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 α-melanocyte-stimulating hormone (α-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 synthetic α-MSH analog Nle4DPhe7 (NDP)-α-MSH. On postoperative day 1, the peptide induced expression of cytoskeleton proteins, intracellular kinases, transcription regulators, metallopeptidases, and protease inhibitors. Conversely, NDP-α-MSH repressed immune, inflammatory, cell cycle, and protein turnover mediators. Later effects of α-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-α-MSH exerted its effects on both Ag-dependent and -independent injury. The results indicate that NDP-α-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.

Acute rejection is a significant obstacle to successful organ transplantation and its prevention is crucial for favorable clinical outcome. Although immunosuppressive molecules can reduce rejection, they are associated with serious side effects such as organ toxicity, increased viral infection, and cancer (1). Because most of these harmful effects are dose-dependent, reduction of immunosuppressive drug treatment necessary to prevent rejection is a major clinical target. As intragraft inflammation is known to promote and accelerate rejection (2), use of anti-inflammatory compounds that enhance effectiveness of immunosuppressive agents could be a successful strategy.

Previous research on experimental heart transplantation showed that treatment with the immunomodulatory peptide α-melanocyte-stimulating hormone (α-MSH)3 prolongs survival and improves allograft histopathology (3). Such beneficial effects were associated with reduced intragraft expression of cytokines, chemokines, and adhesion molecules (3). α-MSH or its synthetic analogues may soon be used clinically as truly novel anti-inflammatory/immunomodulatory compounds (4, 5, 6, 7, 8). Therefore, we designed research to determine the molecular mechanism underlying the protective effects of the peptide. Using complement DNA arrays, an established technique for identification of pathways involved in transplant rejection and its prevention (9), we found multiple protective influences of α-MSH in experimental heart transplantation.

Adult inbred Brown Norway and Lewis male rats (Charles River Laboratories) weighing 200–300 g were used in the research. All animals received care in compliance with the Principles of Laboratory Animal Care, formulated by the National Society of Medical Research, and the Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Sciences and published by the National Institutes of Health (National Institutes of Health Publication No. 86–23).

Rats were anesthetized with a combination of 100 mg/kg ketamine and 6 mg/kg xylazine injected i.p. During anesthesia, heart rate, ventilation rate, and temperature were closely monitored. Brown Norway donor hearts were transplanted into either the MHC incompatible Lewis rats (allografts) or into Brown Norway rats (isografts). The donor heart was transplanted heterotopically into the abdominal cavity of the recipient using the technique described by Ono and Lindsey (10). All cardiac transplants had good initial contractile function. Graft function was monitored by palpation through the abdominal wall twice daily. There were no early deaths nor graft rejections during the study period. At each planned interval, rats were euthanized with thoracotomy under ketamine and xylazine anesthesia. The abdomen was incised and the heart grafts were immediately removed.

Each treatment group included five rats. Allograft recipients assigned to active treatment received i.p. injections of 100 μg of Nle4DPhe7 (NPD)-α-MSH (11) (kindly provided by Prof. P. Grieco, University of Naples, Naples, Italy) dissolved in 0.5 ml of saline, every 12 h. Treatment was started 1 h before transplantation and continued until sacrifice. Untreated allograft recipients and isograft recipients received i.p. parallel injections of 0.5 ml of saline.

Cardiac isografts were used to estimate heart injury caused by surgical procedures alone and were harvested on postoperative day (POD) 1. Allografts were harvested on POD 1 or 4. Two Brown Norway donor hearts were subjected to cold ischemia of similar duration and not transplanted. They served as nontransplanted controls. Heart grafts were sectioned coronally. Two sections were snap-frozen in liquid nitrogen and stored at −80°C for RNA extraction. One section was fixed in 10% buffered formalin and paraffin-embedded for light microscopy examination.

Frozen tissue samples were homogenized with an Ultra-Turrax tissue homogenizer (IKA Labortechnik) and total RNA was isolated using the Atlas Pure Total RNA extraction kit (BD Biosciences/Clontech), according to the manufacturer’s instructions. Analysis of gene expression was performed using Clontech Atlas Rat 1.2 Arrays I and II (BD Biosciences/Clontech). These membrane arrays include 2352 spotted cDNAs of known and functionally annotated genes. cDNAs are 200–600 bp long and selected for low homology to other genes, and gene-specific primers are used in probe syntheses. A complete list of all the genes on the arrays, including array coordinates and GenBank accession numbers, is available at the BD Biosciences/Clontech Bioinformatics web site AtlasInfo 3.2 (〈http:// bioinfo.clontech.com/atlasinfo/〉).

Radiolabeled complex probes were generated by reverse transcription using total RNA, [α-32P]dATP (Amersham Biosciences) and the Atlas gene-specific mix of oligonucleotide primers (BD Biosciences/Clontech). Unincorporated radiolabeled nucleotides were removed with Nucleospin Extraction spin columns (BD Biosciences/Clontech), and probe yields were quantified by liquid scintillation counting.

Array membranes for each experimental condition were separately prehybridized in ExpressHyb buffer (BD Biosciences/Clontech). The 32P-labeled probes were denatured, diluted with carrier DNA, and an equal amount added to each membrane. Hybridization was allowed to proceed for 18 h at 68°C. After three high-stringency washes, membranes were exposed to a storage phosphor screen (Molecular Dynamics) for 48–72 h. Phosphor screens were scanned at 100 μm resolution and images were acquired using a 8600 Typhoon Variable Mode Imager (Amersham Biosciences).

Based on previous evidence (12), five biological replicates for each treatment group were considered adequate to ensure statistical power and stability of the results. Further, to assess reproducibility of the technique, we performed a second, independent hybridization for two randomly chosen samples for each allograft group and obtained consistent results (data not shown).

Normalization.

Phosphorimager scans were analyzed using AtlasImage software (version 2.7; BD Biosciences/Clontech). A given gene was considered to be detectable if its intensity was at least twice the global external background of the array. The background level was subtracted from the intensity of each spot to generate the raw data for each gene. Raw data were normalized according to the sum of the intensities global normalization method. The normalization coefficient was obtained by dividing the global intensity of each array by the global intensity of a reference array. The reference array was the array hybridized with a pool of RNAs from the control hearts (i.e., hearts subjected to cold ischemia and not transplanted). The relative expression level for each gene was calculated as the ratio: gene intensity/intensity of the same gene in the control hearts. Total intensities for each experimental condition were then scaled and mean intensities were calculated and used for scatter plot visualization of fold changes.

Filtering and statistical analysis.

Hierarchical agglomerative clustering of the array data was performed using a modified version of the Cluster/TreeView software (version 3.0) (13), originally developed by Eisen et al. (14) (〈http://rana.lbl.gov/〉). Data were filtered to include only genes detected in at least 80% of the replicates. Relative expression values (see above) were log-transformed (log base 2), genes and arrays were median centered and clustered by correlation (uncentered) centroid linkage. The hierarchical clustering was visualized with TreeView.

Primary statistical analysis of the filtered data was performed using the significance analysis of microarrays procedure (SAM, Excel Add-In version 1.21; 〈www-stat.stanford.edu/∼tibs/SAM/〉) (15). Only data that passed the quality assurance criteria were included in the analysis. A median false discovery rate (FDR) of <2%, in a two-class unpaired sample analysis on log2-transformed ratios followed by 100 random permutations of the data, was used to identify genes differentially expressed between comparison groups.

Treatment-related fold change was used to identify genes consistently up- or down-regulated in response to NDP-α-MSH. The ratio-mean relative expression for a given gene in treated allograft/mean relative expression of the same gene in untreated allografts provided the fold change measure. Genes were sorted on the basis of this ratio. A ratio of 1.6-fold up- or down-regulation (i.e., the fold change value used in SAM) was required to include genes in the subsequent analysis. Genes that satisfied this fold-change parameter were then analyzed using the unpaired two-tailed Student t test; a probability value <0.05 was considered significant. Genes identified using this method were compared with those identified by the SAM analysis. Only genes that passed both analyses were considered significant.

Gene classification.

Annotation of gene functions was performed combining information from several public databases. The selected genes were first analyzed using the web-based, client/server application Database for Annotation, Visualization and Integrated Discovery (DAVID version 2.0, 〈http://david.niaid.nih.gov/david/version2/index.htm〉) (16). Genes that remained unclassified were assigned manually based on information retrieved from the National Center for Biotechnology Information Entrez Gene Database (〈www.ncbi.nlm.nih.gov/entrez/query.fcgi?db = gene〉), or from the Stanford Online Universal Resource for Clones and Expressed sequence tags (SOURCE) (〈http://genome-www5.stanford.edu/cgi-bin/source/sourceSearch〉) (17).

Pattern identification.

Overrepresentation analysis was performed on genes identified by SAM and unpaired t test using the LocusLink identifiers and the Expression Analysis Systematic Explorer tool (EASE version 2.2, 〈http://apps1.niaid.nih.gov/david/ease1.htm〉) (18). EASE was used to test Gene Ontology terms (〈www.geneontology.org〉) (19) for “biological process” and to identify significantly overrepresented biological themes based on KEGG (〈www.genome.ad.jp/kegg〉) and GenMAPP (〈www.genmapp.org〉) (20) pathways.

Expression of six mRNAs in each treatment group was evaluated by real-time RT-PCR based on TaqMan methodology. PCR was performed in an ABI PRISM 7000 sequence detection system (Applied Biosystems). The assay identification numbers for selected genes were: Rn00563162_m1 for adenylyl cyclase 6 (Adcy6), Rn00586403_m1 for Cxcl2, Rn00571500_m1 for glucose-dependent insulinotropic peptide (Gip), Rn00561661_m1 for natriuretic peptide precursor type A (Nppa), Rn00566108_m1 for phospholipase Cγ1 (Plcg1), and Rn00565502_m1 for sodium channel voltage-gated type V α polypeptide (Scn5a). Three PCR amplification replicates were performed and averaged for each transcript. To normalize for differences in the amount of sample RNA added to each reaction mixture, GAPDH was selected as an endogenous control. RNA isolated from control hearts was used as calibrator. Relative quantitation of gene expression (fold change) was performed using the comparative cycle threshold (CT) method (ΔΔCT): the amount of target, normalized to the endogenous reference and relative to the calibrator, is given by the formula 2−ΔΔCT (21). The unpaired Student t test was used to compare differences in mean fold changes; a probability value <0.05 was considered statistically significant.

NDP-α-MSH treatment reduced the marked pathology observed in untreated heart grafts (Fig. 1). Heart grafts from untreated rats showed interstitial and perivascular edema and severe inflammatory cell infiltration. Both intragraft edema and inflammatory cell infiltration were much less in grafts from NDP-α-MSH-treated animals: inflammation and edema were confined to the subendocardial region and no abscesses were evident.

FIGURE 1.

Histology of cardiac grafts. H&E staining (×120) of a control nontransplanted heart (A); POD4 cardiac allograft from a saline-treated rat (B) and from an NDP-α-MSH-treated animal (C).

FIGURE 1.

Histology of cardiac grafts. H&E staining (×120) of a control nontransplanted heart (A); POD4 cardiac allograft from a saline-treated rat (B) and from an NDP-α-MSH-treated animal (C).

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Unsupervised hierarchical agglomerative clustering of array data from POD4 allografts indicated that the global expression profile correctly discriminates treated from untreated rats. All samples from NDP-α-MSH-treated animals clustered separately from saline-treated allografts (Fig. 2, left). The global gene expression profile identified two main gene clusters with opposite trend in their expression level (Fig. 2, left): genes overexpressed in treated and reduced in untreated allografts (cluster I) and genes decreased in treated and increased in untreated animals (cluster II). This observation suggests a distinctive global expression profile associated with NDP-α-MSH treatment.

FIGURE 2.

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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-α-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-α-MSH therapy (cell adhesion, cell growth, hormones, inflammatory and oxidative stress response, proteasome components, and ribosomal proteins).

FIGURE 2.

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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-α-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-α-MSH therapy (cell adhesion, cell growth, hormones, inflammatory and oxidative stress response, proteasome components, and ribosomal proteins).

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SAM analysis and the fold-change method (Table I) identified 53 genes whose expression was significantly altered by NDP-α-MSH treatment on POD1. Differences were even more marked on POD4 (Fig. 3): at this interval 172 genes were modulated by peptide treatment. Thirty genes were clearly enhanced on POD1 in treated allografts, whereas 104 genes were up-regulated on POD4 (2.4 and 8.2% of the spotted cDNAs included in the analysis, respectively). The proportion of genes down-regulated by treatment was 23 on POD1 and 68 on POD4 (1.8 and 5.4%, respectively).

FIGURE 3.

Identification of genes whose expression change on POD4 was potentially significant at SAM analysis. Scatter plot of the observed relative expression difference d(i) vs the expected relative difference dε(i) of genes altered by NDP-α-MSH treatment. The solid line indicates where genes would align if their d(i) = dε(i). At the threshold Δ = 0.60 (distance from the solid line drawn as dotted lines) and fold change ≥1.60, SAM predicts 179 genes as being differentially regulated. The FDR was <2%. The scatter plot shows significantly up-regulated genes as r, and significantly down-regulated genes as □. Genes that passed also the second analysis based on unpaired two-tailed Student’s t test are reported in Table I.

FIGURE 3.

Identification of genes whose expression change on POD4 was potentially significant at SAM analysis. Scatter plot of the observed relative expression difference d(i) vs the expected relative difference dε(i) of genes altered by NDP-α-MSH treatment. The solid line indicates where genes would align if their d(i) = dε(i). At the threshold Δ = 0.60 (distance from the solid line drawn as dotted lines) and fold change ≥1.60, SAM predicts 179 genes as being differentially regulated. The FDR was <2%. The scatter plot shows significantly up-regulated genes as r, and significantly down-regulated genes as □. Genes that passed also the second analysis based on unpaired two-tailed Student’s t test are reported in Table I.

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With regard to the classes of genes affected by treatment, NDP-α-MSH-treated allografts showed increased expression of cytoskeleton components (plectin, dystrophin, espin, and Ppp1r9b), receptors (Igf1r, Grm7, and Ptprd), molecules associated with signal transduction and intracellular signaling cascade (Rgs14, Rgs19ip1, Map3k1, Map2k5, Pkn1, Prkce, Dusp1, and Jak3), regulation of transcription (Fosl2, Stat3, and St18), glycolipidic metabolism (Pfkm, Lipf, and Acox2), and metallopeptidases and protease inhibitors (Ace, Ece1, Timp3, and Serpina4), both on POD1 and POD4. The range of mean fold change varied from +1.6 to +3.9. Conversely, NDP-α-MSH treatment down-regulated transcripts related to cell proliferation (Ccng1, Cdk7, Cd53, and H2afz), protein biosynthesis and turnover (laminin receptor, ribosomal protein L5, Erp29, and proteasome subunit β8), immune and inflammatory and/or cell infiltration responses (Cxcl2, Cxcr4, IL-1β, lysozyme, Hmgb1, mucin 3, and Arpc1b), oxide-reduction reactions (Cox6c, peroxiredoxins, and Hsd17b4), and hormones (Nppa and Gip). The mean fold-change ranged from −1.7 to −6.2.

To identify subsets of coregulated genes, we applied hierarchical agglomerative clustering to the 172 genes differentially expressed on POD4 using the log2 transformed expression data. Three gene clusters could be identified (Fig. 2, right): cluster A included genes induced in untreated and unchanged in treated allografts; cluster B contained genes up-regulated in untreated and down-regulated in treated allografts; cluster C consisted of genes down-regulated in untreated and enhanced in treated allografts.

To separate effects of NDP-α-MSH treatment on the rejection process from the Ag-independent graft damage due to transplant procedures, gene expression was estimated as the ratio to isografts (Table II). The peptide inhibited changes specific for mismatched allotransplantation, which were only evident in allografts, but it also reduced transcriptional modifications related to transplantation procedures, which was similar in allografts and isografts (Table II).

Table II.

Gene expression in untreated and NDP-α-MSH-treated allografts relative to isograftsa

ClassificationUntreated Allo./Isograft Fold ChangeTreated Allo./Isograft Fold ChangeClassificationUntreated Allo./Isograft Fold ChangeTreated Allo./Isograft Fold Change
Gene SymbolGene Symbol
Cell adhesion/extracellular matrix   Neurophysiological process   
Nlgn2 −2.4∗ Sema6b 2.8∗ 
Ddr1 −2.4∗ Cplx2 3.2∗ 
Lamb2 2.9∗∗ Cspg4 3.5∗ 11.5∗∗ 
Itga1 −5.6 Protein biosynthesis   
Cell growth and/or maintenance   Rps12 2.0∗ 
S100a4 −1.6∗ Rpl12 2.6∗∗ 
Atp2a2 1.9∗∗ Rps10 5.0∗ 2.7∗ 
Kif1c −4.7∗∗ −2.1∗ Rpl29 −2.0∗∗ 
Cytoskeleton   Rpl13 −2.1∗ 
Plec1 −3.2∗∗ −1.7∗ Rpl5 −1.8∗ 
Electron transport   Rps19 2.5∗ 
Cox6c 3.6∗ Rpl11 −1.6∗ 
Cyb5 −1.6∗ Rpl10a −1.9∗∗∗ 
Hormones/cytokines   Arbp −1.6∗ 
Nppa −2.0∗ Rpl32 −2.5∗ 
Vegf 2.2∗∗ Rps4x −1.6∗ 
Immune response   Rps8 −1.6∗ 
Cxcl2 3.0∗ Rpl18 −1.8∗ 
Fcer1a −2.3∗∗ Rps7 −1.6∗ 
Lyz 7.8∗∗ 4.5∗∗ Protein turnover   
Intracellular signaling cascade   Erp29 8.0∗∗ 4.0∗∗ 
Psen1 −1.7∗∗ Hspb1 −7.7∗ −3.8∗ 
Dusp1 −1.9∗ Rnpep 1.6∗∗ 
Adcy5 −2.1∗ Ece1 −1.6∗ 
Adcy6 2.4∗ Cpd 2.5∗∗∗∗∗ 
Limk2 −4.0∗ −2.3∗ Timp3 1.8∗ 3.4∗∗ 
Map2k5 −2.7∗∗∗ −1.6∗∗ Response to oxidative stress   
Camk4 1.6∗ Ucn −3.5∗∗ 
Prkch 1.6∗ Sod1 1.8∗∗ 
Prkce −2.9∗∗ Prdx2 −3.0∗ 
Pik4cb −3.5∗∗∗∗ −1.7∗∗∗ Mgst1 −1.6∗ 
Itpkb 2.8∗ Sepw1 −2.0∗∗∗ −3.4∗∗∗ 
Pik3r1 1.7 4.0∗ Prdx1 4.0∗ 2.3∗ 
Plcg1 3.2∗∗ Gpx5 1.6∗ 
Ion channels   Signal transduction   
Kcnh2 1.9∗ Agt 3.2∗∗ 2.0∗ 
Kcnj5 −6.2∗∗∗ −2.7∗ Ptprd −1.6∗ 
Gja1 −4.5∗∗ −2.3∗∗ Igflr 2.2∗ 
Scn5a −4.4∗ Hrasls3 34.7∗ 13.7∗ 
Metabolism, carbohydrate   Dlc1 1.7∗ 
Gpd2 1.9∗ 3.6∗∗ Rgs14 1.7∗ 
Pfkfb3 2.4∗ 5.0∗∗ Transcription   
Pfkm 1.6∗ Hmgb1 −1.8∗∗∗ 
Metabolism, lipid   Mef2d −1.7∗ 
Hsd17b4 3.3∗ 1.6∗ Fosl2 2.4∗ 
Tpi1 7.8∗∗ 4.2∗ Stat3 2.2∗ 
Prkab1 1.9∗ St18 −3.9∗∗ 
Lipf 2.0∗∗ Transport/trafficking   
Acad1 −2.7∗∗ Fabp4 −4.5∗ −18.5∗∗ 
Cpt1b −2.4∗ Fabp5 3.4∗ 
Metabolism, nucleic acid   Cltc 5.5∗ 2.9∗ 
Nme1 −3.3∗∗ Cd63 −1.6∗ −2.8∗∗∗ 
Nme2 −2.2∗∗ Ntt4 −4.6∗∗∗ −2.4∗∗∗∗ 
Atic 2.7∗∗ 4.6∗∗∗ Slc20a2 −2.7∗ 
Metabolism, other   Phldb1 −1.7∗ 
Oplah 3.4∗ 6.7∗ Akap1 3.0∗∗ 
Dio2 −6.2∗∗ −2.2∗∗ Rph3a1 −3.6∗ 
ClassificationUntreated Allo./Isograft Fold ChangeTreated Allo./Isograft Fold ChangeClassificationUntreated Allo./Isograft Fold ChangeTreated Allo./Isograft Fold Change
Gene SymbolGene Symbol
Cell adhesion/extracellular matrix   Neurophysiological process   
Nlgn2 −2.4∗ Sema6b 2.8∗ 
Ddr1 −2.4∗ Cplx2 3.2∗ 
Lamb2 2.9∗∗ Cspg4 3.5∗ 11.5∗∗ 
Itga1 −5.6 Protein biosynthesis   
Cell growth and/or maintenance   Rps12 2.0∗ 
S100a4 −1.6∗ Rpl12 2.6∗∗ 
Atp2a2 1.9∗∗ Rps10 5.0∗ 2.7∗ 
Kif1c −4.7∗∗ −2.1∗ Rpl29 −2.0∗∗ 
Cytoskeleton   Rpl13 −2.1∗ 
Plec1 −3.2∗∗ −1.7∗ Rpl5 −1.8∗ 
Electron transport   Rps19 2.5∗ 
Cox6c 3.6∗ Rpl11 −1.6∗ 
Cyb5 −1.6∗ Rpl10a −1.9∗∗∗ 
Hormones/cytokines   Arbp −1.6∗ 
Nppa −2.0∗ Rpl32 −2.5∗ 
Vegf 2.2∗∗ Rps4x −1.6∗ 
Immune response   Rps8 −1.6∗ 
Cxcl2 3.0∗ Rpl18 −1.8∗ 
Fcer1a −2.3∗∗ Rps7 −1.6∗ 
Lyz 7.8∗∗ 4.5∗∗ Protein turnover   
Intracellular signaling cascade   Erp29 8.0∗∗ 4.0∗∗ 
Psen1 −1.7∗∗ Hspb1 −7.7∗ −3.8∗ 
Dusp1 −1.9∗ Rnpep 1.6∗∗ 
Adcy5 −2.1∗ Ece1 −1.6∗ 
Adcy6 2.4∗ Cpd 2.5∗∗∗∗∗ 
Limk2 −4.0∗ −2.3∗ Timp3 1.8∗ 3.4∗∗ 
Map2k5 −2.7∗∗∗ −1.6∗∗ Response to oxidative stress   
Camk4 1.6∗ Ucn −3.5∗∗ 
Prkch 1.6∗ Sod1 1.8∗∗ 
Prkce −2.9∗∗ Prdx2 −3.0∗ 
Pik4cb −3.5∗∗∗∗ −1.7∗∗∗ Mgst1 −1.6∗ 
Itpkb 2.8∗ Sepw1 −2.0∗∗∗ −3.4∗∗∗ 
Pik3r1 1.7 4.0∗ Prdx1 4.0∗ 2.3∗ 
Plcg1 3.2∗∗ Gpx5 1.6∗ 
Ion channels   Signal transduction   
Kcnh2 1.9∗ Agt 3.2∗∗ 2.0∗ 
Kcnj5 −6.2∗∗∗ −2.7∗ Ptprd −1.6∗ 
Gja1 −4.5∗∗ −2.3∗∗ Igflr 2.2∗ 
Scn5a −4.4∗ Hrasls3 34.7∗ 13.7∗ 
Metabolism, carbohydrate   Dlc1 1.7∗ 
Gpd2 1.9∗ 3.6∗∗ Rgs14 1.7∗ 
Pfkfb3 2.4∗ 5.0∗∗ Transcription   
Pfkm 1.6∗ Hmgb1 −1.8∗∗∗ 
Metabolism, lipid   Mef2d −1.7∗ 
Hsd17b4 3.3∗ 1.6∗ Fosl2 2.4∗ 
Tpi1 7.8∗∗ 4.2∗ Stat3 2.2∗ 
Prkab1 1.9∗ St18 −3.9∗∗ 
Lipf 2.0∗∗ Transport/trafficking   
Acad1 −2.7∗∗ Fabp4 −4.5∗ −18.5∗∗ 
Cpt1b −2.4∗ Fabp5 3.4∗ 
Metabolism, nucleic acid   Cltc 5.5∗ 2.9∗ 
Nme1 −3.3∗∗ Cd63 −1.6∗ −2.8∗∗∗ 
Nme2 −2.2∗∗ Ntt4 −4.6∗∗∗ −2.4∗∗∗∗ 
Atic 2.7∗∗ 4.6∗∗∗ Slc20a2 −2.7∗ 
Metabolism, other   Phldb1 −1.7∗ 
Oplah 3.4∗ 6.7∗ Akap1 3.0∗∗ 
Dio2 −6.2∗∗ −2.2∗∗ Rph3a1 −3.6∗ 
a

∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; ∗∗∗∗∗, p < 0.00001.

An independent evaluation of six array-identified genes was performed using real-time RT-PCR. Three of them (Adcy6, Plcg1, and Scn5a) were enhanced by NDP-α-MSH-treatment and three were down-regulated (Cxcl2, Gip, and Nppa). The RT-PCR data confirmed all the changes in gene expression disclosed by the macroarray method, although there were small disparities in magnitude (Fig. 4). Expressions of Cxcl2, Gip, and Nppa were significantly inhibited by NDP-α-MSH-treatment on both POD1 and POD4, but expression was still greater relative to the control level. The peptide totally prevented decrease in expression of Adcy6 on POD4.

FIGURE 4.

Verification of array data by real-time RT-PCR. Consistent with the macroarray data, Adcy6, Scn5a, and Plcg1 transcripts were down-regulated in allografts relative to isografts and controls; NDP-α-MSH did not alter expression of these transcripts in treated allografts on POD1, but did induce them on POD4 Cxcl2; Gip and Nppa were up-regulated in allografts compared with isografts and controls, and were significantly inhibited by NDP-α-MSH treatment in allografts on both POD1 and POD4. Data are expressed as fold change of the targeted gene relative to control hearts. Bars denote mean ± SEM of specific mRNA. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

FIGURE 4.

Verification of array data by real-time RT-PCR. Consistent with the macroarray data, Adcy6, Scn5a, and Plcg1 transcripts were down-regulated in allografts relative to isografts and controls; NDP-α-MSH did not alter expression of these transcripts in treated allografts on POD1, but did induce them on POD4 Cxcl2; Gip and Nppa were up-regulated in allografts compared with isografts and controls, and were significantly inhibited by NDP-α-MSH treatment in allografts on both POD1 and POD4. Data are expressed as fold change of the targeted gene relative to control hearts. Bars denote mean ± SEM of specific mRNA. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

Close modal

Gene classification (Table I) was performed using DAVID and public databases. An EASE overrepresentation analysis of functional gene categories was used to identify biological pathways or gene groups. Changes in five functional categories/biological processes were significantly associated with NDP-α-MSH treatment: ribosome biogenesis and assembly (p < 0.0001), oxidative stress response (p < 0.05), protein amino acid phosphorylation (p < 0.01), intracellular signaling cascade (p < 0.01), and lipid metabolism (p < 0.01). Transcription of two cellular/metabolic pathways was significantly activated by treatment: the phosphatidylinositol signaling system (p < 0.001) and the fatty acid degradation (p < 0.05).

The present data, based on gene expression profiling, reveal multiple protective influences exerted by NDP-α-MSH that could account for the reduced damage in transplanted heart grafts. Indeed, peptide treatment caused substantial up-regulation of several salutary molecules including signal transduction mediators, metalloproteinases, serine proteases, energy pathway mediators, and ion channels. Concurrent down-regulation of growth factors, cytokines, chemokines, oxidative stress mediators, and ribosomal proteins likely contributes to preserve myocardium from injury.

The main finding of the present investigation is that the protective influences of NDP-α-MSH in heart transplantation are not restricted to the anti-inflammatory/anti-cytokine effects of the pep-tide (4, 5, 6, 7, 8, 22). Indeed, treatment preserved molecules of paramount importance for myocardial function. At least five metabolic/regulatory pathways were significantly altered by NDP-α-MSH treatment (Table I, Fig. 2): three of them were enhanced–intracellular signaling cascade, protein amino acid phosphorylation, and glycolipidic metabolism; two were repressed–ribosome biogenesis and response to oxidative stress. In addition, NDP-α-MSH markedly inhibited expression of Hmgb1 and S100a4, proteins belonging to the family of damage-associated molecular pattern molecules. These are a recently recognized group of molecules, naturally expressed in the nucleus or cytosol, that are released upon tissue damage or injury; they are believed to initiate inflammation and innate immune responses (23) and are significant targets for novel anti-inflammatory/immunomodulatory treatments (24).

The effects of treatment were very broad. NDP-α-MSH preserved Atp2a2 expression that was reduced in both allografts and isografts (Tables I and II). The cardiac Ca2+-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 Ca2+ channel kinetics (25). Atp2a2 improves cardiac muscle contractility in vivo and in vitro (26) and its expression in cardiomyocytes is selectively regulated by protein kinase C (PKC) isoenzymes PKCε and PKCδ (27). A decrease in Atp2a2 and the consequent impaired Ca2+ 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-α-MSH.

The increase in phospholipase C (Plc) isoenzyme mRNA observed in NDP-α-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 transduction pathways. Their physiological substrate, phosphatidylinositol 4,5-bisphosphate, is converted to two messenger molecules, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, which participate in many different physiological processes within cardiomyocytes, including Ca2+ movements (31, 32).

Decreased adenylyl cyclase activity was observed in human myocardium after orthotopic cardiac transplantation (33). Recent research indicates that adenylyl cyclase VI (Adcy6) expression improves heart function and abrogates myocardial hypertrophy (34, 35, 36). The present investigation confirms a substantial reduction of Adcy6 in transplanted hearts relative to control hearts and indicates that Adcy6 is normalized by NDP-α-MSH treatment (Table I, Fig. 4).

Further, there is evidence that α-MSH participates in calcium regulation in both the cytosol and sarcoplasmic reticulum of cardiac cells. This likely occurs via coordinated up-regulation of cytoplasmic, cytosolic, and sarcoplasmic proteins (Table I). Indeed, the muscarinic receptor Chrm2, the G protein-controlled inwardlyrectifying potassium channel Kcnj5, the adenylyl cyclases Adcy5 and Adcy6, the receptor regulated cation channel Trpc4, and the gap junction component Gja1 are all proteins integral to cytoplasmic membrane that were induced by NDP-α-MSH. The regulator of G-protein signaling Rgs14, protein kinase C η, and ε isoenzymes, Plc β and γ isoenzymes, and calcium/calmodulin-dependent protein kinases Camk2d and Camk4 are cytosolic proteins collectively involved in the regulation of Ca2+ influx. Their expression was clearly restored by NDP-α-MSH. Finally, NDP-α-MSH treatment induced Atp2a2 and the IP3 receptor Itpr1 that are key sarcoplasmic genes involved in the Ca2+ channel kinetics.

The voltage-gated sodium channel Scn5a drives the initial depolarization phase of the cardiac action potential and, therefore, participates in conduction of excitation through the heart (37, 38). Deletions or loss-of-function mutations of the human gene SCN5A have been associated with a wide range of arrhythmias (39, 40), and targeted disruption of murine Scn5a slowed conduction and caused ventricular tachycardia (41). The present data indicate a reduction of Scn5a in cardiac allografts relative to isografts and control hearts (Table II, Fig. 4), and show virtual normalization of Scn5a by NDP-α-MSH treatment on POD4 (Table I, Fig. 4).

JAKs, STATs, and PI3K provide a critical survival pathway to cardiomyocytes in vivo. Recent research shows that activation of the JAK/STAT pathway transduces cytoprotective signals in rat hearts subjected to acute pressure overload, myocardial infarction (42), or doxorubicin-induced cardiomyopathy (43). Conversely, patients with end-stage dilated cardiomyopathy had impaired downstream activation of the JAK/STAT pathway (44). This pathway, involved in the synthesis of key myocardial molecules, is transcriptionally regulated by NDP-α-MSH as suggested by the increase in Jak3, Stat3, Stat5a, and Pik3r1 mRNA in treated allografts (Table I, Fig. 2).

Another critical pathway for myocardial function involves induction, activation and translocation of PKC isoenzymes, and, in particular, of the myofilament-associated Prkce. In vivo and in vitro experiments indicate that Prkce plays a major role in cardioprotection against hypoxic or ischemia/reperfusion injury in the heart (45, 46, 47). NDP-α-MSH treatment preserved expression of Prkce that was reduced in both allografts and isografts (Tables I and II, Fig. 2).

Temporal analysis of transcriptional changes (Table I) allowed distinction between early and late effects of the peptide. The early response to NDP-α-MSH treatment includes induction of most cytoskeleton components, intracellular kinase network members, signal transduction receptors and transcription regulators, metallopeptidases, and protease inhibitors. Repression of immune and inflammatory response, cell cycle, Nppa, and proteasome components likewise occurs in the early phase of peptide treatment. Among the early effects of NDP-α-MSH treatment, the restoration of mRNA levels of key cytoskeleton components is of particular interest. Indeed, dystrophin (Dmd) is a vital component of a muscle sarcolemma membrane-spanning complex that connects cytoskeleton to basal lamina. Loss of intracellular dystrophin is believed to contribute to myocardial reperfusion injury (48), and restoration of its production can therefore protect the heart from this early injury after transplantation. Conversely, alterations in cell adhesion and extracellular matrix proteins, induction of phosphatidylinositol signaling system, glycolipidic metabolism, ion channels, and the anti-inflammatory cytokine IL-10, and repression of ribosome biosynthetic pathway and response to oxidative stress are late effects of NDP-α-MSH treatment.

The differences in gene expression profiles of allografts and isografts (Table II) allowed discrimination of damage caused by transplantation procedures from injury linked to genetic mismatch. Indeed, allografts–but not isografts–showed increased expression of immune response mediators, neuro-related proteins, and certain ribosomal genes. Further, some structural proteins, intracellular kinase network members, ion channels, and fatty acid degradation proteins that were decreased in allografts remained unaffected in isografts. Treatment with NDP-α-MSH prevented most of the changes induced by genetic mismatch in allografts, but the peptide also improved Ag-independent gene expression, linked to mechanical damage and reperfusion (Fig. 5).

FIGURE 5.

Effects of NDP-α-MSH treatment on Ag-dependent and independent injury.

FIGURE 5.

Effects of NDP-α-MSH treatment on Ag-dependent and independent injury.

Close modal

Despite its anti-inflammatory and cardioprotective influences, NDP-α-MSH did not eventually prevent rejection (3). A possible reason for this failure is that the peptide did not abolish intragraft expression of certain chemokines that have been associated with cardiac allograft rejection (49), including chemokines Ccl3, Ccl4, and Cxcl10. In addition, the peptide did not reduce expression of other putative mediators of acute rejection, including allograft inflammatory factor 1, IFN-γ, IFN regulatory factor 1, and leukocyte common Ag (50, 51) (data not shown). Therefore, it appears that there are mediators or pathways that escape the inhibitory effects of NDP-α-MSH. This is not surprising as rejection prevention requires profound immunosuppression that is clearly not exerted by NDP-α-MSH.

NDP-α-MSH is very safe. The peptide had no toxic effects in preclinical studies (52); further, the peptide was injected s.c. in human subjects over 12 days and blood tests revealed no changes (53). The present research indicates multiple protective influences of the peptide that could enhance effectiveness of immunosuppressive drugs in transplantation.

J. M. Lipton’s participation in this research is based on a long-standing scientific cooperation with Dr. Anna Catania on α-MSH research.

J. M. Lipton currently serves on the Board of Directors of Zengen.

Table I.

Genes regulated by NDP-α-MSH treatment in rat cardiac allografts

ClassificationGene IDGene SymbolGene NamePOD1 T/UaPOD4 T/Ua
GenBank accession no.Fold ChangeFold Change
Cell adhesion/extracellular matrix      
 U57362 25683 Col12a1 Procollagen, type XII, α1 −2.5∗∗ 
 U41662 117096 Nlgn2 Neuroligin 2 −2.3∗∗ 
 U78889 84010 DII1 δ-like 1 (Drosophila−2.1∗∗ 
 U76551 24573 Muc3 Mucin 3 −1.7∗∗ −2.0∗∗ 
 D50568 58826 Prg2 Proteoglycan 2, bone marrow −2.0∗∗ 
 L26525 25678 Ddr1 Discoidin domain receptor family, member 1 1.8∗∗∗ 
 X16563 25473 Lamb2 Laminin, β2 2.4∗∗∗ 
 L20468 171517 Gpc2 Glypican 2 3.4∗ 
 X52140 25118 Itga1 Integrin α1 3.7∗∗∗ 
Cell growth and/or maintenance      
 X70871 25405 Ccng1 Cyclin G1 −1.8∗ −4.1∗∗ 
 X83579 171150 Cdk7 Cyclin-dependent kinase 7 −1.9∗ −2.2∗∗ 
 M57276 24251 Cd53 CD53 Ag −2.0∗∗ −2.2∗∗ 
 J03628 24615 S100a4 S100 calcium-binding protein A4 −2.0∗∗∗ 
 M37584 58940 H2afz H2A histone family, member Z −2.2∗∗ −2.0∗∗∗ 
 L15618 116549 Csnk2a1 Casein kinase II, α 1 polypeptide 2.0∗∗ 
 M75146 171041 Klc1 Kinesin L chain 1 2.0∗∗∗∗ 
 J04022 29693 Atp2a2 ATPase, Ca2+ transporting, cardiac muscle, slow twitch 2 2.1∗∗ 
 AJ000696 113886 Klf1c Kinesin 1C 2.3∗∗ 
 AJ223599 85248 Kif3c Kinesin family member 3C 2.4∗∗ 
Cytoskeleton      
 AF083269 54227 Arpc1b Actin-related protein 2/3 complex, subunit 1B −1.8∗∗∗ 
 X59601 64204 Plec1 Plectin 2.0∗ 1.9∗∗ 
 X69767 24907 Dmd Dystrophin 2.1∗ 2.2∗∗∗ 
 U46007 56227 Espn Espin 1.6∗ 2.4∗∗ 
 AF016252 84686 Ppp1r9b Protein phosphatase 1, regulatory subunit 9B 2.9∗ 2.4∗∗∗∗ 
Electron transport      
 M27466 54322 Cox6c Cytochrome oxidase subunit Vlc −1.7∗ −2.6∗∗ 
 D13205 64001 Cyb5 Cytochrome b5 −1.8∗ 
 X79991 252931 Cyp3a18 Cytochrome P450, 3a18 3.0∗∗ 
Hormones/cytokines      
 L08831 25040 Gip Glucose-dependent insulinotropic peptide −2.1∗ −6.2∗∗ 
 L06441 24315 Dtprp Decidual/trophoblast prolactin-related protein −2.3∗∗∗ 
 D78591 29201 Ctf1 Cardiotrophin 1 −2.3∗ 
 X01118 24602 Nppa Natriuretic peptide precursor type A −2.4∗ −2.2∗ 
 K02809 24952 Gcg Glucagon 2.1∗∗∗∗ 
 M32167 83785 Vegf Vascular endothelial growth factor A 2.3∗∗∗∗ 
 M31603 24695 Pthlh Parathyroid hormone-like peptide 2.4∗∗∗ 
Immune response      
 U45965 114105 Cxcl2 Chemokine (CXC motif) ligand 2 −2.7∗ −2.4∗∗ 
 X75305 24812 Tap2 Transporter 2, ATP-binding cassette, subfamily B (MDR/TAP) −2.3∗∗∗∗ 
 U54791 60628 Cxcr4 Chemokine (CXC motif) receptor 4 −1.7∗ −2.0∗∗ 
 M98820 24494 Il1b IL-1β −3.3∗∗ −1.9∗∗ 
 J03606 25047 Fcer1a FcR, IgE, high affinity I, α polypeptide −1.9∗∗ 
 L12458 25211 Lyz lysozyme −1.9∗∗ −1.9∗∗∗ 
 U49066 171106 Il1rl2 IL-1 receptor-like 2 2.0∗ 
 L02926 25325 Il10 IL-10 nd 3.2∗ 
Intracellular-signaling cascade      
 D82363 29192 Psen1 Presenilin 1 1.7∗∗ 
 U28356 246781 Heptp Protein-tyrosine phosphatase, nonreceptor type 7 1.9∗∗ 
 X84004 114856 Dusp1 Dual specificity phosphatase 1 1.9∗ 2.1∗∗ 
 U25281 259242 Cr16 SH3 domain-binding protein CR16 1.6∗ 2.4∗ 
 M96159 64532 Adcy5 Adenylyl cyclase 5 2.6∗∗∗ 
 L01115 25289 Adcy6 Adenylyl cyclase 6 4.4∗∗∗∗∗ 
Intracellular kinase network members      
 D31874 29524 Limk2 LIM motif-containing protein kinase 2 1.7∗∗ 
 U37462 29568 Map2k5 MAPK kinase 5 1.9∗ 1.7∗∗∗ 
 D28508 25326 Jak 3 JAK 3 1.7∗∗∗∗ 
 X94351 171305 Clk3 CDC-like kinase 3 1.8∗∗ 
 M63334 25050 Camk4 Calcium/calmodulin-dependent protein kinase IV 1.8∗∗ 
 X68400 81749 Prkch PKCη 1.8∗∗ 
 M18331 29340 Prkce PKCε 1.6∗ 1.9∗∗∗ 
 U48596 116667 Map3k1 MAPK kinase kinase 1 1.7∗ 2.1∗∗ 
 D26180 29355 Pkn1 Protein kinase N1 1.9∗ 2.1∗∗∗ 
 L13408 24246 Camk2d Calcium/calmodulin-dependent protein kinase II, δ 2.1∗∗∗ 
 M19007 25023 Prkcb1 PKCβ1 2.6∗∗∗ 
Phosphatidylinositol-signaling system      
 D84667 81747 Pik4cb Phosphatidylinositol 4-kinase, catalytic, β polypeptide 2.0∗∗∗∗∗ 
(Table continues     
ClassificationGene IDGene SymbolGene NamePOD1 T/UaPOD4 T/Ua
GenBank accession no.Fold ChangeFold Change
Cell adhesion/extracellular matrix      
 U57362 25683 Col12a1 Procollagen, type XII, α1 −2.5∗∗ 
 U41662 117096 Nlgn2 Neuroligin 2 −2.3∗∗ 
 U78889 84010 DII1 δ-like 1 (Drosophila−2.1∗∗ 
 U76551 24573 Muc3 Mucin 3 −1.7∗∗ −2.0∗∗ 
 D50568 58826 Prg2 Proteoglycan 2, bone marrow −2.0∗∗ 
 L26525 25678 Ddr1 Discoidin domain receptor family, member 1 1.8∗∗∗ 
 X16563 25473 Lamb2 Laminin, β2 2.4∗∗∗ 
 L20468 171517 Gpc2 Glypican 2 3.4∗ 
 X52140 25118 Itga1 Integrin α1 3.7∗∗∗ 
Cell growth and/or maintenance      
 X70871 25405 Ccng1 Cyclin G1 −1.8∗ −4.1∗∗ 
 X83579 171150 Cdk7 Cyclin-dependent kinase 7 −1.9∗ −2.2∗∗ 
 M57276 24251 Cd53 CD53 Ag −2.0∗∗ −2.2∗∗ 
 J03628 24615 S100a4 S100 calcium-binding protein A4 −2.0∗∗∗ 
 M37584 58940 H2afz H2A histone family, member Z −2.2∗∗ −2.0∗∗∗ 
 L15618 116549 Csnk2a1 Casein kinase II, α 1 polypeptide 2.0∗∗ 
 M75146 171041 Klc1 Kinesin L chain 1 2.0∗∗∗∗ 
 J04022 29693 Atp2a2 ATPase, Ca2+ transporting, cardiac muscle, slow twitch 2 2.1∗∗ 
 AJ000696 113886 Klf1c Kinesin 1C 2.3∗∗ 
 AJ223599 85248 Kif3c Kinesin family member 3C 2.4∗∗ 
Cytoskeleton      
 AF083269 54227 Arpc1b Actin-related protein 2/3 complex, subunit 1B −1.8∗∗∗ 
 X59601 64204 Plec1 Plectin 2.0∗ 1.9∗∗ 
 X69767 24907 Dmd Dystrophin 2.1∗ 2.2∗∗∗ 
 U46007 56227 Espn Espin 1.6∗ 2.4∗∗ 
 AF016252 84686 Ppp1r9b Protein phosphatase 1, regulatory subunit 9B 2.9∗ 2.4∗∗∗∗ 
Electron transport      
 M27466 54322 Cox6c Cytochrome oxidase subunit Vlc −1.7∗ −2.6∗∗ 
 D13205 64001 Cyb5 Cytochrome b5 −1.8∗ 
 X79991 252931 Cyp3a18 Cytochrome P450, 3a18 3.0∗∗ 
Hormones/cytokines      
 L08831 25040 Gip Glucose-dependent insulinotropic peptide −2.1∗ −6.2∗∗ 
 L06441 24315 Dtprp Decidual/trophoblast prolactin-related protein −2.3∗∗∗ 
 D78591 29201 Ctf1 Cardiotrophin 1 −2.3∗ 
 X01118 24602 Nppa Natriuretic peptide precursor type A −2.4∗ −2.2∗ 
 K02809 24952 Gcg Glucagon 2.1∗∗∗∗ 
 M32167 83785 Vegf Vascular endothelial growth factor A 2.3∗∗∗∗ 
 M31603 24695 Pthlh Parathyroid hormone-like peptide 2.4∗∗∗ 
Immune response      
 U45965 114105 Cxcl2 Chemokine (CXC motif) ligand 2 −2.7∗ −2.4∗∗ 
 X75305 24812 Tap2 Transporter 2, ATP-binding cassette, subfamily B (MDR/TAP) −2.3∗∗∗∗ 
 U54791 60628 Cxcr4 Chemokine (CXC motif) receptor 4 −1.7∗ −2.0∗∗ 
 M98820 24494 Il1b IL-1β −3.3∗∗ −1.9∗∗ 
 J03606 25047 Fcer1a FcR, IgE, high affinity I, α polypeptide −1.9∗∗ 
 L12458 25211 Lyz lysozyme −1.9∗∗ −1.9∗∗∗ 
 U49066 171106 Il1rl2 IL-1 receptor-like 2 2.0∗ 
 L02926 25325 Il10 IL-10 nd 3.2∗ 
Intracellular-signaling cascade      
 D82363 29192 Psen1 Presenilin 1 1.7∗∗ 
 U28356 246781 Heptp Protein-tyrosine phosphatase, nonreceptor type 7 1.9∗∗ 
 X84004 114856 Dusp1 Dual specificity phosphatase 1 1.9∗ 2.1∗∗ 
 U25281 259242 Cr16 SH3 domain-binding protein CR16 1.6∗ 2.4∗ 
 M96159 64532 Adcy5 Adenylyl cyclase 5 2.6∗∗∗ 
 L01115 25289 Adcy6 Adenylyl cyclase 6 4.4∗∗∗∗∗ 
Intracellular kinase network members      
 D31874 29524 Limk2 LIM motif-containing protein kinase 2 1.7∗∗ 
 U37462 29568 Map2k5 MAPK kinase 5 1.9∗ 1.7∗∗∗ 
 D28508 25326 Jak 3 JAK 3 1.7∗∗∗∗ 
 X94351 171305 Clk3 CDC-like kinase 3 1.8∗∗ 
 M63334 25050 Camk4 Calcium/calmodulin-dependent protein kinase IV 1.8∗∗ 
 X68400 81749 Prkch PKCη 1.8∗∗ 
 M18331 29340 Prkce PKCε 1.6∗ 1.9∗∗∗ 
 U48596 116667 Map3k1 MAPK kinase kinase 1 1.7∗ 2.1∗∗ 
 D26180 29355 Pkn1 Protein kinase N1 1.9∗ 2.1∗∗∗ 
 L13408 24246 Camk2d Calcium/calmodulin-dependent protein kinase II, δ 2.1∗∗∗ 
 M19007 25023 Prkcb1 PKCβ1 2.6∗∗∗ 
Phosphatidylinositol-signaling system      
 D84667 81747 Pik4cb Phosphatidylinositol 4-kinase, catalytic, β polypeptide 2.0∗∗∗∗∗ 
(Table continues     
Table IA.

Continued

ClassificationGene IDGene SymbolGene NamePOD1 T/UaPOD4 T/Ua
GenBank accession no.Fold ChangeFold Change
 X74227 54260 Itpkb Inositol 1,4,5-trisphosphate 3-kinase B 2.1∗∗∗∗ 
 D64045 25513 Pik3r1 PI3K regulatory subunit, polypeptide 1 2.3∗∗ 
 AF033355 89812 Pip5k2b Phosphatidylinositol-4-phosphate 5-kinase, type II, β ND 2.4∗∗ 
 U55192 54259 Inpp5d Inositol polyphosphate-5-phosphatase D ND 2.4∗∗∗ 
 U96920 116699 Inpp4b Inositol polyphosphate-4-phosphatase, type II, 105 kDa ND 2.6∗ 
 J05155 29337 Plcg2 Plc, γ2 2.0∗∗ 
 M20636 24654 Plcb1 Plc, β1 2.0∗ 
 AJ011035 85240 Plcb2 Plc, β2 ND 2.3∗ 
 J03806 25738 Plcg1 Plc, γ1 2.6∗∗∗∗∗ 
Ion channels      
 U38665 25262 Itpr1 Inositol 1,4,5-triphosphate receptor 1 1.9∗∗∗∗ 
 Z96106 117018 Kcnh2 Potassium voltage-gated channel, subfamily H (eag-related), member 2 2.3∗∗∗ 
 Z67744 29233 Clcn7 Chloride channel 7 1.7∗ 2.4∗∗∗ 
 L35771 29713 Kcnj5 Potassium inwardly-rectifying channel, subfamily J, member 5 2.5∗ 
 X06656 24392 Gja1 Gap junction membrane channel protein α1 2.6∗∗ 
 AB008889 84494 Trpc4 Transient receptor potential cation channel, subfamily C, member 4 2.6∗∗∗ 
 M27902 25665 Scn5a Sodium channel, voltage-gated, type V, α, polypeptide 3.6∗∗∗∗ 
Metabolism, carbohydrate      
 U08027 25062 Gpd2 Glycerol-3-phosphate dehydrogenase 2 1.9∗∗∗ 
 U25651 65152 Pfkm Phosphofructokinase, muscle 1.7∗ 2.0∗∗ 
 D87240 117276 Pfkfb3 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 2.0∗∗∗ 
 D16102 79223 Gyk Glycerol kinase ND 3.3∗ 
Metabolism, lipid      
 U37486 79244 Hsd17b4 Peroxisomal multifunctional enzyme type II −1.8∗∗ −2.1∗ 
 L36250 24849 Tpi1 Triosephosphate isomerase 1 −1.9∗∗ 
 U42411 83803 Prkab1 Protein kinase, AMP-activated, β1 noncatalytic subunit 1.7∗∗ 
 X02309 50682 Lipf Lipase, gastric 1.6∗ 2.0∗∗∗∗ 
 J05029 25287 Acadl Acetyl-coenzyme A dehydrogenase, long-chain 2.0∗∗ 
 U32314 25104 Pc Pyruvate carboxylase 2.0∗∗∗ 
 D43623 25756 Cpt1b Carnitine palmitoyltransferase 1b 2.1∗∗ 
 U36771 29653 Gpam Glycerol-3-phosphate acyltransferase, mitochondrial 2.2∗∗ 
 X95189 252898 Acox2 Acyl-coenzyme A oxidase 2, branched chain 1.8∗ 2.8∗∗ 
Metabolism, nucleic acid      
 D13374 191575 Nme1 Expressed in nonmetastatic cells 1 −2.7∗∗∗∗ 
 M91597 83782 Nme2 Nucleoside diphosphate kinase −2.2∗∗∗∗ 
 D89514 81643 Atic 5-Aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase 1.7∗∗∗∗ 
 U18942 81635 Adar Adenosine deaminase, RNA-specific 1.7∗∗∗∗ 
Metabolism, other      
 U40803 25120 Aanat Arylalkylamine N-acetyltransferase 1.8∗∗ 
 M58364 29244 Gch GTP cyclohydrolase 1 1.6∗∗ 1.9∗∗ 
 U70825 116684 Oplah 5-oxoprolinase (ATP-hydrolysing) 1.9∗ 
 D83479 81718 Cdo1 Cytosolic cysteine dioxygenase 1 2.1∗∗∗ 
 L20427 29309 Coq3 Coenzyme q (ubiquinone) biosynthetic enzyme 3 2.2∗ 
 U53505 65162 Dio2 Deiodinase, iodothyronine, type II 2.9∗∗ 
Neurophysiological process      
 AB000776 84609 Sema6b Semaphorin 6B −2.3∗∗ 
 U35099 116657 Cplx2 Complexin 2 −2.0∗∗ 
 AF007583 29755 Colq Collagen-like tail subunit of asymmetric acetylcholinesterase 2.1∗∗∗∗ 
 AF044201 246274 Faim2 Fas apoptotic inhibitory molecule 2 2.5∗∗ 
 X56541 81651 Cspg4 Membrane-spanning proteoglycan NG2 3.3∗∗∗∗∗ 
Protein biosynthesis      
 M18547 65139 Rps12 Ribosomal protein S12 −2.6∗∗ 
 X53504  Rpl12 Ribosomal protein L12 −2.2∗∗∗∗ 
 X13549 81773 Rps10 Ribosomal protein S10 −2.1∗∗ 
 M27798 29236 Lamr1 Laminin receptor 1 (67 kDa ribosomal protein SA) −2.6∗∗ −2.1∗∗ 
 X68283 29283 Rpl29 Ribosomal protein L29 −2.1∗∗∗ 
 X78327 81765 Rpl13 Ribosomal protein L13 −2.0∗∗ 
 X06148 81763 Rpl5 Ribosomal protein L5 −1.8∗ −1.9∗ 
 X51707 29287 Rps19 Ribosomal protein S19 −1.9∗∗ 
 X62146  Rpl11 Ribosomal protein L11 −1.9∗∗∗ 
 X93352 81729 Rpl10a Ribosomal protein L10a −1.9∗∗∗ 
 X52445 81776 Rps24 Ribosomal protein S24 −1.6∗ −1.9∗ 
 Z29530 64205 Arbp Acidic ribosomal protein P0 −1.9∗∗ 
 M19635 81769 Rpl36a Large subunit ribosomal protein L36a −1.8∗∗ 
 X06483 28298 Rpl32 Ribosomal protein L32 −1.8∗∗ 
 X14210 29426 Rps4x Ribosomal protein S4, X-linked −1.8∗∗ 
(Table continues     
ClassificationGene IDGene SymbolGene NamePOD1 T/UaPOD4 T/Ua
GenBank accession no.Fold ChangeFold Change
 X74227 54260 Itpkb Inositol 1,4,5-trisphosphate 3-kinase B 2.1∗∗∗∗ 
 D64045 25513 Pik3r1 PI3K regulatory subunit, polypeptide 1 2.3∗∗ 
 AF033355 89812 Pip5k2b Phosphatidylinositol-4-phosphate 5-kinase, type II, β ND 2.4∗∗ 
 U55192 54259 Inpp5d Inositol polyphosphate-5-phosphatase D ND 2.4∗∗∗ 
 U96920 116699 Inpp4b Inositol polyphosphate-4-phosphatase, type II, 105 kDa ND 2.6∗ 
 J05155 29337 Plcg2 Plc, γ2 2.0∗∗ 
 M20636 24654 Plcb1 Plc, β1 2.0∗ 
 AJ011035 85240 Plcb2 Plc, β2 ND 2.3∗ 
 J03806 25738 Plcg1 Plc, γ1 2.6∗∗∗∗∗ 
Ion channels      
 U38665 25262 Itpr1 Inositol 1,4,5-triphosphate receptor 1 1.9∗∗∗∗ 
 Z96106 117018 Kcnh2 Potassium voltage-gated channel, subfamily H (eag-related), member 2 2.3∗∗∗ 
 Z67744 29233 Clcn7 Chloride channel 7 1.7∗ 2.4∗∗∗ 
 L35771 29713 Kcnj5 Potassium inwardly-rectifying channel, subfamily J, member 5 2.5∗ 
 X06656 24392 Gja1 Gap junction membrane channel protein α1 2.6∗∗ 
 AB008889 84494 Trpc4 Transient receptor potential cation channel, subfamily C, member 4 2.6∗∗∗ 
 M27902 25665 Scn5a Sodium channel, voltage-gated, type V, α, polypeptide 3.6∗∗∗∗ 
Metabolism, carbohydrate      
 U08027 25062 Gpd2 Glycerol-3-phosphate dehydrogenase 2 1.9∗∗∗ 
 U25651 65152 Pfkm Phosphofructokinase, muscle 1.7∗ 2.0∗∗ 
 D87240 117276 Pfkfb3 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 2.0∗∗∗ 
 D16102 79223 Gyk Glycerol kinase ND 3.3∗ 
Metabolism, lipid      
 U37486 79244 Hsd17b4 Peroxisomal multifunctional enzyme type II −1.8∗∗ −2.1∗ 
 L36250 24849 Tpi1 Triosephosphate isomerase 1 −1.9∗∗ 
 U42411 83803 Prkab1 Protein kinase, AMP-activated, β1 noncatalytic subunit 1.7∗∗ 
 X02309 50682 Lipf Lipase, gastric 1.6∗ 2.0∗∗∗∗ 
 J05029 25287 Acadl Acetyl-coenzyme A dehydrogenase, long-chain 2.0∗∗ 
 U32314 25104 Pc Pyruvate carboxylase 2.0∗∗∗ 
 D43623 25756 Cpt1b Carnitine palmitoyltransferase 1b 2.1∗∗ 
 U36771 29653 Gpam Glycerol-3-phosphate acyltransferase, mitochondrial 2.2∗∗ 
 X95189 252898 Acox2 Acyl-coenzyme A oxidase 2, branched chain 1.8∗ 2.8∗∗ 
Metabolism, nucleic acid      
 D13374 191575 Nme1 Expressed in nonmetastatic cells 1 −2.7∗∗∗∗ 
 M91597 83782 Nme2 Nucleoside diphosphate kinase −2.2∗∗∗∗ 
 D89514 81643 Atic 5-Aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase 1.7∗∗∗∗ 
 U18942 81635 Adar Adenosine deaminase, RNA-specific 1.7∗∗∗∗ 
Metabolism, other      
 U40803 25120 Aanat Arylalkylamine N-acetyltransferase 1.8∗∗ 
 M58364 29244 Gch GTP cyclohydrolase 1 1.6∗∗ 1.9∗∗ 
 U70825 116684 Oplah 5-oxoprolinase (ATP-hydrolysing) 1.9∗ 
 D83479 81718 Cdo1 Cytosolic cysteine dioxygenase 1 2.1∗∗∗ 
 L20427 29309 Coq3 Coenzyme q (ubiquinone) biosynthetic enzyme 3 2.2∗ 
 U53505 65162 Dio2 Deiodinase, iodothyronine, type II 2.9∗∗ 
Neurophysiological process      
 AB000776 84609 Sema6b Semaphorin 6B −2.3∗∗ 
 U35099 116657 Cplx2 Complexin 2 −2.0∗∗ 
 AF007583 29755 Colq Collagen-like tail subunit of asymmetric acetylcholinesterase 2.1∗∗∗∗ 
 AF044201 246274 Faim2 Fas apoptotic inhibitory molecule 2 2.5∗∗ 
 X56541 81651 Cspg4 Membrane-spanning proteoglycan NG2 3.3∗∗∗∗∗ 
Protein biosynthesis      
 M18547 65139 Rps12 Ribosomal protein S12 −2.6∗∗ 
 X53504  Rpl12 Ribosomal protein L12 −2.2∗∗∗∗ 
 X13549 81773 Rps10 Ribosomal protein S10 −2.1∗∗ 
 M27798 29236 Lamr1 Laminin receptor 1 (67 kDa ribosomal protein SA) −2.6∗∗ −2.1∗∗ 
 X68283 29283 Rpl29 Ribosomal protein L29 −2.1∗∗∗ 
 X78327 81765 Rpl13 Ribosomal protein L13 −2.0∗∗ 
 X06148 81763 Rpl5 Ribosomal protein L5 −1.8∗ −1.9∗ 
 X51707 29287 Rps19 Ribosomal protein S19 −1.9∗∗ 
 X62146  Rpl11 Ribosomal protein L11 −1.9∗∗∗ 
 X93352 81729 Rpl10a Ribosomal protein L10a −1.9∗∗∗ 
 X52445 81776 Rps24 Ribosomal protein S24 −1.6∗ −1.9∗ 
 Z29530 64205 Arbp Acidic ribosomal protein P0 −1.9∗∗ 
 M19635 81769 Rpl36a Large subunit ribosomal protein L36a −1.8∗∗ 
 X06483 28298 Rpl32 Ribosomal protein L32 −1.8∗∗ 
 X14210 29426 Rps4x Ribosomal protein S4, X-linked −1.8∗∗ 
(Table continues     
Table IB.

Continued

ClassificationGene IDGene SymbolGene NamePOD1 T/UaPOD4 T/Ua
GenBank accession no.Fold ChangeFold Change
 X06423 65136 Rps8 Ribosomal protein S8 −1.8∗∗∗∗ 
 M20156 81766 Rpl18 Ribosomal protein L18 −1.8∗∗ 
 X53377 29258 Rps7 Ribosomal protein S7 −1.7∗ 
Protein turnover      
 U36482 117030 Erp29 Endoplasmic reticulum protein 29 −2.2∗ −2.0∗ 
 D10729 24968 Psmb8 Proteasome (prosome, macropain) subunit, β type 8 −2.0∗ −2.0∗∗ 
 D10754 29666 Psmb6 Proteasome (prosome, macropain) subunit, β type 6 −1.7∗∗ 
 M86389 24471 Hspb1 Heat shock 27-kDa protein 1 1.6∗ 2.1∗∗∗∗ 
 U56407 79435 Ube2d2 Ubiquitin-conjugating enzyme E2D 2 2.2∗∗ 
 Metallopeptidase      
 M15944 24590 Mme Membrane metalloendopeptidase −2.0∗∗∗ 
 U61696 81761 Rnpep Aminopeptidase B 1.7∗∗∗∗ 
 U03734 24310 Ace Angiotensin 1-converting enzyme 1 2.3∗ 2.0∗∗ 
 D29683 94204 Ece1 Endothelin-converting enzyme 1 1.7∗ 2.0∗∗∗∗ 
 U62897 25306 Cpd Carboxypeptidase D 1.6∗ 2.4∗∗ 
 Protease inhibitor      
 U27201 25358 Timp3 Tissue inhibitor of metalloproteinase 3 1.7∗∗ 1.9∗∗∗ 
 U51017 246328 Serpina4 Serine (or cysteine) proteinase inhibitor, clade, member 4 2.0∗ 2.3∗∗ 
Response to oxidative stress      
 U33935 29151 Ucn Urocortin −2.5∗∗ 
 Y00404 24786 Sod1 Superoxide dismutase 1 −2.1∗∗∗∗ 
 U06099 29338 Prdx2 Peroxiredoxin 2 −1.7∗ −2.1∗∗∗∗ 
 J03752 171341 Mgst1 Microsomal glutathione S-transferase 1 −2.0∗∗ 
 U25264 25545 Sepw1 Selenoprotein W, muscle 1 −1.9∗∗∗ 
 D30035 117254 Prdx1 Peroxiredoxin 1 −2.4∗∗ −1.9∗∗ 
 AF058787  Ho3 Heme oxygenase 3 −1.6∗ −1.7∗∗ 
 X62404 113919 Gpx5 Glutathione peroxidase 5 −1.7∗∗∗ 
Signal transduction      
 Receptors      
 U35025 245921 Acvr1c Activin receptor-like kinase 7 −2.2∗∗ 
 L00091 24179 Agt Angiotensinogen −1.7∗∗∗∗ 
 L19181 25529 Ptprd Protein tyrosine phosphatase, receptor type, D 1.7∗∗ 2.0∗ 
 J03025 81645 Chrm2 Cholinergic receptor, muscarinic 2 2.4∗∗ 
 L29232 25718 Igflr Insulin-like growth factor 1 receptor 2.0∗∗∗ 2.5∗∗∗∗∗ 
 D16817 81672 Grm7 Glutamate receptor, metabotropic 7 2.3∗ 2.9∗ 
 G-protein/GTPase activity modulators      
 X76453 24913 Hrasls3 HRAS like suppressor −2.5∗ 
 D31962 58834 Dlc1 Deleted in liver cancer 1 2.0∗∗∗∗ 
 U92279 114705 Rgs14 Regulator of G-protein signaling 14 1.6∗ 2.1∗∗ 
 AF044673 114559 Pak3bp PAK-interacting exchange factor β 2.1∗∗ 
 AF058789 192117 Syngap1 Synaptic Ras GTPase-activating protein 1 2.5∗∗∗ 
 AF032120 83823 Rgs19ip1 Regulator of G-protein signaling 19-interacting protein 1 2.4∗ 3.8∗∗∗ 
Transcription      
 M64986 25459 Hmgb1 High mobility group box 1 −2.2∗ −1.7∗∗ 
 AJ005425 81518 Mef2d Myocyte enhancer factor 2D 1.7∗∗∗ 
 U18913 25446 Fosl2 fos-like Ag 2 1.8∗ 1.9∗∗ 
 X91810 25125 Stat3 Signal transducer and activator of transcription 3 1.9∗∗ 2.0∗∗ 
 X14788 81646 Creb1 cAMP response element binding protein 1 2.0∗∗∗ 
 U24175 24918 Stat5a Signal transducer and activator of transcription 5A 2.3∗∗ 
 J03933 24831 Thrb Thyroid hormone receptor β 2.4∗∗ 
 U67080 266680 Stl8 Suppression of tumorigenicity 18 3.1∗∗∗ 2.9∗ 
Transport/trafficking      
 U75581 79451 Fabp4 Fatty acid binding protein 4, adipocyte −4.8∗ 
 U13253 140868 Fabp5 Fatty acid binding protein 5, epidermal −2.4∗ −2.3∗ 
 J03583 54241 Cltc Clathrin, heavy polypeptide (Hc) −2.0∗∗ 
 X61654 29186 Cd63 CD63 Ag −2.0∗∗∗ 
 U21871 266601 Tomm20 Translocase of outer mitochondrial membrane 20 homologue (yeast) −1.7∗∗∗ 
 S68944  Ntt4 Sodium/chloride-dependent neurotransmitter transporter 1.9∗∗∗ 
 L19931 29502 Slc20a2 Solute carrier family 20, member 2 2.0∗∗ 
 X74226 171434 Phldb1 Pleckstrin homology-like domain, family B, member 1 2.1∗ 
 AF068202 114124 Akap1 A kinase (PRKA) anchor protein 1 2.1∗∗∗ 
 AF022774 171123 Rph3al Rabphilin 3A-like (without C2 domains) 2.2∗∗ 2.2∗∗∗ 
ClassificationGene IDGene SymbolGene NamePOD1 T/UaPOD4 T/Ua
GenBank accession no.Fold ChangeFold Change
 X06423 65136 Rps8 Ribosomal protein S8 −1.8∗∗∗∗ 
 M20156 81766 Rpl18 Ribosomal protein L18 −1.8∗∗ 
 X53377 29258 Rps7 Ribosomal protein S7 −1.7∗ 
Protein turnover      
 U36482 117030 Erp29 Endoplasmic reticulum protein 29 −2.2∗ −2.0∗ 
 D10729 24968 Psmb8 Proteasome (prosome, macropain) subunit, β type 8 −2.0∗ −2.0∗∗ 
 D10754 29666 Psmb6 Proteasome (prosome, macropain) subunit, β type 6 −1.7∗∗ 
 M86389 24471 Hspb1 Heat shock 27-kDa protein 1 1.6∗ 2.1∗∗∗∗ 
 U56407 79435 Ube2d2 Ubiquitin-conjugating enzyme E2D 2 2.2∗∗ 
 Metallopeptidase      
 M15944 24590 Mme Membrane metalloendopeptidase −2.0∗∗∗ 
 U61696 81761 Rnpep Aminopeptidase B 1.7∗∗∗∗ 
 U03734 24310 Ace Angiotensin 1-converting enzyme 1 2.3∗ 2.0∗∗ 
 D29683 94204 Ece1 Endothelin-converting enzyme 1 1.7∗ 2.0∗∗∗∗ 
 U62897 25306 Cpd Carboxypeptidase D 1.6∗ 2.4∗∗ 
 Protease inhibitor      
 U27201 25358 Timp3 Tissue inhibitor of metalloproteinase 3 1.7∗∗ 1.9∗∗∗ 
 U51017 246328 Serpina4 Serine (or cysteine) proteinase inhibitor, clade, member 4 2.0∗ 2.3∗∗ 
Response to oxidative stress      
 U33935 29151 Ucn Urocortin −2.5∗∗ 
 Y00404 24786 Sod1 Superoxide dismutase 1 −2.1∗∗∗∗ 
 U06099 29338 Prdx2 Peroxiredoxin 2 −1.7∗ −2.1∗∗∗∗ 
 J03752 171341 Mgst1 Microsomal glutathione S-transferase 1 −2.0∗∗ 
 U25264 25545 Sepw1 Selenoprotein W, muscle 1 −1.9∗∗∗ 
 D30035 117254 Prdx1 Peroxiredoxin 1 −2.4∗∗ −1.9∗∗ 
 AF058787  Ho3 Heme oxygenase 3 −1.6∗ −1.7∗∗ 
 X62404 113919 Gpx5 Glutathione peroxidase 5 −1.7∗∗∗ 
Signal transduction      
 Receptors      
 U35025 245921 Acvr1c Activin receptor-like kinase 7 −2.2∗∗ 
 L00091 24179 Agt Angiotensinogen −1.7∗∗∗∗ 
 L19181 25529 Ptprd Protein tyrosine phosphatase, receptor type, D 1.7∗∗ 2.0∗ 
 J03025 81645 Chrm2 Cholinergic receptor, muscarinic 2 2.4∗∗ 
 L29232 25718 Igflr Insulin-like growth factor 1 receptor 2.0∗∗∗ 2.5∗∗∗∗∗ 
 D16817 81672 Grm7 Glutamate receptor, metabotropic 7 2.3∗ 2.9∗ 
 G-protein/GTPase activity modulators      
 X76453 24913 Hrasls3 HRAS like suppressor −2.5∗ 
 D31962 58834 Dlc1 Deleted in liver cancer 1 2.0∗∗∗∗ 
 U92279 114705 Rgs14 Regulator of G-protein signaling 14 1.6∗ 2.1∗∗ 
 AF044673 114559 Pak3bp PAK-interacting exchange factor β 2.1∗∗ 
 AF058789 192117 Syngap1 Synaptic Ras GTPase-activating protein 1 2.5∗∗∗ 
 AF032120 83823 Rgs19ip1 Regulator of G-protein signaling 19-interacting protein 1 2.4∗ 3.8∗∗∗ 
Transcription      
 M64986 25459 Hmgb1 High mobility group box 1 −2.2∗ −1.7∗∗ 
 AJ005425 81518 Mef2d Myocyte enhancer factor 2D 1.7∗∗∗ 
 U18913 25446 Fosl2 fos-like Ag 2 1.8∗ 1.9∗∗ 
 X91810 25125 Stat3 Signal transducer and activator of transcription 3 1.9∗∗ 2.0∗∗ 
 X14788 81646 Creb1 cAMP response element binding protein 1 2.0∗∗∗ 
 U24175 24918 Stat5a Signal transducer and activator of transcription 5A 2.3∗∗ 
 J03933 24831 Thrb Thyroid hormone receptor β 2.4∗∗ 
 U67080 266680 Stl8 Suppression of tumorigenicity 18 3.1∗∗∗ 2.9∗ 
Transport/trafficking      
 U75581 79451 Fabp4 Fatty acid binding protein 4, adipocyte −4.8∗ 
 U13253 140868 Fabp5 Fatty acid binding protein 5, epidermal −2.4∗ −2.3∗ 
 J03583 54241 Cltc Clathrin, heavy polypeptide (Hc) −2.0∗∗ 
 X61654 29186 Cd63 CD63 Ag −2.0∗∗∗ 
 U21871 266601 Tomm20 Translocase of outer mitochondrial membrane 20 homologue (yeast) −1.7∗∗∗ 
 S68944  Ntt4 Sodium/chloride-dependent neurotransmitter transporter 1.9∗∗∗ 
 L19931 29502 Slc20a2 Solute carrier family 20, member 2 2.0∗∗ 
 X74226 171434 Phldb1 Pleckstrin homology-like domain, family B, member 1 2.1∗ 
 AF068202 114124 Akap1 A kinase (PRKA) anchor protein 1 2.1∗∗∗ 
 AF022774 171123 Rph3al Rabphilin 3A-like (without C2 domains) 2.2∗∗ 2.2∗∗∗ 
a

Treated/untreated allografts. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; ∗∗∗∗∗, p < 0.00001.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by Progetto di Ricerca “Meccanismi molecolari del danno nel trapianto singenico e nell’allotrapianto”, Ospedale Maggiore di Milano, Italy, and Progetto di Ricerca Finalizzata “Strategie innovative per il trapianto di fegato (SITF)”, Ministero della Salute, Italy.

3

Abbreviations used in this paper: α-MSH, α-melanocyte stimulating hormone; NDP, Nle4DPhe7; POD, postoperative day; SAM, significance analysis of microarrays; FDR, false discovery rate; CT, cycle threshold; PKC, protein kinase C; Plc, phospholipase C; Adcy6, adenylyl cyclase VI.

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