cardiac-restricted igf-1ea overexpression reduces …ethicon-johnson & johnson, usa). ... and...
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Research ArticleCardiac-Restricted IGF-1Ea Overexpression Reducesthe Early Accumulation of Inflammatory MyeloidCells and Mediates Expression of Extracellular MatrixRemodelling Genes after Myocardial Infarction
Enrique Gallego-Colon1 Robert D Sampson1 Susanne Sattler1
Michael D Schneider1 Nadia Rosenthal123 and Joanne Tonkin1
1National Heart and Lung Institute Imperial College London London UK2Australian Regenerative Medicine Institute EMBL Australia Monash University Clayton Melbourne VIC Australia3The Jackson Laboratory Bar Harbor ME USA
Correspondence should be addressed to Joanne Tonkin jtonkinimperialacuk
Received 23 July 2015 Accepted 13 September 2015
Academic Editor Michael Schnoor
Copyright copy 2015 Enrique Gallego-Colon et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Strategies to limit damage and improve repair after myocardial infarct remain a major therapeutic goal in cardiology Our previousstudies have shown that constitutive expression of a locally acting insulin-like growth factor-1 Ea (IGF-1Ea) propeptide promotesfunctional restoration after cardiac injury associated with decreased scar formation In the current study we investigated theunderlying molecular and cellular mechanisms behind the enhanced functional recovery We observed improved cardiac functioninmice overexpressing cardiac-specific IGF-1Ea as early as day 7 aftermyocardial infarction Analysis of gene transcription revealedthat supplemental IGF-1Ea regulated expression of key metalloproteinases (MMP-2 and MMP-9) their inhibitors (TIMP-1 andTIMP-2) and collagen types (Col 11205721 and Col 11205723) in the first week after injury Infiltration of inflammatory cells which direct theremodelling process was also altered in particular there was a notable reduction in inflammatory Ly6C+ monocytes at day 3 andan increase in anti-inflammatory CD206+ macrophages at day 7 Taken together these results indicate that the IGF-1Ea transgeneshifts the balance of innate immune cell populations early after infarction favouring a reduction in inflammatory myeloid cellsThis correlates with reduced extracellular matrix remodelling and changes in collagen composition that may confer enhanced scarelasticity and improved cardiac function
1 Introduction
Cardiovascular diseases (CVD) are the major cause of deathglobally with myocardial infarction (MI) being one ofthe main causes of mortality [1] After MI the damagedmyocardium releases inflammatory signals that trigger a cas-cade of cellular processes in order to repair damaged tissueleading to the formation of scar tissue and left ventricular(LV) dysfunction [2 3]
Our laboratory has characterised the therapeutic proper-ties of the insulin-like growth factor-1 Ea (IGF-1Ea) propep-tide during wound healingregeneration and pathological
inflammationThe IGF-1 gene is encoded in 70 kb of genomicDNA distributed over six exons and five introns [4 5]Use of alternative start codons generates proteins with N-terminal variability while different exon use at the 31015840 endgenerates multiple C-terminal extension-peptides termed E-peptides The most predominant is a 35-amino-acid-long E-peptide termed Ea alternating with a far less abundant E-peptide termed Eb or mechanogrowth factor (MGF) [6 7]TheE-peptides control local IGF-1 bioavailability by adheringstrongly to the extracellular matrix (ECM) retaining thepropeptides locally and preventing their release into the cir-culation [8] Expressed as a cardiomyocyte-specific transgene
Hindawi Publishing CorporationMediators of InflammationVolume 2015 Article ID 484357 10 pageshttpdxdoiorg1011552015484357
2 Mediators of Inflammation
or delivered locally to the mouse heart IGF-1Ea improvesfunctional recovery after cardiac injury [9 10] however theunderlying mechanisms are not fully understood
Tissue restructuring after infarction involves the break-down of the ECM by proteolytic enzymes mainly the matrixmetalloproteinases (MMP) MMP-2 and MMP-9 balancedby interaction with tissue inhibitors of metalloproteinases(TIMPs) [11] Initially a temporary matrix is formed pri-marily composed of collagen type III (Col I1205723) providing ascaffold for replacement cells and structural integrity to theheart thereby reducing the risk of LVdilation and rupture [1213] This is later replaced by collagen type I (Col I1205721) whichwill constitute the permanent ECM [14] Col I1205721 conferstensile strength and resistance to stretch and deformationwhile Col I1205723 confers compliance Their balance determinescardiac tissue stiffness with increased Col I1205723 to Col I1205721 ratiogenerating a more resilient left ventricle [13 14]
Innate immune cells recruited to the injuredmyocardiumfrom the blood include neutrophils monocytes macro-phages and dendritic cells [15ndash17]Theyplay a prominent rolein remodelling producing the MMPs that break down thematrix synthesising new ECM components and activatingfibroblasts to myofibroblasts which will later in the inflam-matory process be the main producers of matrix proteins[14] The tissue microenvironment at a given time afterMI influences the recruitment of immune cells as well astheir phenotypic and functional properties This is especiallyrelevant for the macrophage population which undergoes atime-dependent shift between inflammatory and reparativefunctions [3 18] At early time points after injury themajorityof macrophages produce inflammatory cytokines and reac-tive oxygen species including interleukin- (IL-) 1120573 IL-12MMP-9 and nitric oxide These are termed inflammatoryor M1-polarised macrophages which express high levelsof Ly6C and function to recruit more inflammatory cellsand phagocytose cellular debris and produce growth factors[12] As the inflammation progresses reparativemacrophagesaccumulate in the infarct area In contrast to the inflamma-torymacrophages these cells many of which express CD206are involved in the suppression of inflammation due to highproduction of IL-10 and TGF-120573 [3 19] and assist in theprogression from inflammation to repair
They also perform reparative roles promoting cell growthangiogenesis and remodelling of the ECM Additionallydifferentmonocyte populations can be distinguished by Ly6Cin themouse andmay preferentially give rise to inflammatoryversus reparative macrophages
We have previously shown that IGF-1Ea and its maturecirculating form IGF-1 can modulate immune responses andsuppress pathological inflammation by inducing regulatorycytokines and immune cell types [20 21] In the heart IGF-1Ea increased expression of IL-10 after cardiotoxin injury anddecreased levels of IL-1120573 suggesting that a shift in immunecell populations may also occur in the heart [9] In thepresent study we investigated whether there was a differencein the immune cell dynamics after MI in transgenic IGF-1Ea hearts and whether this had a carry-on effect on tissueremodelling
2 Materials and Methods
21 Cardiac Injury Model Myocardial infarction by perma-nent left coronary artery occlusion was induced in wild-type (WT) and 120572MHCIGF-1Ea male mice which were 8to 12 weeks old Surgeries were performed under mechan-ical ventilation with 1ndash25 isofluorane The chest cav-ity was opened through the left fourth intercostal spaceThe heart was exposed and the left coronary artery wasligated using an 80mm non-absorbable suture (Ethicon-Johnson amp Johnson USA) below the left atrium to producean ischemic region of 20ndash30 of the left ventricle area Thechest cavity and skin were sutured with 60mm silk sutures(Ethicon-Johnson amp Johnson USA) Analgesic treatmentwith buprenorphine (03mgkg sc) was provided beforeand after surgery They were housed in individually venti-lated cages in temperature-controlled facilities on a 12-hourlightdark cycle on standard diet All mouse procedures wereapproved by the Imperial College London Ethical Committeeand were in accordance with national and internationalregulations (UK Home Office Project license 707589)
22 Echocardiography Echocardiographicmeasurements weretaken using a high-frequency ultrasound system Vevo 770(VisualSonics Inc Canada) with a 30MHz linear transducerand recorded images were analyzed by using the Vevo 770workstation software Mice were anesthetised with 1-2isofluorane and the anesthetic flow rate was adjusted tomaintain heart rate of approximately 450 plusmn 50 beats perminute Furthermore warmed ultrasound gel and a heatingplatform were used to maintain body temperature at 37 plusmn05∘C to minimise variation between mice This analysis wasperformed at basal level 1 3 4 7 and 28 days after MI toevaluate left ventricle cardiac function chamber dimensionsand infarct size
23 Massonrsquos Trichrome Staining Samples were fixed inparaformaldehyde (4 in PBS) for 48 h washed in PBSdehydrated and embedded in paraffin wax Five-micron-thick sections were stained with Celestine Blue for 5 minuteswashed in tap water and then incubated in haematoxylinfor 5 minutes Slides were then incubated with Acid Fuchsinfor 5 minutes rinsed in distilled water incubated in phos-phomolybdic acid (1) and then rinsed in distilled waterbefore staining with Methyl Blue for 2-3 minutes Slides weredehydrated in ascending concentrations of ethanol cleared inxylene and mounted in DPX (VWR UK)
24 Cell Isolation To analyse neutrophils monocytesmacrophages and dendritic cells a single cell suspensionwas prepared from hearts before or at various time pointsafter MI (days 1 3 5 7 and 28 after operation) The heartswere mechanically dissociated using surgical scissors andsubsequently treated with a 1x Hankrsquos Balanced Salt Solution(HBSS) (Invitrogen USA) enzymatic dissociation buffercontaining 01mgmL Liberase TH Research Grade (RocheDiagnostics UK) 50 120583gmL of DNaseI (Roche DiagnosticsUK) 10mM HEPES (Invitrogen USA) and 30mM Taurine(Sigma UK) for 4 cycles of 10min at 37∘C After each 10min
Mediators of Inflammation 3
incubation cycle the cells were collected and filtered usinga 70 120583m cell strainer (BD Pharmingen USA) and an equalvolume of ice cold 1xHBSS containing 10mMHEPES 30mMTaurine (Sigma UK) and 20 Fetal Bovine Serum (FBS)(GE Healthcare USA) was added to the enzyme dissociationbuffer The cells were pelleted at 320 g for 7min at 4∘C andwashed with the 1x HBSSmedia solution containing 20 FBSas described above Under these isolation conditions adultcardiomyocytes are predominately lysed as the enzymaticdissociation buffer is toxic to these large fragile cells [22] Analiquot of the cell suspension was used to quantify the cellconcentrationmL using a Beckman Coulter Vi-Cell XR cellcounter (Beckman Coulter High Wycombe UK)
25 Flow Cytometry and Cell Sorting Isolated cells wereincubated in a 1x Dulbeccorsquos Modified Eagle Medium(DMEM) solution (Gibco Life Technologies UK) containing2 FBS and 10mM HEPES for 30min on ice in thedark with the following primary antibodies CD11b-PE (BDBiosciences catalogue 553311) F4-80-Biotin (eBiosciencecatalogue 13-4801-85) CD45-APC-Cy7 (BioLegend cata-logue 103116) CD206-PerCP-Cy55 (BioLegend catalogue141716) Ly6C-APC (eBioscience catalogue 17-5932-82) andLy6G-AlexaFluor700 (BioLegend catalogue 127622) Sam-ples stainedwith biotin-labelled primary antibodywere incu-bated with a streptavidin-PE-Cy7 (eBioscience catalogue 25-4317-82) secondary antibody for 30min on ice in the darkwith the 1x DMEM media solution as mentioned above Thesamples were washed and resuspended in fresh 1x DMEMmedia solution with 15 120583M Sytox Blue (Invitrogen USA)dead cell stain and refiltered using 5mL 35 120583m filter captubes (BD Falcon) just prior to sample acquisition Flowcytometric cell sorting was performed using a BD FAC-SAriaI cell sorter (BD Biosciences Oxford UK) equippedwith a 355 nm UV laser a 405 nm Violet laser a 488 nmBlue laser a 561 nm Yellow-Green laser and a 640 nm Redlaser The antibody cocktail fluorescence minus one (FMO)controls were used as gating controls for analyses to distin-guish positive from negative fluorescence signal (see Sup-plementary 4ndash6 in Supplementary Material available onlineat httpdxdoiorg1011552015484357) Total leukocytes(CD45+) neutrophils (CD45+ CD11b+ F480minus CD11cminusand Ly6G+) and monocytes (CD45+ CD11b+ CD11cminusLy6Gminus and F480minus) were analysed Monocytes were furtherclassified as Ly6Chigh and Ly6Clow monocytes Macrophageswere defined as CD45+ CD11b+ CD11cminus Ly6Gminus andF480+ and further characterised on the basis of Ly6C andCD206 expression (ie Ly6ChighCD206low (inflammatorymacrophages) and Ly6ClowCD206high (reparative macro-phages)) Dendritic cells were defined as CD45+ CD11b+F480minus CD11c+ and Ly6Gminus Flow Jo software (version 9 85)(Tree Star Ashland OR) was used for analysis
26 RNA Isolation and cDNAGeneration Mouse heart tissuewas harvested and flushed with cold PBS Samples from theinfarct and remote (non-infarct) myocardium were placedin a 15mL tube and homogenised in TRIzol (InvitrogenUSA) reagent using a rotor-stator homogeniser (Polytron PT
2500 E) Total RNA was isolated according to the TRIzolmanufacturerrsquos instructions RNA was pelleted air-driedand resuspended in DNaseRNase free water and the yieldquantified using Nanodrop (Thermo Scientific USA) at260 nm One microgram of RNA was reverse-transcribedinto cDNA using the Quantitect Reverse Transcription Kit(QIAGEN Crawley UK)
27 Quantitative Real-Time PCR Following cDNA synthesisquantitative RT-PCR using TaqMan probes (InvitrogenUSA) was performed on an ABI 7900HT SequenceDetection System (Applied Biosystems Carlsbad CAUSA) The probes used were IGF-1Ea (Mm00710307 m1)IL-10 (Mm00439614 m1) IL-1120573 (Mm00434228 m1)CCL2 (Mm00441242 m1) CCL5 (Mm01302427 m1) TGF120573(Mm03024053 m1) collagen I1205721 (Mm00801666 g1) collagenI1205723 (Mm01254476 m1) Lox (Mm00495386 m1) MMP2(Mm00439498 m1) MMP9 (Mm00442991 m1) TIMP1(Mm00441818 m1) TIMP2 (Mm004418225 m1) actinalpha 1 skeletal muscle ACTA (Mm00808218 g1) AtrialNatriuretic Peptide and ANP (Mm01255747 g1) Geneexpression was determined as fold induction over uninjuredhearts after normalising to the reference gene GAPDH
28 Statistics Data are presented as mean plusmn SEM Two-tailed Studentrsquos t-test was performed to compare WT and120572MHCIGF-1Ea mice at selected time points after MI Datawere analysedwithGraphPad-Prism 50 (Graphpad SoftwareInc wwwgraphpadcom) and differences were consideredstatistically significant at 119875 lt 005
3 Results
31 IGF-1Ea Improves Cardiac Function after MyocardialInfarction Endogenous IGF-1Ea expression in WT heartswas measured 1 3 5 7 and 28 days after MI in both ischemicand remote (nonischemic) regions IGF-1Ea levels increasedin both the ischemic and remote regions (Figure 1(a)) with asubstantially stronger induction in the ischemic area (8-foldover uninjured levels) These results indicate that similar toother organs endogenous IGF1-Ea expression is upregulatedafter cardiac tissue damage [23 24] In 120572MHCIGF-1Eahearts the baseline expression of transgenic IGF-1Ea wasmuch higher than the expression of endogenous IGF-1Eain WT hearts (average 286-fold) at all experimental timepoints (Supplementary 1A and 1B) Although far exceedingthe expression of endogenous IGF-1Ea the 120572MHCIGF-1Ea transgenic mice provide a suitable model of IGF-1Eaat supraphysiological concentrations of possible therapeuticrelevance
Previously our group showed an improvement in cardiacfunction in 120572MHCIGF-1Ea compared to WT mice onemonth after MI [25] To pinpoint the start of functionalimprovement we extended this analysis and performedechocardiography before and 1 3 5 7 and 28 days after MI(Supplementary Table 1) One day followingMI both groupsdisplayed a reduction in ejection fraction (EF) however120572MHCIGF-1Ea hearts showed significant improvement in
4 Mediators of Inflammation
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Figure 1 120572MHCIGF-1Ea improves cardiac function and reduces dilation as early as 7 days after myocardial infarction (a) Levels of IGF-1Eain the ischemic and in the remote area ofWT hearts at 1 3 5 7 and 28 days followingMI (b) Left ventricular ejection fraction (LVEF) and (c)left ventricular end systolic volume (LVESV) after MI Solid lines represent WTmice Dashed lines represent 120572MHCIGF-1Ea mice Levels of(d) actin-alpha 1 skeletal muscle (ACTA 1) and (e) atrial natriuretic peptide (ANP) mRNA expression 28 days after MI Results are expressedas mean fold induction plusmn SEM over the values of uninjured hearts 119899 = 4ndash6 per group Two-tailed Studentrsquos t-test was performed to compareWT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
left ventricular EF by day 7 after MI (Figure 1(b) and Sup-plementary Table 1) Left ventricular end systolicdiastolicvolumes significantly increased after MI in WT heartsindicating left ventricular dilation while the 120572MHCIGF-1Ea hearts did not display any such signs (Figure 1(c) andSupplementary Table 1) In support of the functional datathe expression of molecular markers for cardiac damage suchas actin-alpha 1 skeletal muscle (ACTA) and atrial natriureticpeptide (ANP) was significantly reduced in 120572MHCIGF-1Eacompared to WT mice 28 days after MI (Figures 1(d) and1(e))The peak of endogenous IGF-1Ea in the ischemic regionof WT mice by day 7 along with the improvement in cardiacfunction in 120572MHCIGF-1Ea as early as day 7 indicates thatIGF-1Ea signalling at early time points is key for cardiacrepair
32 Altered Tissue Remodelling in120572MHCIGF-1Ea after InjuryThree days after MI WT and 120572MHCIGF-1Ea hearts dis-played infarcts of equal size quantified byMassonrsquos trichromestaining (Figure 2(a)) However by 28 days after infarction120572MHCIGF-1Ea hearts exhibited smaller scar areas compared
to WT (Figures 2(b) 2(c) and 2(d)) as previously reported[25] This was observed as a reduction in scar length butincreased scar thickness consistent with reduced infarctexpansion As amolecular measurement of fibrosis we quan-tified TGF-120573 mRNA levels which were significantly lowerin 120572MHCIGF-1Ea than in WT mice after MI (Figure 2(e))We therefore measured expression of genes involved in ECMturnover and synthesis MMP-2 MMP-9 TIMP-1 TIMP-2Col I1205721 Col I1205723 and lysyl oxidase (Lox) at 1 3 5 7 and28 days after MI In WT hearts MMP-9 was upregulated3 days after MI followed by MMP-2 at day 7 (Figures 2(f)and 2(g)) Interestingly neither MMP-2 nor MMP-9 wassignificantly upregulated in 120572MHCIGF-1Ea hearts at anytime point Inhibitors of matrix degradation TIMP-1 andTIMP-2 had similar expression in WT and 120572MHCIGF-1Eahearts at all time points except for day 7 when TIMP-2 wassignificantly upregulated in120572MHCIGF-1Ea hearts comparedto WT (Figures 2(h) and 2(i)) Taken together this supportsthe idea of a net overall reduction in matrix breakdownwhich may contribute to the reduced fibrosis observed in120572MHCIGF-1Ea hearts
Mediators of Inflammation 5
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Figure 2 Positive remodeling in the 120572MHCIGF-1Ea infarcted region after myocardial infarction Quantification of the infarcted area at (a)3 days and (b) 28 days after MI 119899 = 3 Representative histological sections of (c) WT and (d) 120572MHCIGF-1Ea hearts stained with Massonrsquostrichrome 28 days after MI Scale bar 1mm ((e)ndash(l)) mRNA relative expression of (e) transforming growth factor beta (TGF-120573) (f) matrixmetalloproteinase 9 (MMP-9) (g) matrix metalloproteinase 2 (MMP-2) (h) tissue inhibitor of metalloproteinase (TIMP) 2 (i) TIMP-1 (j)collagen (Col) I1205721 (k) Col I1205723 (l) ColI1205721ColI1205723 ratio (m) lysyl oxidase in the infarct area Results are expressed as mean fold induction plusmnSEM Solid lines representWTmice Dashed lines represent 120572MHCIGF-1Eamice 119899 = 3 per group Two-tailed Studentrsquos t-test was performedto compare WT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005 lowastlowastlowast119875 lt 0001
6 Mediators of Inflammation
We next analysed the composition of the newly synthe-sised matrix by measuring mRNA expression of the mostabundant cardiac ECM collagen Col I1205721 and Col I1205723 aswell as the collagen cross-linker lox which increases matrixstiffness Upregulation of both collagen types was detectedby day 3 peaking at day 7 (Figures 2(j) and 2(k)) At thistime point WT hearts expressed significantly more of bothcollagen types than 120572MHCIGF-1Ea hearts We also noted adifference in the ratio of the two collagen types 120572MHCIGF-1Ea hearts had a reduced Col I1205721Col I1205723 ratio compared toWT which was significant at days 3 7 and 28 (Figure 2(l)) Atthe peak of collagen upregulation lox was also upregulatedin WT hearts yet this was not observed in 120572MHCIGF-1Ea hearts (Figure 2(m)) These results indicate that IGF-1Eaoverexpression reduces ECM turnover afterMI and alters thecomposition of thematrix with preferential expression of ColI1205723 over Col I1205721 and less cross-linking which likely alters themechanical properties of the fibrotic area
33 Distinct Chemokine and Cytokine Production in 120572MHC-IGF-1Ea Hearts after Myocardial Infarct Tissue remodellingafter injury is closely tied to the inflammatory processWe therefore compared the inflammatory status of the120572MHCIGF-1Ea and WT hearts monitoring the productionof key immune genes IL-1120573 IL-10 MCP-1 and CCL5Expression of the inflammatory cytokine IL-1120573 was rapidlyinduced upon injury in WT hearts (66-fold over uninjured)yet was not upregulated as strongly in 120572MHCIGF-1Ea hearts(19-fold over uninjured Figure 3(a)) Similarly the strongupregulation of MCP-1 in WT hearts was not observed in120572MHCIGF-1Ea hearts (Figure 3(b)) IL-10 was also rapidlyupregulated peaking 1 day after MI In contrast to IL-1120573and MCP-1 this immunosuppressive cytokine was upreg-ulated 3-fold higher in 120572MHCIGF-1Ea hearts comparedto WT controls Although a second peak of IL-10 mRNAat 7 days was comparable in both WT and 120572MHCIGF-1Ea hearts 7 days after MI (Figure 3(c)) CCL5 which isinvolved in the recruitment of Ly6Clow monocytes [26 27]was upregulated in 120572MHCIGF-1Ea hearts 7 days after MI(Figure 3(d)) These data suggest an early bias towards aless inflammatory environment potentially modulating therecruitment of monocytes in 120572MHCIGF-1Ea hearts aftermyocardial infraction
34 IGF-1Ea Modulates Myeloid Cell Recruitment after Myo-cardial Infarction To document accumulation of the maininnate immune cell populations involved in cardiac inflam-mation after infarct single cell suspensions were preparedfromwholemouse hearts and analysed by flow cytometry Allcell populations described in this work were identified usingthe gating strategy shown in Supplementary Figures 3ndash6
In WT and 120572MHCIGF-1Ea hearts the total numberof infiltrating leukocytes (CD45+) gradually increased afterMI both reaching comparable peak numbers at day 5(Figure 4(a)) However at the earlier 3-day time point120572MHCIGF-1Ea hearts contained 49 less leukocytes permilligram of tissue than WT hearts In examining spe-cific immune cell populations this difference was partlyexplained by a 75 reduction in neutrophils (CD45+
CD11b+ F480minus CD11cminus and Ly6G+ 186 versus 46 cellsmgof tissue Figure 4(b)) and a 67 reduction in monocytes(CD45+ CD11b+ CD11cminus Ly6Gminus and F480minus 191 versus 62cellsmg of heart Figure 4(c)) however it was mostly due toreduced presence of macrophages (CD45+ CD11b+ CD11cminusLy6Gminus and F480+ 58 2276 versus 949 cellsmg of heartFigure 4(f)) These data agree with the reduced MCP-1expression observed in 120572MHCIGF-1Ea hearts (Figure 3(b))as it is the principal chemokine involved in monocyterecruitment [26 28]
The Ly6C surface marker distinguishes two differentsubsets ofmonocytes [2 3] Analysis of the Ly6Chigh (CD45+CD11b+ CD11cminus Ly6Gminus F480minus and Ly6Chigh) and Ly6Clow
(CD45+ CD11b+ CD11cminus Ly6Gminus F480minus and Ly6Clow)populations revealed that the reduction of total monocytenumbers at day 3 was attributable to a 20 decrease of theLy6Chigh population (102 versus 32 cellsmgof heart) whereasthe Ly6Clow population was not significantly different in WTcompared to 120572MHCIGF-1Ea (Figures 4(d) and 4(e)) hearts
To differentiate between inflammatory and reparativemacrophage populations we used the markers Ly6C andCD206 For this work only cells that were either Ly6C+CD206minus or Ly6CminusCD206+ were analysed although wenoted a double positive cell population (ie CD206+Ly6C+) However no changes were observed over timebetween WT and 120572MHCIGF-1Ea for the double positivepopulation (Supplementary 2) Both Ly6C+ inflammato-ry macrophage (CD45+ CD11b+ CD11cminus Ly6Gminus F480+and Ly6C+CD206minus) and CD206+ reparative macro-phage (CD45+ CD11b+ CD11cminus Ly6Gminus F480+ andLy6CminusCD206+) normalised cell numbers were reduced by71 and 48 respectively in 120572MHCIGF-1Ea hearts at theday 3 time point (522 versus 153 and 596 versus 310 cellsmgof tissue of heart resp Figures 4(g) and 4(h)) howeverthis was significant only for the Ly6C+ population By day 7after MI macrophage dynamics changed and we observeda greater number of total macrophages in 120572MHCIGF-1Eahearts compared to WT which was mainly due to a 155preferential increase in the CD206+ population (575 versus1468 cellsmg heart Figure 4(h)) Dendritic cells (CD45+CD11b+ F480minus CD11c+ and Ly6Gminus) were increased by48 in 120572MHCIGF-1Ea hearts compared to WT 5 days afterMI (168 versus 248 cellsmg heart Figure 4(i))
In summary cardiac-restricted expression of an IGF-1Eatransgene limited the early accumulation of innate immunecells at day 3 after MI with a bias towards the reduction ofinflammatory myeloid populations rather than regenerativepopulations This reduction corresponds with the lowerexpression of myeloid chemokines and the less inflammatorymilieu observed in the 120572MHCIGF-1Ea hearts
4 Discussion
Previous work in our lab showed that local expression of IGF-1Ea promoted functional restoration after MI and observedreduced infarct expansion thinning and dilation of the leftventricular wall [25] We now demonstrate transcriptionalmodulation of key ECM remodelling genes in the IGF-1Ea hearts associated with tempering of the inflammatorymyeloid cell response
Mediators of Inflammation 7
0
100
200
300
400
IL-1120573
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
d
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
lowast
WT120572MHCIGF-1Ea
(a)
0
100
200
300
MC
P-1
GA
PD
H f
old
in
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
(b)
0
50
100
150
200
IL-1
0G
AP
DH
fo
ldin
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
(c)
0
20
40
60
80
CC
L5
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
d
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
(d)
Figure 3 Cytokine dynamics in the infarcted 120572MHCIGF-1Ea hearts (a) Interleukin- (IL-) 1120573 (b) MCP-1 (c) IL-10 (d) CCL5 at several timepoints after permanent ligation Solid lines represent WT mice Dashed lines represent 120572MHCIGF-1Ea mice Results are expressed as meanfold induction plusmn SEM over the values of uninjured hearts 119899 = 3 per group Two-tailed Studentrsquos t-test was performed to compare WT versus120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
Increased MMP expression after injury has been impli-cated in contributing to scar destabilisation as transgenic ani-mal model knockouts of either MMP-2 or MMP-9 have beenshown to attenuate LV dilation rupture and impairment ofcardiac function [29 30] While MMP-2 and MMP-9 mRNAexpression levels were upregulated in injured WT heartsthis increase was not observed in 120572MHCIGF-1Ea hearts Inline with reduced matrix breakdown mRNA expression ofthe MMP inhibitor TIMP-2 was significantly increased in120572MHCIGF-1Ea hearts at day 7 after MI Thus IGF-1Ea mayprevent adverse cardiac remodelling in part by modulatingtranscription of MMPsTIMPs
The production of new matrix components was alsoaltered by the presence of the IGF-1Ea transgenewith an over-all reduction in collagen synthesis confirmed in histologicalstains and a bias towards expression of Col I1205723 over ColI1205721 In MI patients turnover of cardiac extracellular matrixcan be assessed by using circulating collagen peptides asblood biomarkers [31] and high type I collagen is associatedwith adverse clinical outcome [32] A prospectivemulticentrestudy further showed that a low type IIItype I collagen ratioespecially at 1 month after MI is predictive of detrimental
left ventricular remodelling as well as cardiovascular deathsand hospitalisation cases for heart failure [33] Changes incollagen ratios are known to affect the strength and tensileproperties of the ECM It would be interesting to determinewhether these properties are modified in the 120572MHCIGF-1Eahearts
IGF-1Ea could promote the changes in collagen depo-sition by directly acting on fibroblasts as it promotes boththeir proliferation and activation to myofibroblasts [3435] Alternatively IGF-1Ea may influence accumulation andactivation of immune cells present at the infarct which inturn regulate myofibroblast activation Indeed we observedmodulation of the inflammatory process in 120572MHCIGF-1Ea hearts with less monocyte (Ly6Chigh) infiltration intothe injured myocardium This effect is associated withreduced expression of themonocyte chemoattractantMCP-1although no significant changes were observed in the Ly6Clow
monocyte population in infarcted hearts possibly due toCCL5 upregulation
It is interesting that while complete depletion of mono-cytes at any stage of repair leads to poor recovery [36]a more subtle modulation of the monocyte population in
8 Mediators of Inflammation
0
2000
4000
6000
8000
Cel
lsm
g h
eart
Total leukocytes
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
(a)
0
200
400
600
800Neutrophils
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(b)
0
100
200
300Monocytes
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(c)
0
50
100
150
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
Ly-6Chigh monocytes
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(d)
0
50
100
150
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
Ly-6Clow monocytes
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(e)
0
1000
2000
3000
4000
5000Macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
lowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(f)
0
500
1000
1500
2000Ly6C+ macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(g)
0
500
1000
1500
2000CD206+ macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(h)
0
100
200
300Dendritic cells
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(i)
Figure 4 Characterisation and temporal dynamics of immune cell populations in 120572MHCIGF-1Ea hearts after MI Quantification of (a) totalleukocytes (CD45+) (b) neutrophils (CD45+ CD11b+ F480minus CD11cminus and Ly6G+) and (c) monocytes (CD45+ CD11b+ CD11cminus Ly6Gminusand F480minus) which were further classified as (d) Ly6Chigh and (e) Ly6Clow monocytes (f) Macrophages (CD45+ CD11b+ CD11cminus Ly6Gminusand F480+) were further characterised on the basis of Ly6C and CD206 expression as (g) Ly6ChighCD206low (inflammatory macrophages)and (h) Ly6ClowCD206high (reparative macrophages) For this work only cells that were either Ly6ChighCD206minus or Ly6CminusCD206+ wereanalysed although we noted a double positive cell population (ie CD206+ Ly6C+) (i) Dendritic cells were defined as CD45+ CD11b+F480minus CD11c+ and Ly6Gminus Data is presented as the total number of cells per mg of heart 119899 = 4ndash6 per group Two-tailed Studentrsquos t-testwas performed to compare WT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
the 120572MHCIGF-1Ea hearts is associated with improved heartfunction Studies abrogating monocyte recruitment usinga selective CCR2 inhibitor have resulted in reduced IL-1120573 IL-6 MCP-1 and TNF120572 levels [37] IGF-1Ea influ-ences macrophage polarisation in skeletal muscle [38]
and we similarly found reduced Ly6Chigh monocyte andLy6C+ macrophage normalised cell numbers while CD206+macrophage numbers were increased by day 7 suggestingthat IGF-1Ea promoted a quick progression to the reparativephase of repair by modulating macrophage phenotype In
Mediators of Inflammation 9
the 120572MHCIGF-1Ea hearts we observed a decrease in MCP-1which is the chemokine for CCR2 recently shown to distin-guish infiltrating monocytes from the resident macrophagepopulation which has a different embryological origin andexpresses CD206 [39ndash41] It is therefore possible that in our120572MHCIGF-1Ea transgenic mouse model IGF-1Ea reducesthe infiltration of inflammatory Ly6Chigh monocytes bypreventing upregulation of MCP-1 while still allowing forexpansion of the resident macrophage population
In summary we show that the 120572MHCIGF-1Ea mousemodel can modulate several associated aspects of the cellularrepair process after MI including immune cell recruit-ment cytokine expression and matrix turnover All of thesechanges occur within the first 7 days after infarct at whichtime a functional improvement can already be measured in120572MHCIGF-1Ea hearts compared toWT controlsThese dataprovide new insights into the mechanism of IGF-1Ea andsuggest that early immunomodulation is key to successfulcardiac repair after injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Robert D Sampson and Susanne Sattler contributed equallyto this work
Acknowledgments
The authors are grateful to members of the RosenthalLaboratory for advice and critique This work was sup-ported by grants from the European Unionrsquos Seventh Frame-work Programme Marie Curie Initial Training Network-CardioNeT GA-289600 and British Heart Foundation GrantRM13130157 to Nadia Rosenthal Robert D Sampson andMichael D Schneider are supported by the British HeartFoundation Centre of Research Excellence and British HeartFoundation Simon Marks Chair We gratefully acknowledgethe support from the British Spanish Society (BSS) for theBritish Spanish SocietySantander Universities 2015 scholar-ship that partially supported this project
References
[1] A S Go D Mozaffarian V L Roger et al ldquoHeart disease andstroke statisticsmdash2014 update a report from theAmerican heartassociationrdquo Circulation vol 129 pp e28ndashe292 2014
[2] N G Frangogiannis ldquoRegulation of the inflammatory responsein cardiac repairrdquo Circulation Research vol 110 no 1 pp 159ndash173 2012
[3] N G Frangogiannis ldquoThe inflammatory response in myocar-dial injury repair and remodellingrdquoNature Reviews Cardiologyvol 11 no 5 pp 255ndash265 2014
[4] T Shavlakadze N Winn N Rosenthal and M D GroundsldquoReconciling data from transgenic mice that overexpress IGF-I specifically in skeletal musclerdquo Growth Hormone and IGFResearch vol 15 no 1 pp 4ndash18 2005
[5] P Rotwein K M Pollock D K Didier and G G KrivildquoOrganization and sequence of the human insulin-like growthfactor I gene Alternative RNAprocessing produces two insulin-like growth factor I precursor peptidesrdquo Journal of BiologicalChemistry vol 261 no 11 pp 4828ndash4832 1986
[6] J G Simmons J J Van Wyk E C Hoyt and P K LundldquoMultiple transcription start sites in the rat insulin-like growthfactor-I gene Give rise to IGF-I mRNAs that encode differentIGF-I precursors and are processed differently in vitvordquoGrowthFactors vol 9 no 3 pp 205ndash221 1993
[7] G I Bell M M Stempien N M Fong and L B RallldquoSequences of liver cDNAs encoding two different mouseinsulin-like growth factor I precursorsrdquo Nucleic Acids Researchvol 14 no 20 pp 7873ndash7882 1986
[8] M S Hede E Salimova A Piszczek et al ldquoE-peptides controlbioavailability of IGF-1rdquo PLoS ONE vol 7 no 12 Article IDe51152 2012
[9] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[10] M Vinciguerra M P Santini W C Claycomb A G Ladurnerand N Rosenthal ldquoLocal IGF-1 isoform protects cardiomy-ocytes from hypertrophic and oxidative stresses via SirT1activityrdquo Aging vol 2 no 1 pp 43ndash62 2010
[11] D Vanhoutte M Schellings Y Pinto and S Heymans ldquoRel-evance of matrix metalloproteinases and their inhibitors aftermyocardial infarction a temporal and spatial windowrdquo Cardio-vascular Research vol 69 no 3 pp 604ndash613 2006
[12] J B Caulfield and T K Borg ldquoThe collagen network of theheartrdquo Laboratory Investigation vol 40 no 3 pp 364ndash372 1979
[13] B I Jugdutt ldquoRemodeling of the myocardium and potentialtargets in the collagen degradation and synthesis pathwaysrdquoCurrent Drug TargetsmdashCardiovascular amp Haematological Dis-orders vol 3 no 1 pp 1ndash30 2003
[14] B I Jugdutt ldquoVentricular remodeling after infarction andthe extracellular collagen matrix when is enough enoughrdquoCirculation vol 108 no 11 pp 1395ndash1403 2003
[15] M Nahrendorf and F K Swirski ldquoMonocyte and macrophageheterogeneity in the heartrdquo Circulation Research vol 112 no 12pp 1624ndash1633 2013
[16] F Leuschner P J Rauch T Ueno et al ldquoRapid mono-cyte kinetics in acute myocardial infarction are sustained byextramedullary monocytopoiesisrdquo The Journal of ExperimentalMedicine vol 209 no 1 pp 123ndash137 2012
[17] X Yan A Anzai Y Katsumata et al ldquoTemporal dynamics ofcardiac immune cell accumulation following acute myocardialinfarctionrdquo Journal ofMolecular andCellular Cardiology vol 62pp 24ndash35 2013
[18] S Frantz and M Nahrendorf ldquoCardiac macrophages and theirrole in ischemic heart diseaserdquo Cardiovascular Research vol102 no 2 pp 240ndash248 2014
[19] Q Cao Y Wang D Zheng et al ldquoIL-10TGF-120573-modifiedmacrophages induce regulatory T cells and protect againstadriamycin nephrosisrdquo Journal of the American Society ofNephrology vol 21 no 6 pp 933ndash942 2010
[20] B Johannesson S Sattler E Semenova et al ldquoInsulin-likegrowth factor-1 induces regulatory T cell-mediated suppressionof allergic contact dermatitis in micerdquo Disease Models ampMechanisms vol 7 no 8 pp 977ndash985 2014
10 Mediators of Inflammation
[21] D Bilbao L Luciani B Johannesson A Piszczek and NRosenthal ldquoInsulin-like growth factor-1 stimulates regulatoryT cells and suppresses autoimmune diseaserdquo EMBO MolecularMedicine vol 6 no 11 pp 1423ndash1435 2005
[22] H Oh S B Bradfute T D Gallardo et al ldquoCardiac progenitorcells from adult myocardium homing differentiation andfusion after infarctionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 21 pp12313ndash12318 2003
[23] A Philippou E Papageorgiou G Bogdanis et al ldquoExpressionof IGF-1 isoforms after exercise-induced muscle damage inhumans characterization of theMGFEpeptide actions in vitrordquoIn Vivo vol 23 no 4 pp 567ndash576 2009
[24] S Sanz J B Pucilowska S Liu et al ldquoExpression of insulin-like growth factor I by activated hepatic stellate cells reducesfibrogenesis and enhances regeneration after liver injuryrdquo Gutvol 54 no 1 pp 134ndash141 2005
[25] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[26] F Tacke D Alvarez T J Kaplan et al ldquoMonocyte subsetsdifferentially employ CCR2 CCR5 and CX3CR1 to accumulatewithin atherosclerotic plaquesrdquo Journal of Clinical Investigationvol 117 no 1 pp 185ndash194 2007
[27] L M Carlin E G Stamatiades C Auffray et al ldquoNr4a1-dependent Ly6Clow monocytes monitor endothelial cells andorchestrate their disposalrdquoCell vol 153 no 2 pp 362ndash375 2013
[28] N V Serbina and E G Pamer ldquoMonocyte emigration frombone marrow during bacterial infection requires signals medi-ated by chemokine receptor CCR2rdquo Nature Immunology vol 7no 3 pp 311ndash317 2006
[29] S Hayashidani H Tsutsui M Ikeuchi et al ldquoTargeted deletionofMMP-2 attenuates early LV rupture and late remodeling afterexperimental myocardial infarctionrdquo The American Journal ofPhysiologymdashHeart and Circulatory Physiology vol 285 no 3pp H1229ndashH1235 2003
[30] A Ducharme S Frantz M Aikawa et al ldquoTargeted deletion ofmatrix metalloproteinase-9 attenuates left ventricular enlarge-ment and collagen accumulation after experimental myocardialinfarctionrdquo Journal of Clinical Investigation vol 106 no 1 pp55ndash62 2000
[31] K TWeber ldquoExtracellular matrix remodeling in heart failure arole for de novo angiotensin II generationrdquo Circulation vol 96no 11 pp 4065ndash4082 1997
[32] W Iraqi P Rossignol M Angioi et al ldquoExtracellular cardiacmatrix biomarkers in patients with acute myocardial infarctioncomplicated by left ventricular dysfunction and heart failureinsights from the Eplerenone Post-Acute Myocardial InfarctionHeart Failure Efficacy and Survival Study (EPHESUS) studyrdquoCirculation vol 119 no 18 pp 2471ndash2479 2009
[33] R Eschalier M Fertin R Fay et al ldquoExtracellular matrixturnover biomarkers predict long-term left ventricular remod-eling aftermyocardial infarction insights from the reve-2 studyrdquoCirculation Heart Failure vol 6 no 6 pp 1199ndash1205 2013
[34] J G Simmons J B Pucilowska T O Keku and P K LundldquoIGF-I and TGF-1205731 have distinct effects on phenotype andproliferation of intestinal fibroblastsrdquo American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 283 no3 pp G809ndashG818 2002
[35] C F Hung M G Rohani S-S Lee P Chen and L MSchnapp ldquoRole of IGF-1 pathway in lung fibroblast activationrdquoRespiratory Research vol 14 no 1 article 102 2013
[36] M Nahrendorf F K Swirski E Aikawa et al ldquoThe healingmyocardium sequentially mobilizes twomonocyte subsets withdivergent and complementary functionsrdquoThe Journal of Exper-imental Medicine vol 204 no 12 pp 3037ndash3047 2007
[37] F L Chen Z H Yang Y Liu et al ldquoBerberine inhibits theexpression of TNF120572 MCP-1 and IL-6 in AcLDL-stimulatedmacrophages through PPAR120574 pathwayrdquo Endocrine vol 33 no3 pp 331ndash337 2008
[38] J Tonkin L Temmerman R D Sampson et alldquoMonocytemacrophage-derived IGF-1 orchestrates murineskeletal muscle regeneration and modulates autocrinepolarizationrdquo Molecular Therapy vol 23 no 7 pp 1189ndash1200 2015
[39] T Heidt G Courties P Dutta et al ldquoDifferential contributionof monocytes to heart macrophages in steady-state and aftermyocardial infarctionrdquo Circulation Research vol 115 no 2 pp284ndash295 2014
[40] S Epelman K J Lavine and G J Randolph ldquoOrigin andfunctions of tissue macrophagesrdquo Immunity vol 41 no 1 pp21ndash35 2014
[41] A R Pinto J W Godwin and N A Rosenthal ldquoMacrophagesin cardiac homeostasis injury responses and progenitor cellmobilisationrdquo Stem Cell Research vol 13 no 3 pp 705ndash7142014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
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Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 Mediators of Inflammation
or delivered locally to the mouse heart IGF-1Ea improvesfunctional recovery after cardiac injury [9 10] however theunderlying mechanisms are not fully understood
Tissue restructuring after infarction involves the break-down of the ECM by proteolytic enzymes mainly the matrixmetalloproteinases (MMP) MMP-2 and MMP-9 balancedby interaction with tissue inhibitors of metalloproteinases(TIMPs) [11] Initially a temporary matrix is formed pri-marily composed of collagen type III (Col I1205723) providing ascaffold for replacement cells and structural integrity to theheart thereby reducing the risk of LVdilation and rupture [1213] This is later replaced by collagen type I (Col I1205721) whichwill constitute the permanent ECM [14] Col I1205721 conferstensile strength and resistance to stretch and deformationwhile Col I1205723 confers compliance Their balance determinescardiac tissue stiffness with increased Col I1205723 to Col I1205721 ratiogenerating a more resilient left ventricle [13 14]
Innate immune cells recruited to the injuredmyocardiumfrom the blood include neutrophils monocytes macro-phages and dendritic cells [15ndash17]Theyplay a prominent rolein remodelling producing the MMPs that break down thematrix synthesising new ECM components and activatingfibroblasts to myofibroblasts which will later in the inflam-matory process be the main producers of matrix proteins[14] The tissue microenvironment at a given time afterMI influences the recruitment of immune cells as well astheir phenotypic and functional properties This is especiallyrelevant for the macrophage population which undergoes atime-dependent shift between inflammatory and reparativefunctions [3 18] At early time points after injury themajorityof macrophages produce inflammatory cytokines and reac-tive oxygen species including interleukin- (IL-) 1120573 IL-12MMP-9 and nitric oxide These are termed inflammatoryor M1-polarised macrophages which express high levelsof Ly6C and function to recruit more inflammatory cellsand phagocytose cellular debris and produce growth factors[12] As the inflammation progresses reparativemacrophagesaccumulate in the infarct area In contrast to the inflamma-torymacrophages these cells many of which express CD206are involved in the suppression of inflammation due to highproduction of IL-10 and TGF-120573 [3 19] and assist in theprogression from inflammation to repair
They also perform reparative roles promoting cell growthangiogenesis and remodelling of the ECM Additionallydifferentmonocyte populations can be distinguished by Ly6Cin themouse andmay preferentially give rise to inflammatoryversus reparative macrophages
We have previously shown that IGF-1Ea and its maturecirculating form IGF-1 can modulate immune responses andsuppress pathological inflammation by inducing regulatorycytokines and immune cell types [20 21] In the heart IGF-1Ea increased expression of IL-10 after cardiotoxin injury anddecreased levels of IL-1120573 suggesting that a shift in immunecell populations may also occur in the heart [9] In thepresent study we investigated whether there was a differencein the immune cell dynamics after MI in transgenic IGF-1Ea hearts and whether this had a carry-on effect on tissueremodelling
2 Materials and Methods
21 Cardiac Injury Model Myocardial infarction by perma-nent left coronary artery occlusion was induced in wild-type (WT) and 120572MHCIGF-1Ea male mice which were 8to 12 weeks old Surgeries were performed under mechan-ical ventilation with 1ndash25 isofluorane The chest cav-ity was opened through the left fourth intercostal spaceThe heart was exposed and the left coronary artery wasligated using an 80mm non-absorbable suture (Ethicon-Johnson amp Johnson USA) below the left atrium to producean ischemic region of 20ndash30 of the left ventricle area Thechest cavity and skin were sutured with 60mm silk sutures(Ethicon-Johnson amp Johnson USA) Analgesic treatmentwith buprenorphine (03mgkg sc) was provided beforeand after surgery They were housed in individually venti-lated cages in temperature-controlled facilities on a 12-hourlightdark cycle on standard diet All mouse procedures wereapproved by the Imperial College London Ethical Committeeand were in accordance with national and internationalregulations (UK Home Office Project license 707589)
22 Echocardiography Echocardiographicmeasurements weretaken using a high-frequency ultrasound system Vevo 770(VisualSonics Inc Canada) with a 30MHz linear transducerand recorded images were analyzed by using the Vevo 770workstation software Mice were anesthetised with 1-2isofluorane and the anesthetic flow rate was adjusted tomaintain heart rate of approximately 450 plusmn 50 beats perminute Furthermore warmed ultrasound gel and a heatingplatform were used to maintain body temperature at 37 plusmn05∘C to minimise variation between mice This analysis wasperformed at basal level 1 3 4 7 and 28 days after MI toevaluate left ventricle cardiac function chamber dimensionsand infarct size
23 Massonrsquos Trichrome Staining Samples were fixed inparaformaldehyde (4 in PBS) for 48 h washed in PBSdehydrated and embedded in paraffin wax Five-micron-thick sections were stained with Celestine Blue for 5 minuteswashed in tap water and then incubated in haematoxylinfor 5 minutes Slides were then incubated with Acid Fuchsinfor 5 minutes rinsed in distilled water incubated in phos-phomolybdic acid (1) and then rinsed in distilled waterbefore staining with Methyl Blue for 2-3 minutes Slides weredehydrated in ascending concentrations of ethanol cleared inxylene and mounted in DPX (VWR UK)
24 Cell Isolation To analyse neutrophils monocytesmacrophages and dendritic cells a single cell suspensionwas prepared from hearts before or at various time pointsafter MI (days 1 3 5 7 and 28 after operation) The heartswere mechanically dissociated using surgical scissors andsubsequently treated with a 1x Hankrsquos Balanced Salt Solution(HBSS) (Invitrogen USA) enzymatic dissociation buffercontaining 01mgmL Liberase TH Research Grade (RocheDiagnostics UK) 50 120583gmL of DNaseI (Roche DiagnosticsUK) 10mM HEPES (Invitrogen USA) and 30mM Taurine(Sigma UK) for 4 cycles of 10min at 37∘C After each 10min
Mediators of Inflammation 3
incubation cycle the cells were collected and filtered usinga 70 120583m cell strainer (BD Pharmingen USA) and an equalvolume of ice cold 1xHBSS containing 10mMHEPES 30mMTaurine (Sigma UK) and 20 Fetal Bovine Serum (FBS)(GE Healthcare USA) was added to the enzyme dissociationbuffer The cells were pelleted at 320 g for 7min at 4∘C andwashed with the 1x HBSSmedia solution containing 20 FBSas described above Under these isolation conditions adultcardiomyocytes are predominately lysed as the enzymaticdissociation buffer is toxic to these large fragile cells [22] Analiquot of the cell suspension was used to quantify the cellconcentrationmL using a Beckman Coulter Vi-Cell XR cellcounter (Beckman Coulter High Wycombe UK)
25 Flow Cytometry and Cell Sorting Isolated cells wereincubated in a 1x Dulbeccorsquos Modified Eagle Medium(DMEM) solution (Gibco Life Technologies UK) containing2 FBS and 10mM HEPES for 30min on ice in thedark with the following primary antibodies CD11b-PE (BDBiosciences catalogue 553311) F4-80-Biotin (eBiosciencecatalogue 13-4801-85) CD45-APC-Cy7 (BioLegend cata-logue 103116) CD206-PerCP-Cy55 (BioLegend catalogue141716) Ly6C-APC (eBioscience catalogue 17-5932-82) andLy6G-AlexaFluor700 (BioLegend catalogue 127622) Sam-ples stainedwith biotin-labelled primary antibodywere incu-bated with a streptavidin-PE-Cy7 (eBioscience catalogue 25-4317-82) secondary antibody for 30min on ice in the darkwith the 1x DMEM media solution as mentioned above Thesamples were washed and resuspended in fresh 1x DMEMmedia solution with 15 120583M Sytox Blue (Invitrogen USA)dead cell stain and refiltered using 5mL 35 120583m filter captubes (BD Falcon) just prior to sample acquisition Flowcytometric cell sorting was performed using a BD FAC-SAriaI cell sorter (BD Biosciences Oxford UK) equippedwith a 355 nm UV laser a 405 nm Violet laser a 488 nmBlue laser a 561 nm Yellow-Green laser and a 640 nm Redlaser The antibody cocktail fluorescence minus one (FMO)controls were used as gating controls for analyses to distin-guish positive from negative fluorescence signal (see Sup-plementary 4ndash6 in Supplementary Material available onlineat httpdxdoiorg1011552015484357) Total leukocytes(CD45+) neutrophils (CD45+ CD11b+ F480minus CD11cminusand Ly6G+) and monocytes (CD45+ CD11b+ CD11cminusLy6Gminus and F480minus) were analysed Monocytes were furtherclassified as Ly6Chigh and Ly6Clow monocytes Macrophageswere defined as CD45+ CD11b+ CD11cminus Ly6Gminus andF480+ and further characterised on the basis of Ly6C andCD206 expression (ie Ly6ChighCD206low (inflammatorymacrophages) and Ly6ClowCD206high (reparative macro-phages)) Dendritic cells were defined as CD45+ CD11b+F480minus CD11c+ and Ly6Gminus Flow Jo software (version 9 85)(Tree Star Ashland OR) was used for analysis
26 RNA Isolation and cDNAGeneration Mouse heart tissuewas harvested and flushed with cold PBS Samples from theinfarct and remote (non-infarct) myocardium were placedin a 15mL tube and homogenised in TRIzol (InvitrogenUSA) reagent using a rotor-stator homogeniser (Polytron PT
2500 E) Total RNA was isolated according to the TRIzolmanufacturerrsquos instructions RNA was pelleted air-driedand resuspended in DNaseRNase free water and the yieldquantified using Nanodrop (Thermo Scientific USA) at260 nm One microgram of RNA was reverse-transcribedinto cDNA using the Quantitect Reverse Transcription Kit(QIAGEN Crawley UK)
27 Quantitative Real-Time PCR Following cDNA synthesisquantitative RT-PCR using TaqMan probes (InvitrogenUSA) was performed on an ABI 7900HT SequenceDetection System (Applied Biosystems Carlsbad CAUSA) The probes used were IGF-1Ea (Mm00710307 m1)IL-10 (Mm00439614 m1) IL-1120573 (Mm00434228 m1)CCL2 (Mm00441242 m1) CCL5 (Mm01302427 m1) TGF120573(Mm03024053 m1) collagen I1205721 (Mm00801666 g1) collagenI1205723 (Mm01254476 m1) Lox (Mm00495386 m1) MMP2(Mm00439498 m1) MMP9 (Mm00442991 m1) TIMP1(Mm00441818 m1) TIMP2 (Mm004418225 m1) actinalpha 1 skeletal muscle ACTA (Mm00808218 g1) AtrialNatriuretic Peptide and ANP (Mm01255747 g1) Geneexpression was determined as fold induction over uninjuredhearts after normalising to the reference gene GAPDH
28 Statistics Data are presented as mean plusmn SEM Two-tailed Studentrsquos t-test was performed to compare WT and120572MHCIGF-1Ea mice at selected time points after MI Datawere analysedwithGraphPad-Prism 50 (Graphpad SoftwareInc wwwgraphpadcom) and differences were consideredstatistically significant at 119875 lt 005
3 Results
31 IGF-1Ea Improves Cardiac Function after MyocardialInfarction Endogenous IGF-1Ea expression in WT heartswas measured 1 3 5 7 and 28 days after MI in both ischemicand remote (nonischemic) regions IGF-1Ea levels increasedin both the ischemic and remote regions (Figure 1(a)) with asubstantially stronger induction in the ischemic area (8-foldover uninjured levels) These results indicate that similar toother organs endogenous IGF1-Ea expression is upregulatedafter cardiac tissue damage [23 24] In 120572MHCIGF-1Eahearts the baseline expression of transgenic IGF-1Ea wasmuch higher than the expression of endogenous IGF-1Eain WT hearts (average 286-fold) at all experimental timepoints (Supplementary 1A and 1B) Although far exceedingthe expression of endogenous IGF-1Ea the 120572MHCIGF-1Ea transgenic mice provide a suitable model of IGF-1Eaat supraphysiological concentrations of possible therapeuticrelevance
Previously our group showed an improvement in cardiacfunction in 120572MHCIGF-1Ea compared to WT mice onemonth after MI [25] To pinpoint the start of functionalimprovement we extended this analysis and performedechocardiography before and 1 3 5 7 and 28 days after MI(Supplementary Table 1) One day followingMI both groupsdisplayed a reduction in ejection fraction (EF) however120572MHCIGF-1Ea hearts showed significant improvement in
4 Mediators of Inflammation
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Figure 1 120572MHCIGF-1Ea improves cardiac function and reduces dilation as early as 7 days after myocardial infarction (a) Levels of IGF-1Eain the ischemic and in the remote area ofWT hearts at 1 3 5 7 and 28 days followingMI (b) Left ventricular ejection fraction (LVEF) and (c)left ventricular end systolic volume (LVESV) after MI Solid lines represent WTmice Dashed lines represent 120572MHCIGF-1Ea mice Levels of(d) actin-alpha 1 skeletal muscle (ACTA 1) and (e) atrial natriuretic peptide (ANP) mRNA expression 28 days after MI Results are expressedas mean fold induction plusmn SEM over the values of uninjured hearts 119899 = 4ndash6 per group Two-tailed Studentrsquos t-test was performed to compareWT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
left ventricular EF by day 7 after MI (Figure 1(b) and Sup-plementary Table 1) Left ventricular end systolicdiastolicvolumes significantly increased after MI in WT heartsindicating left ventricular dilation while the 120572MHCIGF-1Ea hearts did not display any such signs (Figure 1(c) andSupplementary Table 1) In support of the functional datathe expression of molecular markers for cardiac damage suchas actin-alpha 1 skeletal muscle (ACTA) and atrial natriureticpeptide (ANP) was significantly reduced in 120572MHCIGF-1Eacompared to WT mice 28 days after MI (Figures 1(d) and1(e))The peak of endogenous IGF-1Ea in the ischemic regionof WT mice by day 7 along with the improvement in cardiacfunction in 120572MHCIGF-1Ea as early as day 7 indicates thatIGF-1Ea signalling at early time points is key for cardiacrepair
32 Altered Tissue Remodelling in120572MHCIGF-1Ea after InjuryThree days after MI WT and 120572MHCIGF-1Ea hearts dis-played infarcts of equal size quantified byMassonrsquos trichromestaining (Figure 2(a)) However by 28 days after infarction120572MHCIGF-1Ea hearts exhibited smaller scar areas compared
to WT (Figures 2(b) 2(c) and 2(d)) as previously reported[25] This was observed as a reduction in scar length butincreased scar thickness consistent with reduced infarctexpansion As amolecular measurement of fibrosis we quan-tified TGF-120573 mRNA levels which were significantly lowerin 120572MHCIGF-1Ea than in WT mice after MI (Figure 2(e))We therefore measured expression of genes involved in ECMturnover and synthesis MMP-2 MMP-9 TIMP-1 TIMP-2Col I1205721 Col I1205723 and lysyl oxidase (Lox) at 1 3 5 7 and28 days after MI In WT hearts MMP-9 was upregulated3 days after MI followed by MMP-2 at day 7 (Figures 2(f)and 2(g)) Interestingly neither MMP-2 nor MMP-9 wassignificantly upregulated in 120572MHCIGF-1Ea hearts at anytime point Inhibitors of matrix degradation TIMP-1 andTIMP-2 had similar expression in WT and 120572MHCIGF-1Eahearts at all time points except for day 7 when TIMP-2 wassignificantly upregulated in120572MHCIGF-1Ea hearts comparedto WT (Figures 2(h) and 2(i)) Taken together this supportsthe idea of a net overall reduction in matrix breakdownwhich may contribute to the reduced fibrosis observed in120572MHCIGF-1Ea hearts
Mediators of Inflammation 5
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Figure 2 Positive remodeling in the 120572MHCIGF-1Ea infarcted region after myocardial infarction Quantification of the infarcted area at (a)3 days and (b) 28 days after MI 119899 = 3 Representative histological sections of (c) WT and (d) 120572MHCIGF-1Ea hearts stained with Massonrsquostrichrome 28 days after MI Scale bar 1mm ((e)ndash(l)) mRNA relative expression of (e) transforming growth factor beta (TGF-120573) (f) matrixmetalloproteinase 9 (MMP-9) (g) matrix metalloproteinase 2 (MMP-2) (h) tissue inhibitor of metalloproteinase (TIMP) 2 (i) TIMP-1 (j)collagen (Col) I1205721 (k) Col I1205723 (l) ColI1205721ColI1205723 ratio (m) lysyl oxidase in the infarct area Results are expressed as mean fold induction plusmnSEM Solid lines representWTmice Dashed lines represent 120572MHCIGF-1Eamice 119899 = 3 per group Two-tailed Studentrsquos t-test was performedto compare WT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005 lowastlowastlowast119875 lt 0001
6 Mediators of Inflammation
We next analysed the composition of the newly synthe-sised matrix by measuring mRNA expression of the mostabundant cardiac ECM collagen Col I1205721 and Col I1205723 aswell as the collagen cross-linker lox which increases matrixstiffness Upregulation of both collagen types was detectedby day 3 peaking at day 7 (Figures 2(j) and 2(k)) At thistime point WT hearts expressed significantly more of bothcollagen types than 120572MHCIGF-1Ea hearts We also noted adifference in the ratio of the two collagen types 120572MHCIGF-1Ea hearts had a reduced Col I1205721Col I1205723 ratio compared toWT which was significant at days 3 7 and 28 (Figure 2(l)) Atthe peak of collagen upregulation lox was also upregulatedin WT hearts yet this was not observed in 120572MHCIGF-1Ea hearts (Figure 2(m)) These results indicate that IGF-1Eaoverexpression reduces ECM turnover afterMI and alters thecomposition of thematrix with preferential expression of ColI1205723 over Col I1205721 and less cross-linking which likely alters themechanical properties of the fibrotic area
33 Distinct Chemokine and Cytokine Production in 120572MHC-IGF-1Ea Hearts after Myocardial Infarct Tissue remodellingafter injury is closely tied to the inflammatory processWe therefore compared the inflammatory status of the120572MHCIGF-1Ea and WT hearts monitoring the productionof key immune genes IL-1120573 IL-10 MCP-1 and CCL5Expression of the inflammatory cytokine IL-1120573 was rapidlyinduced upon injury in WT hearts (66-fold over uninjured)yet was not upregulated as strongly in 120572MHCIGF-1Ea hearts(19-fold over uninjured Figure 3(a)) Similarly the strongupregulation of MCP-1 in WT hearts was not observed in120572MHCIGF-1Ea hearts (Figure 3(b)) IL-10 was also rapidlyupregulated peaking 1 day after MI In contrast to IL-1120573and MCP-1 this immunosuppressive cytokine was upreg-ulated 3-fold higher in 120572MHCIGF-1Ea hearts comparedto WT controls Although a second peak of IL-10 mRNAat 7 days was comparable in both WT and 120572MHCIGF-1Ea hearts 7 days after MI (Figure 3(c)) CCL5 which isinvolved in the recruitment of Ly6Clow monocytes [26 27]was upregulated in 120572MHCIGF-1Ea hearts 7 days after MI(Figure 3(d)) These data suggest an early bias towards aless inflammatory environment potentially modulating therecruitment of monocytes in 120572MHCIGF-1Ea hearts aftermyocardial infraction
34 IGF-1Ea Modulates Myeloid Cell Recruitment after Myo-cardial Infarction To document accumulation of the maininnate immune cell populations involved in cardiac inflam-mation after infarct single cell suspensions were preparedfromwholemouse hearts and analysed by flow cytometry Allcell populations described in this work were identified usingthe gating strategy shown in Supplementary Figures 3ndash6
In WT and 120572MHCIGF-1Ea hearts the total numberof infiltrating leukocytes (CD45+) gradually increased afterMI both reaching comparable peak numbers at day 5(Figure 4(a)) However at the earlier 3-day time point120572MHCIGF-1Ea hearts contained 49 less leukocytes permilligram of tissue than WT hearts In examining spe-cific immune cell populations this difference was partlyexplained by a 75 reduction in neutrophils (CD45+
CD11b+ F480minus CD11cminus and Ly6G+ 186 versus 46 cellsmgof tissue Figure 4(b)) and a 67 reduction in monocytes(CD45+ CD11b+ CD11cminus Ly6Gminus and F480minus 191 versus 62cellsmg of heart Figure 4(c)) however it was mostly due toreduced presence of macrophages (CD45+ CD11b+ CD11cminusLy6Gminus and F480+ 58 2276 versus 949 cellsmg of heartFigure 4(f)) These data agree with the reduced MCP-1expression observed in 120572MHCIGF-1Ea hearts (Figure 3(b))as it is the principal chemokine involved in monocyterecruitment [26 28]
The Ly6C surface marker distinguishes two differentsubsets ofmonocytes [2 3] Analysis of the Ly6Chigh (CD45+CD11b+ CD11cminus Ly6Gminus F480minus and Ly6Chigh) and Ly6Clow
(CD45+ CD11b+ CD11cminus Ly6Gminus F480minus and Ly6Clow)populations revealed that the reduction of total monocytenumbers at day 3 was attributable to a 20 decrease of theLy6Chigh population (102 versus 32 cellsmgof heart) whereasthe Ly6Clow population was not significantly different in WTcompared to 120572MHCIGF-1Ea (Figures 4(d) and 4(e)) hearts
To differentiate between inflammatory and reparativemacrophage populations we used the markers Ly6C andCD206 For this work only cells that were either Ly6C+CD206minus or Ly6CminusCD206+ were analysed although wenoted a double positive cell population (ie CD206+Ly6C+) However no changes were observed over timebetween WT and 120572MHCIGF-1Ea for the double positivepopulation (Supplementary 2) Both Ly6C+ inflammato-ry macrophage (CD45+ CD11b+ CD11cminus Ly6Gminus F480+and Ly6C+CD206minus) and CD206+ reparative macro-phage (CD45+ CD11b+ CD11cminus Ly6Gminus F480+ andLy6CminusCD206+) normalised cell numbers were reduced by71 and 48 respectively in 120572MHCIGF-1Ea hearts at theday 3 time point (522 versus 153 and 596 versus 310 cellsmgof tissue of heart resp Figures 4(g) and 4(h)) howeverthis was significant only for the Ly6C+ population By day 7after MI macrophage dynamics changed and we observeda greater number of total macrophages in 120572MHCIGF-1Eahearts compared to WT which was mainly due to a 155preferential increase in the CD206+ population (575 versus1468 cellsmg heart Figure 4(h)) Dendritic cells (CD45+CD11b+ F480minus CD11c+ and Ly6Gminus) were increased by48 in 120572MHCIGF-1Ea hearts compared to WT 5 days afterMI (168 versus 248 cellsmg heart Figure 4(i))
In summary cardiac-restricted expression of an IGF-1Eatransgene limited the early accumulation of innate immunecells at day 3 after MI with a bias towards the reduction ofinflammatory myeloid populations rather than regenerativepopulations This reduction corresponds with the lowerexpression of myeloid chemokines and the less inflammatorymilieu observed in the 120572MHCIGF-1Ea hearts
4 Discussion
Previous work in our lab showed that local expression of IGF-1Ea promoted functional restoration after MI and observedreduced infarct expansion thinning and dilation of the leftventricular wall [25] We now demonstrate transcriptionalmodulation of key ECM remodelling genes in the IGF-1Ea hearts associated with tempering of the inflammatorymyeloid cell response
Mediators of Inflammation 7
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Figure 3 Cytokine dynamics in the infarcted 120572MHCIGF-1Ea hearts (a) Interleukin- (IL-) 1120573 (b) MCP-1 (c) IL-10 (d) CCL5 at several timepoints after permanent ligation Solid lines represent WT mice Dashed lines represent 120572MHCIGF-1Ea mice Results are expressed as meanfold induction plusmn SEM over the values of uninjured hearts 119899 = 3 per group Two-tailed Studentrsquos t-test was performed to compare WT versus120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
Increased MMP expression after injury has been impli-cated in contributing to scar destabilisation as transgenic ani-mal model knockouts of either MMP-2 or MMP-9 have beenshown to attenuate LV dilation rupture and impairment ofcardiac function [29 30] While MMP-2 and MMP-9 mRNAexpression levels were upregulated in injured WT heartsthis increase was not observed in 120572MHCIGF-1Ea hearts Inline with reduced matrix breakdown mRNA expression ofthe MMP inhibitor TIMP-2 was significantly increased in120572MHCIGF-1Ea hearts at day 7 after MI Thus IGF-1Ea mayprevent adverse cardiac remodelling in part by modulatingtranscription of MMPsTIMPs
The production of new matrix components was alsoaltered by the presence of the IGF-1Ea transgenewith an over-all reduction in collagen synthesis confirmed in histologicalstains and a bias towards expression of Col I1205723 over ColI1205721 In MI patients turnover of cardiac extracellular matrixcan be assessed by using circulating collagen peptides asblood biomarkers [31] and high type I collagen is associatedwith adverse clinical outcome [32] A prospectivemulticentrestudy further showed that a low type IIItype I collagen ratioespecially at 1 month after MI is predictive of detrimental
left ventricular remodelling as well as cardiovascular deathsand hospitalisation cases for heart failure [33] Changes incollagen ratios are known to affect the strength and tensileproperties of the ECM It would be interesting to determinewhether these properties are modified in the 120572MHCIGF-1Eahearts
IGF-1Ea could promote the changes in collagen depo-sition by directly acting on fibroblasts as it promotes boththeir proliferation and activation to myofibroblasts [3435] Alternatively IGF-1Ea may influence accumulation andactivation of immune cells present at the infarct which inturn regulate myofibroblast activation Indeed we observedmodulation of the inflammatory process in 120572MHCIGF-1Ea hearts with less monocyte (Ly6Chigh) infiltration intothe injured myocardium This effect is associated withreduced expression of themonocyte chemoattractantMCP-1although no significant changes were observed in the Ly6Clow
monocyte population in infarcted hearts possibly due toCCL5 upregulation
It is interesting that while complete depletion of mono-cytes at any stage of repair leads to poor recovery [36]a more subtle modulation of the monocyte population in
8 Mediators of Inflammation
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red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(i)
Figure 4 Characterisation and temporal dynamics of immune cell populations in 120572MHCIGF-1Ea hearts after MI Quantification of (a) totalleukocytes (CD45+) (b) neutrophils (CD45+ CD11b+ F480minus CD11cminus and Ly6G+) and (c) monocytes (CD45+ CD11b+ CD11cminus Ly6Gminusand F480minus) which were further classified as (d) Ly6Chigh and (e) Ly6Clow monocytes (f) Macrophages (CD45+ CD11b+ CD11cminus Ly6Gminusand F480+) were further characterised on the basis of Ly6C and CD206 expression as (g) Ly6ChighCD206low (inflammatory macrophages)and (h) Ly6ClowCD206high (reparative macrophages) For this work only cells that were either Ly6ChighCD206minus or Ly6CminusCD206+ wereanalysed although we noted a double positive cell population (ie CD206+ Ly6C+) (i) Dendritic cells were defined as CD45+ CD11b+F480minus CD11c+ and Ly6Gminus Data is presented as the total number of cells per mg of heart 119899 = 4ndash6 per group Two-tailed Studentrsquos t-testwas performed to compare WT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
the 120572MHCIGF-1Ea hearts is associated with improved heartfunction Studies abrogating monocyte recruitment usinga selective CCR2 inhibitor have resulted in reduced IL-1120573 IL-6 MCP-1 and TNF120572 levels [37] IGF-1Ea influ-ences macrophage polarisation in skeletal muscle [38]
and we similarly found reduced Ly6Chigh monocyte andLy6C+ macrophage normalised cell numbers while CD206+macrophage numbers were increased by day 7 suggestingthat IGF-1Ea promoted a quick progression to the reparativephase of repair by modulating macrophage phenotype In
Mediators of Inflammation 9
the 120572MHCIGF-1Ea hearts we observed a decrease in MCP-1which is the chemokine for CCR2 recently shown to distin-guish infiltrating monocytes from the resident macrophagepopulation which has a different embryological origin andexpresses CD206 [39ndash41] It is therefore possible that in our120572MHCIGF-1Ea transgenic mouse model IGF-1Ea reducesthe infiltration of inflammatory Ly6Chigh monocytes bypreventing upregulation of MCP-1 while still allowing forexpansion of the resident macrophage population
In summary we show that the 120572MHCIGF-1Ea mousemodel can modulate several associated aspects of the cellularrepair process after MI including immune cell recruit-ment cytokine expression and matrix turnover All of thesechanges occur within the first 7 days after infarct at whichtime a functional improvement can already be measured in120572MHCIGF-1Ea hearts compared toWT controlsThese dataprovide new insights into the mechanism of IGF-1Ea andsuggest that early immunomodulation is key to successfulcardiac repair after injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Robert D Sampson and Susanne Sattler contributed equallyto this work
Acknowledgments
The authors are grateful to members of the RosenthalLaboratory for advice and critique This work was sup-ported by grants from the European Unionrsquos Seventh Frame-work Programme Marie Curie Initial Training Network-CardioNeT GA-289600 and British Heart Foundation GrantRM13130157 to Nadia Rosenthal Robert D Sampson andMichael D Schneider are supported by the British HeartFoundation Centre of Research Excellence and British HeartFoundation Simon Marks Chair We gratefully acknowledgethe support from the British Spanish Society (BSS) for theBritish Spanish SocietySantander Universities 2015 scholar-ship that partially supported this project
References
[1] A S Go D Mozaffarian V L Roger et al ldquoHeart disease andstroke statisticsmdash2014 update a report from theAmerican heartassociationrdquo Circulation vol 129 pp e28ndashe292 2014
[2] N G Frangogiannis ldquoRegulation of the inflammatory responsein cardiac repairrdquo Circulation Research vol 110 no 1 pp 159ndash173 2012
[3] N G Frangogiannis ldquoThe inflammatory response in myocar-dial injury repair and remodellingrdquoNature Reviews Cardiologyvol 11 no 5 pp 255ndash265 2014
[4] T Shavlakadze N Winn N Rosenthal and M D GroundsldquoReconciling data from transgenic mice that overexpress IGF-I specifically in skeletal musclerdquo Growth Hormone and IGFResearch vol 15 no 1 pp 4ndash18 2005
[5] P Rotwein K M Pollock D K Didier and G G KrivildquoOrganization and sequence of the human insulin-like growthfactor I gene Alternative RNAprocessing produces two insulin-like growth factor I precursor peptidesrdquo Journal of BiologicalChemistry vol 261 no 11 pp 4828ndash4832 1986
[6] J G Simmons J J Van Wyk E C Hoyt and P K LundldquoMultiple transcription start sites in the rat insulin-like growthfactor-I gene Give rise to IGF-I mRNAs that encode differentIGF-I precursors and are processed differently in vitvordquoGrowthFactors vol 9 no 3 pp 205ndash221 1993
[7] G I Bell M M Stempien N M Fong and L B RallldquoSequences of liver cDNAs encoding two different mouseinsulin-like growth factor I precursorsrdquo Nucleic Acids Researchvol 14 no 20 pp 7873ndash7882 1986
[8] M S Hede E Salimova A Piszczek et al ldquoE-peptides controlbioavailability of IGF-1rdquo PLoS ONE vol 7 no 12 Article IDe51152 2012
[9] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[10] M Vinciguerra M P Santini W C Claycomb A G Ladurnerand N Rosenthal ldquoLocal IGF-1 isoform protects cardiomy-ocytes from hypertrophic and oxidative stresses via SirT1activityrdquo Aging vol 2 no 1 pp 43ndash62 2010
[11] D Vanhoutte M Schellings Y Pinto and S Heymans ldquoRel-evance of matrix metalloproteinases and their inhibitors aftermyocardial infarction a temporal and spatial windowrdquo Cardio-vascular Research vol 69 no 3 pp 604ndash613 2006
[12] J B Caulfield and T K Borg ldquoThe collagen network of theheartrdquo Laboratory Investigation vol 40 no 3 pp 364ndash372 1979
[13] B I Jugdutt ldquoRemodeling of the myocardium and potentialtargets in the collagen degradation and synthesis pathwaysrdquoCurrent Drug TargetsmdashCardiovascular amp Haematological Dis-orders vol 3 no 1 pp 1ndash30 2003
[14] B I Jugdutt ldquoVentricular remodeling after infarction andthe extracellular collagen matrix when is enough enoughrdquoCirculation vol 108 no 11 pp 1395ndash1403 2003
[15] M Nahrendorf and F K Swirski ldquoMonocyte and macrophageheterogeneity in the heartrdquo Circulation Research vol 112 no 12pp 1624ndash1633 2013
[16] F Leuschner P J Rauch T Ueno et al ldquoRapid mono-cyte kinetics in acute myocardial infarction are sustained byextramedullary monocytopoiesisrdquo The Journal of ExperimentalMedicine vol 209 no 1 pp 123ndash137 2012
[17] X Yan A Anzai Y Katsumata et al ldquoTemporal dynamics ofcardiac immune cell accumulation following acute myocardialinfarctionrdquo Journal ofMolecular andCellular Cardiology vol 62pp 24ndash35 2013
[18] S Frantz and M Nahrendorf ldquoCardiac macrophages and theirrole in ischemic heart diseaserdquo Cardiovascular Research vol102 no 2 pp 240ndash248 2014
[19] Q Cao Y Wang D Zheng et al ldquoIL-10TGF-120573-modifiedmacrophages induce regulatory T cells and protect againstadriamycin nephrosisrdquo Journal of the American Society ofNephrology vol 21 no 6 pp 933ndash942 2010
[20] B Johannesson S Sattler E Semenova et al ldquoInsulin-likegrowth factor-1 induces regulatory T cell-mediated suppressionof allergic contact dermatitis in micerdquo Disease Models ampMechanisms vol 7 no 8 pp 977ndash985 2014
10 Mediators of Inflammation
[21] D Bilbao L Luciani B Johannesson A Piszczek and NRosenthal ldquoInsulin-like growth factor-1 stimulates regulatoryT cells and suppresses autoimmune diseaserdquo EMBO MolecularMedicine vol 6 no 11 pp 1423ndash1435 2005
[22] H Oh S B Bradfute T D Gallardo et al ldquoCardiac progenitorcells from adult myocardium homing differentiation andfusion after infarctionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 21 pp12313ndash12318 2003
[23] A Philippou E Papageorgiou G Bogdanis et al ldquoExpressionof IGF-1 isoforms after exercise-induced muscle damage inhumans characterization of theMGFEpeptide actions in vitrordquoIn Vivo vol 23 no 4 pp 567ndash576 2009
[24] S Sanz J B Pucilowska S Liu et al ldquoExpression of insulin-like growth factor I by activated hepatic stellate cells reducesfibrogenesis and enhances regeneration after liver injuryrdquo Gutvol 54 no 1 pp 134ndash141 2005
[25] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[26] F Tacke D Alvarez T J Kaplan et al ldquoMonocyte subsetsdifferentially employ CCR2 CCR5 and CX3CR1 to accumulatewithin atherosclerotic plaquesrdquo Journal of Clinical Investigationvol 117 no 1 pp 185ndash194 2007
[27] L M Carlin E G Stamatiades C Auffray et al ldquoNr4a1-dependent Ly6Clow monocytes monitor endothelial cells andorchestrate their disposalrdquoCell vol 153 no 2 pp 362ndash375 2013
[28] N V Serbina and E G Pamer ldquoMonocyte emigration frombone marrow during bacterial infection requires signals medi-ated by chemokine receptor CCR2rdquo Nature Immunology vol 7no 3 pp 311ndash317 2006
[29] S Hayashidani H Tsutsui M Ikeuchi et al ldquoTargeted deletionofMMP-2 attenuates early LV rupture and late remodeling afterexperimental myocardial infarctionrdquo The American Journal ofPhysiologymdashHeart and Circulatory Physiology vol 285 no 3pp H1229ndashH1235 2003
[30] A Ducharme S Frantz M Aikawa et al ldquoTargeted deletion ofmatrix metalloproteinase-9 attenuates left ventricular enlarge-ment and collagen accumulation after experimental myocardialinfarctionrdquo Journal of Clinical Investigation vol 106 no 1 pp55ndash62 2000
[31] K TWeber ldquoExtracellular matrix remodeling in heart failure arole for de novo angiotensin II generationrdquo Circulation vol 96no 11 pp 4065ndash4082 1997
[32] W Iraqi P Rossignol M Angioi et al ldquoExtracellular cardiacmatrix biomarkers in patients with acute myocardial infarctioncomplicated by left ventricular dysfunction and heart failureinsights from the Eplerenone Post-Acute Myocardial InfarctionHeart Failure Efficacy and Survival Study (EPHESUS) studyrdquoCirculation vol 119 no 18 pp 2471ndash2479 2009
[33] R Eschalier M Fertin R Fay et al ldquoExtracellular matrixturnover biomarkers predict long-term left ventricular remod-eling aftermyocardial infarction insights from the reve-2 studyrdquoCirculation Heart Failure vol 6 no 6 pp 1199ndash1205 2013
[34] J G Simmons J B Pucilowska T O Keku and P K LundldquoIGF-I and TGF-1205731 have distinct effects on phenotype andproliferation of intestinal fibroblastsrdquo American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 283 no3 pp G809ndashG818 2002
[35] C F Hung M G Rohani S-S Lee P Chen and L MSchnapp ldquoRole of IGF-1 pathway in lung fibroblast activationrdquoRespiratory Research vol 14 no 1 article 102 2013
[36] M Nahrendorf F K Swirski E Aikawa et al ldquoThe healingmyocardium sequentially mobilizes twomonocyte subsets withdivergent and complementary functionsrdquoThe Journal of Exper-imental Medicine vol 204 no 12 pp 3037ndash3047 2007
[37] F L Chen Z H Yang Y Liu et al ldquoBerberine inhibits theexpression of TNF120572 MCP-1 and IL-6 in AcLDL-stimulatedmacrophages through PPAR120574 pathwayrdquo Endocrine vol 33 no3 pp 331ndash337 2008
[38] J Tonkin L Temmerman R D Sampson et alldquoMonocytemacrophage-derived IGF-1 orchestrates murineskeletal muscle regeneration and modulates autocrinepolarizationrdquo Molecular Therapy vol 23 no 7 pp 1189ndash1200 2015
[39] T Heidt G Courties P Dutta et al ldquoDifferential contributionof monocytes to heart macrophages in steady-state and aftermyocardial infarctionrdquo Circulation Research vol 115 no 2 pp284ndash295 2014
[40] S Epelman K J Lavine and G J Randolph ldquoOrigin andfunctions of tissue macrophagesrdquo Immunity vol 41 no 1 pp21ndash35 2014
[41] A R Pinto J W Godwin and N A Rosenthal ldquoMacrophagesin cardiac homeostasis injury responses and progenitor cellmobilisationrdquo Stem Cell Research vol 13 no 3 pp 705ndash7142014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
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Research and TreatmentAIDS
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Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Mediators of Inflammation 3
incubation cycle the cells were collected and filtered usinga 70 120583m cell strainer (BD Pharmingen USA) and an equalvolume of ice cold 1xHBSS containing 10mMHEPES 30mMTaurine (Sigma UK) and 20 Fetal Bovine Serum (FBS)(GE Healthcare USA) was added to the enzyme dissociationbuffer The cells were pelleted at 320 g for 7min at 4∘C andwashed with the 1x HBSSmedia solution containing 20 FBSas described above Under these isolation conditions adultcardiomyocytes are predominately lysed as the enzymaticdissociation buffer is toxic to these large fragile cells [22] Analiquot of the cell suspension was used to quantify the cellconcentrationmL using a Beckman Coulter Vi-Cell XR cellcounter (Beckman Coulter High Wycombe UK)
25 Flow Cytometry and Cell Sorting Isolated cells wereincubated in a 1x Dulbeccorsquos Modified Eagle Medium(DMEM) solution (Gibco Life Technologies UK) containing2 FBS and 10mM HEPES for 30min on ice in thedark with the following primary antibodies CD11b-PE (BDBiosciences catalogue 553311) F4-80-Biotin (eBiosciencecatalogue 13-4801-85) CD45-APC-Cy7 (BioLegend cata-logue 103116) CD206-PerCP-Cy55 (BioLegend catalogue141716) Ly6C-APC (eBioscience catalogue 17-5932-82) andLy6G-AlexaFluor700 (BioLegend catalogue 127622) Sam-ples stainedwith biotin-labelled primary antibodywere incu-bated with a streptavidin-PE-Cy7 (eBioscience catalogue 25-4317-82) secondary antibody for 30min on ice in the darkwith the 1x DMEM media solution as mentioned above Thesamples were washed and resuspended in fresh 1x DMEMmedia solution with 15 120583M Sytox Blue (Invitrogen USA)dead cell stain and refiltered using 5mL 35 120583m filter captubes (BD Falcon) just prior to sample acquisition Flowcytometric cell sorting was performed using a BD FAC-SAriaI cell sorter (BD Biosciences Oxford UK) equippedwith a 355 nm UV laser a 405 nm Violet laser a 488 nmBlue laser a 561 nm Yellow-Green laser and a 640 nm Redlaser The antibody cocktail fluorescence minus one (FMO)controls were used as gating controls for analyses to distin-guish positive from negative fluorescence signal (see Sup-plementary 4ndash6 in Supplementary Material available onlineat httpdxdoiorg1011552015484357) Total leukocytes(CD45+) neutrophils (CD45+ CD11b+ F480minus CD11cminusand Ly6G+) and monocytes (CD45+ CD11b+ CD11cminusLy6Gminus and F480minus) were analysed Monocytes were furtherclassified as Ly6Chigh and Ly6Clow monocytes Macrophageswere defined as CD45+ CD11b+ CD11cminus Ly6Gminus andF480+ and further characterised on the basis of Ly6C andCD206 expression (ie Ly6ChighCD206low (inflammatorymacrophages) and Ly6ClowCD206high (reparative macro-phages)) Dendritic cells were defined as CD45+ CD11b+F480minus CD11c+ and Ly6Gminus Flow Jo software (version 9 85)(Tree Star Ashland OR) was used for analysis
26 RNA Isolation and cDNAGeneration Mouse heart tissuewas harvested and flushed with cold PBS Samples from theinfarct and remote (non-infarct) myocardium were placedin a 15mL tube and homogenised in TRIzol (InvitrogenUSA) reagent using a rotor-stator homogeniser (Polytron PT
2500 E) Total RNA was isolated according to the TRIzolmanufacturerrsquos instructions RNA was pelleted air-driedand resuspended in DNaseRNase free water and the yieldquantified using Nanodrop (Thermo Scientific USA) at260 nm One microgram of RNA was reverse-transcribedinto cDNA using the Quantitect Reverse Transcription Kit(QIAGEN Crawley UK)
27 Quantitative Real-Time PCR Following cDNA synthesisquantitative RT-PCR using TaqMan probes (InvitrogenUSA) was performed on an ABI 7900HT SequenceDetection System (Applied Biosystems Carlsbad CAUSA) The probes used were IGF-1Ea (Mm00710307 m1)IL-10 (Mm00439614 m1) IL-1120573 (Mm00434228 m1)CCL2 (Mm00441242 m1) CCL5 (Mm01302427 m1) TGF120573(Mm03024053 m1) collagen I1205721 (Mm00801666 g1) collagenI1205723 (Mm01254476 m1) Lox (Mm00495386 m1) MMP2(Mm00439498 m1) MMP9 (Mm00442991 m1) TIMP1(Mm00441818 m1) TIMP2 (Mm004418225 m1) actinalpha 1 skeletal muscle ACTA (Mm00808218 g1) AtrialNatriuretic Peptide and ANP (Mm01255747 g1) Geneexpression was determined as fold induction over uninjuredhearts after normalising to the reference gene GAPDH
28 Statistics Data are presented as mean plusmn SEM Two-tailed Studentrsquos t-test was performed to compare WT and120572MHCIGF-1Ea mice at selected time points after MI Datawere analysedwithGraphPad-Prism 50 (Graphpad SoftwareInc wwwgraphpadcom) and differences were consideredstatistically significant at 119875 lt 005
3 Results
31 IGF-1Ea Improves Cardiac Function after MyocardialInfarction Endogenous IGF-1Ea expression in WT heartswas measured 1 3 5 7 and 28 days after MI in both ischemicand remote (nonischemic) regions IGF-1Ea levels increasedin both the ischemic and remote regions (Figure 1(a)) with asubstantially stronger induction in the ischemic area (8-foldover uninjured levels) These results indicate that similar toother organs endogenous IGF1-Ea expression is upregulatedafter cardiac tissue damage [23 24] In 120572MHCIGF-1Eahearts the baseline expression of transgenic IGF-1Ea wasmuch higher than the expression of endogenous IGF-1Eain WT hearts (average 286-fold) at all experimental timepoints (Supplementary 1A and 1B) Although far exceedingthe expression of endogenous IGF-1Ea the 120572MHCIGF-1Ea transgenic mice provide a suitable model of IGF-1Eaat supraphysiological concentrations of possible therapeuticrelevance
Previously our group showed an improvement in cardiacfunction in 120572MHCIGF-1Ea compared to WT mice onemonth after MI [25] To pinpoint the start of functionalimprovement we extended this analysis and performedechocardiography before and 1 3 5 7 and 28 days after MI(Supplementary Table 1) One day followingMI both groupsdisplayed a reduction in ejection fraction (EF) however120572MHCIGF-1Ea hearts showed significant improvement in
4 Mediators of Inflammation
WT ischemicWT remote
0
5
15
10
20
Isch
emic
ver
sus
rem
ote
IGF
-1E
aG
AP
DH
fold
in
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
(a)
0
20
40
60
80
100
LVE
F (
)
WT120572MHCIGF-1Ea
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast lowast
(b)
0
50
100
150
LVE
SV (120583
L)
WT120572MHCIGF-1Ea
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowastlowast
(c)
0
2
4
6
AC
TA
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
dlowast
WT
120572M
HC
IG
F-
1E
a
(d)
0
5
10
15
AN
PG
AP
DH
fo
ldin
du
ctio
n o
ver
WT
un
inju
red
lowastlowast
WT
120572M
HC
IG
F-
1E
a
(e)
Figure 1 120572MHCIGF-1Ea improves cardiac function and reduces dilation as early as 7 days after myocardial infarction (a) Levels of IGF-1Eain the ischemic and in the remote area ofWT hearts at 1 3 5 7 and 28 days followingMI (b) Left ventricular ejection fraction (LVEF) and (c)left ventricular end systolic volume (LVESV) after MI Solid lines represent WTmice Dashed lines represent 120572MHCIGF-1Ea mice Levels of(d) actin-alpha 1 skeletal muscle (ACTA 1) and (e) atrial natriuretic peptide (ANP) mRNA expression 28 days after MI Results are expressedas mean fold induction plusmn SEM over the values of uninjured hearts 119899 = 4ndash6 per group Two-tailed Studentrsquos t-test was performed to compareWT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
left ventricular EF by day 7 after MI (Figure 1(b) and Sup-plementary Table 1) Left ventricular end systolicdiastolicvolumes significantly increased after MI in WT heartsindicating left ventricular dilation while the 120572MHCIGF-1Ea hearts did not display any such signs (Figure 1(c) andSupplementary Table 1) In support of the functional datathe expression of molecular markers for cardiac damage suchas actin-alpha 1 skeletal muscle (ACTA) and atrial natriureticpeptide (ANP) was significantly reduced in 120572MHCIGF-1Eacompared to WT mice 28 days after MI (Figures 1(d) and1(e))The peak of endogenous IGF-1Ea in the ischemic regionof WT mice by day 7 along with the improvement in cardiacfunction in 120572MHCIGF-1Ea as early as day 7 indicates thatIGF-1Ea signalling at early time points is key for cardiacrepair
32 Altered Tissue Remodelling in120572MHCIGF-1Ea after InjuryThree days after MI WT and 120572MHCIGF-1Ea hearts dis-played infarcts of equal size quantified byMassonrsquos trichromestaining (Figure 2(a)) However by 28 days after infarction120572MHCIGF-1Ea hearts exhibited smaller scar areas compared
to WT (Figures 2(b) 2(c) and 2(d)) as previously reported[25] This was observed as a reduction in scar length butincreased scar thickness consistent with reduced infarctexpansion As amolecular measurement of fibrosis we quan-tified TGF-120573 mRNA levels which were significantly lowerin 120572MHCIGF-1Ea than in WT mice after MI (Figure 2(e))We therefore measured expression of genes involved in ECMturnover and synthesis MMP-2 MMP-9 TIMP-1 TIMP-2Col I1205721 Col I1205723 and lysyl oxidase (Lox) at 1 3 5 7 and28 days after MI In WT hearts MMP-9 was upregulated3 days after MI followed by MMP-2 at day 7 (Figures 2(f)and 2(g)) Interestingly neither MMP-2 nor MMP-9 wassignificantly upregulated in 120572MHCIGF-1Ea hearts at anytime point Inhibitors of matrix degradation TIMP-1 andTIMP-2 had similar expression in WT and 120572MHCIGF-1Eahearts at all time points except for day 7 when TIMP-2 wassignificantly upregulated in120572MHCIGF-1Ea hearts comparedto WT (Figures 2(h) and 2(i)) Taken together this supportsthe idea of a net overall reduction in matrix breakdownwhich may contribute to the reduced fibrosis observed in120572MHCIGF-1Ea hearts
Mediators of Inflammation 5
WT
0
10
20
30In
farc
ted
are
a (L
V)
()
3 days after MI
120572MHCIGF-1Ea
(a)
0
10
20
30
Infa
rcte
d a
rea
(LV
) (
)
28 days after MI
lowastlowastlowast
WT 120572MHCIGF-1Ea
(b)
WT
1mm
(c)
120572MHC-IGF-1Ea
1mm
(d)
0
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
10
20
30
40
50
TG
F120573
GA
PD
H f
old
i
nd
uct
ion
ove
r W
T u
nin
jure
d lowastlowast
lowast
WT120572MHCIGF-1Ea
(e)
0
5
10
15
20
MM
P-9
GA
PD
H f
old
in
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
(f)
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
0
10
20
30
40
MM
P-2
GA
PD
Hfo
ld o
ver
WT
un
inju
red
lowastlowast
WT120572MHCIGF-1Ea
(g)
TIM
P-2
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
d
0
10
20
30
40
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
(h)
0
200
400
600
800
1000
TIM
P-1
GA
PD
Hfo
ld o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
WT120572MHCIGF-1Ea
(i)
0
20
40
60
80
100
Co
l 1120572
1G
AP
DH
fo
ldin
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
lowast
WT120572MHCIGF-1Ea
(j)
0
20
40
60
80
100
Co
l 1120572
3G
AP
DH
fo
ldin
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
WT120572MHCIGF-1Ea
lowast
lowastlowast
(k)
WT120572MHCIGF-1Ea
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
0
1
2
3
4
5
Co
l1120572
1 ve
rsu
s C
ol1120572
3 r
atio
lowast
lowast
lowastlowast
(l)
WT120572MHCIGF-1Ea
0
200
400
600
Lo
xG
AP
DH
fo
ld i
nd
uct
ion
ove
r W
T u
nin
jure
d
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowastlowast
(m)
Figure 2 Positive remodeling in the 120572MHCIGF-1Ea infarcted region after myocardial infarction Quantification of the infarcted area at (a)3 days and (b) 28 days after MI 119899 = 3 Representative histological sections of (c) WT and (d) 120572MHCIGF-1Ea hearts stained with Massonrsquostrichrome 28 days after MI Scale bar 1mm ((e)ndash(l)) mRNA relative expression of (e) transforming growth factor beta (TGF-120573) (f) matrixmetalloproteinase 9 (MMP-9) (g) matrix metalloproteinase 2 (MMP-2) (h) tissue inhibitor of metalloproteinase (TIMP) 2 (i) TIMP-1 (j)collagen (Col) I1205721 (k) Col I1205723 (l) ColI1205721ColI1205723 ratio (m) lysyl oxidase in the infarct area Results are expressed as mean fold induction plusmnSEM Solid lines representWTmice Dashed lines represent 120572MHCIGF-1Eamice 119899 = 3 per group Two-tailed Studentrsquos t-test was performedto compare WT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005 lowastlowastlowast119875 lt 0001
6 Mediators of Inflammation
We next analysed the composition of the newly synthe-sised matrix by measuring mRNA expression of the mostabundant cardiac ECM collagen Col I1205721 and Col I1205723 aswell as the collagen cross-linker lox which increases matrixstiffness Upregulation of both collagen types was detectedby day 3 peaking at day 7 (Figures 2(j) and 2(k)) At thistime point WT hearts expressed significantly more of bothcollagen types than 120572MHCIGF-1Ea hearts We also noted adifference in the ratio of the two collagen types 120572MHCIGF-1Ea hearts had a reduced Col I1205721Col I1205723 ratio compared toWT which was significant at days 3 7 and 28 (Figure 2(l)) Atthe peak of collagen upregulation lox was also upregulatedin WT hearts yet this was not observed in 120572MHCIGF-1Ea hearts (Figure 2(m)) These results indicate that IGF-1Eaoverexpression reduces ECM turnover afterMI and alters thecomposition of thematrix with preferential expression of ColI1205723 over Col I1205721 and less cross-linking which likely alters themechanical properties of the fibrotic area
33 Distinct Chemokine and Cytokine Production in 120572MHC-IGF-1Ea Hearts after Myocardial Infarct Tissue remodellingafter injury is closely tied to the inflammatory processWe therefore compared the inflammatory status of the120572MHCIGF-1Ea and WT hearts monitoring the productionof key immune genes IL-1120573 IL-10 MCP-1 and CCL5Expression of the inflammatory cytokine IL-1120573 was rapidlyinduced upon injury in WT hearts (66-fold over uninjured)yet was not upregulated as strongly in 120572MHCIGF-1Ea hearts(19-fold over uninjured Figure 3(a)) Similarly the strongupregulation of MCP-1 in WT hearts was not observed in120572MHCIGF-1Ea hearts (Figure 3(b)) IL-10 was also rapidlyupregulated peaking 1 day after MI In contrast to IL-1120573and MCP-1 this immunosuppressive cytokine was upreg-ulated 3-fold higher in 120572MHCIGF-1Ea hearts comparedto WT controls Although a second peak of IL-10 mRNAat 7 days was comparable in both WT and 120572MHCIGF-1Ea hearts 7 days after MI (Figure 3(c)) CCL5 which isinvolved in the recruitment of Ly6Clow monocytes [26 27]was upregulated in 120572MHCIGF-1Ea hearts 7 days after MI(Figure 3(d)) These data suggest an early bias towards aless inflammatory environment potentially modulating therecruitment of monocytes in 120572MHCIGF-1Ea hearts aftermyocardial infraction
34 IGF-1Ea Modulates Myeloid Cell Recruitment after Myo-cardial Infarction To document accumulation of the maininnate immune cell populations involved in cardiac inflam-mation after infarct single cell suspensions were preparedfromwholemouse hearts and analysed by flow cytometry Allcell populations described in this work were identified usingthe gating strategy shown in Supplementary Figures 3ndash6
In WT and 120572MHCIGF-1Ea hearts the total numberof infiltrating leukocytes (CD45+) gradually increased afterMI both reaching comparable peak numbers at day 5(Figure 4(a)) However at the earlier 3-day time point120572MHCIGF-1Ea hearts contained 49 less leukocytes permilligram of tissue than WT hearts In examining spe-cific immune cell populations this difference was partlyexplained by a 75 reduction in neutrophils (CD45+
CD11b+ F480minus CD11cminus and Ly6G+ 186 versus 46 cellsmgof tissue Figure 4(b)) and a 67 reduction in monocytes(CD45+ CD11b+ CD11cminus Ly6Gminus and F480minus 191 versus 62cellsmg of heart Figure 4(c)) however it was mostly due toreduced presence of macrophages (CD45+ CD11b+ CD11cminusLy6Gminus and F480+ 58 2276 versus 949 cellsmg of heartFigure 4(f)) These data agree with the reduced MCP-1expression observed in 120572MHCIGF-1Ea hearts (Figure 3(b))as it is the principal chemokine involved in monocyterecruitment [26 28]
The Ly6C surface marker distinguishes two differentsubsets ofmonocytes [2 3] Analysis of the Ly6Chigh (CD45+CD11b+ CD11cminus Ly6Gminus F480minus and Ly6Chigh) and Ly6Clow
(CD45+ CD11b+ CD11cminus Ly6Gminus F480minus and Ly6Clow)populations revealed that the reduction of total monocytenumbers at day 3 was attributable to a 20 decrease of theLy6Chigh population (102 versus 32 cellsmgof heart) whereasthe Ly6Clow population was not significantly different in WTcompared to 120572MHCIGF-1Ea (Figures 4(d) and 4(e)) hearts
To differentiate between inflammatory and reparativemacrophage populations we used the markers Ly6C andCD206 For this work only cells that were either Ly6C+CD206minus or Ly6CminusCD206+ were analysed although wenoted a double positive cell population (ie CD206+Ly6C+) However no changes were observed over timebetween WT and 120572MHCIGF-1Ea for the double positivepopulation (Supplementary 2) Both Ly6C+ inflammato-ry macrophage (CD45+ CD11b+ CD11cminus Ly6Gminus F480+and Ly6C+CD206minus) and CD206+ reparative macro-phage (CD45+ CD11b+ CD11cminus Ly6Gminus F480+ andLy6CminusCD206+) normalised cell numbers were reduced by71 and 48 respectively in 120572MHCIGF-1Ea hearts at theday 3 time point (522 versus 153 and 596 versus 310 cellsmgof tissue of heart resp Figures 4(g) and 4(h)) howeverthis was significant only for the Ly6C+ population By day 7after MI macrophage dynamics changed and we observeda greater number of total macrophages in 120572MHCIGF-1Eahearts compared to WT which was mainly due to a 155preferential increase in the CD206+ population (575 versus1468 cellsmg heart Figure 4(h)) Dendritic cells (CD45+CD11b+ F480minus CD11c+ and Ly6Gminus) were increased by48 in 120572MHCIGF-1Ea hearts compared to WT 5 days afterMI (168 versus 248 cellsmg heart Figure 4(i))
In summary cardiac-restricted expression of an IGF-1Eatransgene limited the early accumulation of innate immunecells at day 3 after MI with a bias towards the reduction ofinflammatory myeloid populations rather than regenerativepopulations This reduction corresponds with the lowerexpression of myeloid chemokines and the less inflammatorymilieu observed in the 120572MHCIGF-1Ea hearts
4 Discussion
Previous work in our lab showed that local expression of IGF-1Ea promoted functional restoration after MI and observedreduced infarct expansion thinning and dilation of the leftventricular wall [25] We now demonstrate transcriptionalmodulation of key ECM remodelling genes in the IGF-1Ea hearts associated with tempering of the inflammatorymyeloid cell response
Mediators of Inflammation 7
0
100
200
300
400
IL-1120573
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
d
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
lowast
WT120572MHCIGF-1Ea
(a)
0
100
200
300
MC
P-1
GA
PD
H f
old
in
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
(b)
0
50
100
150
200
IL-1
0G
AP
DH
fo
ldin
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
(c)
0
20
40
60
80
CC
L5
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
d
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
(d)
Figure 3 Cytokine dynamics in the infarcted 120572MHCIGF-1Ea hearts (a) Interleukin- (IL-) 1120573 (b) MCP-1 (c) IL-10 (d) CCL5 at several timepoints after permanent ligation Solid lines represent WT mice Dashed lines represent 120572MHCIGF-1Ea mice Results are expressed as meanfold induction plusmn SEM over the values of uninjured hearts 119899 = 3 per group Two-tailed Studentrsquos t-test was performed to compare WT versus120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
Increased MMP expression after injury has been impli-cated in contributing to scar destabilisation as transgenic ani-mal model knockouts of either MMP-2 or MMP-9 have beenshown to attenuate LV dilation rupture and impairment ofcardiac function [29 30] While MMP-2 and MMP-9 mRNAexpression levels were upregulated in injured WT heartsthis increase was not observed in 120572MHCIGF-1Ea hearts Inline with reduced matrix breakdown mRNA expression ofthe MMP inhibitor TIMP-2 was significantly increased in120572MHCIGF-1Ea hearts at day 7 after MI Thus IGF-1Ea mayprevent adverse cardiac remodelling in part by modulatingtranscription of MMPsTIMPs
The production of new matrix components was alsoaltered by the presence of the IGF-1Ea transgenewith an over-all reduction in collagen synthesis confirmed in histologicalstains and a bias towards expression of Col I1205723 over ColI1205721 In MI patients turnover of cardiac extracellular matrixcan be assessed by using circulating collagen peptides asblood biomarkers [31] and high type I collagen is associatedwith adverse clinical outcome [32] A prospectivemulticentrestudy further showed that a low type IIItype I collagen ratioespecially at 1 month after MI is predictive of detrimental
left ventricular remodelling as well as cardiovascular deathsand hospitalisation cases for heart failure [33] Changes incollagen ratios are known to affect the strength and tensileproperties of the ECM It would be interesting to determinewhether these properties are modified in the 120572MHCIGF-1Eahearts
IGF-1Ea could promote the changes in collagen depo-sition by directly acting on fibroblasts as it promotes boththeir proliferation and activation to myofibroblasts [3435] Alternatively IGF-1Ea may influence accumulation andactivation of immune cells present at the infarct which inturn regulate myofibroblast activation Indeed we observedmodulation of the inflammatory process in 120572MHCIGF-1Ea hearts with less monocyte (Ly6Chigh) infiltration intothe injured myocardium This effect is associated withreduced expression of themonocyte chemoattractantMCP-1although no significant changes were observed in the Ly6Clow
monocyte population in infarcted hearts possibly due toCCL5 upregulation
It is interesting that while complete depletion of mono-cytes at any stage of repair leads to poor recovery [36]a more subtle modulation of the monocyte population in
8 Mediators of Inflammation
0
2000
4000
6000
8000
Cel
lsm
g h
eart
Total leukocytes
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
(a)
0
200
400
600
800Neutrophils
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(b)
0
100
200
300Monocytes
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(c)
0
50
100
150
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
Ly-6Chigh monocytes
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(d)
0
50
100
150
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
Ly-6Clow monocytes
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(e)
0
1000
2000
3000
4000
5000Macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
lowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(f)
0
500
1000
1500
2000Ly6C+ macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(g)
0
500
1000
1500
2000CD206+ macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(h)
0
100
200
300Dendritic cells
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(i)
Figure 4 Characterisation and temporal dynamics of immune cell populations in 120572MHCIGF-1Ea hearts after MI Quantification of (a) totalleukocytes (CD45+) (b) neutrophils (CD45+ CD11b+ F480minus CD11cminus and Ly6G+) and (c) monocytes (CD45+ CD11b+ CD11cminus Ly6Gminusand F480minus) which were further classified as (d) Ly6Chigh and (e) Ly6Clow monocytes (f) Macrophages (CD45+ CD11b+ CD11cminus Ly6Gminusand F480+) were further characterised on the basis of Ly6C and CD206 expression as (g) Ly6ChighCD206low (inflammatory macrophages)and (h) Ly6ClowCD206high (reparative macrophages) For this work only cells that were either Ly6ChighCD206minus or Ly6CminusCD206+ wereanalysed although we noted a double positive cell population (ie CD206+ Ly6C+) (i) Dendritic cells were defined as CD45+ CD11b+F480minus CD11c+ and Ly6Gminus Data is presented as the total number of cells per mg of heart 119899 = 4ndash6 per group Two-tailed Studentrsquos t-testwas performed to compare WT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
the 120572MHCIGF-1Ea hearts is associated with improved heartfunction Studies abrogating monocyte recruitment usinga selective CCR2 inhibitor have resulted in reduced IL-1120573 IL-6 MCP-1 and TNF120572 levels [37] IGF-1Ea influ-ences macrophage polarisation in skeletal muscle [38]
and we similarly found reduced Ly6Chigh monocyte andLy6C+ macrophage normalised cell numbers while CD206+macrophage numbers were increased by day 7 suggestingthat IGF-1Ea promoted a quick progression to the reparativephase of repair by modulating macrophage phenotype In
Mediators of Inflammation 9
the 120572MHCIGF-1Ea hearts we observed a decrease in MCP-1which is the chemokine for CCR2 recently shown to distin-guish infiltrating monocytes from the resident macrophagepopulation which has a different embryological origin andexpresses CD206 [39ndash41] It is therefore possible that in our120572MHCIGF-1Ea transgenic mouse model IGF-1Ea reducesthe infiltration of inflammatory Ly6Chigh monocytes bypreventing upregulation of MCP-1 while still allowing forexpansion of the resident macrophage population
In summary we show that the 120572MHCIGF-1Ea mousemodel can modulate several associated aspects of the cellularrepair process after MI including immune cell recruit-ment cytokine expression and matrix turnover All of thesechanges occur within the first 7 days after infarct at whichtime a functional improvement can already be measured in120572MHCIGF-1Ea hearts compared toWT controlsThese dataprovide new insights into the mechanism of IGF-1Ea andsuggest that early immunomodulation is key to successfulcardiac repair after injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Robert D Sampson and Susanne Sattler contributed equallyto this work
Acknowledgments
The authors are grateful to members of the RosenthalLaboratory for advice and critique This work was sup-ported by grants from the European Unionrsquos Seventh Frame-work Programme Marie Curie Initial Training Network-CardioNeT GA-289600 and British Heart Foundation GrantRM13130157 to Nadia Rosenthal Robert D Sampson andMichael D Schneider are supported by the British HeartFoundation Centre of Research Excellence and British HeartFoundation Simon Marks Chair We gratefully acknowledgethe support from the British Spanish Society (BSS) for theBritish Spanish SocietySantander Universities 2015 scholar-ship that partially supported this project
References
[1] A S Go D Mozaffarian V L Roger et al ldquoHeart disease andstroke statisticsmdash2014 update a report from theAmerican heartassociationrdquo Circulation vol 129 pp e28ndashe292 2014
[2] N G Frangogiannis ldquoRegulation of the inflammatory responsein cardiac repairrdquo Circulation Research vol 110 no 1 pp 159ndash173 2012
[3] N G Frangogiannis ldquoThe inflammatory response in myocar-dial injury repair and remodellingrdquoNature Reviews Cardiologyvol 11 no 5 pp 255ndash265 2014
[4] T Shavlakadze N Winn N Rosenthal and M D GroundsldquoReconciling data from transgenic mice that overexpress IGF-I specifically in skeletal musclerdquo Growth Hormone and IGFResearch vol 15 no 1 pp 4ndash18 2005
[5] P Rotwein K M Pollock D K Didier and G G KrivildquoOrganization and sequence of the human insulin-like growthfactor I gene Alternative RNAprocessing produces two insulin-like growth factor I precursor peptidesrdquo Journal of BiologicalChemistry vol 261 no 11 pp 4828ndash4832 1986
[6] J G Simmons J J Van Wyk E C Hoyt and P K LundldquoMultiple transcription start sites in the rat insulin-like growthfactor-I gene Give rise to IGF-I mRNAs that encode differentIGF-I precursors and are processed differently in vitvordquoGrowthFactors vol 9 no 3 pp 205ndash221 1993
[7] G I Bell M M Stempien N M Fong and L B RallldquoSequences of liver cDNAs encoding two different mouseinsulin-like growth factor I precursorsrdquo Nucleic Acids Researchvol 14 no 20 pp 7873ndash7882 1986
[8] M S Hede E Salimova A Piszczek et al ldquoE-peptides controlbioavailability of IGF-1rdquo PLoS ONE vol 7 no 12 Article IDe51152 2012
[9] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[10] M Vinciguerra M P Santini W C Claycomb A G Ladurnerand N Rosenthal ldquoLocal IGF-1 isoform protects cardiomy-ocytes from hypertrophic and oxidative stresses via SirT1activityrdquo Aging vol 2 no 1 pp 43ndash62 2010
[11] D Vanhoutte M Schellings Y Pinto and S Heymans ldquoRel-evance of matrix metalloproteinases and their inhibitors aftermyocardial infarction a temporal and spatial windowrdquo Cardio-vascular Research vol 69 no 3 pp 604ndash613 2006
[12] J B Caulfield and T K Borg ldquoThe collagen network of theheartrdquo Laboratory Investigation vol 40 no 3 pp 364ndash372 1979
[13] B I Jugdutt ldquoRemodeling of the myocardium and potentialtargets in the collagen degradation and synthesis pathwaysrdquoCurrent Drug TargetsmdashCardiovascular amp Haematological Dis-orders vol 3 no 1 pp 1ndash30 2003
[14] B I Jugdutt ldquoVentricular remodeling after infarction andthe extracellular collagen matrix when is enough enoughrdquoCirculation vol 108 no 11 pp 1395ndash1403 2003
[15] M Nahrendorf and F K Swirski ldquoMonocyte and macrophageheterogeneity in the heartrdquo Circulation Research vol 112 no 12pp 1624ndash1633 2013
[16] F Leuschner P J Rauch T Ueno et al ldquoRapid mono-cyte kinetics in acute myocardial infarction are sustained byextramedullary monocytopoiesisrdquo The Journal of ExperimentalMedicine vol 209 no 1 pp 123ndash137 2012
[17] X Yan A Anzai Y Katsumata et al ldquoTemporal dynamics ofcardiac immune cell accumulation following acute myocardialinfarctionrdquo Journal ofMolecular andCellular Cardiology vol 62pp 24ndash35 2013
[18] S Frantz and M Nahrendorf ldquoCardiac macrophages and theirrole in ischemic heart diseaserdquo Cardiovascular Research vol102 no 2 pp 240ndash248 2014
[19] Q Cao Y Wang D Zheng et al ldquoIL-10TGF-120573-modifiedmacrophages induce regulatory T cells and protect againstadriamycin nephrosisrdquo Journal of the American Society ofNephrology vol 21 no 6 pp 933ndash942 2010
[20] B Johannesson S Sattler E Semenova et al ldquoInsulin-likegrowth factor-1 induces regulatory T cell-mediated suppressionof allergic contact dermatitis in micerdquo Disease Models ampMechanisms vol 7 no 8 pp 977ndash985 2014
10 Mediators of Inflammation
[21] D Bilbao L Luciani B Johannesson A Piszczek and NRosenthal ldquoInsulin-like growth factor-1 stimulates regulatoryT cells and suppresses autoimmune diseaserdquo EMBO MolecularMedicine vol 6 no 11 pp 1423ndash1435 2005
[22] H Oh S B Bradfute T D Gallardo et al ldquoCardiac progenitorcells from adult myocardium homing differentiation andfusion after infarctionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 21 pp12313ndash12318 2003
[23] A Philippou E Papageorgiou G Bogdanis et al ldquoExpressionof IGF-1 isoforms after exercise-induced muscle damage inhumans characterization of theMGFEpeptide actions in vitrordquoIn Vivo vol 23 no 4 pp 567ndash576 2009
[24] S Sanz J B Pucilowska S Liu et al ldquoExpression of insulin-like growth factor I by activated hepatic stellate cells reducesfibrogenesis and enhances regeneration after liver injuryrdquo Gutvol 54 no 1 pp 134ndash141 2005
[25] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[26] F Tacke D Alvarez T J Kaplan et al ldquoMonocyte subsetsdifferentially employ CCR2 CCR5 and CX3CR1 to accumulatewithin atherosclerotic plaquesrdquo Journal of Clinical Investigationvol 117 no 1 pp 185ndash194 2007
[27] L M Carlin E G Stamatiades C Auffray et al ldquoNr4a1-dependent Ly6Clow monocytes monitor endothelial cells andorchestrate their disposalrdquoCell vol 153 no 2 pp 362ndash375 2013
[28] N V Serbina and E G Pamer ldquoMonocyte emigration frombone marrow during bacterial infection requires signals medi-ated by chemokine receptor CCR2rdquo Nature Immunology vol 7no 3 pp 311ndash317 2006
[29] S Hayashidani H Tsutsui M Ikeuchi et al ldquoTargeted deletionofMMP-2 attenuates early LV rupture and late remodeling afterexperimental myocardial infarctionrdquo The American Journal ofPhysiologymdashHeart and Circulatory Physiology vol 285 no 3pp H1229ndashH1235 2003
[30] A Ducharme S Frantz M Aikawa et al ldquoTargeted deletion ofmatrix metalloproteinase-9 attenuates left ventricular enlarge-ment and collagen accumulation after experimental myocardialinfarctionrdquo Journal of Clinical Investigation vol 106 no 1 pp55ndash62 2000
[31] K TWeber ldquoExtracellular matrix remodeling in heart failure arole for de novo angiotensin II generationrdquo Circulation vol 96no 11 pp 4065ndash4082 1997
[32] W Iraqi P Rossignol M Angioi et al ldquoExtracellular cardiacmatrix biomarkers in patients with acute myocardial infarctioncomplicated by left ventricular dysfunction and heart failureinsights from the Eplerenone Post-Acute Myocardial InfarctionHeart Failure Efficacy and Survival Study (EPHESUS) studyrdquoCirculation vol 119 no 18 pp 2471ndash2479 2009
[33] R Eschalier M Fertin R Fay et al ldquoExtracellular matrixturnover biomarkers predict long-term left ventricular remod-eling aftermyocardial infarction insights from the reve-2 studyrdquoCirculation Heart Failure vol 6 no 6 pp 1199ndash1205 2013
[34] J G Simmons J B Pucilowska T O Keku and P K LundldquoIGF-I and TGF-1205731 have distinct effects on phenotype andproliferation of intestinal fibroblastsrdquo American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 283 no3 pp G809ndashG818 2002
[35] C F Hung M G Rohani S-S Lee P Chen and L MSchnapp ldquoRole of IGF-1 pathway in lung fibroblast activationrdquoRespiratory Research vol 14 no 1 article 102 2013
[36] M Nahrendorf F K Swirski E Aikawa et al ldquoThe healingmyocardium sequentially mobilizes twomonocyte subsets withdivergent and complementary functionsrdquoThe Journal of Exper-imental Medicine vol 204 no 12 pp 3037ndash3047 2007
[37] F L Chen Z H Yang Y Liu et al ldquoBerberine inhibits theexpression of TNF120572 MCP-1 and IL-6 in AcLDL-stimulatedmacrophages through PPAR120574 pathwayrdquo Endocrine vol 33 no3 pp 331ndash337 2008
[38] J Tonkin L Temmerman R D Sampson et alldquoMonocytemacrophage-derived IGF-1 orchestrates murineskeletal muscle regeneration and modulates autocrinepolarizationrdquo Molecular Therapy vol 23 no 7 pp 1189ndash1200 2015
[39] T Heidt G Courties P Dutta et al ldquoDifferential contributionof monocytes to heart macrophages in steady-state and aftermyocardial infarctionrdquo Circulation Research vol 115 no 2 pp284ndash295 2014
[40] S Epelman K J Lavine and G J Randolph ldquoOrigin andfunctions of tissue macrophagesrdquo Immunity vol 41 no 1 pp21ndash35 2014
[41] A R Pinto J W Godwin and N A Rosenthal ldquoMacrophagesin cardiac homeostasis injury responses and progenitor cellmobilisationrdquo Stem Cell Research vol 13 no 3 pp 705ndash7142014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Research and TreatmentAIDS
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 Mediators of Inflammation
WT ischemicWT remote
0
5
15
10
20
Isch
emic
ver
sus
rem
ote
IGF
-1E
aG
AP
DH
fold
in
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
(a)
0
20
40
60
80
100
LVE
F (
)
WT120572MHCIGF-1Ea
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast lowast
(b)
0
50
100
150
LVE
SV (120583
L)
WT120572MHCIGF-1Ea
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowastlowast
(c)
0
2
4
6
AC
TA
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
dlowast
WT
120572M
HC
IG
F-
1E
a
(d)
0
5
10
15
AN
PG
AP
DH
fo
ldin
du
ctio
n o
ver
WT
un
inju
red
lowastlowast
WT
120572M
HC
IG
F-
1E
a
(e)
Figure 1 120572MHCIGF-1Ea improves cardiac function and reduces dilation as early as 7 days after myocardial infarction (a) Levels of IGF-1Eain the ischemic and in the remote area ofWT hearts at 1 3 5 7 and 28 days followingMI (b) Left ventricular ejection fraction (LVEF) and (c)left ventricular end systolic volume (LVESV) after MI Solid lines represent WTmice Dashed lines represent 120572MHCIGF-1Ea mice Levels of(d) actin-alpha 1 skeletal muscle (ACTA 1) and (e) atrial natriuretic peptide (ANP) mRNA expression 28 days after MI Results are expressedas mean fold induction plusmn SEM over the values of uninjured hearts 119899 = 4ndash6 per group Two-tailed Studentrsquos t-test was performed to compareWT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
left ventricular EF by day 7 after MI (Figure 1(b) and Sup-plementary Table 1) Left ventricular end systolicdiastolicvolumes significantly increased after MI in WT heartsindicating left ventricular dilation while the 120572MHCIGF-1Ea hearts did not display any such signs (Figure 1(c) andSupplementary Table 1) In support of the functional datathe expression of molecular markers for cardiac damage suchas actin-alpha 1 skeletal muscle (ACTA) and atrial natriureticpeptide (ANP) was significantly reduced in 120572MHCIGF-1Eacompared to WT mice 28 days after MI (Figures 1(d) and1(e))The peak of endogenous IGF-1Ea in the ischemic regionof WT mice by day 7 along with the improvement in cardiacfunction in 120572MHCIGF-1Ea as early as day 7 indicates thatIGF-1Ea signalling at early time points is key for cardiacrepair
32 Altered Tissue Remodelling in120572MHCIGF-1Ea after InjuryThree days after MI WT and 120572MHCIGF-1Ea hearts dis-played infarcts of equal size quantified byMassonrsquos trichromestaining (Figure 2(a)) However by 28 days after infarction120572MHCIGF-1Ea hearts exhibited smaller scar areas compared
to WT (Figures 2(b) 2(c) and 2(d)) as previously reported[25] This was observed as a reduction in scar length butincreased scar thickness consistent with reduced infarctexpansion As amolecular measurement of fibrosis we quan-tified TGF-120573 mRNA levels which were significantly lowerin 120572MHCIGF-1Ea than in WT mice after MI (Figure 2(e))We therefore measured expression of genes involved in ECMturnover and synthesis MMP-2 MMP-9 TIMP-1 TIMP-2Col I1205721 Col I1205723 and lysyl oxidase (Lox) at 1 3 5 7 and28 days after MI In WT hearts MMP-9 was upregulated3 days after MI followed by MMP-2 at day 7 (Figures 2(f)and 2(g)) Interestingly neither MMP-2 nor MMP-9 wassignificantly upregulated in 120572MHCIGF-1Ea hearts at anytime point Inhibitors of matrix degradation TIMP-1 andTIMP-2 had similar expression in WT and 120572MHCIGF-1Eahearts at all time points except for day 7 when TIMP-2 wassignificantly upregulated in120572MHCIGF-1Ea hearts comparedto WT (Figures 2(h) and 2(i)) Taken together this supportsthe idea of a net overall reduction in matrix breakdownwhich may contribute to the reduced fibrosis observed in120572MHCIGF-1Ea hearts
Mediators of Inflammation 5
WT
0
10
20
30In
farc
ted
are
a (L
V)
()
3 days after MI
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(a)
0
10
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rcte
d a
rea
(LV
) (
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28 days after MI
lowastlowastlowast
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(b)
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1mm
(c)
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1mm
(d)
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inju
red
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F120573
GA
PD
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old
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nd
uct
ion
ove
r W
T u
nin
jure
d lowastlowast
lowast
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(e)
0
5
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P-9
GA
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old
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du
ctio
n o
ver
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inju
red
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inju
red
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(f)
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inju
red
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10
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30
40
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P-2
GA
PD
Hfo
ld o
ver
WT
un
inju
red
lowastlowast
WT120572MHCIGF-1Ea
(g)
TIM
P-2
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
d
0
10
20
30
40
Un
inju
red
Day
1
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Day
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lowastlowast
WT120572MHCIGF-1Ea
(h)
0
200
400
600
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1000
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P-1
GA
PD
Hfo
ld o
ver
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un
inju
red
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inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
WT120572MHCIGF-1Ea
(i)
0
20
40
60
80
100
Co
l 1120572
1G
AP
DH
fo
ldin
du
ctio
n o
ver
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un
inju
red
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inju
red
Day
1
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lowast
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(j)
0
20
40
60
80
100
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l 1120572
3G
AP
DH
fo
ldin
du
ctio
n o
ver
WT
un
inju
red
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inju
red
Day
1
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3
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5
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7
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28
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lowast
lowastlowast
(k)
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inju
red
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1
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3
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5
Day
7
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28
0
1
2
3
4
5
Co
l1120572
1 ve
rsu
s C
ol1120572
3 r
atio
lowast
lowast
lowastlowast
(l)
WT120572MHCIGF-1Ea
0
200
400
600
Lo
xG
AP
DH
fo
ld i
nd
uct
ion
ove
r W
T u
nin
jure
d
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowastlowast
(m)
Figure 2 Positive remodeling in the 120572MHCIGF-1Ea infarcted region after myocardial infarction Quantification of the infarcted area at (a)3 days and (b) 28 days after MI 119899 = 3 Representative histological sections of (c) WT and (d) 120572MHCIGF-1Ea hearts stained with Massonrsquostrichrome 28 days after MI Scale bar 1mm ((e)ndash(l)) mRNA relative expression of (e) transforming growth factor beta (TGF-120573) (f) matrixmetalloproteinase 9 (MMP-9) (g) matrix metalloproteinase 2 (MMP-2) (h) tissue inhibitor of metalloproteinase (TIMP) 2 (i) TIMP-1 (j)collagen (Col) I1205721 (k) Col I1205723 (l) ColI1205721ColI1205723 ratio (m) lysyl oxidase in the infarct area Results are expressed as mean fold induction plusmnSEM Solid lines representWTmice Dashed lines represent 120572MHCIGF-1Eamice 119899 = 3 per group Two-tailed Studentrsquos t-test was performedto compare WT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005 lowastlowastlowast119875 lt 0001
6 Mediators of Inflammation
We next analysed the composition of the newly synthe-sised matrix by measuring mRNA expression of the mostabundant cardiac ECM collagen Col I1205721 and Col I1205723 aswell as the collagen cross-linker lox which increases matrixstiffness Upregulation of both collagen types was detectedby day 3 peaking at day 7 (Figures 2(j) and 2(k)) At thistime point WT hearts expressed significantly more of bothcollagen types than 120572MHCIGF-1Ea hearts We also noted adifference in the ratio of the two collagen types 120572MHCIGF-1Ea hearts had a reduced Col I1205721Col I1205723 ratio compared toWT which was significant at days 3 7 and 28 (Figure 2(l)) Atthe peak of collagen upregulation lox was also upregulatedin WT hearts yet this was not observed in 120572MHCIGF-1Ea hearts (Figure 2(m)) These results indicate that IGF-1Eaoverexpression reduces ECM turnover afterMI and alters thecomposition of thematrix with preferential expression of ColI1205723 over Col I1205721 and less cross-linking which likely alters themechanical properties of the fibrotic area
33 Distinct Chemokine and Cytokine Production in 120572MHC-IGF-1Ea Hearts after Myocardial Infarct Tissue remodellingafter injury is closely tied to the inflammatory processWe therefore compared the inflammatory status of the120572MHCIGF-1Ea and WT hearts monitoring the productionof key immune genes IL-1120573 IL-10 MCP-1 and CCL5Expression of the inflammatory cytokine IL-1120573 was rapidlyinduced upon injury in WT hearts (66-fold over uninjured)yet was not upregulated as strongly in 120572MHCIGF-1Ea hearts(19-fold over uninjured Figure 3(a)) Similarly the strongupregulation of MCP-1 in WT hearts was not observed in120572MHCIGF-1Ea hearts (Figure 3(b)) IL-10 was also rapidlyupregulated peaking 1 day after MI In contrast to IL-1120573and MCP-1 this immunosuppressive cytokine was upreg-ulated 3-fold higher in 120572MHCIGF-1Ea hearts comparedto WT controls Although a second peak of IL-10 mRNAat 7 days was comparable in both WT and 120572MHCIGF-1Ea hearts 7 days after MI (Figure 3(c)) CCL5 which isinvolved in the recruitment of Ly6Clow monocytes [26 27]was upregulated in 120572MHCIGF-1Ea hearts 7 days after MI(Figure 3(d)) These data suggest an early bias towards aless inflammatory environment potentially modulating therecruitment of monocytes in 120572MHCIGF-1Ea hearts aftermyocardial infraction
34 IGF-1Ea Modulates Myeloid Cell Recruitment after Myo-cardial Infarction To document accumulation of the maininnate immune cell populations involved in cardiac inflam-mation after infarct single cell suspensions were preparedfromwholemouse hearts and analysed by flow cytometry Allcell populations described in this work were identified usingthe gating strategy shown in Supplementary Figures 3ndash6
In WT and 120572MHCIGF-1Ea hearts the total numberof infiltrating leukocytes (CD45+) gradually increased afterMI both reaching comparable peak numbers at day 5(Figure 4(a)) However at the earlier 3-day time point120572MHCIGF-1Ea hearts contained 49 less leukocytes permilligram of tissue than WT hearts In examining spe-cific immune cell populations this difference was partlyexplained by a 75 reduction in neutrophils (CD45+
CD11b+ F480minus CD11cminus and Ly6G+ 186 versus 46 cellsmgof tissue Figure 4(b)) and a 67 reduction in monocytes(CD45+ CD11b+ CD11cminus Ly6Gminus and F480minus 191 versus 62cellsmg of heart Figure 4(c)) however it was mostly due toreduced presence of macrophages (CD45+ CD11b+ CD11cminusLy6Gminus and F480+ 58 2276 versus 949 cellsmg of heartFigure 4(f)) These data agree with the reduced MCP-1expression observed in 120572MHCIGF-1Ea hearts (Figure 3(b))as it is the principal chemokine involved in monocyterecruitment [26 28]
The Ly6C surface marker distinguishes two differentsubsets ofmonocytes [2 3] Analysis of the Ly6Chigh (CD45+CD11b+ CD11cminus Ly6Gminus F480minus and Ly6Chigh) and Ly6Clow
(CD45+ CD11b+ CD11cminus Ly6Gminus F480minus and Ly6Clow)populations revealed that the reduction of total monocytenumbers at day 3 was attributable to a 20 decrease of theLy6Chigh population (102 versus 32 cellsmgof heart) whereasthe Ly6Clow population was not significantly different in WTcompared to 120572MHCIGF-1Ea (Figures 4(d) and 4(e)) hearts
To differentiate between inflammatory and reparativemacrophage populations we used the markers Ly6C andCD206 For this work only cells that were either Ly6C+CD206minus or Ly6CminusCD206+ were analysed although wenoted a double positive cell population (ie CD206+Ly6C+) However no changes were observed over timebetween WT and 120572MHCIGF-1Ea for the double positivepopulation (Supplementary 2) Both Ly6C+ inflammato-ry macrophage (CD45+ CD11b+ CD11cminus Ly6Gminus F480+and Ly6C+CD206minus) and CD206+ reparative macro-phage (CD45+ CD11b+ CD11cminus Ly6Gminus F480+ andLy6CminusCD206+) normalised cell numbers were reduced by71 and 48 respectively in 120572MHCIGF-1Ea hearts at theday 3 time point (522 versus 153 and 596 versus 310 cellsmgof tissue of heart resp Figures 4(g) and 4(h)) howeverthis was significant only for the Ly6C+ population By day 7after MI macrophage dynamics changed and we observeda greater number of total macrophages in 120572MHCIGF-1Eahearts compared to WT which was mainly due to a 155preferential increase in the CD206+ population (575 versus1468 cellsmg heart Figure 4(h)) Dendritic cells (CD45+CD11b+ F480minus CD11c+ and Ly6Gminus) were increased by48 in 120572MHCIGF-1Ea hearts compared to WT 5 days afterMI (168 versus 248 cellsmg heart Figure 4(i))
In summary cardiac-restricted expression of an IGF-1Eatransgene limited the early accumulation of innate immunecells at day 3 after MI with a bias towards the reduction ofinflammatory myeloid populations rather than regenerativepopulations This reduction corresponds with the lowerexpression of myeloid chemokines and the less inflammatorymilieu observed in the 120572MHCIGF-1Ea hearts
4 Discussion
Previous work in our lab showed that local expression of IGF-1Ea promoted functional restoration after MI and observedreduced infarct expansion thinning and dilation of the leftventricular wall [25] We now demonstrate transcriptionalmodulation of key ECM remodelling genes in the IGF-1Ea hearts associated with tempering of the inflammatorymyeloid cell response
Mediators of Inflammation 7
0
100
200
300
400
IL-1120573
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
d
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
lowast
WT120572MHCIGF-1Ea
(a)
0
100
200
300
MC
P-1
GA
PD
H f
old
in
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
(b)
0
50
100
150
200
IL-1
0G
AP
DH
fo
ldin
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
(c)
0
20
40
60
80
CC
L5
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
d
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
(d)
Figure 3 Cytokine dynamics in the infarcted 120572MHCIGF-1Ea hearts (a) Interleukin- (IL-) 1120573 (b) MCP-1 (c) IL-10 (d) CCL5 at several timepoints after permanent ligation Solid lines represent WT mice Dashed lines represent 120572MHCIGF-1Ea mice Results are expressed as meanfold induction plusmn SEM over the values of uninjured hearts 119899 = 3 per group Two-tailed Studentrsquos t-test was performed to compare WT versus120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
Increased MMP expression after injury has been impli-cated in contributing to scar destabilisation as transgenic ani-mal model knockouts of either MMP-2 or MMP-9 have beenshown to attenuate LV dilation rupture and impairment ofcardiac function [29 30] While MMP-2 and MMP-9 mRNAexpression levels were upregulated in injured WT heartsthis increase was not observed in 120572MHCIGF-1Ea hearts Inline with reduced matrix breakdown mRNA expression ofthe MMP inhibitor TIMP-2 was significantly increased in120572MHCIGF-1Ea hearts at day 7 after MI Thus IGF-1Ea mayprevent adverse cardiac remodelling in part by modulatingtranscription of MMPsTIMPs
The production of new matrix components was alsoaltered by the presence of the IGF-1Ea transgenewith an over-all reduction in collagen synthesis confirmed in histologicalstains and a bias towards expression of Col I1205723 over ColI1205721 In MI patients turnover of cardiac extracellular matrixcan be assessed by using circulating collagen peptides asblood biomarkers [31] and high type I collagen is associatedwith adverse clinical outcome [32] A prospectivemulticentrestudy further showed that a low type IIItype I collagen ratioespecially at 1 month after MI is predictive of detrimental
left ventricular remodelling as well as cardiovascular deathsand hospitalisation cases for heart failure [33] Changes incollagen ratios are known to affect the strength and tensileproperties of the ECM It would be interesting to determinewhether these properties are modified in the 120572MHCIGF-1Eahearts
IGF-1Ea could promote the changes in collagen depo-sition by directly acting on fibroblasts as it promotes boththeir proliferation and activation to myofibroblasts [3435] Alternatively IGF-1Ea may influence accumulation andactivation of immune cells present at the infarct which inturn regulate myofibroblast activation Indeed we observedmodulation of the inflammatory process in 120572MHCIGF-1Ea hearts with less monocyte (Ly6Chigh) infiltration intothe injured myocardium This effect is associated withreduced expression of themonocyte chemoattractantMCP-1although no significant changes were observed in the Ly6Clow
monocyte population in infarcted hearts possibly due toCCL5 upregulation
It is interesting that while complete depletion of mono-cytes at any stage of repair leads to poor recovery [36]a more subtle modulation of the monocyte population in
8 Mediators of Inflammation
0
2000
4000
6000
8000
Cel
lsm
g h
eart
Total leukocytes
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
(a)
0
200
400
600
800Neutrophils
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(b)
0
100
200
300Monocytes
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(c)
0
50
100
150
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
Ly-6Chigh monocytes
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(d)
0
50
100
150
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
Ly-6Clow monocytes
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(e)
0
1000
2000
3000
4000
5000Macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
lowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(f)
0
500
1000
1500
2000Ly6C+ macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(g)
0
500
1000
1500
2000CD206+ macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(h)
0
100
200
300Dendritic cells
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(i)
Figure 4 Characterisation and temporal dynamics of immune cell populations in 120572MHCIGF-1Ea hearts after MI Quantification of (a) totalleukocytes (CD45+) (b) neutrophils (CD45+ CD11b+ F480minus CD11cminus and Ly6G+) and (c) monocytes (CD45+ CD11b+ CD11cminus Ly6Gminusand F480minus) which were further classified as (d) Ly6Chigh and (e) Ly6Clow monocytes (f) Macrophages (CD45+ CD11b+ CD11cminus Ly6Gminusand F480+) were further characterised on the basis of Ly6C and CD206 expression as (g) Ly6ChighCD206low (inflammatory macrophages)and (h) Ly6ClowCD206high (reparative macrophages) For this work only cells that were either Ly6ChighCD206minus or Ly6CminusCD206+ wereanalysed although we noted a double positive cell population (ie CD206+ Ly6C+) (i) Dendritic cells were defined as CD45+ CD11b+F480minus CD11c+ and Ly6Gminus Data is presented as the total number of cells per mg of heart 119899 = 4ndash6 per group Two-tailed Studentrsquos t-testwas performed to compare WT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
the 120572MHCIGF-1Ea hearts is associated with improved heartfunction Studies abrogating monocyte recruitment usinga selective CCR2 inhibitor have resulted in reduced IL-1120573 IL-6 MCP-1 and TNF120572 levels [37] IGF-1Ea influ-ences macrophage polarisation in skeletal muscle [38]
and we similarly found reduced Ly6Chigh monocyte andLy6C+ macrophage normalised cell numbers while CD206+macrophage numbers were increased by day 7 suggestingthat IGF-1Ea promoted a quick progression to the reparativephase of repair by modulating macrophage phenotype In
Mediators of Inflammation 9
the 120572MHCIGF-1Ea hearts we observed a decrease in MCP-1which is the chemokine for CCR2 recently shown to distin-guish infiltrating monocytes from the resident macrophagepopulation which has a different embryological origin andexpresses CD206 [39ndash41] It is therefore possible that in our120572MHCIGF-1Ea transgenic mouse model IGF-1Ea reducesthe infiltration of inflammatory Ly6Chigh monocytes bypreventing upregulation of MCP-1 while still allowing forexpansion of the resident macrophage population
In summary we show that the 120572MHCIGF-1Ea mousemodel can modulate several associated aspects of the cellularrepair process after MI including immune cell recruit-ment cytokine expression and matrix turnover All of thesechanges occur within the first 7 days after infarct at whichtime a functional improvement can already be measured in120572MHCIGF-1Ea hearts compared toWT controlsThese dataprovide new insights into the mechanism of IGF-1Ea andsuggest that early immunomodulation is key to successfulcardiac repair after injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Robert D Sampson and Susanne Sattler contributed equallyto this work
Acknowledgments
The authors are grateful to members of the RosenthalLaboratory for advice and critique This work was sup-ported by grants from the European Unionrsquos Seventh Frame-work Programme Marie Curie Initial Training Network-CardioNeT GA-289600 and British Heart Foundation GrantRM13130157 to Nadia Rosenthal Robert D Sampson andMichael D Schneider are supported by the British HeartFoundation Centre of Research Excellence and British HeartFoundation Simon Marks Chair We gratefully acknowledgethe support from the British Spanish Society (BSS) for theBritish Spanish SocietySantander Universities 2015 scholar-ship that partially supported this project
References
[1] A S Go D Mozaffarian V L Roger et al ldquoHeart disease andstroke statisticsmdash2014 update a report from theAmerican heartassociationrdquo Circulation vol 129 pp e28ndashe292 2014
[2] N G Frangogiannis ldquoRegulation of the inflammatory responsein cardiac repairrdquo Circulation Research vol 110 no 1 pp 159ndash173 2012
[3] N G Frangogiannis ldquoThe inflammatory response in myocar-dial injury repair and remodellingrdquoNature Reviews Cardiologyvol 11 no 5 pp 255ndash265 2014
[4] T Shavlakadze N Winn N Rosenthal and M D GroundsldquoReconciling data from transgenic mice that overexpress IGF-I specifically in skeletal musclerdquo Growth Hormone and IGFResearch vol 15 no 1 pp 4ndash18 2005
[5] P Rotwein K M Pollock D K Didier and G G KrivildquoOrganization and sequence of the human insulin-like growthfactor I gene Alternative RNAprocessing produces two insulin-like growth factor I precursor peptidesrdquo Journal of BiologicalChemistry vol 261 no 11 pp 4828ndash4832 1986
[6] J G Simmons J J Van Wyk E C Hoyt and P K LundldquoMultiple transcription start sites in the rat insulin-like growthfactor-I gene Give rise to IGF-I mRNAs that encode differentIGF-I precursors and are processed differently in vitvordquoGrowthFactors vol 9 no 3 pp 205ndash221 1993
[7] G I Bell M M Stempien N M Fong and L B RallldquoSequences of liver cDNAs encoding two different mouseinsulin-like growth factor I precursorsrdquo Nucleic Acids Researchvol 14 no 20 pp 7873ndash7882 1986
[8] M S Hede E Salimova A Piszczek et al ldquoE-peptides controlbioavailability of IGF-1rdquo PLoS ONE vol 7 no 12 Article IDe51152 2012
[9] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[10] M Vinciguerra M P Santini W C Claycomb A G Ladurnerand N Rosenthal ldquoLocal IGF-1 isoform protects cardiomy-ocytes from hypertrophic and oxidative stresses via SirT1activityrdquo Aging vol 2 no 1 pp 43ndash62 2010
[11] D Vanhoutte M Schellings Y Pinto and S Heymans ldquoRel-evance of matrix metalloproteinases and their inhibitors aftermyocardial infarction a temporal and spatial windowrdquo Cardio-vascular Research vol 69 no 3 pp 604ndash613 2006
[12] J B Caulfield and T K Borg ldquoThe collagen network of theheartrdquo Laboratory Investigation vol 40 no 3 pp 364ndash372 1979
[13] B I Jugdutt ldquoRemodeling of the myocardium and potentialtargets in the collagen degradation and synthesis pathwaysrdquoCurrent Drug TargetsmdashCardiovascular amp Haematological Dis-orders vol 3 no 1 pp 1ndash30 2003
[14] B I Jugdutt ldquoVentricular remodeling after infarction andthe extracellular collagen matrix when is enough enoughrdquoCirculation vol 108 no 11 pp 1395ndash1403 2003
[15] M Nahrendorf and F K Swirski ldquoMonocyte and macrophageheterogeneity in the heartrdquo Circulation Research vol 112 no 12pp 1624ndash1633 2013
[16] F Leuschner P J Rauch T Ueno et al ldquoRapid mono-cyte kinetics in acute myocardial infarction are sustained byextramedullary monocytopoiesisrdquo The Journal of ExperimentalMedicine vol 209 no 1 pp 123ndash137 2012
[17] X Yan A Anzai Y Katsumata et al ldquoTemporal dynamics ofcardiac immune cell accumulation following acute myocardialinfarctionrdquo Journal ofMolecular andCellular Cardiology vol 62pp 24ndash35 2013
[18] S Frantz and M Nahrendorf ldquoCardiac macrophages and theirrole in ischemic heart diseaserdquo Cardiovascular Research vol102 no 2 pp 240ndash248 2014
[19] Q Cao Y Wang D Zheng et al ldquoIL-10TGF-120573-modifiedmacrophages induce regulatory T cells and protect againstadriamycin nephrosisrdquo Journal of the American Society ofNephrology vol 21 no 6 pp 933ndash942 2010
[20] B Johannesson S Sattler E Semenova et al ldquoInsulin-likegrowth factor-1 induces regulatory T cell-mediated suppressionof allergic contact dermatitis in micerdquo Disease Models ampMechanisms vol 7 no 8 pp 977ndash985 2014
10 Mediators of Inflammation
[21] D Bilbao L Luciani B Johannesson A Piszczek and NRosenthal ldquoInsulin-like growth factor-1 stimulates regulatoryT cells and suppresses autoimmune diseaserdquo EMBO MolecularMedicine vol 6 no 11 pp 1423ndash1435 2005
[22] H Oh S B Bradfute T D Gallardo et al ldquoCardiac progenitorcells from adult myocardium homing differentiation andfusion after infarctionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 21 pp12313ndash12318 2003
[23] A Philippou E Papageorgiou G Bogdanis et al ldquoExpressionof IGF-1 isoforms after exercise-induced muscle damage inhumans characterization of theMGFEpeptide actions in vitrordquoIn Vivo vol 23 no 4 pp 567ndash576 2009
[24] S Sanz J B Pucilowska S Liu et al ldquoExpression of insulin-like growth factor I by activated hepatic stellate cells reducesfibrogenesis and enhances regeneration after liver injuryrdquo Gutvol 54 no 1 pp 134ndash141 2005
[25] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[26] F Tacke D Alvarez T J Kaplan et al ldquoMonocyte subsetsdifferentially employ CCR2 CCR5 and CX3CR1 to accumulatewithin atherosclerotic plaquesrdquo Journal of Clinical Investigationvol 117 no 1 pp 185ndash194 2007
[27] L M Carlin E G Stamatiades C Auffray et al ldquoNr4a1-dependent Ly6Clow monocytes monitor endothelial cells andorchestrate their disposalrdquoCell vol 153 no 2 pp 362ndash375 2013
[28] N V Serbina and E G Pamer ldquoMonocyte emigration frombone marrow during bacterial infection requires signals medi-ated by chemokine receptor CCR2rdquo Nature Immunology vol 7no 3 pp 311ndash317 2006
[29] S Hayashidani H Tsutsui M Ikeuchi et al ldquoTargeted deletionofMMP-2 attenuates early LV rupture and late remodeling afterexperimental myocardial infarctionrdquo The American Journal ofPhysiologymdashHeart and Circulatory Physiology vol 285 no 3pp H1229ndashH1235 2003
[30] A Ducharme S Frantz M Aikawa et al ldquoTargeted deletion ofmatrix metalloproteinase-9 attenuates left ventricular enlarge-ment and collagen accumulation after experimental myocardialinfarctionrdquo Journal of Clinical Investigation vol 106 no 1 pp55ndash62 2000
[31] K TWeber ldquoExtracellular matrix remodeling in heart failure arole for de novo angiotensin II generationrdquo Circulation vol 96no 11 pp 4065ndash4082 1997
[32] W Iraqi P Rossignol M Angioi et al ldquoExtracellular cardiacmatrix biomarkers in patients with acute myocardial infarctioncomplicated by left ventricular dysfunction and heart failureinsights from the Eplerenone Post-Acute Myocardial InfarctionHeart Failure Efficacy and Survival Study (EPHESUS) studyrdquoCirculation vol 119 no 18 pp 2471ndash2479 2009
[33] R Eschalier M Fertin R Fay et al ldquoExtracellular matrixturnover biomarkers predict long-term left ventricular remod-eling aftermyocardial infarction insights from the reve-2 studyrdquoCirculation Heart Failure vol 6 no 6 pp 1199ndash1205 2013
[34] J G Simmons J B Pucilowska T O Keku and P K LundldquoIGF-I and TGF-1205731 have distinct effects on phenotype andproliferation of intestinal fibroblastsrdquo American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 283 no3 pp G809ndashG818 2002
[35] C F Hung M G Rohani S-S Lee P Chen and L MSchnapp ldquoRole of IGF-1 pathway in lung fibroblast activationrdquoRespiratory Research vol 14 no 1 article 102 2013
[36] M Nahrendorf F K Swirski E Aikawa et al ldquoThe healingmyocardium sequentially mobilizes twomonocyte subsets withdivergent and complementary functionsrdquoThe Journal of Exper-imental Medicine vol 204 no 12 pp 3037ndash3047 2007
[37] F L Chen Z H Yang Y Liu et al ldquoBerberine inhibits theexpression of TNF120572 MCP-1 and IL-6 in AcLDL-stimulatedmacrophages through PPAR120574 pathwayrdquo Endocrine vol 33 no3 pp 331ndash337 2008
[38] J Tonkin L Temmerman R D Sampson et alldquoMonocytemacrophage-derived IGF-1 orchestrates murineskeletal muscle regeneration and modulates autocrinepolarizationrdquo Molecular Therapy vol 23 no 7 pp 1189ndash1200 2015
[39] T Heidt G Courties P Dutta et al ldquoDifferential contributionof monocytes to heart macrophages in steady-state and aftermyocardial infarctionrdquo Circulation Research vol 115 no 2 pp284ndash295 2014
[40] S Epelman K J Lavine and G J Randolph ldquoOrigin andfunctions of tissue macrophagesrdquo Immunity vol 41 no 1 pp21ndash35 2014
[41] A R Pinto J W Godwin and N A Rosenthal ldquoMacrophagesin cardiac homeostasis injury responses and progenitor cellmobilisationrdquo Stem Cell Research vol 13 no 3 pp 705ndash7142014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Mediators of Inflammation 5
WT
0
10
20
30In
farc
ted
are
a (L
V)
()
3 days after MI
120572MHCIGF-1Ea
(a)
0
10
20
30
Infa
rcte
d a
rea
(LV
) (
)
28 days after MI
lowastlowastlowast
WT 120572MHCIGF-1Ea
(b)
WT
1mm
(c)
120572MHC-IGF-1Ea
1mm
(d)
0
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
10
20
30
40
50
TG
F120573
GA
PD
H f
old
i
nd
uct
ion
ove
r W
T u
nin
jure
d lowastlowast
lowast
WT120572MHCIGF-1Ea
(e)
0
5
10
15
20
MM
P-9
GA
PD
H f
old
in
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
(f)
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
0
10
20
30
40
MM
P-2
GA
PD
Hfo
ld o
ver
WT
un
inju
red
lowastlowast
WT120572MHCIGF-1Ea
(g)
TIM
P-2
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
d
0
10
20
30
40
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
(h)
0
200
400
600
800
1000
TIM
P-1
GA
PD
Hfo
ld o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
WT120572MHCIGF-1Ea
(i)
0
20
40
60
80
100
Co
l 1120572
1G
AP
DH
fo
ldin
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
lowast
WT120572MHCIGF-1Ea
(j)
0
20
40
60
80
100
Co
l 1120572
3G
AP
DH
fo
ldin
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
WT120572MHCIGF-1Ea
lowast
lowastlowast
(k)
WT120572MHCIGF-1Ea
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
0
1
2
3
4
5
Co
l1120572
1 ve
rsu
s C
ol1120572
3 r
atio
lowast
lowast
lowastlowast
(l)
WT120572MHCIGF-1Ea
0
200
400
600
Lo
xG
AP
DH
fo
ld i
nd
uct
ion
ove
r W
T u
nin
jure
d
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowastlowast
(m)
Figure 2 Positive remodeling in the 120572MHCIGF-1Ea infarcted region after myocardial infarction Quantification of the infarcted area at (a)3 days and (b) 28 days after MI 119899 = 3 Representative histological sections of (c) WT and (d) 120572MHCIGF-1Ea hearts stained with Massonrsquostrichrome 28 days after MI Scale bar 1mm ((e)ndash(l)) mRNA relative expression of (e) transforming growth factor beta (TGF-120573) (f) matrixmetalloproteinase 9 (MMP-9) (g) matrix metalloproteinase 2 (MMP-2) (h) tissue inhibitor of metalloproteinase (TIMP) 2 (i) TIMP-1 (j)collagen (Col) I1205721 (k) Col I1205723 (l) ColI1205721ColI1205723 ratio (m) lysyl oxidase in the infarct area Results are expressed as mean fold induction plusmnSEM Solid lines representWTmice Dashed lines represent 120572MHCIGF-1Eamice 119899 = 3 per group Two-tailed Studentrsquos t-test was performedto compare WT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005 lowastlowastlowast119875 lt 0001
6 Mediators of Inflammation
We next analysed the composition of the newly synthe-sised matrix by measuring mRNA expression of the mostabundant cardiac ECM collagen Col I1205721 and Col I1205723 aswell as the collagen cross-linker lox which increases matrixstiffness Upregulation of both collagen types was detectedby day 3 peaking at day 7 (Figures 2(j) and 2(k)) At thistime point WT hearts expressed significantly more of bothcollagen types than 120572MHCIGF-1Ea hearts We also noted adifference in the ratio of the two collagen types 120572MHCIGF-1Ea hearts had a reduced Col I1205721Col I1205723 ratio compared toWT which was significant at days 3 7 and 28 (Figure 2(l)) Atthe peak of collagen upregulation lox was also upregulatedin WT hearts yet this was not observed in 120572MHCIGF-1Ea hearts (Figure 2(m)) These results indicate that IGF-1Eaoverexpression reduces ECM turnover afterMI and alters thecomposition of thematrix with preferential expression of ColI1205723 over Col I1205721 and less cross-linking which likely alters themechanical properties of the fibrotic area
33 Distinct Chemokine and Cytokine Production in 120572MHC-IGF-1Ea Hearts after Myocardial Infarct Tissue remodellingafter injury is closely tied to the inflammatory processWe therefore compared the inflammatory status of the120572MHCIGF-1Ea and WT hearts monitoring the productionof key immune genes IL-1120573 IL-10 MCP-1 and CCL5Expression of the inflammatory cytokine IL-1120573 was rapidlyinduced upon injury in WT hearts (66-fold over uninjured)yet was not upregulated as strongly in 120572MHCIGF-1Ea hearts(19-fold over uninjured Figure 3(a)) Similarly the strongupregulation of MCP-1 in WT hearts was not observed in120572MHCIGF-1Ea hearts (Figure 3(b)) IL-10 was also rapidlyupregulated peaking 1 day after MI In contrast to IL-1120573and MCP-1 this immunosuppressive cytokine was upreg-ulated 3-fold higher in 120572MHCIGF-1Ea hearts comparedto WT controls Although a second peak of IL-10 mRNAat 7 days was comparable in both WT and 120572MHCIGF-1Ea hearts 7 days after MI (Figure 3(c)) CCL5 which isinvolved in the recruitment of Ly6Clow monocytes [26 27]was upregulated in 120572MHCIGF-1Ea hearts 7 days after MI(Figure 3(d)) These data suggest an early bias towards aless inflammatory environment potentially modulating therecruitment of monocytes in 120572MHCIGF-1Ea hearts aftermyocardial infraction
34 IGF-1Ea Modulates Myeloid Cell Recruitment after Myo-cardial Infarction To document accumulation of the maininnate immune cell populations involved in cardiac inflam-mation after infarct single cell suspensions were preparedfromwholemouse hearts and analysed by flow cytometry Allcell populations described in this work were identified usingthe gating strategy shown in Supplementary Figures 3ndash6
In WT and 120572MHCIGF-1Ea hearts the total numberof infiltrating leukocytes (CD45+) gradually increased afterMI both reaching comparable peak numbers at day 5(Figure 4(a)) However at the earlier 3-day time point120572MHCIGF-1Ea hearts contained 49 less leukocytes permilligram of tissue than WT hearts In examining spe-cific immune cell populations this difference was partlyexplained by a 75 reduction in neutrophils (CD45+
CD11b+ F480minus CD11cminus and Ly6G+ 186 versus 46 cellsmgof tissue Figure 4(b)) and a 67 reduction in monocytes(CD45+ CD11b+ CD11cminus Ly6Gminus and F480minus 191 versus 62cellsmg of heart Figure 4(c)) however it was mostly due toreduced presence of macrophages (CD45+ CD11b+ CD11cminusLy6Gminus and F480+ 58 2276 versus 949 cellsmg of heartFigure 4(f)) These data agree with the reduced MCP-1expression observed in 120572MHCIGF-1Ea hearts (Figure 3(b))as it is the principal chemokine involved in monocyterecruitment [26 28]
The Ly6C surface marker distinguishes two differentsubsets ofmonocytes [2 3] Analysis of the Ly6Chigh (CD45+CD11b+ CD11cminus Ly6Gminus F480minus and Ly6Chigh) and Ly6Clow
(CD45+ CD11b+ CD11cminus Ly6Gminus F480minus and Ly6Clow)populations revealed that the reduction of total monocytenumbers at day 3 was attributable to a 20 decrease of theLy6Chigh population (102 versus 32 cellsmgof heart) whereasthe Ly6Clow population was not significantly different in WTcompared to 120572MHCIGF-1Ea (Figures 4(d) and 4(e)) hearts
To differentiate between inflammatory and reparativemacrophage populations we used the markers Ly6C andCD206 For this work only cells that were either Ly6C+CD206minus or Ly6CminusCD206+ were analysed although wenoted a double positive cell population (ie CD206+Ly6C+) However no changes were observed over timebetween WT and 120572MHCIGF-1Ea for the double positivepopulation (Supplementary 2) Both Ly6C+ inflammato-ry macrophage (CD45+ CD11b+ CD11cminus Ly6Gminus F480+and Ly6C+CD206minus) and CD206+ reparative macro-phage (CD45+ CD11b+ CD11cminus Ly6Gminus F480+ andLy6CminusCD206+) normalised cell numbers were reduced by71 and 48 respectively in 120572MHCIGF-1Ea hearts at theday 3 time point (522 versus 153 and 596 versus 310 cellsmgof tissue of heart resp Figures 4(g) and 4(h)) howeverthis was significant only for the Ly6C+ population By day 7after MI macrophage dynamics changed and we observeda greater number of total macrophages in 120572MHCIGF-1Eahearts compared to WT which was mainly due to a 155preferential increase in the CD206+ population (575 versus1468 cellsmg heart Figure 4(h)) Dendritic cells (CD45+CD11b+ F480minus CD11c+ and Ly6Gminus) were increased by48 in 120572MHCIGF-1Ea hearts compared to WT 5 days afterMI (168 versus 248 cellsmg heart Figure 4(i))
In summary cardiac-restricted expression of an IGF-1Eatransgene limited the early accumulation of innate immunecells at day 3 after MI with a bias towards the reduction ofinflammatory myeloid populations rather than regenerativepopulations This reduction corresponds with the lowerexpression of myeloid chemokines and the less inflammatorymilieu observed in the 120572MHCIGF-1Ea hearts
4 Discussion
Previous work in our lab showed that local expression of IGF-1Ea promoted functional restoration after MI and observedreduced infarct expansion thinning and dilation of the leftventricular wall [25] We now demonstrate transcriptionalmodulation of key ECM remodelling genes in the IGF-1Ea hearts associated with tempering of the inflammatorymyeloid cell response
Mediators of Inflammation 7
0
100
200
300
400
IL-1120573
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
d
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
lowast
WT120572MHCIGF-1Ea
(a)
0
100
200
300
MC
P-1
GA
PD
H f
old
in
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
(b)
0
50
100
150
200
IL-1
0G
AP
DH
fo
ldin
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
(c)
0
20
40
60
80
CC
L5
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
d
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
(d)
Figure 3 Cytokine dynamics in the infarcted 120572MHCIGF-1Ea hearts (a) Interleukin- (IL-) 1120573 (b) MCP-1 (c) IL-10 (d) CCL5 at several timepoints after permanent ligation Solid lines represent WT mice Dashed lines represent 120572MHCIGF-1Ea mice Results are expressed as meanfold induction plusmn SEM over the values of uninjured hearts 119899 = 3 per group Two-tailed Studentrsquos t-test was performed to compare WT versus120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
Increased MMP expression after injury has been impli-cated in contributing to scar destabilisation as transgenic ani-mal model knockouts of either MMP-2 or MMP-9 have beenshown to attenuate LV dilation rupture and impairment ofcardiac function [29 30] While MMP-2 and MMP-9 mRNAexpression levels were upregulated in injured WT heartsthis increase was not observed in 120572MHCIGF-1Ea hearts Inline with reduced matrix breakdown mRNA expression ofthe MMP inhibitor TIMP-2 was significantly increased in120572MHCIGF-1Ea hearts at day 7 after MI Thus IGF-1Ea mayprevent adverse cardiac remodelling in part by modulatingtranscription of MMPsTIMPs
The production of new matrix components was alsoaltered by the presence of the IGF-1Ea transgenewith an over-all reduction in collagen synthesis confirmed in histologicalstains and a bias towards expression of Col I1205723 over ColI1205721 In MI patients turnover of cardiac extracellular matrixcan be assessed by using circulating collagen peptides asblood biomarkers [31] and high type I collagen is associatedwith adverse clinical outcome [32] A prospectivemulticentrestudy further showed that a low type IIItype I collagen ratioespecially at 1 month after MI is predictive of detrimental
left ventricular remodelling as well as cardiovascular deathsand hospitalisation cases for heart failure [33] Changes incollagen ratios are known to affect the strength and tensileproperties of the ECM It would be interesting to determinewhether these properties are modified in the 120572MHCIGF-1Eahearts
IGF-1Ea could promote the changes in collagen depo-sition by directly acting on fibroblasts as it promotes boththeir proliferation and activation to myofibroblasts [3435] Alternatively IGF-1Ea may influence accumulation andactivation of immune cells present at the infarct which inturn regulate myofibroblast activation Indeed we observedmodulation of the inflammatory process in 120572MHCIGF-1Ea hearts with less monocyte (Ly6Chigh) infiltration intothe injured myocardium This effect is associated withreduced expression of themonocyte chemoattractantMCP-1although no significant changes were observed in the Ly6Clow
monocyte population in infarcted hearts possibly due toCCL5 upregulation
It is interesting that while complete depletion of mono-cytes at any stage of repair leads to poor recovery [36]a more subtle modulation of the monocyte population in
8 Mediators of Inflammation
0
2000
4000
6000
8000
Cel
lsm
g h
eart
Total leukocytes
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
(a)
0
200
400
600
800Neutrophils
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(b)
0
100
200
300Monocytes
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(c)
0
50
100
150
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
Ly-6Chigh monocytes
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(d)
0
50
100
150
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
Ly-6Clow monocytes
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(e)
0
1000
2000
3000
4000
5000Macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
lowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(f)
0
500
1000
1500
2000Ly6C+ macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(g)
0
500
1000
1500
2000CD206+ macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(h)
0
100
200
300Dendritic cells
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(i)
Figure 4 Characterisation and temporal dynamics of immune cell populations in 120572MHCIGF-1Ea hearts after MI Quantification of (a) totalleukocytes (CD45+) (b) neutrophils (CD45+ CD11b+ F480minus CD11cminus and Ly6G+) and (c) monocytes (CD45+ CD11b+ CD11cminus Ly6Gminusand F480minus) which were further classified as (d) Ly6Chigh and (e) Ly6Clow monocytes (f) Macrophages (CD45+ CD11b+ CD11cminus Ly6Gminusand F480+) were further characterised on the basis of Ly6C and CD206 expression as (g) Ly6ChighCD206low (inflammatory macrophages)and (h) Ly6ClowCD206high (reparative macrophages) For this work only cells that were either Ly6ChighCD206minus or Ly6CminusCD206+ wereanalysed although we noted a double positive cell population (ie CD206+ Ly6C+) (i) Dendritic cells were defined as CD45+ CD11b+F480minus CD11c+ and Ly6Gminus Data is presented as the total number of cells per mg of heart 119899 = 4ndash6 per group Two-tailed Studentrsquos t-testwas performed to compare WT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
the 120572MHCIGF-1Ea hearts is associated with improved heartfunction Studies abrogating monocyte recruitment usinga selective CCR2 inhibitor have resulted in reduced IL-1120573 IL-6 MCP-1 and TNF120572 levels [37] IGF-1Ea influ-ences macrophage polarisation in skeletal muscle [38]
and we similarly found reduced Ly6Chigh monocyte andLy6C+ macrophage normalised cell numbers while CD206+macrophage numbers were increased by day 7 suggestingthat IGF-1Ea promoted a quick progression to the reparativephase of repair by modulating macrophage phenotype In
Mediators of Inflammation 9
the 120572MHCIGF-1Ea hearts we observed a decrease in MCP-1which is the chemokine for CCR2 recently shown to distin-guish infiltrating monocytes from the resident macrophagepopulation which has a different embryological origin andexpresses CD206 [39ndash41] It is therefore possible that in our120572MHCIGF-1Ea transgenic mouse model IGF-1Ea reducesthe infiltration of inflammatory Ly6Chigh monocytes bypreventing upregulation of MCP-1 while still allowing forexpansion of the resident macrophage population
In summary we show that the 120572MHCIGF-1Ea mousemodel can modulate several associated aspects of the cellularrepair process after MI including immune cell recruit-ment cytokine expression and matrix turnover All of thesechanges occur within the first 7 days after infarct at whichtime a functional improvement can already be measured in120572MHCIGF-1Ea hearts compared toWT controlsThese dataprovide new insights into the mechanism of IGF-1Ea andsuggest that early immunomodulation is key to successfulcardiac repair after injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Robert D Sampson and Susanne Sattler contributed equallyto this work
Acknowledgments
The authors are grateful to members of the RosenthalLaboratory for advice and critique This work was sup-ported by grants from the European Unionrsquos Seventh Frame-work Programme Marie Curie Initial Training Network-CardioNeT GA-289600 and British Heart Foundation GrantRM13130157 to Nadia Rosenthal Robert D Sampson andMichael D Schneider are supported by the British HeartFoundation Centre of Research Excellence and British HeartFoundation Simon Marks Chair We gratefully acknowledgethe support from the British Spanish Society (BSS) for theBritish Spanish SocietySantander Universities 2015 scholar-ship that partially supported this project
References
[1] A S Go D Mozaffarian V L Roger et al ldquoHeart disease andstroke statisticsmdash2014 update a report from theAmerican heartassociationrdquo Circulation vol 129 pp e28ndashe292 2014
[2] N G Frangogiannis ldquoRegulation of the inflammatory responsein cardiac repairrdquo Circulation Research vol 110 no 1 pp 159ndash173 2012
[3] N G Frangogiannis ldquoThe inflammatory response in myocar-dial injury repair and remodellingrdquoNature Reviews Cardiologyvol 11 no 5 pp 255ndash265 2014
[4] T Shavlakadze N Winn N Rosenthal and M D GroundsldquoReconciling data from transgenic mice that overexpress IGF-I specifically in skeletal musclerdquo Growth Hormone and IGFResearch vol 15 no 1 pp 4ndash18 2005
[5] P Rotwein K M Pollock D K Didier and G G KrivildquoOrganization and sequence of the human insulin-like growthfactor I gene Alternative RNAprocessing produces two insulin-like growth factor I precursor peptidesrdquo Journal of BiologicalChemistry vol 261 no 11 pp 4828ndash4832 1986
[6] J G Simmons J J Van Wyk E C Hoyt and P K LundldquoMultiple transcription start sites in the rat insulin-like growthfactor-I gene Give rise to IGF-I mRNAs that encode differentIGF-I precursors and are processed differently in vitvordquoGrowthFactors vol 9 no 3 pp 205ndash221 1993
[7] G I Bell M M Stempien N M Fong and L B RallldquoSequences of liver cDNAs encoding two different mouseinsulin-like growth factor I precursorsrdquo Nucleic Acids Researchvol 14 no 20 pp 7873ndash7882 1986
[8] M S Hede E Salimova A Piszczek et al ldquoE-peptides controlbioavailability of IGF-1rdquo PLoS ONE vol 7 no 12 Article IDe51152 2012
[9] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[10] M Vinciguerra M P Santini W C Claycomb A G Ladurnerand N Rosenthal ldquoLocal IGF-1 isoform protects cardiomy-ocytes from hypertrophic and oxidative stresses via SirT1activityrdquo Aging vol 2 no 1 pp 43ndash62 2010
[11] D Vanhoutte M Schellings Y Pinto and S Heymans ldquoRel-evance of matrix metalloproteinases and their inhibitors aftermyocardial infarction a temporal and spatial windowrdquo Cardio-vascular Research vol 69 no 3 pp 604ndash613 2006
[12] J B Caulfield and T K Borg ldquoThe collagen network of theheartrdquo Laboratory Investigation vol 40 no 3 pp 364ndash372 1979
[13] B I Jugdutt ldquoRemodeling of the myocardium and potentialtargets in the collagen degradation and synthesis pathwaysrdquoCurrent Drug TargetsmdashCardiovascular amp Haematological Dis-orders vol 3 no 1 pp 1ndash30 2003
[14] B I Jugdutt ldquoVentricular remodeling after infarction andthe extracellular collagen matrix when is enough enoughrdquoCirculation vol 108 no 11 pp 1395ndash1403 2003
[15] M Nahrendorf and F K Swirski ldquoMonocyte and macrophageheterogeneity in the heartrdquo Circulation Research vol 112 no 12pp 1624ndash1633 2013
[16] F Leuschner P J Rauch T Ueno et al ldquoRapid mono-cyte kinetics in acute myocardial infarction are sustained byextramedullary monocytopoiesisrdquo The Journal of ExperimentalMedicine vol 209 no 1 pp 123ndash137 2012
[17] X Yan A Anzai Y Katsumata et al ldquoTemporal dynamics ofcardiac immune cell accumulation following acute myocardialinfarctionrdquo Journal ofMolecular andCellular Cardiology vol 62pp 24ndash35 2013
[18] S Frantz and M Nahrendorf ldquoCardiac macrophages and theirrole in ischemic heart diseaserdquo Cardiovascular Research vol102 no 2 pp 240ndash248 2014
[19] Q Cao Y Wang D Zheng et al ldquoIL-10TGF-120573-modifiedmacrophages induce regulatory T cells and protect againstadriamycin nephrosisrdquo Journal of the American Society ofNephrology vol 21 no 6 pp 933ndash942 2010
[20] B Johannesson S Sattler E Semenova et al ldquoInsulin-likegrowth factor-1 induces regulatory T cell-mediated suppressionof allergic contact dermatitis in micerdquo Disease Models ampMechanisms vol 7 no 8 pp 977ndash985 2014
10 Mediators of Inflammation
[21] D Bilbao L Luciani B Johannesson A Piszczek and NRosenthal ldquoInsulin-like growth factor-1 stimulates regulatoryT cells and suppresses autoimmune diseaserdquo EMBO MolecularMedicine vol 6 no 11 pp 1423ndash1435 2005
[22] H Oh S B Bradfute T D Gallardo et al ldquoCardiac progenitorcells from adult myocardium homing differentiation andfusion after infarctionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 21 pp12313ndash12318 2003
[23] A Philippou E Papageorgiou G Bogdanis et al ldquoExpressionof IGF-1 isoforms after exercise-induced muscle damage inhumans characterization of theMGFEpeptide actions in vitrordquoIn Vivo vol 23 no 4 pp 567ndash576 2009
[24] S Sanz J B Pucilowska S Liu et al ldquoExpression of insulin-like growth factor I by activated hepatic stellate cells reducesfibrogenesis and enhances regeneration after liver injuryrdquo Gutvol 54 no 1 pp 134ndash141 2005
[25] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[26] F Tacke D Alvarez T J Kaplan et al ldquoMonocyte subsetsdifferentially employ CCR2 CCR5 and CX3CR1 to accumulatewithin atherosclerotic plaquesrdquo Journal of Clinical Investigationvol 117 no 1 pp 185ndash194 2007
[27] L M Carlin E G Stamatiades C Auffray et al ldquoNr4a1-dependent Ly6Clow monocytes monitor endothelial cells andorchestrate their disposalrdquoCell vol 153 no 2 pp 362ndash375 2013
[28] N V Serbina and E G Pamer ldquoMonocyte emigration frombone marrow during bacterial infection requires signals medi-ated by chemokine receptor CCR2rdquo Nature Immunology vol 7no 3 pp 311ndash317 2006
[29] S Hayashidani H Tsutsui M Ikeuchi et al ldquoTargeted deletionofMMP-2 attenuates early LV rupture and late remodeling afterexperimental myocardial infarctionrdquo The American Journal ofPhysiologymdashHeart and Circulatory Physiology vol 285 no 3pp H1229ndashH1235 2003
[30] A Ducharme S Frantz M Aikawa et al ldquoTargeted deletion ofmatrix metalloproteinase-9 attenuates left ventricular enlarge-ment and collagen accumulation after experimental myocardialinfarctionrdquo Journal of Clinical Investigation vol 106 no 1 pp55ndash62 2000
[31] K TWeber ldquoExtracellular matrix remodeling in heart failure arole for de novo angiotensin II generationrdquo Circulation vol 96no 11 pp 4065ndash4082 1997
[32] W Iraqi P Rossignol M Angioi et al ldquoExtracellular cardiacmatrix biomarkers in patients with acute myocardial infarctioncomplicated by left ventricular dysfunction and heart failureinsights from the Eplerenone Post-Acute Myocardial InfarctionHeart Failure Efficacy and Survival Study (EPHESUS) studyrdquoCirculation vol 119 no 18 pp 2471ndash2479 2009
[33] R Eschalier M Fertin R Fay et al ldquoExtracellular matrixturnover biomarkers predict long-term left ventricular remod-eling aftermyocardial infarction insights from the reve-2 studyrdquoCirculation Heart Failure vol 6 no 6 pp 1199ndash1205 2013
[34] J G Simmons J B Pucilowska T O Keku and P K LundldquoIGF-I and TGF-1205731 have distinct effects on phenotype andproliferation of intestinal fibroblastsrdquo American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 283 no3 pp G809ndashG818 2002
[35] C F Hung M G Rohani S-S Lee P Chen and L MSchnapp ldquoRole of IGF-1 pathway in lung fibroblast activationrdquoRespiratory Research vol 14 no 1 article 102 2013
[36] M Nahrendorf F K Swirski E Aikawa et al ldquoThe healingmyocardium sequentially mobilizes twomonocyte subsets withdivergent and complementary functionsrdquoThe Journal of Exper-imental Medicine vol 204 no 12 pp 3037ndash3047 2007
[37] F L Chen Z H Yang Y Liu et al ldquoBerberine inhibits theexpression of TNF120572 MCP-1 and IL-6 in AcLDL-stimulatedmacrophages through PPAR120574 pathwayrdquo Endocrine vol 33 no3 pp 331ndash337 2008
[38] J Tonkin L Temmerman R D Sampson et alldquoMonocytemacrophage-derived IGF-1 orchestrates murineskeletal muscle regeneration and modulates autocrinepolarizationrdquo Molecular Therapy vol 23 no 7 pp 1189ndash1200 2015
[39] T Heidt G Courties P Dutta et al ldquoDifferential contributionof monocytes to heart macrophages in steady-state and aftermyocardial infarctionrdquo Circulation Research vol 115 no 2 pp284ndash295 2014
[40] S Epelman K J Lavine and G J Randolph ldquoOrigin andfunctions of tissue macrophagesrdquo Immunity vol 41 no 1 pp21ndash35 2014
[41] A R Pinto J W Godwin and N A Rosenthal ldquoMacrophagesin cardiac homeostasis injury responses and progenitor cellmobilisationrdquo Stem Cell Research vol 13 no 3 pp 705ndash7142014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
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Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
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Computational and Mathematical Methods in Medicine
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 Mediators of Inflammation
We next analysed the composition of the newly synthe-sised matrix by measuring mRNA expression of the mostabundant cardiac ECM collagen Col I1205721 and Col I1205723 aswell as the collagen cross-linker lox which increases matrixstiffness Upregulation of both collagen types was detectedby day 3 peaking at day 7 (Figures 2(j) and 2(k)) At thistime point WT hearts expressed significantly more of bothcollagen types than 120572MHCIGF-1Ea hearts We also noted adifference in the ratio of the two collagen types 120572MHCIGF-1Ea hearts had a reduced Col I1205721Col I1205723 ratio compared toWT which was significant at days 3 7 and 28 (Figure 2(l)) Atthe peak of collagen upregulation lox was also upregulatedin WT hearts yet this was not observed in 120572MHCIGF-1Ea hearts (Figure 2(m)) These results indicate that IGF-1Eaoverexpression reduces ECM turnover afterMI and alters thecomposition of thematrix with preferential expression of ColI1205723 over Col I1205721 and less cross-linking which likely alters themechanical properties of the fibrotic area
33 Distinct Chemokine and Cytokine Production in 120572MHC-IGF-1Ea Hearts after Myocardial Infarct Tissue remodellingafter injury is closely tied to the inflammatory processWe therefore compared the inflammatory status of the120572MHCIGF-1Ea and WT hearts monitoring the productionof key immune genes IL-1120573 IL-10 MCP-1 and CCL5Expression of the inflammatory cytokine IL-1120573 was rapidlyinduced upon injury in WT hearts (66-fold over uninjured)yet was not upregulated as strongly in 120572MHCIGF-1Ea hearts(19-fold over uninjured Figure 3(a)) Similarly the strongupregulation of MCP-1 in WT hearts was not observed in120572MHCIGF-1Ea hearts (Figure 3(b)) IL-10 was also rapidlyupregulated peaking 1 day after MI In contrast to IL-1120573and MCP-1 this immunosuppressive cytokine was upreg-ulated 3-fold higher in 120572MHCIGF-1Ea hearts comparedto WT controls Although a second peak of IL-10 mRNAat 7 days was comparable in both WT and 120572MHCIGF-1Ea hearts 7 days after MI (Figure 3(c)) CCL5 which isinvolved in the recruitment of Ly6Clow monocytes [26 27]was upregulated in 120572MHCIGF-1Ea hearts 7 days after MI(Figure 3(d)) These data suggest an early bias towards aless inflammatory environment potentially modulating therecruitment of monocytes in 120572MHCIGF-1Ea hearts aftermyocardial infraction
34 IGF-1Ea Modulates Myeloid Cell Recruitment after Myo-cardial Infarction To document accumulation of the maininnate immune cell populations involved in cardiac inflam-mation after infarct single cell suspensions were preparedfromwholemouse hearts and analysed by flow cytometry Allcell populations described in this work were identified usingthe gating strategy shown in Supplementary Figures 3ndash6
In WT and 120572MHCIGF-1Ea hearts the total numberof infiltrating leukocytes (CD45+) gradually increased afterMI both reaching comparable peak numbers at day 5(Figure 4(a)) However at the earlier 3-day time point120572MHCIGF-1Ea hearts contained 49 less leukocytes permilligram of tissue than WT hearts In examining spe-cific immune cell populations this difference was partlyexplained by a 75 reduction in neutrophils (CD45+
CD11b+ F480minus CD11cminus and Ly6G+ 186 versus 46 cellsmgof tissue Figure 4(b)) and a 67 reduction in monocytes(CD45+ CD11b+ CD11cminus Ly6Gminus and F480minus 191 versus 62cellsmg of heart Figure 4(c)) however it was mostly due toreduced presence of macrophages (CD45+ CD11b+ CD11cminusLy6Gminus and F480+ 58 2276 versus 949 cellsmg of heartFigure 4(f)) These data agree with the reduced MCP-1expression observed in 120572MHCIGF-1Ea hearts (Figure 3(b))as it is the principal chemokine involved in monocyterecruitment [26 28]
The Ly6C surface marker distinguishes two differentsubsets ofmonocytes [2 3] Analysis of the Ly6Chigh (CD45+CD11b+ CD11cminus Ly6Gminus F480minus and Ly6Chigh) and Ly6Clow
(CD45+ CD11b+ CD11cminus Ly6Gminus F480minus and Ly6Clow)populations revealed that the reduction of total monocytenumbers at day 3 was attributable to a 20 decrease of theLy6Chigh population (102 versus 32 cellsmgof heart) whereasthe Ly6Clow population was not significantly different in WTcompared to 120572MHCIGF-1Ea (Figures 4(d) and 4(e)) hearts
To differentiate between inflammatory and reparativemacrophage populations we used the markers Ly6C andCD206 For this work only cells that were either Ly6C+CD206minus or Ly6CminusCD206+ were analysed although wenoted a double positive cell population (ie CD206+Ly6C+) However no changes were observed over timebetween WT and 120572MHCIGF-1Ea for the double positivepopulation (Supplementary 2) Both Ly6C+ inflammato-ry macrophage (CD45+ CD11b+ CD11cminus Ly6Gminus F480+and Ly6C+CD206minus) and CD206+ reparative macro-phage (CD45+ CD11b+ CD11cminus Ly6Gminus F480+ andLy6CminusCD206+) normalised cell numbers were reduced by71 and 48 respectively in 120572MHCIGF-1Ea hearts at theday 3 time point (522 versus 153 and 596 versus 310 cellsmgof tissue of heart resp Figures 4(g) and 4(h)) howeverthis was significant only for the Ly6C+ population By day 7after MI macrophage dynamics changed and we observeda greater number of total macrophages in 120572MHCIGF-1Eahearts compared to WT which was mainly due to a 155preferential increase in the CD206+ population (575 versus1468 cellsmg heart Figure 4(h)) Dendritic cells (CD45+CD11b+ F480minus CD11c+ and Ly6Gminus) were increased by48 in 120572MHCIGF-1Ea hearts compared to WT 5 days afterMI (168 versus 248 cellsmg heart Figure 4(i))
In summary cardiac-restricted expression of an IGF-1Eatransgene limited the early accumulation of innate immunecells at day 3 after MI with a bias towards the reduction ofinflammatory myeloid populations rather than regenerativepopulations This reduction corresponds with the lowerexpression of myeloid chemokines and the less inflammatorymilieu observed in the 120572MHCIGF-1Ea hearts
4 Discussion
Previous work in our lab showed that local expression of IGF-1Ea promoted functional restoration after MI and observedreduced infarct expansion thinning and dilation of the leftventricular wall [25] We now demonstrate transcriptionalmodulation of key ECM remodelling genes in the IGF-1Ea hearts associated with tempering of the inflammatorymyeloid cell response
Mediators of Inflammation 7
0
100
200
300
400
IL-1120573
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
d
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
lowast
WT120572MHCIGF-1Ea
(a)
0
100
200
300
MC
P-1
GA
PD
H f
old
in
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
(b)
0
50
100
150
200
IL-1
0G
AP
DH
fo
ldin
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
(c)
0
20
40
60
80
CC
L5
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
d
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
(d)
Figure 3 Cytokine dynamics in the infarcted 120572MHCIGF-1Ea hearts (a) Interleukin- (IL-) 1120573 (b) MCP-1 (c) IL-10 (d) CCL5 at several timepoints after permanent ligation Solid lines represent WT mice Dashed lines represent 120572MHCIGF-1Ea mice Results are expressed as meanfold induction plusmn SEM over the values of uninjured hearts 119899 = 3 per group Two-tailed Studentrsquos t-test was performed to compare WT versus120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
Increased MMP expression after injury has been impli-cated in contributing to scar destabilisation as transgenic ani-mal model knockouts of either MMP-2 or MMP-9 have beenshown to attenuate LV dilation rupture and impairment ofcardiac function [29 30] While MMP-2 and MMP-9 mRNAexpression levels were upregulated in injured WT heartsthis increase was not observed in 120572MHCIGF-1Ea hearts Inline with reduced matrix breakdown mRNA expression ofthe MMP inhibitor TIMP-2 was significantly increased in120572MHCIGF-1Ea hearts at day 7 after MI Thus IGF-1Ea mayprevent adverse cardiac remodelling in part by modulatingtranscription of MMPsTIMPs
The production of new matrix components was alsoaltered by the presence of the IGF-1Ea transgenewith an over-all reduction in collagen synthesis confirmed in histologicalstains and a bias towards expression of Col I1205723 over ColI1205721 In MI patients turnover of cardiac extracellular matrixcan be assessed by using circulating collagen peptides asblood biomarkers [31] and high type I collagen is associatedwith adverse clinical outcome [32] A prospectivemulticentrestudy further showed that a low type IIItype I collagen ratioespecially at 1 month after MI is predictive of detrimental
left ventricular remodelling as well as cardiovascular deathsand hospitalisation cases for heart failure [33] Changes incollagen ratios are known to affect the strength and tensileproperties of the ECM It would be interesting to determinewhether these properties are modified in the 120572MHCIGF-1Eahearts
IGF-1Ea could promote the changes in collagen depo-sition by directly acting on fibroblasts as it promotes boththeir proliferation and activation to myofibroblasts [3435] Alternatively IGF-1Ea may influence accumulation andactivation of immune cells present at the infarct which inturn regulate myofibroblast activation Indeed we observedmodulation of the inflammatory process in 120572MHCIGF-1Ea hearts with less monocyte (Ly6Chigh) infiltration intothe injured myocardium This effect is associated withreduced expression of themonocyte chemoattractantMCP-1although no significant changes were observed in the Ly6Clow
monocyte population in infarcted hearts possibly due toCCL5 upregulation
It is interesting that while complete depletion of mono-cytes at any stage of repair leads to poor recovery [36]a more subtle modulation of the monocyte population in
8 Mediators of Inflammation
0
2000
4000
6000
8000
Cel
lsm
g h
eart
Total leukocytes
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
(a)
0
200
400
600
800Neutrophils
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(b)
0
100
200
300Monocytes
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(c)
0
50
100
150
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
Ly-6Chigh monocytes
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(d)
0
50
100
150
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
Ly-6Clow monocytes
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(e)
0
1000
2000
3000
4000
5000Macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
lowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(f)
0
500
1000
1500
2000Ly6C+ macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(g)
0
500
1000
1500
2000CD206+ macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(h)
0
100
200
300Dendritic cells
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(i)
Figure 4 Characterisation and temporal dynamics of immune cell populations in 120572MHCIGF-1Ea hearts after MI Quantification of (a) totalleukocytes (CD45+) (b) neutrophils (CD45+ CD11b+ F480minus CD11cminus and Ly6G+) and (c) monocytes (CD45+ CD11b+ CD11cminus Ly6Gminusand F480minus) which were further classified as (d) Ly6Chigh and (e) Ly6Clow monocytes (f) Macrophages (CD45+ CD11b+ CD11cminus Ly6Gminusand F480+) were further characterised on the basis of Ly6C and CD206 expression as (g) Ly6ChighCD206low (inflammatory macrophages)and (h) Ly6ClowCD206high (reparative macrophages) For this work only cells that were either Ly6ChighCD206minus or Ly6CminusCD206+ wereanalysed although we noted a double positive cell population (ie CD206+ Ly6C+) (i) Dendritic cells were defined as CD45+ CD11b+F480minus CD11c+ and Ly6Gminus Data is presented as the total number of cells per mg of heart 119899 = 4ndash6 per group Two-tailed Studentrsquos t-testwas performed to compare WT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
the 120572MHCIGF-1Ea hearts is associated with improved heartfunction Studies abrogating monocyte recruitment usinga selective CCR2 inhibitor have resulted in reduced IL-1120573 IL-6 MCP-1 and TNF120572 levels [37] IGF-1Ea influ-ences macrophage polarisation in skeletal muscle [38]
and we similarly found reduced Ly6Chigh monocyte andLy6C+ macrophage normalised cell numbers while CD206+macrophage numbers were increased by day 7 suggestingthat IGF-1Ea promoted a quick progression to the reparativephase of repair by modulating macrophage phenotype In
Mediators of Inflammation 9
the 120572MHCIGF-1Ea hearts we observed a decrease in MCP-1which is the chemokine for CCR2 recently shown to distin-guish infiltrating monocytes from the resident macrophagepopulation which has a different embryological origin andexpresses CD206 [39ndash41] It is therefore possible that in our120572MHCIGF-1Ea transgenic mouse model IGF-1Ea reducesthe infiltration of inflammatory Ly6Chigh monocytes bypreventing upregulation of MCP-1 while still allowing forexpansion of the resident macrophage population
In summary we show that the 120572MHCIGF-1Ea mousemodel can modulate several associated aspects of the cellularrepair process after MI including immune cell recruit-ment cytokine expression and matrix turnover All of thesechanges occur within the first 7 days after infarct at whichtime a functional improvement can already be measured in120572MHCIGF-1Ea hearts compared toWT controlsThese dataprovide new insights into the mechanism of IGF-1Ea andsuggest that early immunomodulation is key to successfulcardiac repair after injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Robert D Sampson and Susanne Sattler contributed equallyto this work
Acknowledgments
The authors are grateful to members of the RosenthalLaboratory for advice and critique This work was sup-ported by grants from the European Unionrsquos Seventh Frame-work Programme Marie Curie Initial Training Network-CardioNeT GA-289600 and British Heart Foundation GrantRM13130157 to Nadia Rosenthal Robert D Sampson andMichael D Schneider are supported by the British HeartFoundation Centre of Research Excellence and British HeartFoundation Simon Marks Chair We gratefully acknowledgethe support from the British Spanish Society (BSS) for theBritish Spanish SocietySantander Universities 2015 scholar-ship that partially supported this project
References
[1] A S Go D Mozaffarian V L Roger et al ldquoHeart disease andstroke statisticsmdash2014 update a report from theAmerican heartassociationrdquo Circulation vol 129 pp e28ndashe292 2014
[2] N G Frangogiannis ldquoRegulation of the inflammatory responsein cardiac repairrdquo Circulation Research vol 110 no 1 pp 159ndash173 2012
[3] N G Frangogiannis ldquoThe inflammatory response in myocar-dial injury repair and remodellingrdquoNature Reviews Cardiologyvol 11 no 5 pp 255ndash265 2014
[4] T Shavlakadze N Winn N Rosenthal and M D GroundsldquoReconciling data from transgenic mice that overexpress IGF-I specifically in skeletal musclerdquo Growth Hormone and IGFResearch vol 15 no 1 pp 4ndash18 2005
[5] P Rotwein K M Pollock D K Didier and G G KrivildquoOrganization and sequence of the human insulin-like growthfactor I gene Alternative RNAprocessing produces two insulin-like growth factor I precursor peptidesrdquo Journal of BiologicalChemistry vol 261 no 11 pp 4828ndash4832 1986
[6] J G Simmons J J Van Wyk E C Hoyt and P K LundldquoMultiple transcription start sites in the rat insulin-like growthfactor-I gene Give rise to IGF-I mRNAs that encode differentIGF-I precursors and are processed differently in vitvordquoGrowthFactors vol 9 no 3 pp 205ndash221 1993
[7] G I Bell M M Stempien N M Fong and L B RallldquoSequences of liver cDNAs encoding two different mouseinsulin-like growth factor I precursorsrdquo Nucleic Acids Researchvol 14 no 20 pp 7873ndash7882 1986
[8] M S Hede E Salimova A Piszczek et al ldquoE-peptides controlbioavailability of IGF-1rdquo PLoS ONE vol 7 no 12 Article IDe51152 2012
[9] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[10] M Vinciguerra M P Santini W C Claycomb A G Ladurnerand N Rosenthal ldquoLocal IGF-1 isoform protects cardiomy-ocytes from hypertrophic and oxidative stresses via SirT1activityrdquo Aging vol 2 no 1 pp 43ndash62 2010
[11] D Vanhoutte M Schellings Y Pinto and S Heymans ldquoRel-evance of matrix metalloproteinases and their inhibitors aftermyocardial infarction a temporal and spatial windowrdquo Cardio-vascular Research vol 69 no 3 pp 604ndash613 2006
[12] J B Caulfield and T K Borg ldquoThe collagen network of theheartrdquo Laboratory Investigation vol 40 no 3 pp 364ndash372 1979
[13] B I Jugdutt ldquoRemodeling of the myocardium and potentialtargets in the collagen degradation and synthesis pathwaysrdquoCurrent Drug TargetsmdashCardiovascular amp Haematological Dis-orders vol 3 no 1 pp 1ndash30 2003
[14] B I Jugdutt ldquoVentricular remodeling after infarction andthe extracellular collagen matrix when is enough enoughrdquoCirculation vol 108 no 11 pp 1395ndash1403 2003
[15] M Nahrendorf and F K Swirski ldquoMonocyte and macrophageheterogeneity in the heartrdquo Circulation Research vol 112 no 12pp 1624ndash1633 2013
[16] F Leuschner P J Rauch T Ueno et al ldquoRapid mono-cyte kinetics in acute myocardial infarction are sustained byextramedullary monocytopoiesisrdquo The Journal of ExperimentalMedicine vol 209 no 1 pp 123ndash137 2012
[17] X Yan A Anzai Y Katsumata et al ldquoTemporal dynamics ofcardiac immune cell accumulation following acute myocardialinfarctionrdquo Journal ofMolecular andCellular Cardiology vol 62pp 24ndash35 2013
[18] S Frantz and M Nahrendorf ldquoCardiac macrophages and theirrole in ischemic heart diseaserdquo Cardiovascular Research vol102 no 2 pp 240ndash248 2014
[19] Q Cao Y Wang D Zheng et al ldquoIL-10TGF-120573-modifiedmacrophages induce regulatory T cells and protect againstadriamycin nephrosisrdquo Journal of the American Society ofNephrology vol 21 no 6 pp 933ndash942 2010
[20] B Johannesson S Sattler E Semenova et al ldquoInsulin-likegrowth factor-1 induces regulatory T cell-mediated suppressionof allergic contact dermatitis in micerdquo Disease Models ampMechanisms vol 7 no 8 pp 977ndash985 2014
10 Mediators of Inflammation
[21] D Bilbao L Luciani B Johannesson A Piszczek and NRosenthal ldquoInsulin-like growth factor-1 stimulates regulatoryT cells and suppresses autoimmune diseaserdquo EMBO MolecularMedicine vol 6 no 11 pp 1423ndash1435 2005
[22] H Oh S B Bradfute T D Gallardo et al ldquoCardiac progenitorcells from adult myocardium homing differentiation andfusion after infarctionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 21 pp12313ndash12318 2003
[23] A Philippou E Papageorgiou G Bogdanis et al ldquoExpressionof IGF-1 isoforms after exercise-induced muscle damage inhumans characterization of theMGFEpeptide actions in vitrordquoIn Vivo vol 23 no 4 pp 567ndash576 2009
[24] S Sanz J B Pucilowska S Liu et al ldquoExpression of insulin-like growth factor I by activated hepatic stellate cells reducesfibrogenesis and enhances regeneration after liver injuryrdquo Gutvol 54 no 1 pp 134ndash141 2005
[25] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[26] F Tacke D Alvarez T J Kaplan et al ldquoMonocyte subsetsdifferentially employ CCR2 CCR5 and CX3CR1 to accumulatewithin atherosclerotic plaquesrdquo Journal of Clinical Investigationvol 117 no 1 pp 185ndash194 2007
[27] L M Carlin E G Stamatiades C Auffray et al ldquoNr4a1-dependent Ly6Clow monocytes monitor endothelial cells andorchestrate their disposalrdquoCell vol 153 no 2 pp 362ndash375 2013
[28] N V Serbina and E G Pamer ldquoMonocyte emigration frombone marrow during bacterial infection requires signals medi-ated by chemokine receptor CCR2rdquo Nature Immunology vol 7no 3 pp 311ndash317 2006
[29] S Hayashidani H Tsutsui M Ikeuchi et al ldquoTargeted deletionofMMP-2 attenuates early LV rupture and late remodeling afterexperimental myocardial infarctionrdquo The American Journal ofPhysiologymdashHeart and Circulatory Physiology vol 285 no 3pp H1229ndashH1235 2003
[30] A Ducharme S Frantz M Aikawa et al ldquoTargeted deletion ofmatrix metalloproteinase-9 attenuates left ventricular enlarge-ment and collagen accumulation after experimental myocardialinfarctionrdquo Journal of Clinical Investigation vol 106 no 1 pp55ndash62 2000
[31] K TWeber ldquoExtracellular matrix remodeling in heart failure arole for de novo angiotensin II generationrdquo Circulation vol 96no 11 pp 4065ndash4082 1997
[32] W Iraqi P Rossignol M Angioi et al ldquoExtracellular cardiacmatrix biomarkers in patients with acute myocardial infarctioncomplicated by left ventricular dysfunction and heart failureinsights from the Eplerenone Post-Acute Myocardial InfarctionHeart Failure Efficacy and Survival Study (EPHESUS) studyrdquoCirculation vol 119 no 18 pp 2471ndash2479 2009
[33] R Eschalier M Fertin R Fay et al ldquoExtracellular matrixturnover biomarkers predict long-term left ventricular remod-eling aftermyocardial infarction insights from the reve-2 studyrdquoCirculation Heart Failure vol 6 no 6 pp 1199ndash1205 2013
[34] J G Simmons J B Pucilowska T O Keku and P K LundldquoIGF-I and TGF-1205731 have distinct effects on phenotype andproliferation of intestinal fibroblastsrdquo American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 283 no3 pp G809ndashG818 2002
[35] C F Hung M G Rohani S-S Lee P Chen and L MSchnapp ldquoRole of IGF-1 pathway in lung fibroblast activationrdquoRespiratory Research vol 14 no 1 article 102 2013
[36] M Nahrendorf F K Swirski E Aikawa et al ldquoThe healingmyocardium sequentially mobilizes twomonocyte subsets withdivergent and complementary functionsrdquoThe Journal of Exper-imental Medicine vol 204 no 12 pp 3037ndash3047 2007
[37] F L Chen Z H Yang Y Liu et al ldquoBerberine inhibits theexpression of TNF120572 MCP-1 and IL-6 in AcLDL-stimulatedmacrophages through PPAR120574 pathwayrdquo Endocrine vol 33 no3 pp 331ndash337 2008
[38] J Tonkin L Temmerman R D Sampson et alldquoMonocytemacrophage-derived IGF-1 orchestrates murineskeletal muscle regeneration and modulates autocrinepolarizationrdquo Molecular Therapy vol 23 no 7 pp 1189ndash1200 2015
[39] T Heidt G Courties P Dutta et al ldquoDifferential contributionof monocytes to heart macrophages in steady-state and aftermyocardial infarctionrdquo Circulation Research vol 115 no 2 pp284ndash295 2014
[40] S Epelman K J Lavine and G J Randolph ldquoOrigin andfunctions of tissue macrophagesrdquo Immunity vol 41 no 1 pp21ndash35 2014
[41] A R Pinto J W Godwin and N A Rosenthal ldquoMacrophagesin cardiac homeostasis injury responses and progenitor cellmobilisationrdquo Stem Cell Research vol 13 no 3 pp 705ndash7142014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Mediators of Inflammation 7
0
100
200
300
400
IL-1120573
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
d
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
lowast
WT120572MHCIGF-1Ea
(a)
0
100
200
300
MC
P-1
GA
PD
H f
old
in
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
(b)
0
50
100
150
200
IL-1
0G
AP
DH
fo
ldin
du
ctio
n o
ver
WT
un
inju
red
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
(c)
0
20
40
60
80
CC
L5
GA
PD
H f
old
ind
uct
ion
ove
r W
T u
nin
jure
d
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
(d)
Figure 3 Cytokine dynamics in the infarcted 120572MHCIGF-1Ea hearts (a) Interleukin- (IL-) 1120573 (b) MCP-1 (c) IL-10 (d) CCL5 at several timepoints after permanent ligation Solid lines represent WT mice Dashed lines represent 120572MHCIGF-1Ea mice Results are expressed as meanfold induction plusmn SEM over the values of uninjured hearts 119899 = 3 per group Two-tailed Studentrsquos t-test was performed to compare WT versus120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
Increased MMP expression after injury has been impli-cated in contributing to scar destabilisation as transgenic ani-mal model knockouts of either MMP-2 or MMP-9 have beenshown to attenuate LV dilation rupture and impairment ofcardiac function [29 30] While MMP-2 and MMP-9 mRNAexpression levels were upregulated in injured WT heartsthis increase was not observed in 120572MHCIGF-1Ea hearts Inline with reduced matrix breakdown mRNA expression ofthe MMP inhibitor TIMP-2 was significantly increased in120572MHCIGF-1Ea hearts at day 7 after MI Thus IGF-1Ea mayprevent adverse cardiac remodelling in part by modulatingtranscription of MMPsTIMPs
The production of new matrix components was alsoaltered by the presence of the IGF-1Ea transgenewith an over-all reduction in collagen synthesis confirmed in histologicalstains and a bias towards expression of Col I1205723 over ColI1205721 In MI patients turnover of cardiac extracellular matrixcan be assessed by using circulating collagen peptides asblood biomarkers [31] and high type I collagen is associatedwith adverse clinical outcome [32] A prospectivemulticentrestudy further showed that a low type IIItype I collagen ratioespecially at 1 month after MI is predictive of detrimental
left ventricular remodelling as well as cardiovascular deathsand hospitalisation cases for heart failure [33] Changes incollagen ratios are known to affect the strength and tensileproperties of the ECM It would be interesting to determinewhether these properties are modified in the 120572MHCIGF-1Eahearts
IGF-1Ea could promote the changes in collagen depo-sition by directly acting on fibroblasts as it promotes boththeir proliferation and activation to myofibroblasts [3435] Alternatively IGF-1Ea may influence accumulation andactivation of immune cells present at the infarct which inturn regulate myofibroblast activation Indeed we observedmodulation of the inflammatory process in 120572MHCIGF-1Ea hearts with less monocyte (Ly6Chigh) infiltration intothe injured myocardium This effect is associated withreduced expression of themonocyte chemoattractantMCP-1although no significant changes were observed in the Ly6Clow
monocyte population in infarcted hearts possibly due toCCL5 upregulation
It is interesting that while complete depletion of mono-cytes at any stage of repair leads to poor recovery [36]a more subtle modulation of the monocyte population in
8 Mediators of Inflammation
0
2000
4000
6000
8000
Cel
lsm
g h
eart
Total leukocytes
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
(a)
0
200
400
600
800Neutrophils
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(b)
0
100
200
300Monocytes
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(c)
0
50
100
150
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
Ly-6Chigh monocytes
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(d)
0
50
100
150
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
Ly-6Clow monocytes
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(e)
0
1000
2000
3000
4000
5000Macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
lowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(f)
0
500
1000
1500
2000Ly6C+ macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(g)
0
500
1000
1500
2000CD206+ macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(h)
0
100
200
300Dendritic cells
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(i)
Figure 4 Characterisation and temporal dynamics of immune cell populations in 120572MHCIGF-1Ea hearts after MI Quantification of (a) totalleukocytes (CD45+) (b) neutrophils (CD45+ CD11b+ F480minus CD11cminus and Ly6G+) and (c) monocytes (CD45+ CD11b+ CD11cminus Ly6Gminusand F480minus) which were further classified as (d) Ly6Chigh and (e) Ly6Clow monocytes (f) Macrophages (CD45+ CD11b+ CD11cminus Ly6Gminusand F480+) were further characterised on the basis of Ly6C and CD206 expression as (g) Ly6ChighCD206low (inflammatory macrophages)and (h) Ly6ClowCD206high (reparative macrophages) For this work only cells that were either Ly6ChighCD206minus or Ly6CminusCD206+ wereanalysed although we noted a double positive cell population (ie CD206+ Ly6C+) (i) Dendritic cells were defined as CD45+ CD11b+F480minus CD11c+ and Ly6Gminus Data is presented as the total number of cells per mg of heart 119899 = 4ndash6 per group Two-tailed Studentrsquos t-testwas performed to compare WT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
the 120572MHCIGF-1Ea hearts is associated with improved heartfunction Studies abrogating monocyte recruitment usinga selective CCR2 inhibitor have resulted in reduced IL-1120573 IL-6 MCP-1 and TNF120572 levels [37] IGF-1Ea influ-ences macrophage polarisation in skeletal muscle [38]
and we similarly found reduced Ly6Chigh monocyte andLy6C+ macrophage normalised cell numbers while CD206+macrophage numbers were increased by day 7 suggestingthat IGF-1Ea promoted a quick progression to the reparativephase of repair by modulating macrophage phenotype In
Mediators of Inflammation 9
the 120572MHCIGF-1Ea hearts we observed a decrease in MCP-1which is the chemokine for CCR2 recently shown to distin-guish infiltrating monocytes from the resident macrophagepopulation which has a different embryological origin andexpresses CD206 [39ndash41] It is therefore possible that in our120572MHCIGF-1Ea transgenic mouse model IGF-1Ea reducesthe infiltration of inflammatory Ly6Chigh monocytes bypreventing upregulation of MCP-1 while still allowing forexpansion of the resident macrophage population
In summary we show that the 120572MHCIGF-1Ea mousemodel can modulate several associated aspects of the cellularrepair process after MI including immune cell recruit-ment cytokine expression and matrix turnover All of thesechanges occur within the first 7 days after infarct at whichtime a functional improvement can already be measured in120572MHCIGF-1Ea hearts compared toWT controlsThese dataprovide new insights into the mechanism of IGF-1Ea andsuggest that early immunomodulation is key to successfulcardiac repair after injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Robert D Sampson and Susanne Sattler contributed equallyto this work
Acknowledgments
The authors are grateful to members of the RosenthalLaboratory for advice and critique This work was sup-ported by grants from the European Unionrsquos Seventh Frame-work Programme Marie Curie Initial Training Network-CardioNeT GA-289600 and British Heart Foundation GrantRM13130157 to Nadia Rosenthal Robert D Sampson andMichael D Schneider are supported by the British HeartFoundation Centre of Research Excellence and British HeartFoundation Simon Marks Chair We gratefully acknowledgethe support from the British Spanish Society (BSS) for theBritish Spanish SocietySantander Universities 2015 scholar-ship that partially supported this project
References
[1] A S Go D Mozaffarian V L Roger et al ldquoHeart disease andstroke statisticsmdash2014 update a report from theAmerican heartassociationrdquo Circulation vol 129 pp e28ndashe292 2014
[2] N G Frangogiannis ldquoRegulation of the inflammatory responsein cardiac repairrdquo Circulation Research vol 110 no 1 pp 159ndash173 2012
[3] N G Frangogiannis ldquoThe inflammatory response in myocar-dial injury repair and remodellingrdquoNature Reviews Cardiologyvol 11 no 5 pp 255ndash265 2014
[4] T Shavlakadze N Winn N Rosenthal and M D GroundsldquoReconciling data from transgenic mice that overexpress IGF-I specifically in skeletal musclerdquo Growth Hormone and IGFResearch vol 15 no 1 pp 4ndash18 2005
[5] P Rotwein K M Pollock D K Didier and G G KrivildquoOrganization and sequence of the human insulin-like growthfactor I gene Alternative RNAprocessing produces two insulin-like growth factor I precursor peptidesrdquo Journal of BiologicalChemistry vol 261 no 11 pp 4828ndash4832 1986
[6] J G Simmons J J Van Wyk E C Hoyt and P K LundldquoMultiple transcription start sites in the rat insulin-like growthfactor-I gene Give rise to IGF-I mRNAs that encode differentIGF-I precursors and are processed differently in vitvordquoGrowthFactors vol 9 no 3 pp 205ndash221 1993
[7] G I Bell M M Stempien N M Fong and L B RallldquoSequences of liver cDNAs encoding two different mouseinsulin-like growth factor I precursorsrdquo Nucleic Acids Researchvol 14 no 20 pp 7873ndash7882 1986
[8] M S Hede E Salimova A Piszczek et al ldquoE-peptides controlbioavailability of IGF-1rdquo PLoS ONE vol 7 no 12 Article IDe51152 2012
[9] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[10] M Vinciguerra M P Santini W C Claycomb A G Ladurnerand N Rosenthal ldquoLocal IGF-1 isoform protects cardiomy-ocytes from hypertrophic and oxidative stresses via SirT1activityrdquo Aging vol 2 no 1 pp 43ndash62 2010
[11] D Vanhoutte M Schellings Y Pinto and S Heymans ldquoRel-evance of matrix metalloproteinases and their inhibitors aftermyocardial infarction a temporal and spatial windowrdquo Cardio-vascular Research vol 69 no 3 pp 604ndash613 2006
[12] J B Caulfield and T K Borg ldquoThe collagen network of theheartrdquo Laboratory Investigation vol 40 no 3 pp 364ndash372 1979
[13] B I Jugdutt ldquoRemodeling of the myocardium and potentialtargets in the collagen degradation and synthesis pathwaysrdquoCurrent Drug TargetsmdashCardiovascular amp Haematological Dis-orders vol 3 no 1 pp 1ndash30 2003
[14] B I Jugdutt ldquoVentricular remodeling after infarction andthe extracellular collagen matrix when is enough enoughrdquoCirculation vol 108 no 11 pp 1395ndash1403 2003
[15] M Nahrendorf and F K Swirski ldquoMonocyte and macrophageheterogeneity in the heartrdquo Circulation Research vol 112 no 12pp 1624ndash1633 2013
[16] F Leuschner P J Rauch T Ueno et al ldquoRapid mono-cyte kinetics in acute myocardial infarction are sustained byextramedullary monocytopoiesisrdquo The Journal of ExperimentalMedicine vol 209 no 1 pp 123ndash137 2012
[17] X Yan A Anzai Y Katsumata et al ldquoTemporal dynamics ofcardiac immune cell accumulation following acute myocardialinfarctionrdquo Journal ofMolecular andCellular Cardiology vol 62pp 24ndash35 2013
[18] S Frantz and M Nahrendorf ldquoCardiac macrophages and theirrole in ischemic heart diseaserdquo Cardiovascular Research vol102 no 2 pp 240ndash248 2014
[19] Q Cao Y Wang D Zheng et al ldquoIL-10TGF-120573-modifiedmacrophages induce regulatory T cells and protect againstadriamycin nephrosisrdquo Journal of the American Society ofNephrology vol 21 no 6 pp 933ndash942 2010
[20] B Johannesson S Sattler E Semenova et al ldquoInsulin-likegrowth factor-1 induces regulatory T cell-mediated suppressionof allergic contact dermatitis in micerdquo Disease Models ampMechanisms vol 7 no 8 pp 977ndash985 2014
10 Mediators of Inflammation
[21] D Bilbao L Luciani B Johannesson A Piszczek and NRosenthal ldquoInsulin-like growth factor-1 stimulates regulatoryT cells and suppresses autoimmune diseaserdquo EMBO MolecularMedicine vol 6 no 11 pp 1423ndash1435 2005
[22] H Oh S B Bradfute T D Gallardo et al ldquoCardiac progenitorcells from adult myocardium homing differentiation andfusion after infarctionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 21 pp12313ndash12318 2003
[23] A Philippou E Papageorgiou G Bogdanis et al ldquoExpressionof IGF-1 isoforms after exercise-induced muscle damage inhumans characterization of theMGFEpeptide actions in vitrordquoIn Vivo vol 23 no 4 pp 567ndash576 2009
[24] S Sanz J B Pucilowska S Liu et al ldquoExpression of insulin-like growth factor I by activated hepatic stellate cells reducesfibrogenesis and enhances regeneration after liver injuryrdquo Gutvol 54 no 1 pp 134ndash141 2005
[25] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[26] F Tacke D Alvarez T J Kaplan et al ldquoMonocyte subsetsdifferentially employ CCR2 CCR5 and CX3CR1 to accumulatewithin atherosclerotic plaquesrdquo Journal of Clinical Investigationvol 117 no 1 pp 185ndash194 2007
[27] L M Carlin E G Stamatiades C Auffray et al ldquoNr4a1-dependent Ly6Clow monocytes monitor endothelial cells andorchestrate their disposalrdquoCell vol 153 no 2 pp 362ndash375 2013
[28] N V Serbina and E G Pamer ldquoMonocyte emigration frombone marrow during bacterial infection requires signals medi-ated by chemokine receptor CCR2rdquo Nature Immunology vol 7no 3 pp 311ndash317 2006
[29] S Hayashidani H Tsutsui M Ikeuchi et al ldquoTargeted deletionofMMP-2 attenuates early LV rupture and late remodeling afterexperimental myocardial infarctionrdquo The American Journal ofPhysiologymdashHeart and Circulatory Physiology vol 285 no 3pp H1229ndashH1235 2003
[30] A Ducharme S Frantz M Aikawa et al ldquoTargeted deletion ofmatrix metalloproteinase-9 attenuates left ventricular enlarge-ment and collagen accumulation after experimental myocardialinfarctionrdquo Journal of Clinical Investigation vol 106 no 1 pp55ndash62 2000
[31] K TWeber ldquoExtracellular matrix remodeling in heart failure arole for de novo angiotensin II generationrdquo Circulation vol 96no 11 pp 4065ndash4082 1997
[32] W Iraqi P Rossignol M Angioi et al ldquoExtracellular cardiacmatrix biomarkers in patients with acute myocardial infarctioncomplicated by left ventricular dysfunction and heart failureinsights from the Eplerenone Post-Acute Myocardial InfarctionHeart Failure Efficacy and Survival Study (EPHESUS) studyrdquoCirculation vol 119 no 18 pp 2471ndash2479 2009
[33] R Eschalier M Fertin R Fay et al ldquoExtracellular matrixturnover biomarkers predict long-term left ventricular remod-eling aftermyocardial infarction insights from the reve-2 studyrdquoCirculation Heart Failure vol 6 no 6 pp 1199ndash1205 2013
[34] J G Simmons J B Pucilowska T O Keku and P K LundldquoIGF-I and TGF-1205731 have distinct effects on phenotype andproliferation of intestinal fibroblastsrdquo American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 283 no3 pp G809ndashG818 2002
[35] C F Hung M G Rohani S-S Lee P Chen and L MSchnapp ldquoRole of IGF-1 pathway in lung fibroblast activationrdquoRespiratory Research vol 14 no 1 article 102 2013
[36] M Nahrendorf F K Swirski E Aikawa et al ldquoThe healingmyocardium sequentially mobilizes twomonocyte subsets withdivergent and complementary functionsrdquoThe Journal of Exper-imental Medicine vol 204 no 12 pp 3037ndash3047 2007
[37] F L Chen Z H Yang Y Liu et al ldquoBerberine inhibits theexpression of TNF120572 MCP-1 and IL-6 in AcLDL-stimulatedmacrophages through PPAR120574 pathwayrdquo Endocrine vol 33 no3 pp 331ndash337 2008
[38] J Tonkin L Temmerman R D Sampson et alldquoMonocytemacrophage-derived IGF-1 orchestrates murineskeletal muscle regeneration and modulates autocrinepolarizationrdquo Molecular Therapy vol 23 no 7 pp 1189ndash1200 2015
[39] T Heidt G Courties P Dutta et al ldquoDifferential contributionof monocytes to heart macrophages in steady-state and aftermyocardial infarctionrdquo Circulation Research vol 115 no 2 pp284ndash295 2014
[40] S Epelman K J Lavine and G J Randolph ldquoOrigin andfunctions of tissue macrophagesrdquo Immunity vol 41 no 1 pp21ndash35 2014
[41] A R Pinto J W Godwin and N A Rosenthal ldquoMacrophagesin cardiac homeostasis injury responses and progenitor cellmobilisationrdquo Stem Cell Research vol 13 no 3 pp 705ndash7142014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 Mediators of Inflammation
0
2000
4000
6000
8000
Cel
lsm
g h
eart
Total leukocytes
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
(a)
0
200
400
600
800Neutrophils
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(b)
0
100
200
300Monocytes
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(c)
0
50
100
150
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
Ly-6Chigh monocytes
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(d)
0
50
100
150
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
Ly-6Clow monocytes
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(e)
0
1000
2000
3000
4000
5000Macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
lowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(f)
0
500
1000
1500
2000Ly6C+ macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(g)
0
500
1000
1500
2000CD206+ macrophages
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowastlowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(h)
0
100
200
300Dendritic cells
Un
inju
red
Day
1
Day
3
Day
5
Day
7
Day
28
lowast
WT120572MHCIGF-1Ea
Cel
lsm
g h
eart
(i)
Figure 4 Characterisation and temporal dynamics of immune cell populations in 120572MHCIGF-1Ea hearts after MI Quantification of (a) totalleukocytes (CD45+) (b) neutrophils (CD45+ CD11b+ F480minus CD11cminus and Ly6G+) and (c) monocytes (CD45+ CD11b+ CD11cminus Ly6Gminusand F480minus) which were further classified as (d) Ly6Chigh and (e) Ly6Clow monocytes (f) Macrophages (CD45+ CD11b+ CD11cminus Ly6Gminusand F480+) were further characterised on the basis of Ly6C and CD206 expression as (g) Ly6ChighCD206low (inflammatory macrophages)and (h) Ly6ClowCD206high (reparative macrophages) For this work only cells that were either Ly6ChighCD206minus or Ly6CminusCD206+ wereanalysed although we noted a double positive cell population (ie CD206+ Ly6C+) (i) Dendritic cells were defined as CD45+ CD11b+F480minus CD11c+ and Ly6Gminus Data is presented as the total number of cells per mg of heart 119899 = 4ndash6 per group Two-tailed Studentrsquos t-testwas performed to compare WT versus 120572MHCIGF-1Ea at selected time points after MI lowast119875 lt 005 lowastlowast119875 lt 0005
the 120572MHCIGF-1Ea hearts is associated with improved heartfunction Studies abrogating monocyte recruitment usinga selective CCR2 inhibitor have resulted in reduced IL-1120573 IL-6 MCP-1 and TNF120572 levels [37] IGF-1Ea influ-ences macrophage polarisation in skeletal muscle [38]
and we similarly found reduced Ly6Chigh monocyte andLy6C+ macrophage normalised cell numbers while CD206+macrophage numbers were increased by day 7 suggestingthat IGF-1Ea promoted a quick progression to the reparativephase of repair by modulating macrophage phenotype In
Mediators of Inflammation 9
the 120572MHCIGF-1Ea hearts we observed a decrease in MCP-1which is the chemokine for CCR2 recently shown to distin-guish infiltrating monocytes from the resident macrophagepopulation which has a different embryological origin andexpresses CD206 [39ndash41] It is therefore possible that in our120572MHCIGF-1Ea transgenic mouse model IGF-1Ea reducesthe infiltration of inflammatory Ly6Chigh monocytes bypreventing upregulation of MCP-1 while still allowing forexpansion of the resident macrophage population
In summary we show that the 120572MHCIGF-1Ea mousemodel can modulate several associated aspects of the cellularrepair process after MI including immune cell recruit-ment cytokine expression and matrix turnover All of thesechanges occur within the first 7 days after infarct at whichtime a functional improvement can already be measured in120572MHCIGF-1Ea hearts compared toWT controlsThese dataprovide new insights into the mechanism of IGF-1Ea andsuggest that early immunomodulation is key to successfulcardiac repair after injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Robert D Sampson and Susanne Sattler contributed equallyto this work
Acknowledgments
The authors are grateful to members of the RosenthalLaboratory for advice and critique This work was sup-ported by grants from the European Unionrsquos Seventh Frame-work Programme Marie Curie Initial Training Network-CardioNeT GA-289600 and British Heart Foundation GrantRM13130157 to Nadia Rosenthal Robert D Sampson andMichael D Schneider are supported by the British HeartFoundation Centre of Research Excellence and British HeartFoundation Simon Marks Chair We gratefully acknowledgethe support from the British Spanish Society (BSS) for theBritish Spanish SocietySantander Universities 2015 scholar-ship that partially supported this project
References
[1] A S Go D Mozaffarian V L Roger et al ldquoHeart disease andstroke statisticsmdash2014 update a report from theAmerican heartassociationrdquo Circulation vol 129 pp e28ndashe292 2014
[2] N G Frangogiannis ldquoRegulation of the inflammatory responsein cardiac repairrdquo Circulation Research vol 110 no 1 pp 159ndash173 2012
[3] N G Frangogiannis ldquoThe inflammatory response in myocar-dial injury repair and remodellingrdquoNature Reviews Cardiologyvol 11 no 5 pp 255ndash265 2014
[4] T Shavlakadze N Winn N Rosenthal and M D GroundsldquoReconciling data from transgenic mice that overexpress IGF-I specifically in skeletal musclerdquo Growth Hormone and IGFResearch vol 15 no 1 pp 4ndash18 2005
[5] P Rotwein K M Pollock D K Didier and G G KrivildquoOrganization and sequence of the human insulin-like growthfactor I gene Alternative RNAprocessing produces two insulin-like growth factor I precursor peptidesrdquo Journal of BiologicalChemistry vol 261 no 11 pp 4828ndash4832 1986
[6] J G Simmons J J Van Wyk E C Hoyt and P K LundldquoMultiple transcription start sites in the rat insulin-like growthfactor-I gene Give rise to IGF-I mRNAs that encode differentIGF-I precursors and are processed differently in vitvordquoGrowthFactors vol 9 no 3 pp 205ndash221 1993
[7] G I Bell M M Stempien N M Fong and L B RallldquoSequences of liver cDNAs encoding two different mouseinsulin-like growth factor I precursorsrdquo Nucleic Acids Researchvol 14 no 20 pp 7873ndash7882 1986
[8] M S Hede E Salimova A Piszczek et al ldquoE-peptides controlbioavailability of IGF-1rdquo PLoS ONE vol 7 no 12 Article IDe51152 2012
[9] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[10] M Vinciguerra M P Santini W C Claycomb A G Ladurnerand N Rosenthal ldquoLocal IGF-1 isoform protects cardiomy-ocytes from hypertrophic and oxidative stresses via SirT1activityrdquo Aging vol 2 no 1 pp 43ndash62 2010
[11] D Vanhoutte M Schellings Y Pinto and S Heymans ldquoRel-evance of matrix metalloproteinases and their inhibitors aftermyocardial infarction a temporal and spatial windowrdquo Cardio-vascular Research vol 69 no 3 pp 604ndash613 2006
[12] J B Caulfield and T K Borg ldquoThe collagen network of theheartrdquo Laboratory Investigation vol 40 no 3 pp 364ndash372 1979
[13] B I Jugdutt ldquoRemodeling of the myocardium and potentialtargets in the collagen degradation and synthesis pathwaysrdquoCurrent Drug TargetsmdashCardiovascular amp Haematological Dis-orders vol 3 no 1 pp 1ndash30 2003
[14] B I Jugdutt ldquoVentricular remodeling after infarction andthe extracellular collagen matrix when is enough enoughrdquoCirculation vol 108 no 11 pp 1395ndash1403 2003
[15] M Nahrendorf and F K Swirski ldquoMonocyte and macrophageheterogeneity in the heartrdquo Circulation Research vol 112 no 12pp 1624ndash1633 2013
[16] F Leuschner P J Rauch T Ueno et al ldquoRapid mono-cyte kinetics in acute myocardial infarction are sustained byextramedullary monocytopoiesisrdquo The Journal of ExperimentalMedicine vol 209 no 1 pp 123ndash137 2012
[17] X Yan A Anzai Y Katsumata et al ldquoTemporal dynamics ofcardiac immune cell accumulation following acute myocardialinfarctionrdquo Journal ofMolecular andCellular Cardiology vol 62pp 24ndash35 2013
[18] S Frantz and M Nahrendorf ldquoCardiac macrophages and theirrole in ischemic heart diseaserdquo Cardiovascular Research vol102 no 2 pp 240ndash248 2014
[19] Q Cao Y Wang D Zheng et al ldquoIL-10TGF-120573-modifiedmacrophages induce regulatory T cells and protect againstadriamycin nephrosisrdquo Journal of the American Society ofNephrology vol 21 no 6 pp 933ndash942 2010
[20] B Johannesson S Sattler E Semenova et al ldquoInsulin-likegrowth factor-1 induces regulatory T cell-mediated suppressionof allergic contact dermatitis in micerdquo Disease Models ampMechanisms vol 7 no 8 pp 977ndash985 2014
10 Mediators of Inflammation
[21] D Bilbao L Luciani B Johannesson A Piszczek and NRosenthal ldquoInsulin-like growth factor-1 stimulates regulatoryT cells and suppresses autoimmune diseaserdquo EMBO MolecularMedicine vol 6 no 11 pp 1423ndash1435 2005
[22] H Oh S B Bradfute T D Gallardo et al ldquoCardiac progenitorcells from adult myocardium homing differentiation andfusion after infarctionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 21 pp12313ndash12318 2003
[23] A Philippou E Papageorgiou G Bogdanis et al ldquoExpressionof IGF-1 isoforms after exercise-induced muscle damage inhumans characterization of theMGFEpeptide actions in vitrordquoIn Vivo vol 23 no 4 pp 567ndash576 2009
[24] S Sanz J B Pucilowska S Liu et al ldquoExpression of insulin-like growth factor I by activated hepatic stellate cells reducesfibrogenesis and enhances regeneration after liver injuryrdquo Gutvol 54 no 1 pp 134ndash141 2005
[25] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[26] F Tacke D Alvarez T J Kaplan et al ldquoMonocyte subsetsdifferentially employ CCR2 CCR5 and CX3CR1 to accumulatewithin atherosclerotic plaquesrdquo Journal of Clinical Investigationvol 117 no 1 pp 185ndash194 2007
[27] L M Carlin E G Stamatiades C Auffray et al ldquoNr4a1-dependent Ly6Clow monocytes monitor endothelial cells andorchestrate their disposalrdquoCell vol 153 no 2 pp 362ndash375 2013
[28] N V Serbina and E G Pamer ldquoMonocyte emigration frombone marrow during bacterial infection requires signals medi-ated by chemokine receptor CCR2rdquo Nature Immunology vol 7no 3 pp 311ndash317 2006
[29] S Hayashidani H Tsutsui M Ikeuchi et al ldquoTargeted deletionofMMP-2 attenuates early LV rupture and late remodeling afterexperimental myocardial infarctionrdquo The American Journal ofPhysiologymdashHeart and Circulatory Physiology vol 285 no 3pp H1229ndashH1235 2003
[30] A Ducharme S Frantz M Aikawa et al ldquoTargeted deletion ofmatrix metalloproteinase-9 attenuates left ventricular enlarge-ment and collagen accumulation after experimental myocardialinfarctionrdquo Journal of Clinical Investigation vol 106 no 1 pp55ndash62 2000
[31] K TWeber ldquoExtracellular matrix remodeling in heart failure arole for de novo angiotensin II generationrdquo Circulation vol 96no 11 pp 4065ndash4082 1997
[32] W Iraqi P Rossignol M Angioi et al ldquoExtracellular cardiacmatrix biomarkers in patients with acute myocardial infarctioncomplicated by left ventricular dysfunction and heart failureinsights from the Eplerenone Post-Acute Myocardial InfarctionHeart Failure Efficacy and Survival Study (EPHESUS) studyrdquoCirculation vol 119 no 18 pp 2471ndash2479 2009
[33] R Eschalier M Fertin R Fay et al ldquoExtracellular matrixturnover biomarkers predict long-term left ventricular remod-eling aftermyocardial infarction insights from the reve-2 studyrdquoCirculation Heart Failure vol 6 no 6 pp 1199ndash1205 2013
[34] J G Simmons J B Pucilowska T O Keku and P K LundldquoIGF-I and TGF-1205731 have distinct effects on phenotype andproliferation of intestinal fibroblastsrdquo American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 283 no3 pp G809ndashG818 2002
[35] C F Hung M G Rohani S-S Lee P Chen and L MSchnapp ldquoRole of IGF-1 pathway in lung fibroblast activationrdquoRespiratory Research vol 14 no 1 article 102 2013
[36] M Nahrendorf F K Swirski E Aikawa et al ldquoThe healingmyocardium sequentially mobilizes twomonocyte subsets withdivergent and complementary functionsrdquoThe Journal of Exper-imental Medicine vol 204 no 12 pp 3037ndash3047 2007
[37] F L Chen Z H Yang Y Liu et al ldquoBerberine inhibits theexpression of TNF120572 MCP-1 and IL-6 in AcLDL-stimulatedmacrophages through PPAR120574 pathwayrdquo Endocrine vol 33 no3 pp 331ndash337 2008
[38] J Tonkin L Temmerman R D Sampson et alldquoMonocytemacrophage-derived IGF-1 orchestrates murineskeletal muscle regeneration and modulates autocrinepolarizationrdquo Molecular Therapy vol 23 no 7 pp 1189ndash1200 2015
[39] T Heidt G Courties P Dutta et al ldquoDifferential contributionof monocytes to heart macrophages in steady-state and aftermyocardial infarctionrdquo Circulation Research vol 115 no 2 pp284ndash295 2014
[40] S Epelman K J Lavine and G J Randolph ldquoOrigin andfunctions of tissue macrophagesrdquo Immunity vol 41 no 1 pp21ndash35 2014
[41] A R Pinto J W Godwin and N A Rosenthal ldquoMacrophagesin cardiac homeostasis injury responses and progenitor cellmobilisationrdquo Stem Cell Research vol 13 no 3 pp 705ndash7142014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Mediators of Inflammation 9
the 120572MHCIGF-1Ea hearts we observed a decrease in MCP-1which is the chemokine for CCR2 recently shown to distin-guish infiltrating monocytes from the resident macrophagepopulation which has a different embryological origin andexpresses CD206 [39ndash41] It is therefore possible that in our120572MHCIGF-1Ea transgenic mouse model IGF-1Ea reducesthe infiltration of inflammatory Ly6Chigh monocytes bypreventing upregulation of MCP-1 while still allowing forexpansion of the resident macrophage population
In summary we show that the 120572MHCIGF-1Ea mousemodel can modulate several associated aspects of the cellularrepair process after MI including immune cell recruit-ment cytokine expression and matrix turnover All of thesechanges occur within the first 7 days after infarct at whichtime a functional improvement can already be measured in120572MHCIGF-1Ea hearts compared toWT controlsThese dataprovide new insights into the mechanism of IGF-1Ea andsuggest that early immunomodulation is key to successfulcardiac repair after injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Robert D Sampson and Susanne Sattler contributed equallyto this work
Acknowledgments
The authors are grateful to members of the RosenthalLaboratory for advice and critique This work was sup-ported by grants from the European Unionrsquos Seventh Frame-work Programme Marie Curie Initial Training Network-CardioNeT GA-289600 and British Heart Foundation GrantRM13130157 to Nadia Rosenthal Robert D Sampson andMichael D Schneider are supported by the British HeartFoundation Centre of Research Excellence and British HeartFoundation Simon Marks Chair We gratefully acknowledgethe support from the British Spanish Society (BSS) for theBritish Spanish SocietySantander Universities 2015 scholar-ship that partially supported this project
References
[1] A S Go D Mozaffarian V L Roger et al ldquoHeart disease andstroke statisticsmdash2014 update a report from theAmerican heartassociationrdquo Circulation vol 129 pp e28ndashe292 2014
[2] N G Frangogiannis ldquoRegulation of the inflammatory responsein cardiac repairrdquo Circulation Research vol 110 no 1 pp 159ndash173 2012
[3] N G Frangogiannis ldquoThe inflammatory response in myocar-dial injury repair and remodellingrdquoNature Reviews Cardiologyvol 11 no 5 pp 255ndash265 2014
[4] T Shavlakadze N Winn N Rosenthal and M D GroundsldquoReconciling data from transgenic mice that overexpress IGF-I specifically in skeletal musclerdquo Growth Hormone and IGFResearch vol 15 no 1 pp 4ndash18 2005
[5] P Rotwein K M Pollock D K Didier and G G KrivildquoOrganization and sequence of the human insulin-like growthfactor I gene Alternative RNAprocessing produces two insulin-like growth factor I precursor peptidesrdquo Journal of BiologicalChemistry vol 261 no 11 pp 4828ndash4832 1986
[6] J G Simmons J J Van Wyk E C Hoyt and P K LundldquoMultiple transcription start sites in the rat insulin-like growthfactor-I gene Give rise to IGF-I mRNAs that encode differentIGF-I precursors and are processed differently in vitvordquoGrowthFactors vol 9 no 3 pp 205ndash221 1993
[7] G I Bell M M Stempien N M Fong and L B RallldquoSequences of liver cDNAs encoding two different mouseinsulin-like growth factor I precursorsrdquo Nucleic Acids Researchvol 14 no 20 pp 7873ndash7882 1986
[8] M S Hede E Salimova A Piszczek et al ldquoE-peptides controlbioavailability of IGF-1rdquo PLoS ONE vol 7 no 12 Article IDe51152 2012
[9] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[10] M Vinciguerra M P Santini W C Claycomb A G Ladurnerand N Rosenthal ldquoLocal IGF-1 isoform protects cardiomy-ocytes from hypertrophic and oxidative stresses via SirT1activityrdquo Aging vol 2 no 1 pp 43ndash62 2010
[11] D Vanhoutte M Schellings Y Pinto and S Heymans ldquoRel-evance of matrix metalloproteinases and their inhibitors aftermyocardial infarction a temporal and spatial windowrdquo Cardio-vascular Research vol 69 no 3 pp 604ndash613 2006
[12] J B Caulfield and T K Borg ldquoThe collagen network of theheartrdquo Laboratory Investigation vol 40 no 3 pp 364ndash372 1979
[13] B I Jugdutt ldquoRemodeling of the myocardium and potentialtargets in the collagen degradation and synthesis pathwaysrdquoCurrent Drug TargetsmdashCardiovascular amp Haematological Dis-orders vol 3 no 1 pp 1ndash30 2003
[14] B I Jugdutt ldquoVentricular remodeling after infarction andthe extracellular collagen matrix when is enough enoughrdquoCirculation vol 108 no 11 pp 1395ndash1403 2003
[15] M Nahrendorf and F K Swirski ldquoMonocyte and macrophageheterogeneity in the heartrdquo Circulation Research vol 112 no 12pp 1624ndash1633 2013
[16] F Leuschner P J Rauch T Ueno et al ldquoRapid mono-cyte kinetics in acute myocardial infarction are sustained byextramedullary monocytopoiesisrdquo The Journal of ExperimentalMedicine vol 209 no 1 pp 123ndash137 2012
[17] X Yan A Anzai Y Katsumata et al ldquoTemporal dynamics ofcardiac immune cell accumulation following acute myocardialinfarctionrdquo Journal ofMolecular andCellular Cardiology vol 62pp 24ndash35 2013
[18] S Frantz and M Nahrendorf ldquoCardiac macrophages and theirrole in ischemic heart diseaserdquo Cardiovascular Research vol102 no 2 pp 240ndash248 2014
[19] Q Cao Y Wang D Zheng et al ldquoIL-10TGF-120573-modifiedmacrophages induce regulatory T cells and protect againstadriamycin nephrosisrdquo Journal of the American Society ofNephrology vol 21 no 6 pp 933ndash942 2010
[20] B Johannesson S Sattler E Semenova et al ldquoInsulin-likegrowth factor-1 induces regulatory T cell-mediated suppressionof allergic contact dermatitis in micerdquo Disease Models ampMechanisms vol 7 no 8 pp 977ndash985 2014
10 Mediators of Inflammation
[21] D Bilbao L Luciani B Johannesson A Piszczek and NRosenthal ldquoInsulin-like growth factor-1 stimulates regulatoryT cells and suppresses autoimmune diseaserdquo EMBO MolecularMedicine vol 6 no 11 pp 1423ndash1435 2005
[22] H Oh S B Bradfute T D Gallardo et al ldquoCardiac progenitorcells from adult myocardium homing differentiation andfusion after infarctionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 21 pp12313ndash12318 2003
[23] A Philippou E Papageorgiou G Bogdanis et al ldquoExpressionof IGF-1 isoforms after exercise-induced muscle damage inhumans characterization of theMGFEpeptide actions in vitrordquoIn Vivo vol 23 no 4 pp 567ndash576 2009
[24] S Sanz J B Pucilowska S Liu et al ldquoExpression of insulin-like growth factor I by activated hepatic stellate cells reducesfibrogenesis and enhances regeneration after liver injuryrdquo Gutvol 54 no 1 pp 134ndash141 2005
[25] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[26] F Tacke D Alvarez T J Kaplan et al ldquoMonocyte subsetsdifferentially employ CCR2 CCR5 and CX3CR1 to accumulatewithin atherosclerotic plaquesrdquo Journal of Clinical Investigationvol 117 no 1 pp 185ndash194 2007
[27] L M Carlin E G Stamatiades C Auffray et al ldquoNr4a1-dependent Ly6Clow monocytes monitor endothelial cells andorchestrate their disposalrdquoCell vol 153 no 2 pp 362ndash375 2013
[28] N V Serbina and E G Pamer ldquoMonocyte emigration frombone marrow during bacterial infection requires signals medi-ated by chemokine receptor CCR2rdquo Nature Immunology vol 7no 3 pp 311ndash317 2006
[29] S Hayashidani H Tsutsui M Ikeuchi et al ldquoTargeted deletionofMMP-2 attenuates early LV rupture and late remodeling afterexperimental myocardial infarctionrdquo The American Journal ofPhysiologymdashHeart and Circulatory Physiology vol 285 no 3pp H1229ndashH1235 2003
[30] A Ducharme S Frantz M Aikawa et al ldquoTargeted deletion ofmatrix metalloproteinase-9 attenuates left ventricular enlarge-ment and collagen accumulation after experimental myocardialinfarctionrdquo Journal of Clinical Investigation vol 106 no 1 pp55ndash62 2000
[31] K TWeber ldquoExtracellular matrix remodeling in heart failure arole for de novo angiotensin II generationrdquo Circulation vol 96no 11 pp 4065ndash4082 1997
[32] W Iraqi P Rossignol M Angioi et al ldquoExtracellular cardiacmatrix biomarkers in patients with acute myocardial infarctioncomplicated by left ventricular dysfunction and heart failureinsights from the Eplerenone Post-Acute Myocardial InfarctionHeart Failure Efficacy and Survival Study (EPHESUS) studyrdquoCirculation vol 119 no 18 pp 2471ndash2479 2009
[33] R Eschalier M Fertin R Fay et al ldquoExtracellular matrixturnover biomarkers predict long-term left ventricular remod-eling aftermyocardial infarction insights from the reve-2 studyrdquoCirculation Heart Failure vol 6 no 6 pp 1199ndash1205 2013
[34] J G Simmons J B Pucilowska T O Keku and P K LundldquoIGF-I and TGF-1205731 have distinct effects on phenotype andproliferation of intestinal fibroblastsrdquo American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 283 no3 pp G809ndashG818 2002
[35] C F Hung M G Rohani S-S Lee P Chen and L MSchnapp ldquoRole of IGF-1 pathway in lung fibroblast activationrdquoRespiratory Research vol 14 no 1 article 102 2013
[36] M Nahrendorf F K Swirski E Aikawa et al ldquoThe healingmyocardium sequentially mobilizes twomonocyte subsets withdivergent and complementary functionsrdquoThe Journal of Exper-imental Medicine vol 204 no 12 pp 3037ndash3047 2007
[37] F L Chen Z H Yang Y Liu et al ldquoBerberine inhibits theexpression of TNF120572 MCP-1 and IL-6 in AcLDL-stimulatedmacrophages through PPAR120574 pathwayrdquo Endocrine vol 33 no3 pp 331ndash337 2008
[38] J Tonkin L Temmerman R D Sampson et alldquoMonocytemacrophage-derived IGF-1 orchestrates murineskeletal muscle regeneration and modulates autocrinepolarizationrdquo Molecular Therapy vol 23 no 7 pp 1189ndash1200 2015
[39] T Heidt G Courties P Dutta et al ldquoDifferential contributionof monocytes to heart macrophages in steady-state and aftermyocardial infarctionrdquo Circulation Research vol 115 no 2 pp284ndash295 2014
[40] S Epelman K J Lavine and G J Randolph ldquoOrigin andfunctions of tissue macrophagesrdquo Immunity vol 41 no 1 pp21ndash35 2014
[41] A R Pinto J W Godwin and N A Rosenthal ldquoMacrophagesin cardiac homeostasis injury responses and progenitor cellmobilisationrdquo Stem Cell Research vol 13 no 3 pp 705ndash7142014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
10 Mediators of Inflammation
[21] D Bilbao L Luciani B Johannesson A Piszczek and NRosenthal ldquoInsulin-like growth factor-1 stimulates regulatoryT cells and suppresses autoimmune diseaserdquo EMBO MolecularMedicine vol 6 no 11 pp 1423ndash1435 2005
[22] H Oh S B Bradfute T D Gallardo et al ldquoCardiac progenitorcells from adult myocardium homing differentiation andfusion after infarctionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 21 pp12313ndash12318 2003
[23] A Philippou E Papageorgiou G Bogdanis et al ldquoExpressionof IGF-1 isoforms after exercise-induced muscle damage inhumans characterization of theMGFEpeptide actions in vitrordquoIn Vivo vol 23 no 4 pp 567ndash576 2009
[24] S Sanz J B Pucilowska S Liu et al ldquoExpression of insulin-like growth factor I by activated hepatic stellate cells reducesfibrogenesis and enhances regeneration after liver injuryrdquo Gutvol 54 no 1 pp 134ndash141 2005
[25] M P Santini L Tsao L Monassier et al ldquoEnhancing repair ofthe mammalian heartrdquo Circulation Research vol 100 no 12 pp1732ndash1740 2007
[26] F Tacke D Alvarez T J Kaplan et al ldquoMonocyte subsetsdifferentially employ CCR2 CCR5 and CX3CR1 to accumulatewithin atherosclerotic plaquesrdquo Journal of Clinical Investigationvol 117 no 1 pp 185ndash194 2007
[27] L M Carlin E G Stamatiades C Auffray et al ldquoNr4a1-dependent Ly6Clow monocytes monitor endothelial cells andorchestrate their disposalrdquoCell vol 153 no 2 pp 362ndash375 2013
[28] N V Serbina and E G Pamer ldquoMonocyte emigration frombone marrow during bacterial infection requires signals medi-ated by chemokine receptor CCR2rdquo Nature Immunology vol 7no 3 pp 311ndash317 2006
[29] S Hayashidani H Tsutsui M Ikeuchi et al ldquoTargeted deletionofMMP-2 attenuates early LV rupture and late remodeling afterexperimental myocardial infarctionrdquo The American Journal ofPhysiologymdashHeart and Circulatory Physiology vol 285 no 3pp H1229ndashH1235 2003
[30] A Ducharme S Frantz M Aikawa et al ldquoTargeted deletion ofmatrix metalloproteinase-9 attenuates left ventricular enlarge-ment and collagen accumulation after experimental myocardialinfarctionrdquo Journal of Clinical Investigation vol 106 no 1 pp55ndash62 2000
[31] K TWeber ldquoExtracellular matrix remodeling in heart failure arole for de novo angiotensin II generationrdquo Circulation vol 96no 11 pp 4065ndash4082 1997
[32] W Iraqi P Rossignol M Angioi et al ldquoExtracellular cardiacmatrix biomarkers in patients with acute myocardial infarctioncomplicated by left ventricular dysfunction and heart failureinsights from the Eplerenone Post-Acute Myocardial InfarctionHeart Failure Efficacy and Survival Study (EPHESUS) studyrdquoCirculation vol 119 no 18 pp 2471ndash2479 2009
[33] R Eschalier M Fertin R Fay et al ldquoExtracellular matrixturnover biomarkers predict long-term left ventricular remod-eling aftermyocardial infarction insights from the reve-2 studyrdquoCirculation Heart Failure vol 6 no 6 pp 1199ndash1205 2013
[34] J G Simmons J B Pucilowska T O Keku and P K LundldquoIGF-I and TGF-1205731 have distinct effects on phenotype andproliferation of intestinal fibroblastsrdquo American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 283 no3 pp G809ndashG818 2002
[35] C F Hung M G Rohani S-S Lee P Chen and L MSchnapp ldquoRole of IGF-1 pathway in lung fibroblast activationrdquoRespiratory Research vol 14 no 1 article 102 2013
[36] M Nahrendorf F K Swirski E Aikawa et al ldquoThe healingmyocardium sequentially mobilizes twomonocyte subsets withdivergent and complementary functionsrdquoThe Journal of Exper-imental Medicine vol 204 no 12 pp 3037ndash3047 2007
[37] F L Chen Z H Yang Y Liu et al ldquoBerberine inhibits theexpression of TNF120572 MCP-1 and IL-6 in AcLDL-stimulatedmacrophages through PPAR120574 pathwayrdquo Endocrine vol 33 no3 pp 331ndash337 2008
[38] J Tonkin L Temmerman R D Sampson et alldquoMonocytemacrophage-derived IGF-1 orchestrates murineskeletal muscle regeneration and modulates autocrinepolarizationrdquo Molecular Therapy vol 23 no 7 pp 1189ndash1200 2015
[39] T Heidt G Courties P Dutta et al ldquoDifferential contributionof monocytes to heart macrophages in steady-state and aftermyocardial infarctionrdquo Circulation Research vol 115 no 2 pp284ndash295 2014
[40] S Epelman K J Lavine and G J Randolph ldquoOrigin andfunctions of tissue macrophagesrdquo Immunity vol 41 no 1 pp21ndash35 2014
[41] A R Pinto J W Godwin and N A Rosenthal ldquoMacrophagesin cardiac homeostasis injury responses and progenitor cellmobilisationrdquo Stem Cell Research vol 13 no 3 pp 705ndash7142014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom