AbstractThe formation of foamy macrophages by sequestering extracellular modified lipids is a key event in atherosclerosis. However, there is controversy about the effects of lipid loading on macrophage phenotype, with in vitro evidence suggesting either pro- or anti-inflammatory consequences. To investigate this in vivo we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in experimental subcutaneous granulomas in fat-fed ApoE null mice or normal chow-fed wild-type mice, respectively. Consistent with previous studies in peritoneal macrophages from LDL receptor null mice (Spann et al., 2012 [1]), we found that anti-inflammatory LXR/RXR pathway genes were over-represented in the foamy macrophages, but there was no change in M1 or M2 phenotypic markers. Quite unexpectedly, however, we found that genes related to the induction of fibrosis had also been up-regulated (Thomas et al., 2015 [2]). The progression of the foamy macrophages along anti-inflammatory and pro-fibrotic pathways was confirmed using immunohistochemistry (described fully in our primary research article (Thomas et al., 2015 [2]). Here we provide additional details on production of the macrophages and their transcriptomic comparison, with the raw and processed microarray data deposited in GEO (accession number GSE70126). Our observations on these cells are indeed paradoxical, because foamy macrophages have long been implicated in promoting inflammation, extracellular matrix degradation and atherosclerotic plaque rupture, which must be provoked by additional local mediators. Our findings probably explain how very early macrophage-rich lesions maintain their structural integrity

Daemen, Mat

Eigelaar, Wouter

Newby, Andrew C

Thomas, Anita C

Genomics Data

PubMed

The formation of foamy macrophages by sequestering extracellular modified lipids is a key event in atherosclerosis. However, there is controversy about the effects of lipid loading on macrophage phenotype, with in vitro evidence suggesting either pro- or anti-inflammatory consequences. To investigate this in vivo we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in experimental subcutaneous granulomas in fat-fed ApoE null mice or normal chow-fed wild-type mice, respectively. Consistent with previous studies in peritoneal macrophages from LDL receptor null mice (Spann et al., 2012 [1]), we found that anti-inflammatory LXR/RXR pathway genes were over-represented in the foamy macrophages, but there was no change in M1 or M2 phenotypic markers. Quite unexpectedly, however, we found that genes related to the induction of fibrosis had also been up-regulated (Thomas et al., 2015 [2]). The progression of the foamy macrophages along anti-inflammatory and pro-fibrotic pathways was confirmed using immunohistochemistry (described fully in our primary research article (Thomas et al., 2015 [2]). Here we provide additional details on production of the macrophages and their transcriptomic comparison, with the raw and processed microarray data deposited in GEO (accession number GSE70126). Our observations on these cells are indeed paradoxical, because foamy macrophages have long been implicated in promoting inflammation, extracellular matrix degradation and atherosclerotic plaque rupture, which must be provoked by additional local mediators. Our findings probably explain how very early macrophage-rich lesions maintain their structural integrity

Anita C. Thomas

Wouter J. Eijgelaar

Mat J.A.P. Daemen

Andrew C. Newby

Directory of Open Access Journals

The pro-fibrotic and anti-inflammatory foam cell macrophage paradox

The formation of foamy macrophages by sequestering extracellular modified lipids is a key event in atherosclerosis. However, there is controversy about the effects of lipid loading on macrophage phenotype, with in vitro evidence suggesting either pro- or anti-inflammatory consequences. To investigate this in vivo we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in experimental subcutaneous granulomas in fat-fed ApoE null mice or normal chow-fed wild-type mice, respectively. Consistent with previous studies in peritoneal macrophages from LDL receptor null mice (Spann et al., 2012 [1]), we found that anti-inflammatory LXR/RXR pathway genes were over-represented in the foamy macrophages, but there was no change in M1 or M2 phenotypic markers. Quite unexpectedly, however, we found that genes related to the induction of fibrosis had also been up-regulated (Thomas et al., 2015 [2]). The progression of the foamy macrophages along anti-inflammatory and pro-fibrotic pathways was confirmed using immunohistochemistry (described fully in our primary research article (Thomas et al., 2015 [2]). Here we provide additional details on production of the macrophages and their transcriptomic comparison,with the rawand processed microarray data deposited in GEO (accession number GSE70126). Our observations on these cells are indeed paradoxical, because foamy macrophages have long been implicated in promoting inflammation, extracellular matrix degradation and atherosclerotic plaque rupture, which must be provoked by additional local mediators. Our findings probably explain how very early macrophage-rich lesions maintain their structural integrity

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                          Thomas, A. C., Eigelaar, W., Daemen, M., & Newby, A. C. (2015). The pro-fibrotic and anti-inflammatory foam cell macrophage paradox. GenomicsData, 6, 136-138. 10.1016/j.gdata.2015.08.027Publisher's PDF, also known as Final Published VersionLink to published version (if available):10.1016/j.gdata.2015.08.027Link to publication record in Explore Bristol ResearchPDF-documentUniversity of Bristol - Explore Bristol ResearchGeneral rightsThis document is made available in accordance with publisher policies. Please cite only the publishedversion using the reference above. Full terms of use are available:http://www.bristol.ac.uk/pure/about/ebr-terms.htmlTake down policyExplore Bristol Research is a digital archive and the intention is that deposited content should not beremoved. However, if you believe that this version of the work breaches copyright law please contactopen-access@bristol.ac.uk and include the following information in your message:• Your contact details• Bibliographic details for the item, including a URL• An outline of the nature of the complaintOn receipt of your message the Open Access Team will immediately investigate your claim, make aninitial judgement of the validity of the claim and, where appropriate, withdraw the item in questionfrom public view.Data in BriefThe pro-ﬁbrotic and anti-inﬂammatory foam cell macrophage paradoxAnita C. Thomas a,⁎, Wouter J. Eijgelaar b, Mat J.A.P. Daemen b,c, Andrew C. Newby aa Bristol Heart Institute, University of Bristol, Bristol, United Kingdomb Cardiovascular Research Institute Maastricht (CARIM), Maastricht, The Netherlandsc Academisch Medisch Centrum (AMC), Amsterdam, The Netherlandsa b s t r a c ta r t i c l e i n f oArticle history:Received 21 August 2015Accepted 27 August 2015Available online 3 September 2015Keywords:Foam cell macrophageAtherosclerosisInﬂammationFibrosisNon-foamy macrophageThe formation of foamy macrophages by sequestering extracellular modiﬁed lipids is a key event in atheroscle-rosis. However, there is controversy about the effects of lipid loading on macrophage phenotype, with in vitroevidence suggesting either pro- or anti-inﬂammatory consequences. To investigate this in vivo we comparedthe transcriptomes of foamy and non-foamymacrophages that accumulate in experimental subcutaneous gran-ulomas in fat-fed ApoE null mice or normal chow-fed wild-type mice, respectively. Consistent with previousstudies in peritoneal macrophages from LDL receptor null mice (Spann et al., 2012 [1]), we found that anti-inﬂammatory LXR/RXR pathway genes were over-represented in the foamy macrophages, but there was nochange inM1orM2phenotypicmarkers. Quite unexpectedly, however, we found that genes related to the induc-tion of ﬁbrosis had also been up-regulated (Thomas et al., 2015 [2]). The progression of the foamy macrophagesalong anti-inﬂammatory and pro-ﬁbrotic pathways was conﬁrmed using immunohistochemistry (describedfully in our primary research article (Thomas et al., 2015 [2]). Here we provide additional details on productionof themacrophages and their transcriptomic comparison, with the raw and processedmicroarray data depositedin GEO (accession number GSE70126). Our observations on these cells are indeed paradoxical, because foamymacrophages have long been implicated in promoting inﬂammation, extracellular matrix degradation andatherosclerotic plaque rupture, which must be provoked by additional local mediators. Our ﬁndings probablyexplain how very early macrophage-rich lesions maintain their structural integrity.© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).Speciﬁcations1. Direct link to deposited datahttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE70126.2. Experimental design, materials and methods2.1. Experimental groups and conditionsWe generated FCM and NFM in mice in vivo, in order to understandhow the macrophages found in atherosclerotic plaques differ fromnon-foamymacrophages. Consistentwith previous studies in peritonealmacrophages from LDL receptor null mice [1], we found that anti-in-ﬂammatory LXR/RXR pathway genes were over-represented in thefoamy macrophages, but there was no change in M1 or M2 phenotypicmarkers. Quite unexpectedly, however, we found that genes related tothe induction of ﬁbrosis had also been up-regulated [2].Genomics Data 6 (2015) 136–138⁎ Corresponding author at: Bristol Heart Institute, University of Bristol, 7th Floor,Queens Building, Bristol Royal Inﬁrmary, Bristol BS2 8HW, United Kingdom.E-mail address: a.thomas@bristol.ac.uk (A.C. Thomas).Organism/cellline/tissueMus musculus (C57BL background)/NA/sponge granulomamacrophages, atherosclerotic plaqueSex MaleSequencer orarray typeMouseRef8 v2.0 Expression BeadChips, IlluminaData format Raw and analysedExperimentalfactorsSponge granuloma macrophages from fat-fed ApoE−/−mice(FCM: foamy cell macrophages) vs control (ApoE+/+) mice feda normal chow diet (NFM: non-foamy macrophages).ExperimentalfeaturesSponges were surgically placed s.c. in fat-fed ApoE−/−mice orcontrol mice fed a normal chow diet, to produce FCM or NFM,respectively. Four weeks later the sponges were retrieved andthe macrophages isolated and puriﬁed using differentialadherence, with (FCM) or without (NFM) buoyant densitycentrifugation. RNA of high quality was isolated and comparedusing Illumina beadchips. Results were obtained fromIngenuity Pathway Analysis and GO annotation and clustering,and were conﬁrmed using RT-qPCR, immunocytochemistry(on macrophages isolated from the sponges) and immunohis-tochemistry (on sections taken through sponges or diseasedarteries from ApoE−/−mice fed a high-fat diet for 12 weeks).Consent The housing and care of all animals and procedures used inthese studies was in accordance with and under licence of theAnimals (Scientiﬁc Procedures) Act 1986 (London, UnitedKingdom), which transposes EU Directive 2010/63/EU.Sample sourcelocationNAhttp://dx.doi.org/10.1016/j.gdata.2015.08.0272213-5960/© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Contents lists available at ScienceDirectGenomics Dataj ourna l homepage: ht tp : / /www. journa ls .e lsev ie r .com/genomics-data /2.1.1. AnimalsThe housing and care of all animals and procedures used inthese studies was in accordance with and under licence of the Animals(Scientiﬁc Procedures) Act 1986 (London, United Kingdom), whichtransposes EU Directive 2010/63/EU. We obtained homozygousApoE−/− and ApoE+/+ mice on a C57/BL background from Universityof Bristol colonies. Animals were housed conventionally, and weregiven food and water ad libitum throughout the experiments. Animalswere treated as outlined in Fig. 1.2.1.2. Surgical sponge implantation and harvestPolyurethane sponges (Truclean, Merck) were prepared as follows:0.5 cm3 pieces of sponge were boiled for 2 h in ethanol, washed indH2O and autoclaved. Shortly before implantation the sponges wereinjected with ~50 μl Matrigel (BD Biosciences, UK) and soaked in saline(all under sterile conditions). Adult male ApoE−/− mice were placedonto a high-fat diet containing 23% fat from lard, supplemented with0.15% (w/w) cholesterol (821424, Special Diets Services, UK). Age-matched control mice remained on a normal chow diet (3.5% fat,EURodent Diet 22%, LabDiet, USA). Three weeks later the animals wereanaesthetised using halothane, and the sponges inserted subcutaneously.The animals were shaved on their lower back. Using aseptic technique,and with buprenorphine analgesia, a 1-cm long incision was made inthe skin. Six pockets were made under the skin, and a 0.5 cm3 piece ofsterile sponge placed into each pocket and the incision closed. Whenfully recovered the animals were returned to their original cages, andthe diet regimen continued for a further four weeks.2.1.3. Macrophage isolation and puriﬁcation from spongesThe mice were terminated with a lethal dose of anaesthetic and thesponges removed using the aseptic technique. All furthermanipulationsoccurred within a laminar ﬂow cabinet. The sponges were cleaned andincubated in Dispase (BD Biosciences) to partially degrade theMatrigel,and cellular exudates squeezed out of the sponges by twisting. NFM,from control mice fed a normal chow diet, were obtained by differentialadherence in RPMI 1640 media (Life Technologies) without foetal calfserum, after evaluation by Trypan blue exclusion as described previous-ly [3,4]. After 1 h the non-adherent cells were removed and freshmedi-um added. 0.5 h later, the adherent cells were washed in fresh mediumand RNA harvested. FCM were obtained from ApoE−/− mice on thehigh-fat diet. Cellular exudates were obtained as outlined above, andFCM obtained by density centrifugation over a metrizamide gradient(1.3507 refractive index, OPTI-prep, Sigma, USA). Only foam cells ﬂoat,because of the relatively low buoyant density of lipid. Differential adhe-sion steps were subsequently performed as above and RNA harvested.Some isolated FCMandNFMwere placed onto coverslips for assessmentof purity.2.1.4. Assessment of macrophage purityCells that had been adhered onto coverslipswere used to assess theirlipid content (as indicated by Oil-Red-O staining (2% Oil-red-O inisopropanol; Sigma) and identiﬁcation of cell type (using immunocyto-chemistry). All cells from the preparations from sponges fromApoE nullmice contained foamy inclusions, whereas no cells in the preparationsfrom control mice staining positively for lipid. 96 ± 5.8% of cellsfrom sponges obtained from the ApoE null mice were monocyte/macrophages (MOMA2, Biosource International, USA) and 0.86 ± 1.2%were smooth muscle cells (α/γ-SM actin, HHF35, Dako, UK), indicatingthat these preparations were highly pure. Similarly, 87 ± 10% of cellsfrom control mice were MOMA2 positive, and 1.4 ± 1.8% were α/γ-SMactin positive. We conﬁrmed that the preparations had little smoothmuscle cell contamination using RT-qPCR (less than 2.5 copies of SMmyosin/ng RNA). Hence cells isolated from the fat-fed ApoE null micewere indeed foam cell macrophages (FCM), whereas those from thecontrol mice were non-foamy macrophages (NFM).2.1.5. RNA isolation, use and quality control assessmentRNA lysateswere collected using RLT solution (Qiagen Ltd., UK)withβ-mercaptoethanol (Sigma) and the total RNA extracted using theQiagen RNeasy kit (Qiagen Ltd) according to the manufacturer'sinstructions. RNA concentration was determined using NanoDropspectrometry (NanoDrop Technologies, USA), with the samples havingRNA Integrity Numbers (RINs) between 8.0 and 9.7. RNA samples ofhigh quality (A260/280 N 2) (n = 4) were compared using Illuminabeadchips (MouseRef8 v2.0 Expression BeadChips, Illumina, USA)at 1 μg/sample. Samples were labelled and hybridized as describedin the Illumina “Whole-Genome Gene Expression Direct Hybridiza-tion Assay Guide (11322355 A)”. Samples were labelled using theAmbion Illumina TotalPrep RNA Ampliﬁcation Kit (Ambion, USA).The ampliﬁcation of RNA is based on the Eberwine method, andresults in biotinylated, ampliﬁed cRNA. The signal was developedwith streptavidin-Cy3 (Fisher Scientiﬁc, USA) and the BeadChipscanned using the Illumina iScan system. Quality control wasperformed using Illumina BeadStudio software.2.2. Comparison of FCM and NFM2.2.1. Functional annotationAll genes from the data set that met the unadjusted P-value cut-offof 0.01 were uploaded to the Ingenuity Pathways Analysis system(Ingenuity Systems, www.ingenuity.com) and included in the analysis.Each identiﬁer wasmapped to its corresponding object in the IngenuityKnowledge Base. Functional analysis identiﬁed the biological functionsand canonical pathway analysis identiﬁed the pathways that weremost signiﬁcant to the data set. Network maps were also generatedFig. 1.Representation ofmacrophage production inmice. Foam cellmacrophages (FCM) or non-foamymacrophages (NFM)were obtained from subcutaneous sponges inserted intomice.RNA was harvested from isolated macrophages and further samples were taken for examination using immunocytochemistry (ICC) or immunohistochemistry (IHC).137A.C. Thomas et al. / Genomics Data 6 (2015) 136–138within Ingenuity Pathway Analysis. Differentially expressed genes wereoverlaid onto a global molecular network developed from informationcontained in the Ingenuity Knowledge Base and networks of thesemol-ecules algorithmically generated based on their connectivity. Differen-tially expressed genes were also submitted to GO annotation andclustering using DAVID Bioinformatics Resources (National Institute ofAllergy and Infectious Diseases (NIAID) 2008, NIH, http://david.abcc.ncifcrf.gov).2.2.2. Real time quantitative PCR (RT-qPCR) validationThe array resultswere validated and expanded on RNA samples pre-pared in the sameway using RT-qPCR (n= 5–7). For reverse transcrip-tion, 100 ng of total RNA was used to make cDNA, using a QiagenQuantitect Reverse Transcriptase Kit (Qiagen Ltd., UK) according tothe manufacturer's instructions. cDNA was diluted 1:1 in 10 mMTris·HCl, pH 8.0. RT-qPCR was performed in a Roche Light Cycler 1.5(Roche, UK) to quantify the steady-state concentration of RNA, usingthe QuantiTect SYBR Green PCR Kit (Qiagen Ltd). Each reactioncontained 2.5–7 ng RNA and 0.5 μM primers. Initial denaturation(15 min at 95 °C) was followed by 55 cycles of denaturation (15 s at95 °C), annealing (20 s at 60–66 °C) and extension (25 s at 72 °C).Copy numbers of gene transcripts per total ng RNA input were calculat-ed using standard curves constructed as recommended by frompuriﬁedamplicon (Bioline, USA). Sequences of the PCR primer pairs used to am-plify the respective cDNAs were designed using Ensembl and Primer3,and the speciﬁcity of the sequence conﬁrmed using Nucleotide blast(NCBI).2.3. Statistical analysisStatistical analysis of the microarrays was performed using the ‘R’Bioconductor Lumi and Limma packages, applying the linear modelsand empirical Bayes methods included in the package [5]. Variancestabilizing transformation was performed using the Robust SplineNormalization (RSN) algorithm [6]with unsupervised analysismethodssuch as Principal Component Analysis and Hierarchical Clustering usedfor initial data exploration. Statistical analysis of differential expressionconsisted of a univariate model to detect individual genes that aresigniﬁcantly different in abundance between the conditions [5]. P-values were adjusted for multiple comparisons, using the False Discov-ery Rate (FDR) Benjamini–Hochberg method. Differential expressionwas classiﬁed as signiﬁcant (P b 0.01 after FDR correction for multipletesting) or suggestive (P b 0.01 unadjusted). The signiﬁcance of theassociation between the data set and the canonical pathway was mea-sured by either a ratio of the number of molecules from the data setthat map to the pathway divided by the total number of moleculesthat map to the canonical pathway, or by using a right-tailed Fisher'sexact test to calculate a P-value determining the probability that the as-sociation between the genes in the data set and the canonical pathwayis explained by chance alone. For the RT-qPCR, data differences betweenthe two groupswere tested for signiﬁcancewith Student's t-test, using alogarithmic transformation or the Mann–Whitney U-test if the datawere not normally distributed (GraphPad Instat, USA). P-values of lessthan 0.05 were considered statistically signiﬁcant.Third-party ﬁnancial supportThis work was supported by the British Heart Foundation (ACT andACN). The funder had no role in study design, data collection, analysisand interpretation, decision to publish or preparation of themanuscript.References[1] N.J. Spann, L.X. Garmire, J.G. McDonald, D.S. Myers, S.B. Milne, N. Shibata, et al.,Regulated accumulation of desmosterol integrates macrophage lipid metabolismand inﬂammatory responses. Cell 151 (2012) 138–152.[2] A.C. Thomas, W.J. Eijgelaar, M.J.A.P. Daemen, A.C. Newby, Foam cell formation in vivoconverts macrophages to a pro-ﬁbrotic phenotype. PLoS One 10 (2015), e0128163.[3] A.J. Chase, M. Bond, M.F. Crook, A.C. Newby, Role of nuclear factor-κB activation inmetalloproteinase-1, -3, and -9 secretion by human macrophages in vitro and rabbitfoam cells produced in vivo. Arterioscler. Thromb. Vasc. Biol. 22 (2002) 765–771.[4] A.C. Thomas, G.B. Sala-Newby, Y. Ismail, J.L. Johnson, G. Pasterkamp, A.C. Newby,Genomics of foam cells and nonfoamy macrophages from rabbits identiﬁesarginase-I as a differential regulator of nitric oxide production. Arterioscler. Thromb.Vasc. Biol. 27 (2007) 571–577.[5] G.K. Smyth, Linear models and empirical Bayes methods for assessing differentialexpression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3 (2004) Article3.[6] S.M. Lin, P. Du, W. Huber, W.A. Kibbe, Model-based variance-stabilizing transforma-tion for Illumina microarray data. Nucleic Acids Res. 36 (2008), e11.138 A.C. Thomas et al. / Genomics Data 6 (2015) 136–138

Thomas, Anita C.

Eijgelaar, Wouter J.

Daemen, Mat J.A.P.

Newby, Andrew C.

Elsevier - Publisher Connector 

Genomics Data 6 (2015) 136–138
Contents lists available at ScienceDirect
Genomics Data
j ou rna l homepage: ht tp : / /www. journa ls .e lsev ie r .com/genomics-data /Data in BriefThe pro-ﬁbrotic and anti-inﬂammatory foam cell macrophage paradoxAnita C. Thomas a,⁎, Wouter J. Eijgelaar b, Mat J.A.P. Daemen b,c, Andrew C. Newby a
a Bristol Heart Institute, University of Bristol, Bristol, United Kingdom
b Cardiovascular Research Institute Maastricht (CARIM), Maastricht, The Netherlands
c Academisch Medisch Centrum (AMC), Amsterdam, The Netherlands⁎ Corresponding author at: Bristol Heart Institute, U
Queens Building, Bristol Royal Inﬁrmary, Bristol BS2 8HW
E-mail address: a.thomas@bristol.ac.uk (A.C. Thomas)
line/tissue macrophages, atherosclerotic plaq
Sex Male
Sequencer or
array type
MouseRef8 v2.0 Expression BeadC
Data format Raw and analysed
Experimental
factors
Sponge granuloma macrophages f
(FCM: foamy cell macrophages) vs
a normal chow diet (NFM: non-fo
Experimental
features
Sponges were surgically placed s.c
control mice fed a normal chow d
respectively. Four weeks later the
the macrophages isolated and pur
adherence, with (FCM) or withou
centrifugation. RNA of high quality
using Illumina beadchips. Results
(on macrophages isolated from th
tochemistry (on sections taken th
arteries from ApoE−/−mice fed a
http://dx.doi.org/10.1016/j.gdata.2015.08.027
2213-5960/© 2015 The Authors. Published by Elsevier Inca b s t r a c ta r t i c l e i n f oArticle history:
Received 21 August 2015
Accepted 27 August 2015
Available online 3 September 2015
Keywords:
Foam cell macrophage
Atherosclerosis
Inﬂammation
Fibrosis
Non-foamy macrophageThe formation of foamy macrophages by sequestering extracellular modiﬁed lipids is a key event in atheroscle-
rosis. However, there is controversy about the effects of lipid loading on macrophage phenotype, with in vitro
evidence suggesting either pro- or anti-inﬂammatory consequences. To investigate this in vivo we compared
the transcriptomes of foamy and non-foamymacrophages that accumulate in experimental subcutaneous gran-
ulomas in fat-fed ApoE null mice or normal chow-fed wild-type mice, respectively. Consistent with previous
studies in peritoneal macrophages from LDL receptor null mice (Spann et al., 2012 [1]), we found that anti-
inﬂammatory LXR/RXR pathway genes were over-represented in the foamy macrophages, but there was no
change inM1orM2phenotypicmarkers. Quite unexpectedly, however, we found that genes related to the induc-
tion of ﬁbrosis had also been up-regulated (Thomas et al., 2015 [2]). The progression of the foamy macrophages
along anti-inﬂammatory and pro-ﬁbrotic pathways was conﬁrmed using immunohistochemistry (described
fully in our primary research article (Thomas et al., 2015 [2]). Here we provide additional details on production
of themacrophages and their transcriptomic comparison, with the raw and processedmicroarray data deposited
in GEO (accession number GSE70126). Our observations on these cells are indeed paradoxical, because foamy
macrophages have long been implicated in promoting inﬂammation, extracellular matrix degradation and
atherosclerotic plaque rupture, which must be provoked by additional local mediators. Our ﬁndings probably
explain how very early macrophage-rich lesions maintain their structural integrity.
© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).Consent The housing and care of all animals and procedures used in
these studies was in accordance with and under licence of theSpeciﬁcations
Organism/cell Mus musculus (C57BL background)/NA/sponge granuloma
ue
hips, Illumina
rom fat-fed ApoE−/−mice
control (ApoE+/+) mice fed
amy macrophages).
. in fat-fed ApoE−/−mice or
iet, to produce FCM or NFM,
sponges were retrieved and
iﬁed using differential
t (NFM) buoyant density
was isolated and compared
were obtained from
Animals (Scientiﬁc Procedures) Act 1986 (London, United
Kingdom), which transposes EU Directive 2010/63/EU.
Sample source
location
NAIngenuity Pathway Analysis and GO annotation and clustering,
and were conﬁrmed using RT-qPCR, immunocytochemistryniversity of Bristol, 7th Floor,
, United Kingdom.
.
e sponges) and immunohis-
rough sponges or diseased
high-fat diet for 12 weeks).
. This is an open access article under1. Direct link to deposited data
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE70126.
2. Experimental design, materials and methods
2.1. Experimental groups and conditions
We generated FCM and NFM in mice in vivo, in order to understand
how the macrophages found in atherosclerotic plaques differ from
non-foamymacrophages. Consistentwith previous studies in peritoneal
macrophages from LDL receptor null mice [1], we found that anti-in-
ﬂammatory LXR/RXR pathway genes were over-represented in the
foamy macrophages, but there was no change in M1 or M2 phenotypic
markers. Quite unexpectedly, however, we found that genes related to
the induction of ﬁbrosis had also been up-regulated [2].the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
137A.C. Thomas et al. / Genomics Data 6 (2015) 136–1382.1.1. Animals
The housing and care of all animals and procedures used in
these studies was in accordance with and under licence of the Animals
(Scientiﬁc Procedures) Act 1986 (London, United Kingdom), which
transposes EU Directive 2010/63/EU. We obtained homozygous
ApoE−/− and ApoE+/+ mice on a C57/BL background from University
of Bristol colonies. Animals were housed conventionally, and were
given food and water ad libitum throughout the experiments. Animals
were treated as outlined in Fig. 1.
2.1.2. Surgical sponge implantation and harvest
Polyurethane sponges (Truclean, Merck) were prepared as follows:
0.5 cm3 pieces of sponge were boiled for 2 h in ethanol, washed in
dH2O and autoclaved. Shortly before implantation the sponges were
injected with ~50 μl Matrigel (BD Biosciences, UK) and soaked in saline
(all under sterile conditions). Adult male ApoE−/− mice were placed
onto a high-fat diet containing 23% fat from lard, supplemented with
0.15% (w/w) cholesterol (821424, Special Diets Services, UK). Age-
matched control mice remained on a normal chow diet (3.5% fat,
EURodent Diet 22%, LabDiet, USA). Three weeks later the animals were
anaesthetised using halothane, and the sponges inserted subcutaneously.
The animals were shaved on their lower back. Using aseptic technique,
and with buprenorphine analgesia, a 1-cm long incision was made in
the skin. Six pockets were made under the skin, and a 0.5 cm3 piece of
sterile sponge placed into each pocket and the incision closed. When
fully recovered the animals were returned to their original cages, and
the diet regimen continued for a further four weeks.
2.1.3. Macrophage isolation and puriﬁcation from sponges
The mice were terminated with a lethal dose of anaesthetic and the
sponges removed using the aseptic technique. All furthermanipulations
occurred within a laminar ﬂow cabinet. The sponges were cleaned and
incubated in Dispase (BD Biosciences) to partially degrade theMatrigel,
and cellular exudates squeezed out of the sponges by twisting. NFM,
from control mice fed a normal chow diet, were obtained by differential
adherence in RPMI 1640 media (Life Technologies) without foetal calf
serum, after evaluation by Trypan blue exclusion as described previous-
ly [3,4]. After 1 h the non-adherent cells were removed and freshmedi-
um added. 0.5 h later, the adherent cells were washed in fresh medium
and RNA harvested. FCM were obtained from ApoE−/− mice on the
high-fat diet. Cellular exudates were obtained as outlined above, and
FCM obtained by density centrifugation over a metrizamide gradient
(1.3507 refractive index, OPTI-prep, Sigma, USA). Only foam cells ﬂoat,
because of the relatively low buoyant density of lipid. Differential adhe-
sion steps were subsequently performed as above and RNA harvested.
Some isolated FCMandNFMwere placed onto coverslips for assessment
of purity.Fig. 1.Representation ofmacrophage production inmice. Foam cellmacrophages (FCM) or non-
RNA was harvested from isolated macrophages and further samples were taken for examinatio2.1.4. Assessment of macrophage purity
Cells that had been adhered onto coverslipswere used to assess their
lipid content (as indicated by Oil-Red-O staining (2% Oil-red-O in
isopropanol; Sigma) and identiﬁcation of cell type (using immunocyto-
chemistry). All cells from the preparations from sponges fromApoE null
mice contained foamy inclusions, whereas no cells in the preparations
from control mice staining positively for lipid. 96 ± 5.8% of cells
from sponges obtained from the ApoE null mice were monocyte/
macrophages (MOMA2, Biosource International, USA) and 0.86 ± 1.2%
were smooth muscle cells (α/γ-SM actin, HHF35, Dako, UK), indicating
that these preparations were highly pure. Similarly, 87 ± 10% of cells
from control mice were MOMA2 positive, and 1.4 ± 1.8% were α/γ-SM
actin positive. We conﬁrmed that the preparations had little smooth
muscle cell contamination using RT-qPCR (less than 2.5 copies of SM
myosin/ng RNA). Hence cells isolated from the fat-fed ApoE null mice
were indeed foam cell macrophages (FCM), whereas those from the
control mice were non-foamy macrophages (NFM).
2.1.5. RNA isolation, use and quality control assessment
RNA lysateswere collected using RLT solution (Qiagen Ltd., UK)with
β-mercaptoethanol (Sigma) and the total RNA extracted using the
Qiagen RNeasy kit (Qiagen Ltd) according to the manufacturer's
instructions. RNA concentration was determined using NanoDrop
spectrometry (NanoDrop Technologies, USA), with the samples having
RNA Integrity Numbers (RINs) between 8.0 and 9.7. RNA samples of
high quality (A260/280 N 2) (n = 4) were compared using Illumina
beadchips (MouseRef8 v2.0 Expression BeadChips, Illumina, USA)
at 1 μg/sample. Samples were labelled and hybridized as described
in the Illumina “Whole-Genome Gene Expression Direct Hybridiza-
tion Assay Guide (11322355 A)”. Samples were labelled using the
Ambion Illumina TotalPrep RNA Ampliﬁcation Kit (Ambion, USA).
The ampliﬁcation of RNA is based on the Eberwine method, and
results in biotinylated, ampliﬁed cRNA. The signal was developed
with streptavidin-Cy3 (Fisher Scientiﬁc, USA) and the BeadChip
scanned using the Illumina iScan system. Quality control was
performed using Illumina BeadStudio software.
2.2. Comparison of FCM and NFM
2.2.1. Functional annotation
All genes from the data set that met the unadjusted P-value cut-off
of 0.01 were uploaded to the Ingenuity Pathways Analysis system
(Ingenuity Systems, www.ingenuity.com) and included in the analysis.
Each identiﬁer wasmapped to its corresponding object in the Ingenuity
Knowledge Base. Functional analysis identiﬁed the biological functions
and canonical pathway analysis identiﬁed the pathways that were
most signiﬁcant to the data set. Network maps were also generatedfoamymacrophages (NFM)were obtained from subcutaneous sponges inserted intomice.
n using immunocytochemistry (ICC) or immunohistochemistry (IHC).
138 A.C. Thomas et al. / Genomics Data 6 (2015) 136–138within Ingenuity Pathway Analysis. Differentially expressed genes were
overlaid onto a global molecular network developed from information
contained in the Ingenuity Knowledge Base and networks of thesemol-
ecules algorithmically generated based on their connectivity. Differen-
tially expressed genes were also submitted to GO annotation and
clustering using DAVID Bioinformatics Resources (National Institute of
Allergy and Infectious Diseases (NIAID) 2008, NIH, http://david.abcc.
ncifcrf.gov).
2.2.2. Real time quantitative PCR (RT-qPCR) validation
The array resultswere validated and expanded on RNA samples pre-
pared in the sameway using RT-qPCR (n= 5–7). For reverse transcrip-
tion, 100 ng of total RNA was used to make cDNA, using a Qiagen
Quantitect Reverse Transcriptase Kit (Qiagen Ltd., UK) according to
the manufacturer's instructions. cDNA was diluted 1:1 in 10 mM
Tris·HCl, pH 8.0. RT-qPCR was performed in a Roche Light Cycler 1.5
(Roche, UK) to quantify the steady-state concentration of RNA, using
the QuantiTect SYBR Green PCR Kit (Qiagen Ltd). Each reaction
contained 2.5–7 ng RNA and 0.5 μM primers. Initial denaturation
(15 min at 95 °C) was followed by 55 cycles of denaturation (15 s at
95 °C), annealing (20 s at 60–66 °C) and extension (25 s at 72 °C).
Copy numbers of gene transcripts per total ng RNA input were calculat-
ed using standard curves constructed as recommended by frompuriﬁed
amplicon (Bioline, USA). Sequences of the PCR primer pairs used to am-
plify the respective cDNAs were designed using Ensembl and Primer3,
and the speciﬁcity of the sequence conﬁrmed using Nucleotide blast
(NCBI).
2.3. Statistical analysis
Statistical analysis of the microarrays was performed using the ‘R’
Bioconductor Lumi and Limma packages, applying the linear models
and empirical Bayes methods included in the package [5]. Variance
stabilizing transformation was performed using the Robust Spline
Normalization (RSN) algorithm [6]with unsupervised analysismethods
such as Principal Component Analysis and Hierarchical Clustering used
for initial data exploration. Statistical analysis of differential expression
consisted of a univariate model to detect individual genes that aresigniﬁcantly different in abundance between the conditions [5]. P-
values were adjusted for multiple comparisons, using the False Discov-
ery Rate (FDR) Benjamini–Hochberg method. Differential expression
was classiﬁed as signiﬁcant (P b 0.01 after FDR correction for multiple
testing) or suggestive (P b 0.01 unadjusted). The signiﬁcance of the
association between the data set and the canonical pathway was mea-
sured by either a ratio of the number of molecules from the data set
that map to the pathway divided by the total number of molecules
that map to the canonical pathway, or by using a right-tailed Fisher's
exact test to calculate a P-value determining the probability that the as-
sociation between the genes in the data set and the canonical pathway
is explained by chance alone. For the RT-qPCR, data differences between
the two groupswere tested for signiﬁcancewith Student's t-test, using a
logarithmic transformation or the Mann–Whitney U-test if the data
were not normally distributed (GraphPad Instat, USA). P-values of less
than 0.05 were considered statistically signiﬁcant.
Third-party ﬁnancial support
This work was supported by the British Heart Foundation (ACT and
ACN). The funder had no role in study design, data collection, analysis
and interpretation, decision to publish or preparation of themanuscript.
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The pro-fibrotic and anti-inflammatory foam cell macrophage paradox 

The formation of foamy macrophages by sequestering extracellular modified lipids is a key event in atherosclerosis. However, there is controversy about the effects of lipid loading on macrophage phenotype, with in vitro evidence suggesting either pro-or anti-inflammatory consequences. To investigate this in vivo we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in experimental subcutaneous granulomas in fat-fed ApoE null mice or normal chow-fed wild-type mice, respectively. Consistent with previous studies in peritoneal macrophages from LDL receptor null mice (Spann et al., 2012 [1]), we found that anti-inflammatory LXR/RXR pathway genes were over-represented in the foamy macrophages, but there was no change in M1 or M2 phenotypic markers. Quite unexpectedly, however, we found that genes related to the induction of fibrosis had also been up-regulated (Thomas et al., 2015 [2]). The progression of the foamy macrophages along anti-inflammatory and pro-fibrotic pathways was confirmed using immunohistochemistry (described fully in our primary research article (Thomas et al., 2015 [2]). Here we provide additional details on production of the macrophages and their transcriptomic comparison, with the raw and processed microarray data deposited in GEO (accession number GSE70126). Our observations on these cells are indeed paradoxical, because foamy macrophages have long been implicated in promoting inflammation, extracellular matrix degradation and atherosclerotic plaque rupture, which must be provoked by additional local mediators. Our findings probably explain how very early macrophage-rich lesions maintain their structural integrity. (C) 2015 The Authors. Published by Elsevier In

Daemen, Mat J. A. P.

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The pro-fibrotic and anti-inflammatory foam cell macrophage paradox

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