Gene Expression Profiling Reveals Multiple Protective Influences of The

Gene Expression Profiling Reveals Multiple Protective Influences of the Peptide α -Melanocyte-Stimulating Hormone in Experimental Heart Transplantation This information is current as of September 30, 2021. Gualtiero Colombo, Stefano Gatti, Flavia Turcatti, Andrea Sordi, Luigi R. Fassati, Ferruccio Bonino, James M. Lipton and Anna Catania J Immunol 2005; 175:3391-3401; ; doi: 10.4049/jimmunol.175.5.3391 Downloaded from http://www.jimmunol.org/content/175/5/3391

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Gene Expression Proﬁling Reveals Multiple Protective Inﬂuences of the Peptide ␣-Melanocyte-Stimulating Hormone in Experimental Heart Transplantation1

†,Luigi R. Fassati ء,Andrea Sordi ء,Stefano Gatti,† Flavia Turcatti ء,Gualtiero Colombo ءFerruccio Bonino,‡ James M. Lipton,§ and Anna Catania2

Novel therapies are sought to increase efficiency and survival of transplanted organs. Previous research on experimental heart transplantation showed that treatment with the anti-inflammatory peptide ␣-melanocyte-stimulating hormone (␣-MSH) prolongs allograft survival. The aim of the present research was to determine the molecular mechanism of this protective activity. Gene expression profile was examined in heart grafts removed on postoperative days 1 and 4 from rats treated with saline or the 4 7 synthetic ␣-MSH analog Nle DPhe (NDP)-␣-MSH. On postoperative day 1, the peptide induced expression of cytoskeleton Downloaded from proteins, intracellular kinases, transcription regulators, metallopeptidases, and protease inhibitors. Conversely, NDP-␣-MSH repressed immune, inflammatory, cell cycle, and protein turnover mediators. Later effects of ␣-MSH treatment included down- regulation of oxidative stress response and up-regulation of ion channels, calcium regulation proteins, phosphatidylinositol signaling system, and glycolipidic metabolism. NDP-␣-MSH exerted its effects on both Ag-dependent and -independent injury. The results indicate that NDP-␣-MSH preserves heart function through a broad effect on multiple pathways and suggest that the peptide could improve the outcome of organ transplantation in combination with immunosuppressive treatments. The Journal http://www.jimmunol.org/ of Immunology, 2005, 175: 3391–3401.

cute rejection is a significant obstacle to successful or- soon be used clinically as truly novel anti-inflammatory/immuno- gan transplantation and its prevention is crucial for fa- modulatory compounds (4–8). Therefore, we designed research to A vorable clinical outcome. Although immunosuppressive determine the molecular mechanism underlying the protective ef- molecules can reduce rejection, they are associated with serious fects of the peptide. Using complement DNA arrays, an estab- side effects such as organ toxicity, increased viral infection, and lished technique for identification of pathways involved in trans- cancer (1). Because most of these harmful effects are dose-depen- plant rejection and its prevention (9), we found multiple protective dent, reduction of immunosuppressive drug treatment necessary to influences of ␣-MSH in experimental heart transplantation. by guest on September 30, 2021 prevent rejection is a major clinical target. As intragraft inflammation is known to promote and accelerate rejection (2), use of Materials and Methods anti-inflammatory compounds that enhance effectiveness of immu- Animals nosuppressive agents could be a successful strategy. Previous research on experimental heart transplantation showed Adult inbred Brown Norway and Lewis male rats (Charles River Labora- ␣ tories) weighing 200–300 g were used in the research. All animals received that treatment with the immunomodulatory peptide -melanocyte- care in compliance with the Principles of Laboratory Animal Care, formu- stimulating hormone (␣-MSH)3 prolongs survival and improves lated by the National Society of Medical Research, and the Guide for the allograft histopathology (3). Such beneficial effects were associ- Care and Use of Laboratory Animals, prepared by the National Academy ated with reduced intragraft expression of cytokines, chemokines, of Sciences and published by the National Institutes of Health (National Institutes of Health Publication No. 86–23). and adhesion molecules (3). ␣-MSH or its synthetic analogues may Surgical procedures Rats were anesthetized with a combination of 100 mg/kg ketamine and 6 *Division of Internal Medicine, †Division of Liver Transplantation, and ‡Scientific Direction, Fondazione Instituto di Ricovero e Cura a Carattere Scientifico (IRCCS) mg/kg xylazine injected i.p. During anesthesia, heart rate, ventilation rate, Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano, Italy; and §Zen- and temperature were closely monitored. Brown Norway donor hearts were gen, Woodland Hills, CA 91367 transplanted into either the MHC incompatible Lewis rats (allografts) or into Brown Norway rats (isografts). The donor heart was transplanted het- Received for publication May 17, 2005. Accepted for publication June 24, 2005. erotopically into the abdominal cavity of the recipient using the technique The costs of publication of this article were defrayed in part by the payment of page described by Ono and Lindsey (10). All cardiac transplants had good initial charges. This article must therefore be hereby marked advertisement in accordance contractile function. Graft function was monitored by palpation through the with 18 U.S.C. Section 1734 solely to indicate this fact. abdominal wall twice daily. There were no early deaths nor graft rejections 1 This work was supported by Progetto di Ricerca “Meccanismi molecolari del danno during the study period. At each planned interval, rats were euthanized nel trapianto singenico e nell’allotrapianto”, Ospedale Maggiore di Milano, Italy, and with thoracotomy under ketamine and xylazine anesthesia. The abdomen Progetto di Ricerca Finalizzata “Strategie innovative per il trapianto di fegato was incised and the heart grafts were immediately removed. (SITF)”, Ministero della Salute, Italy. 2 Address correspondence and reprint requests to Dr. Anna Catania, Divisione di Treatments Medicina Interna, Pad. Granelli, Ospedale Maggiore Policlinico, Via F. Sforza 35, Milano 20122, Italy. E-mail address: [email protected] Each treatment group included five rats. Allograft recipients assigned to 4 7 3 ␣ ␣ active treatment received i.p. injections of 100 ␮gofNleDPhe (NPD)- Abbreviations used in this paper: -MSH, -melanocyte stimulating hormone; ␣ NDP, Nle4DPhe7; POD, postoperative day; SAM, significance analysis of microar- -MSH (11) (kindly provided by Prof. P. Grieco, University of Naples, rays; FDR, false discovery rate; CT, cycle threshold; PKC, protein kinase C; Plc, Naples, Italy) dissolved in 0.5 ml of saline, every 12 h. Treatment was phospholipase C; Adcy6, adenylyl cyclase VI. started 1 h before transplantation and continued until sacrifice. Untreated

FIGURE 1. Histology of cardiac grafts. H&E staining (ϫ120) of a control nontransplanted heart (A); POD4 cardiac allograft from a saline-treated rat (B) and from an NDP-␣-MSH-treated animal (C). allograft recipients and isograft recipients received i.p. parallel injections sen samples for each allograft group and obtained consistent results (data of 0.5 ml of saline. not shown). Cardiac isografts were used to estimate heart injury caused by surgical

procedures alone and were harvested on postoperative day (POD) 1. Al- Analysis of macroarray data Downloaded from lografts were harvested on POD 1 or 4. Two Brown Norway donor hearts Normalization. Phosphorimager scans were analyzed using AtlasImage were subjected to cold ischemia of similar duration and not transplanted. software (version 2.7; BD Biosciences/Clontech). A given gene was con- They served as nontransplanted controls. Heart grafts were sectioned coro- sidered to be detectable if its intensity was at least twice the global external nally. Two sections were snap-frozen in liquid nitrogen and stored at background of the array. The background level was subtracted from the Ϫ 80°C for RNA extraction. One section was fixed in 10% buffered for- intensity of each spot to generate the raw data for each gene. Raw data were malin and paraffin-embedded for light microscopy examination. normalized according to the sum of the intensities global normalization method. The normalization coefficient was obtained by dividing the global http://www.jimmunol.org/ cDNA macroarray hybridization intensity of each array by the global intensity of a reference array. The Frozen tissue samples were homogenized with an Ultra-Turrax tissue ho- reference array was the array hybridized with a pool of RNAs from the mogenizer (IKA Labortechnik) and total RNA was isolated using the Atlas control hearts (i.e., hearts subjected to cold ischemia and not transplanted). Pure Total RNA extraction kit (BD Biosciences/Clontech), according to the The relative expression level for each gene was calculated as the ratio: gene manufacturer’s instructions. Analysis of gene expression was performed intensity/intensity of the same gene in the control hearts. Total intensities using Clontech Atlas Rat 1.2 Arrays I and II (BD Biosciences/Clontech). for each experimental condition were then scaled and mean intensities were These membrane arrays include 2352 spotted cDNAs of known and func- calculated and used for scatter plot visualization of fold changes. tionally annotated genes. cDNAs are 200–600 bp long and selected for low Filtering and statistical analysis. Hierarchical agglomerative clustering homology to other genes, and gene-specific primers are used in probe syn- of the array data was performed using a modified version of the Cluster/ theses. A complete list of all the genes on the arrays, including array co- TreeView software (version 3.0) (13), originally developed by Eisen et al. ordinates and GenBank accession numbers, is available at the BD Bio- (14) (͗http://rana.lbl.gov/͘). Data were filtered to include only genes de- by guest on September 30, 2021 sciences/Clontech Bioinformatics web site AtlasInfo 3.2 (͗http:// tected in at least 80% of the replicates. Relative expression values (see bioinfo.clontech.com/atlasinfo/͘). above) were log-transformed (log base 2), genes and arrays were median Radiolabeled complex probes were generated by reverse transcription centered and clustered by correlation (uncentered) centroid linkage. The using total RNA, [␣-32P]dATP (Amersham Biosciences) and the Atlas hierarchical clustering was visualized with TreeView. gene-specific mix of oligonucleotide primers (BD Biosciences/Clontech). Primary statistical analysis of the filtered data was performed using the Unincorporated radiolabeled nucleotides were removed with Nucleospin significance analysis of microarrays procedure (SAM, Excel Add-In ver- Extraction spin columns (BD Biosciences/Clontech), and probe yields were sion 1.21; ͗www-stat.stanford.edu/ϳtibs/SAM/͘) (15). Only data that quantified by liquid scintillation counting. passed the quality assurance criteria were included in the analysis. A me- Array membranes for each experimental condition were separately pre- dian false discovery rate (FDR) of Ͻ2%, in a two-class unpaired sample 32 hybridized in ExpressHyb buffer (BD Biosciences/Clontech). The P-la- analysis on log2-transformed ratios followed by 100 random permutations beled probes were denatured, diluted with carrier DNA, and an equal of the data, was used to identify genes differentially expressed between amount added to each membrane. Hybridization was allowed to proceed comparison groups. for 18 h at 68°C. After three high-stringency washes, membranes were Treatment-related fold change was used to identify genes consistently exposed to a storage phosphor screen (Molecular Dynamics) for 48–72 h. up- or down-regulated in response to NDP-␣-MSH. The ratio-mean rela- Phosphor screens were scanned at 100 ␮m resolution and images were tive expression for a given gene in treated allograft/mean relative expres- acquired using a 8600 Typhoon Variable Mode Imager (Amersham sion of the same gene in untreated allografts provided the fold change Biosciences). measure. Genes were sorted on the basis of this ratio. A ratio of 1.6-fold Based on previous evidence (12), five biological replicates for each up- or down-regulation (i.e., the fold change value used in SAM) was treatment group were considered adequate to ensure statistical power and required to include genes in the subsequent analysis. Genes that satisfied stability of the results. Further, to assess reproducibility of the technique, this fold-change parameter were then analyzed using the unpaired two- we performed a second, independent hybridization for two randomly cho- tailed Student t test; a probability value Ͻ0.05 was considered significant.

FIGURE 2. Left, Expression profiles of samples from five untreated and five treated allografts harvested on POD4. Macroarray data were analyzed by hierarchical clustering using 1267 genes that passed the quality assurance criteria. Cluster analysis was performed on log2-transformed values of the fold ratios with Cluster and visualized in Treeview. Each column represents a graft sample from individual rats. Each row represents a single gene. The five untreated allografts (POD4U 1–5) clustered in one group whereas NDP-␣-MSH-treated allografts (POD4T 1–5) clustered in a separate group. Difference in expression level (based on the fold change relative to control nontransplanted hearts) is indicated by the scale at the right side. At least two main gene clusters can be identified: (I) genes overexpressed in treated allografts and underexpressed in untreated allografts; and (II) genes underexpressed in treated allografts and overexpressed in untreated allografts. Right, Cluster analysis of 172 genes selected using SAM and the fold-change method. The name of each gene is shown at the right side of each row. Three main clusters can be identified: A, genes repressed in untreated and normal in treated allografts (ion channels, Atp2a2, adenylyl cyclases, signal transduction proteins, glycolipidic metabolism components, transcription factors, and transport/trafficking proteins); B, genes down-regulated in untreated and up-regulated in treated allografts (cytoskeleton proteins, intracellular kinase network and phosphatidylinositol signaling members, protease inhibitors, and Stat3); C, genes induced in untreated allograft and repressed by NDP-␣-MSH therapy (cell adhesion, cell growth, hormones, inflammatory and oxidative stress response, proteasome components, and ribosomal proteins). The Journal of Immunology 3393 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 2. Facing page 3394 EFFECT OF ␣-MSH ON GRAFT TRANSCRIPTIONAL PROFILE

Genes identified using this method were compared with those identified by treatment. Thirty genes were clearly enhanced on POD1 in treated the SAM analysis. Only genes that passed both analyses were considered allografts, whereas 104 genes were up-regulated on POD4 (2.4 and significant. 8.2% of the spotted cDNAs included in the analysis, respectively). Gene classification. Annotation of gene functions was performed com- The proportion of genes down-regulated by treatment was 23 on bining information from several public databases. The selected genes were first analyzed using the web-based, client/server application Database for POD1 and 68 on POD4 (1.8 and 5.4%, respectively). Annotation, Visualization and Integrated Discovery (DAVID version 2.0, With regard to the classes of genes affected by treatment, NDP- ͗http://david.niaid.nih.gov/david/version2/index.htm͘) (16). Genes that re- ␣-MSH-treated allografts showed increased expression of cy- mained unclassified were assigned manually based on information re- toskeleton components (plectin, dystrophin, espin, and Ppp1r9b), trieved from the National Center for Biotechnology Information Entrez Gene Database (͗www.ncbi.nlm.nih.gov/entrez/query.fcgi?db ϭ gene͘), or receptors (Igf1r, Grm7, and Ptprd), molecules associated with sig- from the Stanford Online Universal Resource for Clones and Expressed nal transduction and intracellular signaling cascade (Rgs14, sequence tags (SOURCE) (͗http://genome-www5.stanford.edu/cgi-bin/ Rgs19ip1, Map3k1, Map2k5, Pkn1, Prkce, Dusp1, and Jak3), reg- source/sourceSearch͘) (17). ulation of transcription (Fosl2, Stat3, and St18), glycolipidic me- Pattern identification. Overrepresentation analysis was performed on tabolism (Pfkm, Lipf, and Acox2), and metallopeptidases and pro- genes identified by SAM and unpaired t test using the LocusLink identifiers tease inhibitors (Ace, Ece1, Timp3, and Serpina4), both on POD1 and the Expression Analysis Systematic Explorer tool (EASE version 2.2, ϩ ͗http://apps1.niaid.nih.gov/david/ease1.htm͘) (18). EASE was used to test and POD4. The range of mean fold change varied from 1.6 to Gene Ontology terms (͗www.geneontology.org͘) (19) for “biological pro- ϩ3.9. Conversely, NDP-␣-MSH treatment down-regulated tran- cess” and to identify significantly overrepresented biological themes based scripts related to cell proliferation (Ccng1, Cdk7, Cd53, and on KEGG (͗www.genome.ad.jp/kegg͘) and GenMAPP (͗www.genmapp. H2afz), protein biosynthesis and turnover (laminin receptor, ri- org͘) (20) pathways. bosomal protein L5, Erp29, and proteasome subunit ␤8), im- Downloaded from Real-time reverse transcription PCR analysis mune and inflammatory and/or cell infiltration responses ␤ Expression of six mRNAs in each treatment group was evaluated by real- (Cxcl2, Cxcr4, IL-1 , lysozyme, Hmgb1, mucin 3, and time RT-PCR based on TaqMan methodology. PCR was performed in an Arpc1b), oxide-reduction reactions (Cox6c, peroxiredoxins, ABI PRISM 7000 sequence detection system (Applied Biosystems). The and Hsd17b4), and hormones (Nppa and Gip). The mean fold- assay identification numbers for selected genes were: Rn00563162_m1 for change ranged from Ϫ1.7 to Ϫ6.2. adenylyl cyclase 6 (Adcy6), Rn00586403_m1 for Cxcl2, Rn00571500_m1 for glucose-dependent insulinotropic peptide (Gip), Rn00561661_m1 for http://www.jimmunol.org/ natriuretic peptide precursor type A (Nppa), Rn00566108_m1 for phos- Hierarchical agglomerative clustering of differentially expressed pholipase C␥1 (Plcg1), and Rn00565502_m1 for sodium channel voltage- genes gated type V ␣ polypeptide (Scn5a). Three PCR amplification replicates were performed and averaged for each transcript. To normalize for differ- To identify subsets of coregulated genes, we applied hierarchical ences in the amount of sample RNA added to each reaction mixture, agglomerative clustering to the 172 genes differentially expressed GAPDH was selected as an endogenous control. RNA isolated from con- on POD4 using the log2 transformed expression data. Three gene trol hearts was used as calibrator. Relative quantitation of gene expression clusters could be identified (Fig. 2, right): cluster A included genes (fold change) was performed using the comparative cycle threshold (CT) ⌬⌬ induced in untreated and unchanged in treated allografts; cluster B method ( CT): the amount of target, normalized to the endogenous ref- Ϫ⌬⌬CT contained genes up-regulated in untreated and down-regulated in erence and relative to the calibrator, is given by the formula 2 (21). by guest on September 30, 2021 The unpaired Student t test was used to compare differences in mean fold treated allografts; cluster C consisted of genes down-regulated in changes; a probability value Ͻ0.05 was considered statistically significant. untreated and enhanced in treated allografts.

Results Gene expression changes in untreated and treated allografts Allograft histology relative to isografts ␣ NDP- -MSH treatment reduced the marked pathology observed in To separate effects of NDP-␣-MSH treatment on the rejection pro- untreated heart grafts (Fig. 1). Heart grafts from untreated rats cess from the Ag-independent graft damage due to transplant pro- showed interstitial and perivascular edema and severe inflamma- cedures, gene expression was estimated as the ratio to isografts tory cell infiltration. Both intragraft edema and inflammatory cell (Table II). The peptide inhibited changes specific for mismatched ␣ infiltration were much less in grafts from NDP- -MSH-treated an- allotransplantation, which were only evident in allografts, but it imals: inflammation and edema were confined to the subendocar- also reduced transcriptional modifications related to transplanta- dial region and no abscesses were evident. tion procedures, which was similar in allografts and isografts Hierarchical agglomerative clustering (Table II).

Unsupervised hierarchical agglomerative clustering of array data Verification of the macroarray data using real-time RT-PCR from POD4 allografts indicated that the global expression profile correctly discriminates treated from untreated rats. All samples An independent evaluation of six array-identified genes was per- from NDP-␣-MSH-treated animals clustered separately from sa- formed using real-time RT-PCR. Three of them (Adcy6, Plcg1, ␣ line-treated allografts (Fig. 2, left). The global gene expression and Scn5a) were enhanced by NDP- -MSH-treatment and three profile identified two main gene clusters with opposite trend in were down-regulated (Cxcl2, Gip, and Nppa). The RT-PCR data their expression level (Fig. 2, left): genes overexpressed in treated confirmed all the changes in gene expression disclosed by the mac- and reduced in untreated allografts (cluster I) and genes decreased roarray method, although there were small disparities in magnitude in treated and increased in untreated animals (cluster II). This ob- (Fig. 4). Expressions of Cxcl2, Gip, and Nppa were significantly ␣ servation suggests a distinctive global expression profile associ- inhibited by NDP- -MSH-treatment on both POD1 and POD4, but ated with NDP-␣-MSH treatment. expression was still greater relative to the control level. The peptide totally prevented decrease in expression of Adcy6 on POD4. Treatment-associated gene expression changes SAM analysis and the fold-change method (Table I) identified 53 Functional classification genes whose expression was significantly altered by NDP-␣-MSH Gene classification (Table I) was performed using DAVID and treatment on POD1. Differences were even more marked on POD4 public databases. An EASE overrepresentation analysis of func- (Fig. 3): at this interval 172 genes were modulated by peptide tional gene categories was used to identify biological pathways or The Journal of Immunology 3395

Table I. Genes regulated by NDP-␣-MSH treatment in rat cardiac allografts

Classiﬁcation POD1 T/Ua POD4 T/Ua

GenBank accession no. Gene ID Gene Symbol Gene Name Fold Change Fold Change

Cell adhesion/extracellular matrix ءءU57362 25683 Col12a1 Procollagen, type XII, ␣1 ϭϪ2.5 ءءU41662 117096 Nlgn2 Neuroligin 2 ϭϪ2.3 ءءU78889 84010 DII1 ␦-like 1 (Drosophila) ϭϪ2.1 ءءϪ2.0 ءءU76551 24573 Muc3 Mucin 3 Ϫ1.7 ءءD50568 58826 Prg2 Proteoglycan 2, bone marrow ϭϪ2.0 ءءءL26525 25678 Ddr1 Discoidin domain receptor family, member 1 ϭ 1.8 ءءءX16563 25473 Lamb2 Laminin, ␤2 ϭ 2.4 ءL20468 171517 Gpc2 Glypican 2 ϭ 3.4 ءءءX52140 25118 Itga1 Integrin ␣1 ϭ 3.7 Cell growth and/or maintenance ءءϪ4.1 ءX70871 25405 Ccng1 Cyclin G1 Ϫ1.8 ءءϪ2.2 ءX83579 171150 Cdk7 Cyclin-dependent kinase 7 Ϫ1.9 ءءϪ2.2 ءءM57276 24251 Cd53 CD53 Ag Ϫ2.0 ءءءJ03628 24615 S100a4 S100 calcium-binding protein A4 ϭϪ2.0

Downloaded from ءءءϪ2.0 ءءM37584 58940 H2afz H2A histone family, member Z Ϫ2.2 ءءL15618 116549 Csnk2a1 Casein kinase II, ␣ 1 polypeptide ϭ 2.0 ءءءءM75146 171041 Klc1 Kinesin L chain 1 ϭ 2.0 ءءJ04022 29693 Atp2a2 ATPase, Ca2ϩ transporting, cardiac muscle, slow twitch 2 ϭ 2.1 ءءAJ000696 113886 Klf1c Kinesin 1C ϭ 2.3 ءءAJ223599 85248 Kif3c Kinesin family member 3C ϭ 2.4 Cytoskeleton ءءءAF083269 54227 Arpc1b Actin-related protein 2/3 complex, subunit 1B ϭϪ1.8 http://www.jimmunol.org/ ءء1.9 ءX59601 64204 Plec1 Plectin 2.0 ءءء2.2 ءX69767 24907 Dmd Dystrophin 2.1 ءء2.4 ءU46007 56227 Espn Espin 1.6 ءءءء2.4 ءAF016252 84686 Ppp1r9b Protein phosphatase 1, regulatory subunit 9B 2.9 Electron transport ءءϪ2.6 ءM27466 54322 Cox6c Cytochrome oxidase subunit Vlc Ϫ1.7 ءD13205 64001 Cyb5 Cytochrome b5 ϭϪ1.8 ءءX79991 252931 Cyp3a18 Cytochrome P450, 3a18 ϭ 3.0 Hormones/cytokines ءءϪ6.2 ءL08831 25040 Gip Glucose-dependent insulinotropic peptide Ϫ2.1 by guest on September 30, 2021 ءءءL06441 24315 Dtprp Decidual/trophoblast prolactin-related protein ϭϪ2.3 ءD78591 29201 Ctf1 Cardiotrophin 1 ϭϪ2.3 ءϪ2.2 ءX01118 24602 Nppa Natriuretic peptide precursor type A Ϫ2.4 ءءءءK02809 24952 Gcg Glucagon ϭ 2.1 ءءءءM32167 83785 Vegf Vascular endothelial growth factor A ϭ 2.3 ءءءM31603 24695 Pthlh Parathyroid hormone-like peptide ϭ 2.4 Immune response ءءϪ2.4 ءU45965 114105 Cxcl2 Chemokine (CXC motif) ligand 2 Ϫ2.7 ءءءءX75305 24812 Tap2 Transporter 2, ATP-binding cassette, subfamily B (MDR/TAP) ϭϪ2.3 ءءϪ2.0 ءU54791 60628 Cxcr4 Chemokine (CXC motif) receptor 4 Ϫ1.7 ءءϪ1.9 ءءM98820 24494 Il1b IL-1␤ Ϫ3.3 ءءJ03606 25047 Fcer1a FcR, IgE, high afﬁnity I, ␣ polypeptide ϭϪ1.9 ءءءϪ1.9 ءءL12458 25211 Lyz lysozyme Ϫ1.9 ءU49066 171106 Il1rl2 IL-1 receptor-like 2 ϭ 2.0 ءL02926 25325 Il10 IL-10 nd 3.2 Intracellular-signaling cascade ءءD82363 29192 Psen1 Presenilin 1 ϭ 1.7 ءءU28356 246781 Heptp Protein-tyrosine phosphatase, nonreceptor type 7 ϭ 1.9 ءء2.1 ءX84004 114856 Dusp1 Dual speciﬁcity phosphatase 1 1.9 ء2.4 ءU25281 259242 Cr16 SH3 domain-binding protein CR16 1.6 ءءءM96159 64532 Adcy5 Adenylyl cyclase 5 ϭ 2.6 ءءءءءL01115 25289 Adcy6 Adenylyl cyclase 6 ϭ 4.4 Intracellular kinase network members ءءD31874 29524 Limk2 LIM motif-containing protein kinase 2 ϭ 1.7 ءءء1.7 ءU37462 29568 Map2k5 MAPK kinase 5 1.9 ءءءءD28508 25326 Jak 3 JAK 3 ϭ 1.7 ءءX94351 171305 Clk3 CDC-like kinase 3 ϭ 1.8 ءءM63334 25050 Camk4 Calcium/calmodulin-dependent protein kinase IV ϭ 1.8 ءءX68400 81749 Prkch PKC␩ ϭ 1.8 ءءء1.9 ءM18331 29340 Prkce PKC␧ 1.6 ءء2.1 ءU48596 116667 Map3k1 MAPK kinase kinase 1 1.7 ءءء2.1 ءD26180 29355 Pkn1 Protein kinase N1 1.9 ءءءL13408 24246 Camk2d Calcium/calmodulin-dependent protein kinase II, ␦ ϭ 2.1 ءءءM19007 25023 Prkcb1 PKC␤1 ϭ 2.6 Phosphatidylinositol-signaling system ءءءءءD84667 81747 Pik4cb Phosphatidylinositol 4-kinase, catalytic, ␤ polypeptide ϭ 2.0 (Table continues) 3396 EFFECT OF ␣-MSH ON GRAFT TRANSCRIPTIONAL PROFILE

Table I. Continued

POD1 POD4 Classiﬁcation T/Ua T/Ua

GenBank accession no. Gene ID Gene Symbol Gene Name Fold Change Fold Change

ءءءءX74227 54260 Itpkb Inositol 1,4,5-trisphosphate 3-kinase B ϭ 2.1 ءءD64045 25513 Pik3r1 PI3K regulatory subunit, polypeptide 1 ϭ 2.3 ءءAF033355 89812 Pip5k2b Phosphatidylinositol-4-phosphate 5-kinase, type II, ␤ ND 2.4 ءءءU55192 54259 Inpp5d Inositol polyphosphate-5-phosphatase D ND 2.4 ءU96920 116699 Inpp4b Inositol polyphosphate-4-phosphatase, type II, 105 kDa ND 2.6 ءءJ05155 29337 Plcg2 Plc, ␥2 ϭ 2.0 ءM20636 24654 Plcb1 Plc, ␤1 ϭ 2.0 ءAJ011035 85240 Plcb2 Plc, ␤2 ND 2.3 ءءءءءJ03806 25738 Plcg1 Plc, ␥1 ϭ 2.6 Ion channels ءءءءU38665 25262 Itpr1 Inositol 1,4,5-triphosphate receptor 1 ϭ 1.9 ءءءZ96106 117018 Kcnh2 Potassium voltage-gated channel, subfamily H (eag-related), ϭ 2.3 member 2 ءءء2.4 ءZ67744 29233 Clcn7 Chloride channel 7 1.7

Downloaded from ءL35771 29713 Kcnj5 Potassium inwardly-rectifying channel, subfamily J, member 5 ϭ 2.5 ءءX06656 24392 Gja1 Gap junction membrane channel protein ␣1 ϭ 2.6 ءءءAB008889 84494 Trpc4 Transient receptor potential cation channel, subfamily C, member 4 ϭ 2.6 ءءءءM27902 25665 Scn5a Sodium channel, voltage-gated, type V, ␣, polypeptide ϭ 3.6 Metabolism, carbohydrate ءءءU08027 25062 Gpd2 Glycerol-3-phosphate dehydrogenase 2 ϭ 1.9 ءء2.0 ءU25651 65152 Pfkm Phosphofructokinase, muscle 1.7 ءءءD87240 117276 Pfkfb3 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 ϭ 2.0 /http://www.jimmunol.org ءD16102 79223 Gyk Glycerol kinase ND 3.3 Metabolism, lipid ءϪ2.1 ءءU37486 79244 Hsd17b4 Peroxisomal multifunctional enzyme type II Ϫ1.8 ءءL36250 24849 Tpi1 Triosephosphate isomerase 1 ϭϪ1.9 ءءU42411 83803 Prkab1 Protein kinase, AMP-activated, ␤1 noncatalytic subunit ϭ 1.7 ءءءء2.0 ءX02309 50682 Lipf Lipase, gastric 1.6 ءءJ05029 25287 Acadl Acetyl-coenzyme A dehydrogenase, long-chain ϭ 2.0 ءءءU32314 25104 Pc Pyruvate carboxylase ϭ 2.0 ءءD43623 25756 Cpt1b Carnitine palmitoyltransferase 1b ϭ 2.1 ءءU36771 29653 Gpam Glycerol-3-phosphate acyltransferase, mitochondrial ϭ 2.2

by guest on September 30, 2021 ءء2.8 ءX95189 252898 Acox2 Acyl-coenzyme A oxidase 2, branched chain 1.8 Metabolism, nucleic acid ءءءءD13374 191575 Nme1 Expressed in nonmetastatic cells 1 ϭϪ2.7 ءءءءM91597 83782 Nme2 Nucleoside diphosphate kinase ϭϪ2.2 ءءءءD89514 81643 Atic 5-Aminoimidazole-4-carboxamide ribonucleotide formyltransferase/ ϭ 1.7 IMP cyclohydrolase ءءءءU18942 81635 Adar Adenosine deaminase, RNA-speciﬁc ϭ 1.7 Metabolism, other ءءU40803 25120 Aanat Arylalkylamine N-acetyltransferase ϭ 1.8 ءء1.9 ءءM58364 29244 Gch GTP cyclohydrolase 1 1.6 ءU70825 116684 Oplah 5-oxoprolinase (ATP-hydrolysing) ϭ 1.9 ءءءD83479 81718 Cdo1 Cytosolic cysteine dioxygenase 1 ϭ 2.1 ءL20427 29309 Coq3 Coenzyme q (ubiquinone) biosynthetic enzyme 3 ϭ 2.2 ءءU53505 65162 Dio2 Deiodinase, iodothyronine, type II ϭ 2.9 Neurophysiological process ءءAB000776 84609 Sema6b Semaphorin 6B ϭϪ2.3 ءءU35099 116657 Cplx2 Complexin 2 ϭϪ2.0 ءءءءAF007583 29755 Colq Collagen-like tail subunit of asymmetric acetylcholinesterase ϭ 2.1 ءءAF044201 246274 Faim2 Fas apoptotic inhibitory molecule 2 ϭ 2.5 ءءءءءX56541 81651 Cspg4 Membrane-spanning proteoglycan NG2 ϭ 3.3 Protein biosynthesis ءءM18547 65139 Rps12 Ribosomal protein S12 ϭϪ2.6 ءءءءX53504 Rpl12 Ribosomal protein L12 ϭϪ2.2 ءءX13549 81773 Rps10 Ribosomal protein S10 ϭϪ2.1 ءءϪ2.1 ءءM27798 29236 Lamr1 Laminin receptor 1 (67 kDa ribosomal protein SA) Ϫ2.6 ءءءX68283 29283 Rpl29 Ribosomal protein L29 ϭϪ2.1 ءءX78327 81765 Rpl13 Ribosomal protein L13 ϭϪ2.0 ءϪ1.9 ءX06148 81763 Rpl5 Ribosomal protein L5 Ϫ1.8 ءءX51707 29287 Rps19 Ribosomal protein S19 ϭϪ1.9 ءءءX62146 Rpl11 Ribosomal protein L11 ϭϪ1.9 ءءءX93352 81729 Rpl10a Ribosomal protein L10a ϭϪ1.9 ءϪ1.9 ءX52445 81776 Rps24 Ribosomal protein S24 Ϫ1.6 ءءZ29530 64205 Arbp Acidic ribosomal protein P0 ϭϪ1.9 ءءM19635 81769 Rpl36a Large subunit ribosomal protein L36a ϭϪ1.8 ءءX06483 28298 Rpl32 Ribosomal protein L32 ϭϪ1.8 ءءX14210 29426 Rps4x Ribosomal protein S4, X-linked ϭϪ1.8 (Table continues) The Journal of Immunology 3397

Table I. Continued

Classiﬁcation POD1 T/Ua POD4 T/Ua

GenBank accession no. Gene ID Gene Symbol Gene Name Fold Change Fold Change

ءءءءX06423 65136 Rps8 Ribosomal protein S8 ϭϪ1.8 ءءM20156 81766 Rpl18 Ribosomal protein L18 ϭϪ1.8 ءX53377 29258 Rps7 Ribosomal protein S7 ϭϪ1.7 Protein turnover ءϪ2.0 ءU36482 117030 Erp29 Endoplasmic reticulum protein 29 Ϫ2.2 ءءϪ2.0 ءD10729 24968 Psmb8 Proteasome (prosome, macropain) subunit, ␤ type 8 Ϫ2.0 ءءD10754 29666 Psmb6 Proteasome (prosome, macropain) subunit, ␤ type 6 ϭϪ1.7 ءءءء2.1 ءM86389 24471 Hspb1 Heat shock 27-kDa protein 1 1.6 ءءU56407 79435 Ube2d2 Ubiquitin-conjugating enzyme E2D 2 ϭ 2.2 Metallopeptidase ءءءM15944 24590 Mme Membrane metalloendopeptidase ϭϪ2.0 ءءءءU61696 81761 Rnpep Aminopeptidase B ϭ 1.7 ءء2.0 ءU03734 24310 Ace Angiotensin 1-converting enzyme 1 2.3 ءءءء2.0 ءD29683 94204 Ece1 Endothelin-converting enzyme 1 1.7 ءء2.4 ءU62897 25306 Cpd Carboxypeptidase D 1.6

Protease inhibitor Downloaded from ءءء1.9 ءءU27201 25358 Timp3 Tissue inhibitor of metalloproteinase 3 1.7 ءء2.3 ءU51017 246328 Serpina4 Serine (or cysteine) proteinase inhibitor, clade, member 4 2.0 Response to oxidative stress ءءU33935 29151 Ucn Urocortin ϭϪ2.5 ءءءءY00404 24786 Sod1 Superoxide dismutase 1 ϭϪ2.1 ءءءءϪ2.1 ءU06099 29338 Prdx2 Peroxiredoxin 2 Ϫ1.7 ءءJ03752 171341 Mgst1 Microsomal glutathione S-transferase 1 ϭϪ2.0 http://www.jimmunol.org/ ءءءU25264 25545 Sepw1 Selenoprotein W, muscle 1 ϭϪ1.9 ءءϪ1.9 ءءD30035 117254 Prdx1 Peroxiredoxin 1 Ϫ2.4 ءءϪ1.7 ءAF058787 Ho3 Heme oxygenase 3 Ϫ1.6 ءءءX62404 113919 Gpx5 Glutathione peroxidase 5 ϭϪ1.7 Signal transduction Receptors ءءU35025 245921 Acvr1c Activin receptor-like kinase 7 ϭϪ2.2 ءءءءL00091 24179 Agt Angiotensinogen ϭϪ1.7 ء2.0 ءءL19181 25529 Ptprd Protein tyrosine phosphatase, receptor type, D 1.7 ءءJ03025 81645 Chrm2 Cholinergic receptor, muscarinic 2 ϭ 2.4 by guest on September 30, 2021 ءءءءء2.5 ءءءL29232 25718 Igﬂr Insulin-like growth factor 1 receptor 2.0 ء2.9 ءD16817 81672 Grm7 Glutamate receptor, metabotropic 7 2.3 G-protein/GTPase activity modulators ءX76453 24913 Hrasls3 HRAS like suppressor ϭϪ2.5 ءءءءD31962 58834 Dlc1 Deleted in liver cancer 1 ϭ 2.0 ءء2.1 ءU92279 114705 Rgs14 Regulator of G-protein signaling 14 1.6 ءءAF044673 114559 Pak3bp PAK-interacting exchange factor ␤ ϭ 2.1 ءءءAF058789 192117 Syngap1 Synaptic Ras GTPase-activating protein 1 ϭ 2.5 ءءء3.8 ءAF032120 83823 Rgs19ip1 Regulator of G-protein signaling 19-interacting protein 1 2.4 Transcription ءءϪ1.7 ءM64986 25459 Hmgb1 High mobility group box 1 Ϫ2.2 ءءءAJ005425 81518 Mef2d Myocyte enhancer factor 2D ϭ 1.7 ءء1.9 ءU18913 25446 Fosl2 fos-like Ag 2 1.8 ءء2.0 ءءX91810 25125 Stat3 Signal transducer and activator of transcription 3 1.9 ءءءX14788 81646 Creb1 cAMP response element binding protein 1 ϭ 2.0 ءءU24175 24918 Stat5a Signal transducer and activator of transcription 5A ϭ 2.3 ءءJ03933 24831 Thrb Thyroid hormone receptor ␤ ϭ 2.4 ء2.9 ءءءU67080 266680 Stl8 Suppression of tumorigenicity 18 3.1 Transport/trafﬁcking ءU75581 79451 Fabp4 Fatty acid binding protein 4, adipocyte ϭϪ4.8 ءϪ2.3 ءU13253 140868 Fabp5 Fatty acid binding protein 5, epidermal Ϫ2.4 ءءJ03583 54241 Cltc Clathrin, heavy polypeptide (Hc) ϭϪ2.0 ءءءX61654 29186 Cd63 CD63 Ag ϭϪ2.0 ءءءU21871 266601 Tomm20 Translocase of outer mitochondrial membrane 20 homologue ϭϪ1.7 (yeast) ءءءS68944 Ntt4 Sodium/chloride-dependent neurotransmitter transporter ϭ 1.9 ءءL19931 29502 Slc20a2 Solute carrier family 20, member 2 ϭ 2.0 ءX74226 171434 Phldb1 Pleckstrin homology-like domain, family B, member 1 ϭ 2.1 ءءءAF068202 114124 Akap1 A kinase (PRKA) anchor protein 1 ϭ 2.1 ءءء2.2 ءءAF022774 171123 Rph3al Rabphilin 3A-like (without C2 domains) 2.2

.p Ͻ 0.00001 ,ءءءءء ;p Ͻ 0.0001 ,ءءءء ;p Ͻ 0.001 ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .a Treated/untreated allografts

gene groups. Changes in ﬁve functional categories/biological pro- response ( p Ͻ 0.05), protein amino acid phosphorylation ( p Ͻ 0.01), cesses were signiﬁcantly associated with NDP-␣-MSH treatment: intracellular signaling cascade ( p Ͻ 0.01), and lipid metabolism ribosome biogenesis and assembly ( p Ͻ 0.0001), oxidative stress ( p Ͻ 0.01). Transcription of two cellular/metabolic pathways was 3398 EFFECT OF ␣-MSH ON GRAFT TRANSCRIPTIONAL PROFILE

isografts (Tables I and II). The cardiac Ca2ϩ-ATPase encoded by Atp2a2 is a sarcoplasmic reticulum protein involved in calcium transport and cycling in the heart. It plays an essential role in myocyte contraction and relaxation and in the Ca2ϩ channel kinetics (25). Atp2a2 improves cardiac muscle contractility in vivo and in vitro (26) and its expression in cardiomyocytes is selec- tively regulated by protein kinase C (PKC) isoenzymes PKC⑀ and PKC␦ (27). A decrease in Atp2a2 and the consequent impaired Ca2ϩ kinetics appear to be associated with ventricular hypertrophy and congestive heart failure (28, 29). Further, decreased Atp2a2 expression was observed in murine heart isografts after prolonged cold ischemia and reperfusion (30). Therefore, these observations point at the importance of normalization of Atp2a2 by NDP-␣-MSH. The increase in phospholipase C (Plc) isoenzyme mRNA observed in NDP-␣-MSH-treated transplanted hearts (Table I, Fig. 4) indicates yet another protective effect on key molecules involved in the regulation of myocardial function. Phosphoinositide-speciﬁc

Plc isoenzymes play a central role in activating intracellular signal Downloaded from transduction pathways. Their physiological substrate, phosphatidylinositol 4,5-bisphosphate, is converted to two messenger mol- FIGURE 3. Identification of genes whose expression change on POD4 was potentially significant at SAM analysis. Scatter plot of the observed ecules, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, which participate in many different physiological processes within cardi- relative expression difference d(i) vs the expected relative difference d⑀(i) 2ϩ of genes altered by NDP-␣-MSH treatment. The solid line indicates where omyocytes, including Ca movements (31, 32). genes would align if their d(i) ϭ d⑀(i). At the threshold ⌬ϭ0.60 (distance Decreased adenylyl cyclase activity was observed in human http://www.jimmunol.org/ from the solid line drawn as dotted lines) and fold change Ն1.60, SAM myocardium after orthotopic cardiac transplantation (33). Recent predicts 179 genes as being differentially regulated. The FDR was Ͻ2%. research indicates that adenylyl cyclase VI (Adcy6) expression The scatter plot shows significantly up-regulated genes as r, and signifi- improves heart function and abrogates myocardial hypertrophy cantly down-regulated genes as Ⅺ. Genes that passed also the second anal- (34–36). The present investigation confirms a substantial reduc- ysis based on unpaired two-tailed Student’s t test are reported in Table I. tion of Adcy6 in transplanted hearts relative to control hearts and indicates that Adcy6 is normalized by NDP-␣-MSH treatment (Ta- significantly activated by treatment: the phosphatidylinositol ble I, Fig. 4). signaling system ( p Ͻ 0.001) and the fatty acid degradation ( p Ͻ Further, there is evidence that ␣-MSH participates in calcium 0.05). regulation in both the cytosol and sarcoplasmic reticulum of car- by guest on September 30, 2021 diac cells. This likely occurs via coordinated up-regulation of cy- Discussion toplasmic, cytosolic, and sarcoplasmic proteins (Table I). Indeed, The present data, based on gene expression profiling, reveal mul- the muscarinic receptor Chrm2, the G protein-controlled inward- tiple protective influences exerted by NDP-␣-MSH that could ac- lyrectifying potassium channel Kcnj5, the adenylyl cyclases count for the reduced damage in transplanted heart grafts. Indeed, Adcy5 and Adcy6, the receptor regulated cation channel Trpc4, peptide treatment caused substantial up-regulation of several sal- and the gap junction component Gja1 are all proteins integral to utary molecules including signal transduction mediators, metallo- cytoplasmic membrane that were induced by NDP-␣-MSH. The proteinases, serine proteases, energy pathway mediators, and ion regulator of G-protein signaling Rgs14, protein kinase C ␩, and ⑀ channels. Concurrent down-regulation of growth factors, cyto- isoenzymes, Plc ␤ and ␥ isoenzymes, and calcium/calmodulin- kines, chemokines, oxidative stress mediators, and ribosomal pro- dependent protein kinases Camk2d and Camk4 are cytosolic proteins likely contributes to preserve myocardium from injury. teins collectively involved in the regulation of Ca2ϩ influx. Their The main finding of the present investigation is that the protec- expression was clearly restored by NDP-␣-MSH. Finally, NDP- tive influences of NDP-␣-MSH in heart transplantation are not ␣-MSH treatment induced Atp2a2 and the IP3 receptor Itpr1 that restricted to the anti-inflammatory/anti-cytokine effects of the pep- are key sarcoplasmic genes involved in the Ca2ϩ channel kinetics. tide (4–8, 22). Indeed, treatment preserved molecules of para- The voltage-gated sodium channel Scn5a drives the initial de- mount importance for myocardial function. At least five metabolic/ polarization phase of the cardiac action potential and, therefore, regulatory pathways were significantly altered by NDP-␣-MSH participates in conduction of excitation through the heart (37, 38). treatment (Table I, Fig. 2): three of them were enhanced–intracel- Deletions or loss-of-function mutations of the human gene SCN5A lular signaling cascade, protein amino acid phosphorylation, and have been associated with a wide range of arrhythmias (39, 40), glycolipidic metabolism; two were repressed–ribosome biogenesis and targeted disruption of murine Scn5a slowed conduction and and response to oxidative stress. In addition, NDP-␣-MSH markedly caused ventricular tachycardia (41). The present data indicate a inhibited expression of Hmgb1 and S100a4, proteins belonging to the reduction of Scn5a in cardiac allografts relative to isografts and family of damage-associated molecular pattern molecules. These are control hearts (Table II, Fig. 4), and show virtual normalization of a recently recognized group of molecules, naturally expressed in Scn5a by NDP-␣-MSH treatment on POD4 (Table I, Fig. 4). the nucleus or cytosol, that are released upon tissue damage or JAKs, STATs, and PI3K provide a critical survival pathway to injury; they are believed to initiate inflammation and innate im- cardiomyocytes in vivo. Recent research shows that activation of mune responses (23) and are significant targets for novel anti- the JAK/STAT pathway transduces cytoprotective signals in rat inflammatory/immunomodulatory treatments (24). hearts subjected to acute pressure overload, myocardial infarction The effects of treatment were very broad. NDP-␣-MSH pre- (42), or doxorubicin-induced cardiomyopathy (43). Conversely, served Atp2a2 expression that was reduced in both allografts and patients with end-stage dilated cardiomyopathy had impaired The Journal of Immunology 3399

Table II. Gene expression in untreated and NDP-␣-MSH-treated allografts relative to isograftsa

Classiﬁcation Untreated Allo./ Treated Allo./ Classiﬁcation Untreated Allo./ Treated Allo./ Isograft Isograft Isograft Isograft Gene SymbolFold Change Fold Change Gene Symbol Fold Change Fold Change

Cell adhesion/extracellular matrix Neurophysiological process ϭ ءSema6b 2.8 ءNlgn2 ϭϪ2.4 ϭ ءϭ Cplx2 3.2 ءDdr1 Ϫ2.4 ءء11.5 ءCspg4 3.5 ءءLamb2 ϭ 2.9 Itga1 Ϫ5.6 ϭ Protein biosynthesis ϭ ءCell growth and/or maintenance Rps12 2.0 ϭ ءءRpl12 2.6 ءS100a4 ϭϪ1.6 ء2.7 ءRps10 5.0 ءءAtp2a2 ϭ 1.9 ءءRpl29 ϭϪ2.0 ءϪ2.1 ءءKif1c Ϫ4.7 ءCytoskeleton Rpl13 ϭϪ2.1 ءRpl5 ϭϪ1.8 ءϪ1.7 ءءPlec1 Ϫ3.2 ϭ ءElectron transport Rps19 2.5 ءϭ Rpl11 ϭϪ1.6 ءCox6c 3.6 ءءءRpl10a ϭϪ1.9 ءCyb5 ϭϪ1.6 ءHormones/cytokines Arbp ϭϪ1.6 ءRpl32 ϭϪ2.5 ءNppa ϭϪ2.0 Downloaded from ءRps4x ϭϪ1.6 ءءVegf ϭ 2.2 ءImmune response Rps8 ϭϪ1.6 ءϭ Rpl18 ϭϪ1.8 ءCxcl2 3.0 ءRps7 ϭϪ1.6 ءءFcer1a ϭϪ2.3 Protein turnover ءء4.5 ءءLyz 7.8 ءء4.0 ءءIntracellular signaling cascade Erp29 8.0 ءϪ3.8 ءϭ Hspb1 Ϫ7.7 ءءPsen1 Ϫ1.7 ءء ϭ ϭ ء Ϫ

Dusp1 1.9 Rnpep 1.6 http://www.jimmunol.org/ ϭ ءϭ Ece1 Ϫ1.6 ءAdcy5 Ϫ2.1 ءءءءءCpd ϭ 2.5 ءAdcy6 ϭ 2.4 ءء3.4 ءTimp3 1.8 ءϪ2.3 ءLimk2 Ϫ4.0 Response to oxidative stress ءءϪ1.6 ءءءMap2k5 Ϫ2.7 ءءUcn ϭϪ3.5 ءCamk4 ϭ 1.6 ϭ ءءSod1 1.8 ءPrkch ϭ 1.6 ءϭ Prdx2 ϭϪ3.0 ءءPrkce Ϫ2.9 ءMgst1 ϭϪ1.6 ءءءϪ1.7 ءءءءPik4cb Ϫ3.5 ءءءϪ3.4 ءءءSepw1 Ϫ2.0 ءItpkb ϭ 2.8 ء2.3 ءPrdx1 4.0 ءPik3r1 1.7 4.0

ϭ by guest on September 30, 2021 ءGpx5 1.6 ءءPlcg1 ϭ 3.2 Ion channels Signal transduction ء2.0 ءءAgt 3.2 ءKcnh2 ϭ 1.9 ϭ ءPtprd Ϫ1.6 ءϪ2.7 ءءءKcnj5 Ϫ6.2 ءIgﬂr ϭ 2.2 ءءϪ2.3 ءءGja1 Ϫ4.5 ء13.7 ءϭ Hrasls3 34.7 ءScn5a Ϫ4.4 ءMetabolism, carbohydrate Dlc1 ϭ 1.7 ءRgs14 ϭ 1.7 ءء3.6 ءGpd2 1.9 Transcription ءء5.0 ءPfkfb3 2.4 ءءءHmgb1 ϭϪ1.8 ءPfkm ϭ 1.6 ϭ ءMetabolism, lipid Mef2d Ϫ1.7 ءFosl2 ϭ 2.4 ء1.6 ءHsd17b4 3.3 ءStat3 ϭ 2.2 ء4.2 ءءTpi1 7.8 ϭ ءءSt18 Ϫ3.9 ءPrkab1 ϭ 1.9 Transport/trafﬁcking ءءLipf ϭ 2.0 ءءϪ18.5 ءϭ Fabp4 Ϫ4.5 ءءAcad1 Ϫ2.7 ϭ ءϭ Fabp5 3.4 ءCpt1b Ϫ2.4 ء2.9 ءMetabolism, nucleic acid Cltc 5.5 ءءءϪ2.8 ءCd63 Ϫ1.6 ءءNme1 ϭϪ3.3 ءءءءϪ2.4 ءءءNtt4 Ϫ4.6 ءءNme2 ϭϪ2.2 ϭ ءSlc20a2 Ϫ2.7 ءءء4.6 ءءAtic 2.7 ϭ ءMetabolism, other Phldb1 Ϫ1.7 ءءAkap1 ϭ 3.0 ء6.7 ءOplah 3.4 ϭ ءRph3a1 Ϫ3.6 ءءϪ2.2 ءءDio2 Ϫ6.2

.p Ͻ 0.00001 ,ءءءءء ;p Ͻ 0.0001 ,ءءءء ;p Ͻ 0.001 ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ءa downstream activation of the JAK/STAT pathway (44). This path- in vitro experiments indicate that Prkce plays a major role in car- way, involved in the synthesis of key myocardial molecules, is dioprotection against hypoxic or ischemia/reperfusion injury in the transcriptionally regulated by NDP-␣-MSH as suggested by the heart (45–47). NDP-␣-MSH treatment preserved expression of increase in Jak3, Stat3, Stat5a, and Pik3r1 mRNA in treated allo- Prkce that was reduced in both allografts and isografts (Tables I grafts (Table I, Fig. 2). and II, Fig. 2). Another critical pathway for myocardial function involves in- Temporal analysis of transcriptional changes (Table I) allowed duction, activation and translocation of PKC isoenzymes, and, in distinction between early and late effects of the peptide. The early particular, of the myoﬁlament-associated Prkce. In vivo and response to NDP-␣-MSH treatment includes induction of most 3400 EFFECT OF ␣-MSH ON GRAFT TRANSCRIPTIONAL PROFILE Downloaded from FIGURE 5. Effects of NDP-␣-MSH treatment on Ag-dependent and independent injury.

proteins that were decreased in allografts remained unaffected in isografts. Treatment with NDP-␣-MSH prevented most of the http://www.jimmunol.org/ changes induced by genetic mismatch in allografts, but the peptide also improved Ag-independent gene expression, linked to mechan- FIGURE 4. Verification of array data by real-time RT-PCR. Consistent ical damage and reperfusion (Fig. 5). with the macroarray data, Adcy6, Scn5a, and Plcg1 transcripts were down- Despite its anti-inflammatory and cardioprotective influences, regulated in allografts relative to isografts and controls; NDP-␣-MSH did NDP-␣-MSH did not eventually prevent rejection (3). A possible not alter expression of these transcripts in treated allografts on POD1, but reason for this failure is that the peptide did not abolish intragraft did induce them on POD4 Cxcl2; Gip and Nppa were up-regulated in expression of certain chemokines that have been associated with allografts compared with isografts and controls, and were significantly in-

cardiac allograft rejection (49), including chemokines Ccl3, Ccl4, by guest on September 30, 2021 hibited by NDP-␣-MSH treatment in allografts on both POD1 and POD4. and Cxcl10. In addition, the peptide did not reduce expression of Data are expressed as fold change of the targeted gene relative to control -p Ͻ other putative mediators of acute rejection, including allograft in ,ءء ;p Ͻ 0.05 ,ء .hearts. Bars denote mean Ϯ SEM of specific mRNA ␥ p Ͻ 0.0001. flammatory factor 1, IFN- , IFN regulatory factor 1, and leukocyte ,ءءءء ;p Ͻ 0.001 ,ءءء ;0.01 common Ag (50, 51) (data not shown). Therefore, it appears that there are mediators or pathways that escape the inhibitory effects of NDP-␣-MSH. This is not surprising as rejection prevention re- cytoskeleton components, intracellular kinase network members, quires profound immunosuppression that is clearly not exerted by signal transduction receptors and transcription regulators, metal- NDP-␣-MSH. lopeptidases, and protease inhibitors. Repression of immune and NDP-␣-MSH is very safe. The peptide had no toxic effects in inflammatory response, cell cycle, Nppa, and proteasome compo- preclinical studies (52); further, the peptide was injected s.c. in nents likewise occurs in the early phase of peptide treatment. human subjects over 12 days and blood tests revealed no changes Among the early effects of NDP-␣-MSH treatment, the restoration (53). The present research indicates multiple protective influences of mRNA levels of key cytoskeleton components is of particular of the peptide that could enhance effectiveness of immunosuppres- interest. Indeed, dystrophin (Dmd) is a vital component of a mus- sive drugs in transplantation. cle sarcolemma membrane-spanning complex that connects cytoskeleton to basal lamina. Loss of intracellular dystrophin is believed to contribute to myocardial reperfusion injury (48), and Acknowledgments restoration of its production can therefore protect the heart from J. M. Lipton’s participation in this research is based on a long-standing scientific cooperation with Dr. Anna Catania on ␣-MSH research. this early injury after transplantation. Conversely, alterations in cell adhesion and extracellular matrix proteins, induction of phos- Disclosures phatidylinositol signaling system, glycolipidic metabolism, ion J. M. Lipton currently serves on the Board of Directors of Zengen. channels, and the anti-inflammatory cytokine IL-10, and repression of ribosome biosynthetic pathway and response to oxidative References stress are late effects of NDP-␣-MSH treatment. 1. Pascual, M., R. D. Swinford, J. R. Ingelfinger, W. W. Williams, A. B. Cosimi, The differences in gene expression profiles of allografts and and N. Tolkoff-Rubin. 1998. Chronic rejection and chronic cyclosporin toxicity isografts (Table II) allowed discrimination of damage caused by in renal allografts. Immunol. Today 19: 514–519. transplantation procedures from injury linked to genetic mismatch. 2. Dallman, M. J. 1999. Immunobiology of graft rejection. In Transplantation. L. C. Ginns, A. B. Cosimi, and P. J. Morris, eds. Blackwell Science, Malden, pp. Indeed, allografts–but not isografts–showed increased expression 23–35. of immune response mediators, neuro-related proteins, and certain 3. Gatti, S., G. Colombo, R. Buffa, F. Turcatti, L. Garofalo, N. Carboni, L. Ferla, L. R. Fassati, J. M. Lipton, and A. Catania. 2002. ␣-Melanocyte-stimulating ribosomal genes. Further, some structural proteins, intracellular hormone protects the allograft in experimental heart transplantation. Transplan- kinase network members, ion channels, and fatty acid degradation tation 74: 1678–1684. The Journal of Immunology 3401

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