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The Journal of Immunology, 2005, 175: 3391-3401.
Copyright © 2005 by The American Association of Immunologists

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

Gualtiero Colombo*, Stefano Gatti{dagger}, Flavia Turcatti*, Andrea Sordi*, Luigi R. Fassati{dagger}, Ferruccio Bonino{ddagger}, James M. Lipton§ and Anna Catania2,*

* Division of Internal Medicine, {dagger} Division of Liver Transplantation, and {ddagger} Scientific Direction, Fondazione Instituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano, Italy; and § Zengen, Woodland Hills, CA 91367


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 {alpha}-melanocyte-stimulating hormone ({alpha}-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 {alpha}-MSH analog Nle4DPhe7 (NDP)-{alpha}-MSH. On postoperative day 1, the peptide induced expression of cytoskeleton proteins, intracellular kinases, transcription regulators, metallopeptidases, and protease inhibitors. Conversely, NDP-{alpha}-MSH repressed immune, inflammatory, cell cycle, and protein turnover mediators. Later effects of {alpha}-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-{alpha}-MSH exerted its effects on both Ag-dependent and -independent injury. The results indicate that NDP-{alpha}-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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 {alpha}-melanocyte-stimulating hormone ({alpha}-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). {alpha}-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 {alpha}-MSH in experimental heart transplantation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

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).

Surgical procedures

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.

Treatments

Each treatment group included five rats. Allograft recipients assigned to active treatment received i.p. injections of 100 µg of Nle4DPhe7 (NPD)-{alpha}-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.

cDNA macroarray hybridization

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, [{alpha}-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).

Analysis of macroarray data

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-{alpha}-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.

Real-time reverse transcription PCR analysis

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 C{gamma}1 (Plcg1), and Rn00565502_m1 for sodium channel voltage-gated type V {alpha} 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 ({Delta}{Delta}CT): the amount of target, normalized to the endogenous reference and relative to the calibrator, is given by the formula 2–{Delta}{Delta}CT (21). The unpaired Student t test was used to compare differences in mean fold changes; a probability value <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Allograft histology

NDP-{alpha}-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-{alpha}-MSH-treated animals: inflammation and edema were confined to the subendocardial region and no abscesses were evident.



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  		FIGURE 1. Histology of cardiac grafts. H&E staining (x120) of a control nontransplanted heart (A); POD4 cardiac allograft from a saline-treated rat (B) and from an NDP-{alpha}-MSH-treated animal (C).

 
Hierarchical agglomerative clustering

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-{alpha}-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-{alpha}-MSH treatment.



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  		FIGURE 2. Facing page

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

 
Treatment-associated gene expression changes

SAM analysis and the fold-change method (Table I) identified 53 genes whose expression was significantly altered by NDP-{alpha}-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).


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  		Table I. Genes regulated by NDP-{alpha}-MSH treatment in rat cardiac allografts

 

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  		Table IA. Continued

 

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  		Table IB. Continued

 


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  		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 d{epsilon}(i) of genes altered by NDP-{alpha}-MSH treatment. The solid line indicates where genes would align if their d(i) = d{epsilon}(i). At the threshold {Delta} = 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 {square}. Genes that passed also the second analysis based on unpaired two-tailed Student’s t test are reported in Table I.

 
With regard to the classes of genes affected by treatment, NDP-{alpha}-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-{alpha}-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 {beta}8), immune and inflammatory and/or cell infiltration responses (Cxcl2, Cxcr4, IL-1{beta}, 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.

Hierarchical agglomerative clustering of differentially expressed genes

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.

Gene expression changes in untreated and treated allografts relative to isografts

To separate effects of NDP-{alpha}-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).


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

 
Verification of the macroarray data using real-time RT-PCR

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-{alpha}-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-{alpha}-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.



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  		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-{alpha}-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-{alpha}-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.

 
Functional classification

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-{alpha}-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).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present data, based on gene expression profiling, reveal multiple protective influences exerted by NDP-{alpha}-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-{alpha}-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-{alpha}-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-{alpha}-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-{alpha}-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 PKC{epsilon} and PKC{delta} (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-{alpha}-MSH.

The increase in phospholipase C (Plc) isoenzyme mRNA observed in NDP-{alpha}-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-{alpha}-MSH treatment (Table I, Fig. 4).

Further, there is evidence that {alpha}-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-{alpha}-MSH. The regulator of G-protein signaling Rgs14, protein kinase C {eta}, and {epsilon} isoenzymes, Plc {beta} and {gamma} 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-{alpha}-MSH. Finally, NDP-{alpha}-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-{alpha}-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-{alpha}-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-{alpha}-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-{alpha}-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-{alpha}-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-{alpha}-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-{alpha}-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).



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  		FIGURE 5. Effects of NDP-{alpha}-MSH treatment on Ag-dependent and independent injury.

 
Despite its anti-inflammatory and cardioprotective influences, NDP-{alpha}-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-{gamma}, 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-{alpha}-MSH. This is not surprising as rejection prevention requires profound immunosuppression that is clearly not exerted by NDP-{alpha}-MSH.

NDP-{alpha}-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.


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


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
J. M. Lipton currently serves on the Board of Directors of Zengen.


   Footnotes
 
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. Back

2 Address correspondence and reprint requests to Dr. Anna Catania, Divisione di Medicina Interna, Pad. Granelli, Ospedale Maggiore Policlinico, Via F. Sforza 35, Milano 20122, Italy. E-mail address: anna.catania{at}unimi.it Back

3 Abbreviations used in this paper: {alpha}-MSH, {alpha}-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. Back

Received for publication May 17, 2005. Accepted for publication June 24, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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