
Patient characteristics. We included 1393 patients; 614 aorticaneurysmsinallhomozygousknockdownanimalsanalyzed
patients (44%) were diagnosed with AA and 779 patients (56%) (Figure 2A). While Fibulin-4+/R aortas are not dilated, careful
with AOD. The majority of AA patients were treated for an histological comparison showed an increased medial thickness,
abdominal aortic aneurysm (AAA). None of the patients in this signs of elastin breakage and increased deposition of amorphous
seriesweretreatedforananeurysmoftheascendingaorta;62/614 cell material between the elastin layers compared to wild type
(10%)ofpatientsweretreatedforananeurysmofthedescending animals (Figure 2A). The downregulation of Fibulin-4 leads to
thoracic aorta (TAA). Clinical characteristics of AA and AOD elastin abnormalities in the ascending aorta accompanied by
patients are presented in Table1. Patients with AA were on extensive remodeling of the ECM presumably through activation
average older and more frequently of male gender. Patients with of matrixmetalloproteinases (MMPs). The increased activity of
AOD had higher rates of diabetes, hypercholesterolemia, and MMPs can be visualized with an MMP-specific activatable near-
cerebrovascular disease. In addition, there were differences in infrared (NIRF) probe developed for in vivo imaging (Perkin
medication use between the two groups: statins and antiplatelet Elmer). Fibulin-4+/R and Fibulin-4R/R mice injected with this
drugsweremorecommonlyusedbypatientswithAOD,whereas proteasesensingprobeshowagradualincreaseinNIRFsignalin
beta-blockers were more often used by patients with AA. thethoracicarea,indicativeofaneurysmformation[14,15].Here,
Importantly,smokingratesweresimilarinthetwopatientgroups. weinjectedthis MMPactivatable NIRFprobeand sacrificedthe
AdifferentiationinclinicalcharacteristicsbetweenAAAandTAA miceafter24hrsafterwhichtheheartswithaortaswereexcised.
patients ispresented inTableS1. Whenwecomparedtheex vivofluorescenceintheaorticarchand
Association between COPD and AA. COPD was more descendingaortausingtheOdysseyimagingsystem,wemeasured
common in AA patients as compared to AOD patients (42% vs. agradualincreaseofMMPactivationinFibulin-4+/RandFibulin-
26%, p,0.001, Figure 1A). COPD rates did not differ between 4R/R compared to wild type Fibulin-4+/+ mice (Figure 2B, upper
TAA and AAA patients (Table S2). Univariate logistic regression row).Sincethiswasconsistentwiththegradualelastindegradation
analysisshowedasignificantassociationbetweenCOPDandAA we noticed histologically, we then used these protease sensing
(odds ratio 2.08, 95%CI: 1.66–2.61, p,0.001; Table S3). Since probes to analyze the abdominal part of the aorta, below the
patients with COPD, AA, and AOD shared a number of diaphragm. Ex-vivo analysis confirmed the in vivo observed
cardiovascular risk factors, we subsequently performed a multi- gradual increase in MMPs within the aneurysmal lesions in the
variable regression analysis. Even after adjustment for potentially thoracicaorta(Th)andshowedaswellagradualincreasedactivity
confounding factors the association between COPD and AA intheabdominalaorta(Ab)inbothFibulin-4+/RandFibulin-4R/R
remained significant (odds ratio 1.56, 95%CI: 1.16–2.10, mice. Thus, decreased expression of fibulin-4 not only leads to
p=0.003;TableS3). MMP activation in the thoracic part of the aorta, but equally
Asinflammationisinvolvedinaneurysmdevelopment,athero- affects the abdominal aorta, predisposing the complete aorta for
sclerosis, and COPD, we measured serum levels of the systemic arterial disease. Since we find extracellular matrix remodeling
inflammatory biomarker high-sensitivity C-reactive protein (hs- activity in both thoracic and abdominal aorta, we conclude that
CRP). The median serum hs-CRP concentration was higher in theFibulin-4+/RandFibulin-4R/Rmousemodelsmimicsdifferent
patients with AA compared to AOD (5.9 [IQR 2.9–12.5] vs stages ofboth TAAandAAA.
4.8 mg/L [IQR 2.1–11.1], p=0.02). However, there were no Alveolar airspace enlargement in Fibulin-4 deficient
differencesinhs-CRPlevelsbetweenarterialdiseasepatientswith mice. Intheclinicalstudy,weobservedasignificantassociation
COPD and those without COPD (median 5.4 vs 5.2mg/L, between COPD and AA. To investigate whether ECM abnor-
p=0.776;Figure 1B).Thesedatastronglysupporttheassociation malities may provide a common ground for aneurysm formation
betweenAAandCOPDinpatientsindependentlyofsmokingand andCOPDwesubsequentlyanalyzedwhetherfibulin-4deficiency
other cardiovascular riskfactors. also predisposes for lung abnormalities in these Fibulin-4
hypomorfic mice.
Experimental study To this end, we first tested whether the transcriptional
Extracellular matrix remodeling in aortas of Fibulin-4 downregulation of Fibulin-4 also occurs in the lungs of these
deficient mice. Complete disruption of Fibulin-4 is incompat- mutant mice. Expression levels of Fibulin-4 mRNA in newborn
ible with life as targeted disruption of Fibulin-4 abolishes and adult lungs of Fibulin-4+/R and Fibulin-4R/R mice were
elastogenesis and causes perinatal lethality in mice [16]. We indeed significantly lower compared to Fibulin-4+/+ mice (Fig-
previously generated a viable mouse model for Fibulin-4 using a ure 3A). Next, we examined whether Fibulin-4 animals display
hypomorphicFibulin-4allele(Fibulin4R)whichresultsinreduced lung emphysema. Assessment of respiratory performance by
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Table1. Clinicalcharacteristics ofpatientswith aortic aneurysm (AA)orarterialocclusive disease (AOD).
AA AOD P-value
n=614 n=779
Baselinecharacteristics
Malegender(%) 525(85.5) 521(66.9) ,0.001
Age(years6SD) 71.467.8 65.6611.0 ,0.001
Bodymassindex(kg/m2,mean6SD) 26.163.9 26.264.3 0.540
Cardiovascularcomorbidities(%)
Congestiveheartfailure 66(10.7) 89(11.4) 0.692
Ischemicheartdisease 272(44.3) 306(39.3) 0.059
Cerebrovasculardisease 89(14.5) 366(47.0) ,0.001
Cardiovascularriskfactors(%)
Kidneydisease 94(15.3) 106(13.6) 0.368
Diabetesmellitus 103(16.8) 225(28.9) ,0.001
Hypertension 408(66.4) 524(67.2) 0.761
Hypercholesterolemia 534(87.0) 706(90.6) 0.030
Smoking–current 236(38.4) 338(43.3) 0.068
Smoking–ever 473(77.0) 613(78.7) 0.459
Medication(%)
Statins 446(72.6) 633(81.2) ,0.001
Beta-blockers 531(86.4) 592(75.9) ,0.001
Renin-angiotensinsysteminhibitors 271(44.1) 369(47.3) 0.247
Diuretics 138(22.4) 211(27.0) 0.052
Antiplatelets 353(57.4) 581(74.5) ,0.001
doi:10.1371/journal.pone.0106054.t001
whole-body plethysmography showed similar breathing frequen- lungs of newborn Fibulin-4R/R mice suggest a developmental
cies and Peak Inspiratory Flows (PIF) in adult Fibulin-4 deficient defect, Fibulin-4+/R mice acquired the COPD phenotype with
mice and Fibulin-4+/+ littermates, whereas the Peak Expiratory age.
Flow (PEF) tended to decrease over time in Fibulin-4R/R mice Transcriptome analysis of Fibulin-4+/R and Fibulin-4R/R
(Figure 3B).Significancecouldnotbedeterminedsince2outof4 lungs. In order to get an idea of the underlying processes
Fibulin-4R/R micedied duringthecourse oftheexperiment. involved, we performed gene expression analysis on mRNA
Downregulation of Fibulin-4 was accompanied by alveolar isolated from Fibulin-4+/+, Fibulin-4+/R and Fibulin-4R/R lung
airspaceenlargementinadultFibulin-4+/RandFibulin-4R/Rlungs tissue, both newborn as well as adult. Comparison of RNA
(Figure 3C). In newborn mice, reduced pulmonary Fibulin-4 expression between newborn Fibulin-4+/+, Fibulin-4+/R and
expression levels coincided with clear alveolar airspace enlarge- Fibulin-4R/R lungs with Significance Analysis of Microarrays
ments in Fibulin-4R/R lungs, but not in Fibulin-4+/R lungs [18] revealed a limited set of differentially regulated genes.
(Figures 3C and 3D, and accompanying Figure S1A and B). Comparison of RNA expression between adult Fibulin-4+/+ and
Importantly,analysisoftheaortasoftheadultmiceusedforlung Fibulin-4R/R lungs using SAM (FDR 10%) revealed 374
analysis showed a gradual thickening of the medial layers of the deregulated genes (both up- and downregulated), whereas no
aorta (Figure S1C). Elastin staining showed sites of complete deregulated genes were found between Fibulin-4+/+ and Fibulin-
fragmentation and disarray of the elastin layers in Fibulin-4R/R 4+/Rlungs.
aortas and increased deposition of amorphous material between Ofthe20mostsignificantlyup-regulatedgenesinadultFibulin-
theelastinlayersinFibulin-4+/Ranimals(FigureS1D),indicating 4R/R lungs, 50% were involved in inflammation processes
that a similar gene-doses decrease in Fibulin-4 expression affects (Table 2andTableS4).NetworkanalysiswithIngenuitypathway
both lungs and the aortic wall in these mice. Immunohistochem- analysis (IPA) on the 374 deregulated probes revealed many
istry on lung tissue with antibodies specific for certain lung cell significantly changed pathways involved in the immune system
markers, including thyroid transcription factor 1 (TTF-1), Clara- (Table S5). This suggests that the severe airspace enlargement in
cell-specificprotein(CC-10),anda-smoothmuscleactin(a-SMA) adult Fibulin-4R/R lungs coincides with overexpression of genes
demonstrated no differences in the presence and relative involved ininflammatory processes.
distribution of the major cell types in the lungs of Fibulin-4+/R Spontaneous inflammation in adult Fibulin-4R/R
and Fibulin-4R/R mice (Figure S2), which may exclude altered lungs. Toinvestigatewhetherindeedtheimmunesystemshows
airway-cell differentiation. significantalterationsinthelungsofFibulin-4R/Ranimals,wedid
These results show that in addition to aortic abnormalities, a flowcytometricanalysisofbroncho-alveolarlavage(BAL)samples.
decrease in Fibulin-4 expression leads to gene dose-dependent This analysis showed more inflammatory cells, in particular
alterations in the lung. While the emphysematous changes in the granulocytes (Gr-1+) and B-cells (CD19+), in lungs of adult
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Figure1.PrevalenceandseverityofCOPDinpatientswithan
aortic aneurysm (AA) or arterial occlusive disease (AOD). (A)
TheprevalenceofCOPDinallGOLDclasseswashigherinAA(n=614)
compared to AOD patients (n=779, ***p,0.001). (B) Serum high-
sensitivityCRPlevelsaccordingtoseverityofCOPDinpatientswithAA
orAOD.Therewasnosignificantdifferencebetweenpatientswithand
withoutCOPD(pfortrend=0.123).
doi:10.1371/journal.pone.0106054.g001
Fibulin-4R/R compared to Fibulin-4+/+ and Fibulin-4+/R mice
(Figure 4A). Cell suspensions of Fibulin-4R/R lungs contained
significantlymoremacrophages(F4/80+)comparedtoFibulin-4+/
+andFibulin-4+/Rlungs(Figure4B),andtendedtocontainmore
T-cells (CD3+) and dendritic cells (CD11c+). Immunohistochem-
Figure 2. Degenerative arterial wall changes and increased
icalanalysisshowedfocalinfiltrationsofinflammatorycellsaround
MMP activity in Fibulin-4 animals. (A) Comparison of the
veinsandbronchiinadultFibulin-4R/Rlungs(Figure4C),mainly architectureoftheaorticwallinFibulin-4+/+,Fibulin-4+/R,andFibulin-
consistingofT-cellsanddendriticcells.CytokineanalysisofBAL 4R/Rmice.Top:Haematoxylin-eosin(HE)stainingofcross-sectionsfrom
samplesshowedsignificantlyhigherlevelsofIL-1binFibulin-4R/R 120day-oldmice.Bottom:AberrationsinelasticlaminaeinFibulin-4R/R
but not in Fibulin-4+/R as compared to Fibulin-4+/+ mice mice, consisting of a fragmented and disorganized appearance of
(Figure 4D) Interestingly, IL-1b is a pro-inflammatory cytokine elastin in the medial layers of the aorta. In heterozygous Fibulin-4+/R
mice, interrupted elastin layers and areas in the aortic wall with a
whichismainlyproducedbyactivatedmacrophagesandwhichis
granular appearance of elastic laminae are present, albeit milder,
increasedinpatientswithCOPD[19,20].Thesedataindicatethat indicatingagenedosageeffect.Scalebarindicates100mm(B)Ex-vivo
thesevereairspaceenlargementobservedinlungsofadultFibulin- analysisshowsagradualincreaseinactiveMMPsasdetectedwiththe
4R/R mice was accompanied by up-regulation of inflammatory activatableNIRFprobeMMPSense680.Withintheaneurysmallesionsin
pathways, whereas the milder lung abnormalities in Fibulin-4+/R thethoracicaorta(Th),aswellasintheabdominalaorta(Ab)ofFibulin-
4+/R and Fibulin-4R/R mice, a graded increase in MMP activity is
animalswerenotassociatedwithanexplicitinflammationprocess.
detected.
Disturbed TGF-b signaling in Fibulin-4 deficient
doi:10.1371/journal.pone.0106054.g002
lungs. Since degradation of the vascular wall in aortic
aneurysms is related to disturbances in the TGF-b signaling 4R/RcomparedtoFibulin-4+/+lungs(1.2-fold,p,0.05,TableS6).
pathway[14,21],wenextinvestigatedtheroleofTGF-bsignaling InadultFibulin-4R/Rlungsweidentifiedup-regulationofTGF-b2
inalveolarwalldegradationinFibulin-4deficientmice.Although and downregulation of the type 2b activin A receptor. In adult
thegeneexpressionanalysisinlungmRNAsamplesonlygaverise Fibulin-4+/Rlungsthe‘SMADspecificE3ubiquitinproteinligase
to a limited set of deregulated genes in newborn Fibulin-4R/R 1’ (Smurf1) gene was significantly downregulated compared to
animals,itdidrevealdownregulationofthePias4geneinFibulin- Fibulin-4+/+lungs.
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Figure 3. Enlarged alveolar airspaces in lungs of Fibulin-4 knockdown mice. (A) Expression levels of Fibulin-4 in lungs isolated from
newborn(n=4,n=4,n=3)andadult(n=4,n=4,n=4)Fibulin-4+/+,Fibulin-4+/RandFibulin-4R/RmicerelativetoFibulin-4+/+lungs(**p,0.01).(B)
Meanpeakinspiratoryflow(PIF)andpeakexpiratoryflow(PEF)valuesforFibulin-4+/+(n=4),Fibulin-4+/R(n=4)andFibulin-4R/Rmice(observedfor
n=4,buttwoanimalsdiedduringtheprocedure)at3-minuteintervals.Aftera9minuteadaptationperiod(thefirstthreetimeintervals),PIFfollows
similartrendsinFibulin-4+/+,Fibulin-4+/RandFibulin-4R/Rmice,whileFibulin-4R/RmiceshowadecreaseinPEF.(C)HEstainedsectionsofformalin
fixedlungsofmalemice.EnlargedalveolarairspacesareobservedinFibulin-4+/R(middle,n=3)andFibulin-4R/Rlungs(right,n=3),withthelatter
beingmorepronounced,comparedtoFibulin-4+/+(n=3).EnlargedalveolarairspacesarealreadypresentinFibulin-4R/Rnewbornlungs(n=3),while
lungs of Fibulin-4+/R littermates (n=5) show no difference compared to Fibulin-4+/+ lungs (n=4). Scale bar 100mm. Magnification 10x. (D) D2
quantification(seemethodsandFigureS1forfurtherexplanation)ofthealveolarairspacesrevealedasignificantdifferencebetweenadultFibulin-4+/
+andFibulin-4+/R(*p,0.05)andbetweenadultFibulin-4+/+andFibulin-4R/Rlungs(**p,0.01)aswellasbetweennewbornFibulin-4+/+andFibulin-4R/
Rlungs(*p,0.05).
doi:10.1371/journal.pone.0106054.g003
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Table2.Themostsignificantlyup-regulatedgenesinadultFibulin-4R/Rlungs.Genesareindicatedwiththeirratioscomparedto
Fibulin-4+/+lungsand the process involved.
Topup-regulatedgenes
Genes Ratio Function
Arg1 3.68 Ureacycle
Slpi 3.37 Inhibitorserineproteases
Ms4a4b 2.38 T-cellregulation
Wisp2 2.26 Inhibitsproliferationofvascularsmoothmusclecells
Prkcb 2.03 B-cellactivation,apoptosis
Emr4 1.98 Mediatebetweenmyeloid-andB-cells
Cd300a 1.92 Leukocytecellsurfaceproteins
Gzma 1.93 CytotoxicT-cellandnaturalkillercellspecificserineproteases
Klra4 1.91 Naturalkillercellreceptor
Nkg7 1.90 Naturalkillercellgranuleprotein
Ctsw 1.84 RegulationofT-cellcytolyticactivity
Wisp1 1.83 Matrixremodeling
Lrat 1.64 Retinoidcycle
Plac8 1.81 Defenseresponsetobacterium
Ccl5 1.78 Chemotacticcytokineandplaysactiveroleinrecruitingleukocytes
Tspan32 1.73 Tumorsuppressingfragment
Cyp51a1 1.70 Productionofsterols
Rbm3 1.68 Temperatureinduced
Bcl2 1.66 Apoptosisregulator
Mef2c 1.65 Transcriptionfactorimportantforvasculardevelopment
doi:10.1371/journal.pone.0106054.t002
To check at the protein level whether changes in the TGF-b a potential common underlying mechanism of the observed lung
system occurred, we performed immunoblot analysis for phos- emphysema phenotype in our Fibulin-4 animals and that in
phorylation of Smad2 (pSmad2), an intracellular mediator of the COPD patients, we compared our mouse dataset to gene lists
TGF-b pathway. These blots showed a gradual increase in related to COPD that we derived from IPA and gene expression
pSmad2 in adult Fibulin-4 deficient lungs, indicating increased datasetsfromlungemphysemapatients.AsearchinIPAwiththe
TGF-b activity (Figure5A). In Fibulin-4+/R lungs we observed a search term ‘chronic obstructive pulmonary disease’ gave 248
1.32-fold change for pSmad2 relative to total Smad and a 1.23- records, which we refer to as ‘COPD-related genes’. Next, gene
foldchangerelativetoactinwhencomparedtoFibulin-4+/+lungs. expression data from the comparison between Fibulin-4+/+ and
In Fibulin-4R/R lungs we observed a 1.67-fold and 1.5-fold Fibulin-4R/R lungs (374 genes) were compared to this list of
change, respectively (Figure 5A and data not shown). Accumula- COPD-related genes derived from IPA, where we found an
tionofphosphorylatedsmad2isageneralreadoutofactivityfor overlapof6genes:PDE3B(1.28q),HCK(1.55q),PRF1(1.47
the TGF-b signaling pathway. Smad4 binding to phosphorylated q), SERPINA1 (1.38 Q), FGFR3 (1.28 Q), and EFEMP2 (i.e.
Smad2 is necessary for translocation to the nucleus Subsequently Fibulin-4, 3.57 Q). In a second analysis, we compared the 374
the smad2/3/4 complex can then bind to the DNA after which deregulated mouse genes to a list of 125 deregulated genes from
transcriptionisinitiated.Wethereforealsodeterminedphosphor- the comparison of GEO dataset GSE8581 (1.5-fold, FDR 30%),
ylationofpSmad3inproteinextractsofthelungsofFibulin-4+/+, consisting of 15 COPD cases (predicted FEV1,70%, FEV1/
Fibulin-4+/RandFibulin-4R/Rmiceandfindagradualincreaseof FVC,0.7) and 18 control cases (predicted FEV1.80%, FEV1/
pSmad3 in the mutant animals confirming the activation of the FVC.0.7). This comparison showed an overlap of ITPKC (1.28
central TGF-b transcription factor Smad2/3/4 (Figure 5B). Q),KIAA1377(1.45Q),andSERPINA1(1.38Q).Remarkably,
Immunohistochemistry confirmed increased pSmad2 expression thesetwoindependentmethodsbothidentifiedSERPINA1asan
in Clara cells lining the bronchioles of the lungs as well as in overlapping downregulated gene. SERPINA1 encodes for the
inflammatorycellinfiltrates(Figure 5C).Togetherthesedatashow serine protease inhibitor a-1 antitrypsin, whose targets include
that TGF-b activity is mildly increased in adult Fibulin-4+/R and elastase. Interestingly, deficiency in a-1 antitrypsin in patients is
Fibulin-4R/R lungs, which may contribute to the breakdown of associated with lung emphysema[22,23].
alveolarwallsinadultFibulin-4deficientmice.Thisupregulation InthemouselungmRNAgeneexpressionanalysisSERPINA1
of TGF-b signaling is reminiscent of the upregulation that has wassignificantlydownregulatedinbothFibulin-4R/Rlungs(1.38-
been observed in the aortas of both mouse models of aortic fold, p,0.01) aswell as in Fibulin-4+/Rlungs (1.59-fold, p,0.01)
aneurysms as wellas inpatients [14,15,27]. compared to Fibulin-4+/+ lungs. We used Path explorer in IPA
Overlapping downregulation of SERPINA1 in lungs of thatidentifiespathwaysbetweendifferentially expressedgenes,in
Fibulin-4deficientmiceandCOPDpatients. Toinvestigate order to determine the relation between Fibulin-4 and SER-
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Figure4.Increasedinflammation in Fibulin-4R/R lungscomparedto Fibulin-4+/+andFibulin-4+/Rlungs.(A)Flowcytometricanalysis
revealedmoreGr1+granulocytesandCD19+B-cellsinBALsamplesfromFibulin-4R/R(n=4)comparedtoFibulin-4+/+mice(n=4,*p,0.05)and(B)
increasednumbersofF4/80macrophagesinFibulin-4R/Rlungs(n=4,*p,0.05).(C)HEstainedsectionsfromadult(n=4,n=4,n=4)Fibulin-4+/+,
Fibulin-4+/RandFibulin-4R/RlungsshowingfocalinfiltrationsaroundvesselsandairwaysinFibulin-4R/Rlungs(blackarrows).(D)StainingforT-cells
(CD3+)and(E)dendriticcells(CD11c+)pointstothepresenceofinflammatorycellswithinthefocalinfiltrations.Magnification20x.Scalebar50mm.
(F)ELISAanalysisshowingincreasedIL-1blevelsinFibulin-4R/Rlungs(n=4,*p,0.05).
doi:10.1371/journal.pone.0106054.g004
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ExtracellularMatrixDefectsCauseAorticAneurysmsandCOPD
Figure5.IncreasedTGF-bsignalinginFibulin-4+/RandFibulin-4R/Rlungs.ImmunoblotanalysisofpSmad2inlunghomogenatesshowsan
increaseintheamountofpSmad2(A)andpSmad3(B)inFibulin-4+/R(n=3)andFibulin-4R/R(n=3)lungs,comparedtothetotalamountofSmad,and
totheirFibulin-4+/+control(n=3).(C)IncreasedpSmad2stainingofinflammatoryandendogenouscellsonFibulin-4+/R(n=3)andFibulin-4R/R(n=3)
lungsections.Magnification2.5x(scalebar1mm)upperpaneland20x(scalebar200mm)lowerpanel.(D)Ingenuitypathwayexplorershowed
MMP9astheshortestconnectionbetweenFibulin-4andSERPINA1.SERPINA1inhibitsneutrophilelastase,whichaffectselastin.MMP9itselfwasnot
deregulatedinFibulin-4R/Rlungs(n=4),butcouldbeconnectedto16deregulatedgenesinFibulin-4R/RcomparedtoFibulin-4+/+lungs(n=4,red,
up-regulated;green,downregulated),suggestionalteredMMP9activity.BlackarrowsindicatetheconnectionbetweenFibulin-4(EFEMP2),MMP9,
SERPINA1,elastaseandELN.GreyarrowsindicatetheconnectionofthesegeneswithderegulatedgenesbetweenFibulin-4+/+andFibulin-4R/Rlungs.
doi:10.1371/journal.pone.0106054.g005
PINA1. By calculating the shortest path between Fibulin-4 revealed, as indicated by the black arrows in Figure 5D
(EFEMP2) and SERPINA1, an indirect connection of Fibulin-4 [14,24,25]. Surprisingly, by connecting Fibulin-4, MMP9 and
to MMP9 and a direct connection of MMP9 to SERPINA1 was SERPINA1 (both direct and indirect) to the significantly
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deregulated genes identified in the SAM comparison between large patient cohort are in line with previous observations of
Fibulin-4+/+ and Fibulin-4R/R lungs (grey arrows), we found an reduced respiratory function in smaller series of AAA patients
interactionbetween16ofthosesignificantlyderegulatedgenesand [6,7,10]. Although some previous studies concluded that the
our dataset with MMP9 (Figure 5C). Importantly, MMPs are association between COPD and aneurysm formation was related
proteins involved in remodeling of the ECM, and play a role in to smoking, medication use or presence of other cardiovascular
aneurysm formation. As we hypothesized that ECM defects risk factors, these associations became non-significant after
provide the link between the relation between AA and COPD, correctioninmultivariableanalyses.[28,29].Ourfindingssuggest
MMPscouldverywellbepartoftheunderlyingmechanism.Since that factors other than cardiovascular risk profiles or systemic
our gene expression analysis did not show a deregulation of inflammation contribute to the association between COPD and
MMP9 at the mRNA level, yet the pathway analysis pointed AA.
towards involvement of MMP9, we next investigated MMP9 Since both diseases, COPD and AA, are characterized by
protein activity inFibulin-4R/R andFibulin-4+/R lungs. breakdown of the ECM in the airways and –spaces and in the
Increased MMP and neutrophil elastase activity in aortic wall, we investigated whether a primary ECM defect
Fibulin-4deficientlungs. Fluorescent imaging using protease provides the pathogenic link between these two diseases.
activatable probes showed increased pulmonary MMP activity in Analogous to the observed degradation of the aortic wall, up-
adultFibulin-4deficientmice(Figure 6A).Theobserveddecrease regulation of MMP activity both thoracic as well as abdominal,
in the elastase inhibitor SERPINA1 and the increased MMP andpreviouslyobservedformationofAAsinmicedeficientinthe
activity, which is also associated with cleavage of a-1 antitrypsin ECM component Fibulin-4, we found that gradual downregula-
[24], might lead to increased activity of neutrophil elastase (NE) tion of Fibulin-4 in the lungs correlated with destruction of
(alsoseeFigure 5Cforthisrelation).Indeed,weobservedagraded alveolar walls and airspace enlargement that is characteristic for
increase in NE activity in adult Fibulin-4+/R and Fibulin-4R/R lung emphysema. Similar to embryonically lethal, complete
lungs compared to Fibulin-4+/+ lungs (Figures6B and C). In line Fibulin-4 knockout mice [16], alveolar breakdown was already
withthis,pulmonaryelastinstainingdemonstratedinterruptionsin present in lungs of newborn Fibulin-4R/R mice, and became
theelastinlayersinFibulin-4+/RlungsandevenmoreinFibulin- progressive with age. In contrast, Fibulin-4+/R mice, which have
4R/RlungscomparedtothoseofFibulin-4+/+animals(Figure 6D), only a 2-fold reduction in the amount of Fibulin-4, had normal
as previously found in the aortas of these mice [14]. Newborn elastin structures and alveolar airspaces at birth, but acquired
Fibulin-4R/R lungs also displayed elastin abnormalities, while alveolar breakdown with ageing.
newbornFibulin-4+/RlungswerecomparabletothoseofFibulin- AnalogoustotheactivationoftheTGF-bpathwayintheaortas
4+/+mice (datanot shown). of Fibulin-4 deficient mice [14], we here demonstrate enhanced
Increased and prolonged inflammatory response in activationoftheTGF-bpathwayinthelungsofFibulin-4+/Rand
lipopolysaccharide (LPS) exposed Fibulin-4+/R Fibulin-4R/R mice. The co-occurrence of lung emphysema and
mice. Fibulin-4+/R mice developed alveolar airspace enlarge- vascular abnormalities in association with deregulated TGF-b
mentwithagetogetherwithincreasedMMP9andNEactivityin signaling has also been shown in another mouse model with a
the absence of inflammation. However, when adult Fibulin-4+/R deficiency in an ECM protein, Fibrillin-1, which is a model for
lungs were triggered by LPS administration, which mimics Marfansyndrome[30].Moreover,thecombinationofpulmonary
bacterial infection in mice by initiating the infiltration of emphysemaandaorticaneurysmscoincidingwithupregulationof
inflammatory cells into the pulmonary alveoli similar to patients TGF-b signaling has also been observed in autosomal recessive
with COPD exacerbation [26,27], flow cytometric analyses of cutis laxa syndrome caused by Fibulin-4 mutations [31–33]. The
BALsamplesandpulmonarycellsuspensionsshowedanincreased role for TGF-b in this process is further supported by the
andprolongedinflammatoryresponseascomparedtoFibulin-4+/ development of progressive airspace enlargement in Smad3
+ mice. There was a significantly greater influx of macrophages knockout mice, which are deficient for an intracellular regulator
(F4/80+) in the lungs 18 hours after LPS exposure and of the TGF-b pathway [34]. Overall, these data point to
significantly higher numbers of dendritic cells (CD11c+), T-cells deregulated TGF-b signaling and ECM defects as common
(CD3+), and granulocytes (GR1+) 72 hours after LPS exposure underlying factors foraortic andpulmonary abnormalities.
(Figure 7 and Figure S3). Moreover, opposite dynamics were Expression analysis further revealed downregulation of the
observed; in the Fibulin-4+/+ lungs the amount of GR1+ cells SERPINA1 gene, encoding for the serine protease inhibitor a-1
decreased after 18 hours, while an increase was observed in antitrypsin whose targets include elastase. Interestingly, overlap-
Fibulin-4+/R lungs 72 hours after LPS exposure. The increase of ping geneexpression profiles of our Fibulin-4 deficient mice with
inflammatory cells after LPS exposure was significantly higher those of COPD patients revealed downregulation of SERPINA1
whencomparedtoPBS.Thelevelsofpro-inflammatorycytokines as a common denominator. As it is known that patients with a-1
released upon LPS exposure, including IL-1b, TNF-a, and antitrypsindeficiencydevelopCOPD[22,35]weexploredthelink
keratinocyte-derived chemokine, were not different between between SERPINA1 and Fibulin-4. Pathway exploration in IPA
groups (data not shown). These data indicate that Fibulin-4+/R revealedadirectlinktoMMP9,TGF-bderegulationand15other
mice exhibitan intensified inflammatory response inthelungs. deregulated genes from our dataset. Although MMP9 itself was
not overexpressed, molecular imaging showed that the MMP
Discussion activity was gradually higher in Fibulin-4+/R and Fibulin-4R/R
lungs. In line with the observed downregulation of SERPINA1,
InthisstudyweshowthatCOPDismorecommoninpatients fluorescentimagingshowedagradualup-regulationofelastasein
withAAthaninpatientswithatheroscleroticarterialdisease.This Fibulin-4+/RandFibulin-4R/Rlungs,whichcorrelatedwithelastin
relationship was independent of cigarette smoking and other fragmentation. The decreased expression of SERPINA1 may
knownriskfactors.Furthermore,therewasnodifferenceinserum eitherbeadirecteffectofFibulin-4deficiency,oranindirecteffect
hs-CRP levels between patients with and without COPD, throughitscleavagebyMMP9[24].Thiscombinationofincreased
indicating that inflammation per se is unlikely to account for the proteaseactivityanddecreasedantiproteaseactivitymayaccount
observed relation between COPD and AA. The findings in this forthebreakdown of alveolar walls, resulting in emphysema.
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Figure6.HigherMMPandNEactivityinFibulin-4+/RandFibulin-4R/Rlungs.In(A)and(C)exvivoimagingofexcisedlungsusingOdyssey
showsincreasedactivityofMMPandNErespectivelyinFibulin-4+/R(n=7)andFibulin-4R/Rlungs(observedforn=5,buttwoanimalsdiedduringthe
procedure)ascomparedtoFibulin-4+/+lungs(n=5),withasignificantupregulationforFibulin-4R/Rlungs(**p,0.01).(B)Open-chestregistrationof
NEactivitywithNeutrophilElastaseFAST680probesshowsincreasedactivityinFibulin-4+/RandFibulin-4R/RlungsascomparedtoFibulin-4+/+lungs.
(D)ElastinstainingofFibulin-4+/+,Fibulin-4+/RandFibulin-4R/Rlungs(n=3,n=3,n=3)showsfragmentedelastinlayersinFibulin-4+/RandFibulin-4R/R
lungs,indicatedbyarrows.Magnification40x.Scalebar10mm.
doi:10.1371/journal.pone.0106054.g006
AnotherhallmarkofadultFibulin-4micewastheinflammatory did not exhibit pulmonary inflammation under baseline condi-
response in the lungs. Lungs of adult Fibulin-4R/R mice already tions, but displayed an enhanced respiratory inflammatory
displayed pulmonary inflammation in a specific pathogen free response upon LPS inhalation. These findings indicate that
environment, including influx of a wide range of inflammatory although inflammation may contribute to the progressive break-
cells with elevated levels of the pro-inflammatory cytokine IL-1b, downofalveolarwallsinadultFibulin-4R/Rmice,itisunlikelyto
which coincided with the overexpression of genes involved in betheprimarycausativefactorinFibulin-4+/Rmice.Conversely,
inflammatory pathways. In contrast, adult Fibulin-4+/R animals ECM degradation by proteases is known to induce the release of
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