protective action of ndp-msh in experimental subarachnoid hemorrhage
TRANSCRIPT
Experimental Neurology 234 (2012) 230–238
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Experimental Neurology
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Regular Article
Protective action of NDP-MSH in experimental subarachnoid hemorrhage☆
Stefano Gatti a, Caterina Lonati a,b, Francesco Acerbi c,d, Andrea Sordi a,b, Patrizia Leonardi b,e,Andrea Carlin b,e, Sergio M. Gaini d, Anna Catania a,b,⁎a Center for Surgical Research, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italyb Center for Preclinical Investigation, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italyc Department of Neurosurgery, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italyd Department of Neurological Sciences, Università degli Studi di Milano, Milan, Italye Department of Internal Medicine, Università degli Studi di Milano, Milan, Italy
☆ The authors declare no conflict of interest.⁎ Corresponding author at: Center for Preclinical Inve
Granda Ospedale Maggiore Policlinico, Via F. Sforza 35,E-mail address: [email protected] (A. Ca
0014-4886/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.expneurol.2011.12.039
a b s t r a c t
a r t i c l e i n f oArticle history:Received 22 September 2011Revised 10 December 2011Accepted 22 December 2011Available online 2 January 2012
Keywords:α-Melanocyte stimulating hormoneBasilar arteryGene expression profilingMelanocortinPreconditioningSubarachnoid hemorrhage
Subarachnoid hemorrhage (SAH) is still a major cause of morbidity and mortality. α-Melanocyte stimulatinghormone (α-MSH) and other melanocortin peptides exert potent neuroprotective action and they mightmodulate key molecules involved in SAH-induced vasospasm. The aim of this research was to determinewhether treatment with the α-MSH analog Nle4,DPhe7-α-MSH (NDP-MSH) exerts protective effects in ex-perimental SAH in the rat. Initial experiments examined effects of NDP-MSH on the basilar artery phenotypein the absence of injury. In these tests intrathecal injection of small concentrations (10 ng) of the peptide in-duced a tolerant phenotype similar to that observed after ischemic preconditioning. Then the effect of sys-temic treatment with NDP-MSH (100 μg i.v.) on experimental SAH was evaluated. SAH was induced by asingle-blood injection into the cisterna magna. The basilar artery phenotype was examined at 4 h and the ar-tery caliber at 5 days following SAH. Expression of 96 genes was analyzed by real-time reverse transcriptionpolymerase chain reaction (RT-PCR) using Custom Taqman Low-Density Arrays. Four hours after SAH, thetranscriptional profile of the basilar artery was deeply disrupted. Transcript alteration included genes in-volved in inflammation, stress response, apoptosis, and vascular remodeling. Treatment with NDP-MSH pre-vented most of these transcription changes and decreased phosphorylation of extracellular-signal-regulatedkinases (ERK1/2) and inhibitor protein IκBα. Vasospasm on day 5 was significantly reduced by NDP-MSH ad-ministration. These results combine with others on CNS inflammation to suggest that the melanocortinscould be safe and effective therapeutic candidates to treat SAH-related complications.
© 2012 Elsevier Inc. All rights reserved.
Introduction
Subarachnoid hemorrhage (SAH) is generally caused by the ruptureof a cerebral artery aneurysmwith discharge of blood into the basal cis-terns. Themost common and severe complication of SAH is cerebral va-sospasm that occurs 3–15 days after hemorrhage (Macdonald et al.,2007). No consistently efficacious therapies have been identified andimplemented in clinical practice for this dire condition (Lubrano-Berthelier et al., 2006).
Although there is no definite explanation for SAH-related cerebralvasospasm, a large number of studies indicate that inflammation sig-nificantly contributes to its pathogenesis (Dumont et al., 2003). Be-cause most of the molecules that have been implicated in delayedvasoconstriction are under control of the transcription factor nuclearfactor-κB (NF-κB), activation of NF-κB is likely involved in the
stigation, Fondazione IRCCS Ca'Milano 20122, Italy.tania).
rights reserved.
disorder (Zhou et al., 2007). It appears, therefore, that correction ofa crucial step such as NF-κB activation after hemorrhage could reducevasoconstriction (Aoki and Nishimura, 2010).
This action could be effectively exploited by treatmentwithmelano-cortin molecules. α-Melanocyte stimulating hormone (α-MSH) andother melanocortins make up a family of endogenous peptides derivedfrom pro-opiomelanocortin (POMC). Their signal transduction occursthrough five melanocortin (MC1 through MC5) guanine nucleotide-binding protein (G-protein)-coupled receptors functionally coupled toadenylyl cyclase (Mountjoy, 2010). Melanocortins exert modulatory ef-fects on local and systemic host reactions including control of fever andinflammation (Brzoska et al., 2008; Catania, 2007; Catania et al., 2004;Gatti et al., 2010). Reduced phosphorylation of the inhibitory moleculeIκBα and, consequently, reduced activation of NF-κB is a well estab-lished mechanism in the anti-inflammatory action of melanocortins(Eves and Haycock, 2010).
Melanocortins are effective agents in preclinical treatment of in-flammatory, traumatic, and vascular brain injury (Catania, 2008;Catania and Lipton, 1998;Muceniece andDambrova, 2010).α-MSH sig-nificantly diminished experimental brain inflammation in the mouse
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through reduced phosphorylation of IκBα and reduced activation of NF-κB (Ichiyama et al., 1999a, 1999b). Further, several investigations indi-cate that melanocortins are promising therapeutic molecules for treat-ment of stroke (Giuliani et al., 2006, 2007; Holloway et al., 2011). Inresearch on amurinemodel of ischemic injury,α-MSH reduced intrace-rebral tumor necrosis factorα (TNF-α) and interleukin 1 β (IL-1β) geneexpression following transient unilateral occlusion and reperfusion(Huang and Tatro, 2002). After transient global cerebral ischemia in ger-bils, melanocortin treatment reduced post-ischemic tissue injury andimproved recovery of behavioral functions, even when treatment wasbegun up to 9 h after ischemia (Giuliani et al., 2006). Protective influ-ences of NDP-α-MSHwere likewise observed in a rat model of focal ce-rebral ischemia induced by intrastriatal microinjection of endothelin-1(Giuliani et al., 2007).
The main objective in the present research was to determinewhether melanocortin administration exerts protective effects in ex-perimental SAH. In addition to the recognized neuroprotective action,the investigation was encouraged by previous observations that cir-culating α-MSH is reduced in patients with acute SAH (Magnoni etal., 2003). The data raised the possibility that restoration of normalblood α-MSH through administration of the peptide could be benefi-cial in this condition.
Initial experiments of the present research determined whetherα-MSH enhances expression of protective molecules in the basilar ar-tery of the rat in the absence of blood injury. This investigation wasbased on our recent observation that α-MSH causes expressionchanges in the heart that resemble the tolerant phenotype producedby transient ischemia or general anesthetics (Catania et al., 2010).The idea was that the peptide could likewise enhance expression ofprotective molecules in cerebral arteries. Treatment was performedusing the potent α-MSH analog Nle4,DPhe7-α-MSH (NDP-MSH), anon specific MC agonist that exerts similar effects relative to the nat-ural α-MSH molecule and is generally preferred for its greater chem-ical stability (Sawyer et al., 1980). In these experiments, theintrathecal route of administration was selected to exclude systemiccircuits based on activation of peripheral MC receptors.
Subsequently, the potential therapeutic action of NDP-MSH wasevaluated in experimental SAH. To this purpose, changes in gene ex-pression profile in the basilar artery were investigated using themodel based on a single injection of autologous blood into the cister-na magna (Prunell et al., 2003). This procedure allows early investiga-tion of the basilar artery that is not feasible with the more commonmethod based on two blood injections given at a 24-hour interval.Early examination is crucial because it appears that delayed vaso-spasm is triggered by changes that occur soon after hemorrhage(Cahill et al., 2006). In these experiments the peptide was injected in-travenously to better reproduce the clinical setting in which the i.v.route of administration would make immediate therapy more realis-tic and to take advantage of the systemic anti-inflammatory effects ofthe peptide.
Materials and methods
All the experiments were performed in compliance with the Prin-ciples of Laboratory Animal Care (NIH Publication No. 86–23, revised1985). The experimental protocol was approved by Istituto Superioredi Sanità, Ministero della Salute, Italy. Adult Sprague–Dawley malerats (Charles River, Calco, Italy) weighing 350–375 g were housed in-dividually in a ventilated cage system (Tecniplast, Buguggiate, Va,Italy) at 22±1 °C, 55±5% humidity, on a 12 h dark/light cycle, andwere allowed free access to rat chow feed and water ad libitum.
Intrathecal NDP-MSH
To evaluate effects of NDP-MSH on the basilar artery phenotype,10 ng of the peptide dissolved in 200 μl saline was injected into the
cisterna magna (n=9). Sham-operated rats received an equal vol-ume of i.t. saline. Anesthesia procedures were identical to those de-scribed below for the SAH model. Animals were sacrificed at 4 hunder deep anesthesia and brains were removed. Basilar arterieswere dissected and snap frozen in liquid nitrogen for molecular biol-ogy analysis.
SAH model and treatments
Anesthesia was induced using 1 mg/kg midazolam plus 100 mg/kgketamine i.p. folllowed by i.m. fentanyl 0.0075 mg/rat for pain con-trol. The depth of anesthesia was carefully monitored. A catheterwas inserted into the femoral artery under sterility procedures towithdraw blood and to measure blood pressure. Two-hundred micro-liters of autologous blood was withdrawn from the femoral artery andinjected into the cisterna magna over a 3 min period. The correct po-sition of the needle was checked by liquor aspiration. Sham-operatedanimals received an equal volume of i.t. saline. Rats were thereaftermaintained head down for 15 min and arterial catheter was removed.During the observation period the rats were monitored regularly andkilled prematurely in the presence of signs of distress (5% mortality).
The animals were randomly divided into four groups (n=9 pergroup): 1) controls; 2) sham-operated; 3) saline-treated SAH; and4) NDP-MSH-treated SAH. At the time of SAH induction, rats ingroup 2 and 3 received i.v. injections of 400 μl saline; rats in group4 received 100 μg i.v of NDP-MSH dissolved in 400 μl saline (the pep-tide was kindly provided by Prof. Paolo Grieco, Università Federico II,Napoli, Italy). The control group received anesthesia only. Rats weresacrificed at 4 h by exsanguination under deep anesthesia inducedby 1 mg/kg midazolam plus 100 mg/kg ketamine i.p. and brainswere removed. With the aid of a microscope, basilar arteries werecarefully dissected, cleared of connective tissue and snap frozen inliquid nitrogen for molecular biology analysis.
RNA purification and cDNA synthesis
To obtain sufficientmaterial formolecular biology andwestern blot-ting, 3 basilar arteries of rats from the same treatment group were ran-domly pooled in 0.4 ml buffer RLT (Qiagen Inc., Hilden, Germany).Samples were rapidly homogenized using MICCRA D-1 tissue homoge-nizer with a 5 mm tip (Labortechnik GmbH, Wasserburg, Germany).Total RNA was isolated by anion exchange chromatography usingRNeasy Micro Kit (Qiagen) according to the manufacturer's instruc-tions. Briefly, Buffer RLT lysates were mixed with 70% ethanol and themixture was transferred to an RNeasy column to bind RNA to thesilica-based membrane. Eluates obtained after centrifugation of col-umns were immediately frozen for protein purification. Genomic DNAcontamination was removed by on-column DNase I treatment (Qia-gen). RNA samples were suspended in RNase-free water and quantifiedspectrophotometrically. The 260 nm/280 nm absorbance ratio readingsof all samples was >1.9. RNA integrity was assessed by electrophoresison denaturing agarose–formaldehyde gels. Single-stranded cDNA wassynthesized from 300 ng total RNA in a volume of 50 μl using High Ca-pacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA). Re-verse transcriptase reaction was performed at 25 °C for 10 min, then37 °C for 2 h, followed by 85 °C for 5 s.
TaqMan Low-Density Array gene expression analysis
Gene expression was evaluated by real-time reverse transcriptionpolymerase chain reaction (RT-PCR) analysis using Custom Low-Density Arrays based on TaqMan chemistry (TLDA, Applied Biosystems)that are microfluidic cards with pre-loaded FAM-labeled Taqmanprobes and primers (Gene Expression Assays, Applied Biosystems). Ar-rays were designed to include genes involved in inflammation, stressresponse, apoptosis, vascular tone regulation, extracellular matrix, and
232 S. Gatti et al. / Experimental Neurology 234 (2012) 230–238
vascular remodeling. Ninety-six-well format cards (PN 4342259, Ap-plied Biosystems) were used to analyze 96 genes, including 8 referencegenes (Supplementary Table 1). Each card was loaded with 2 samples.An amount of cDNA corresponding to 30 ng of reverse transcribedRNA was added to 0.1 ml of Taqman Universal Master Mix 2x (AppliedBiosystems) and then loaded into each port of the array (2 port per sam-ple, 60 ng of cDNA per sample). Cards were spun twice at 1200 rpm for1 min in a Heraeus Multifuge X3 centrifuge (Thermo, Whaltam, MA)and then sealed with the card sealer (Applied Biosystems). Amplifica-tion reactions were performed on ABI PRISM 7900HT Sequence Detec-tion System with Taqman Array Upgrade (Applied Biosystems) withthe following thermal cycling conditions: 50 °C for 2 min, 94.5 °C for10 min and then 45 cycles of 97 °C for 30 s and 59.7 °C for 1 min. Rawdata were analyzed with SDS 2.3 software (Applied Biosystems) andthen imported in a RQ study (RQ Manager 1.2, Applied Biosystems) toconvert fluorescence intensities into threshold cycles (Ct). To optimizeaccuracy and precision, baseline was set automatically whereas thethreshold was adjusted manually to improve comparison of data fromdifferent cards. Only genes whose Ct were lower than 33 were consid-ered significantly expressed. Two genes (Il13 and Il22) were excludedbecause of low detection-threshold signals. Relative quantificationwas obtained with the comparative Ct method (ΔCt), using the averageCt of each gene across all samples as calibrator. mRNA relative quanti-ties (RQ) were then divided by a normalization factor calculated foreach sample with the geNorm VBA applet version 3.4 for Excel(Vandesompele et al., 2002), using Ct geometricalmean of themost sta-ble reference genes (actin beta, Actb; beta-2 microglobulin, B2m;glyceraldehyde-3-phosphate dehydrogenase, Gapdh; glucuronidasebeta, Gusb; ribosomal protein large P2, Rplp2; phosphoglycerate kinase1, Pgk1; TATA box binding protein, Tbp).
Analysis of gene expression data
RQ were log2-transformed and differences among groups were in-vestigated by both unsupervised and supervised methods. In order toreveal trends in gene expression, agglomerative hierarchical clusteranalysis was performed with DNA-chip analyzer program (www.dChip.org) using Spearman's rank correlation-based similarity metricand average linkage clustering technique. To identify differentiallyexpressed genes, log2-RQ were analyzed by permutation t-tests (t-statistics) with “significance analysis of microarrays procedure”(SAM, Excel Add-In version 3.09c; http://www-stat.stanford.edu).Two-class unpaired analysis with 700 random permutations was car-ried out. Genes were considered differentially expressed when thefold change was >1.5 for up-regulation and b0.65 for down-regulation with a false discovery rate (FDR) b5% (qb0.05).
Western blotting
Total proteins were recovered by acetone precipitation fromRNeasyMicro spin column eluates. Briefly, 4 volumes of ice-cold acetone wereadded and the mixture was incubated at−20 °C for 30 min. After cen-trifugation at 16 000×g, 4 °C for 10 min, supernatants were discardedand pellets were washed twice with 100 ml ice-cold ethanol and airdried. Pellets were then resuspended in 200 ml of RIPA buffer supple-mented with protease and phosphatase inhibitor cocktail (SIGMA,Saint Louis, MO). Protein concentration was determined using the BCAProtein Assay (Thermo, Whaltam, MA). Aliquots of tissue lysate con-taining 25 μg of proteins were subjected to mini-SDS-polyacrylamidegel electrophoresis, transferred onto Trans-Blot nitrocellulose mem-branes (Bio-Rad, Hercules, CA) and blocked with SuperBlock BlockingBuffer (Thermo) in TBST (Tris Buffered Saline, 0.1% Tween 20; Bio-Rad) for 1 h at room temperature. After blocking, membranes wereprobed overnight at 4 °C with rabbit monoclonal anti-phospho-IκBα(Ser32) (1:500), anti-IκBα (1:500), or rabbit polyclonal anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) (pErk1/2; 1:500) antibodies (all
from Cell Signaling Technology Inc., Danvers, MA). An horseradish per-oxidase (HRP)-conjugated donkey anti-rabbit IgG (Amersham Biosci-ences, Little Chalfont, UK) was used as secondary antibody (1:50 000).Antibody–antigen complexes were detected with enhanced chemilu-minescence (ECL) AdvancedWestern Blotting System (Amersham Bio-sciences), following manufacturer's instructions. Loading control wasperformed using a goat monoclonal anti-GAPDH antibody (1:5000)(Santa Cruz Biotechnology, Santa Cruz, CA), a secondary HRP-conjugated sheep anti-goat IgG (1:50 000) (Santa Cruz Biotechnology),and ECL reagents. Protein bands were visualized and the luminescentsignals captured with a Kodak Gel Logic 2200 Digital Imaging System(EastmanKodak, Rochester, NY). Densitometric analysiswas performedusing the Kodak Molecular Imaging Software version 4.0.5.
Delayed vasospasm
Delayed vasoconstriction was evaluated 5 days after SAH. Animalswere treated with a single injection of NDP-MSH 100 μg i.v. or anequal volume of i.v. saline at the time of SAH induction (n=6 pergroup). SAH was produced using the same procedure describedabove. On day 5 post-hemorrhage animals were perfused with iso-tonic phospate-buffered saline (PBS), followed by ice cold 4% parafor-maldehyde (PFA) under deep anesthesia (ketamine 100 mg/kg andmidazolam 1 mg/kg) and exsanguinated. The brains were removedand fixed with ice cold phosphate-buffered 4% PFA for 48 h. Thefixed brains were placed in a solution containing 30% sucrose dilutedin PBS overnight at 4 °C, embedded in Optimal Cutting Temperaturecompound (OCT) (Bio Optica, Milan, Italy) and rapidly frozen in iso-pentane pre-cooled in liquid nitrogen. The frozen brains weremounted on a cryostat (bio-Optica, Milan, Italy) and cut in 20 μmthick sections and subsequently stained with hematoxylin–eosin(H&E). For histological evaluation, 3 sections from median level ofthe basilar artery were analyzed in each animal. The sections were ex-amined and photographed using a Nikon Eclipse TE300. The luminalcross-sectional diameter of the arteries was measured in a blind man-ner at 40× magnification using an image analysis program (ZeissAxiovision).
Statistical analysis
Statistical analysis of gene expression in microfluidity cards wasperformed as described above. Western blot data and diameters ofthe basilar arteries were analyzed using OneWay Analysis of Variance(ANOVA) followed by Bonferroni's multiple comparison test (Sigma-Stat 3.1, Systat Software Inc, CA, USA). The gene expression datareported in figures were re-analyzed using this technique as well. Aprobability value of b0.05 was considered statistically significant.
Results
NDP-MSH induced a tolerant phenotype in the basilar artery
Effects of NDP-MSH on the basilar artery in the absence of bloodinjury were evaluated 4 h after injection of 10 ng of the peptide intothe cisterna magna. Genes differentially expressed in arteries fromNDP-MSH-treated rats relative to sham animals were identifiedusing SAM two class unpaired analysis. Based on a fold change >1.5for up-regulation and b0.65 for down-regulation with a FDR b5%,the analysis identified 27 genes significantly enhanced in NDP-MSH-treated rats (qb0.05) (Table 1). Transcripts induced by NDP-MSHwere anti-apoptotic and survival factors including B-cell CLL/lympho-ma 2 (Bcl2), myelocytomatosis oncogene (Myc), fibroblast growthfactor 2 (Fgf2), tumor necrosis factor (Tnf), and rap guanine nucleo-tide exchange factor 3 (Rapgef3), genes involved in stress responseand repair mechanisms such as activating transcription factor 3(Atf3), early growth response 1 (Egr1), tumor protein p53 (p53),
233S. Gatti et al. / Experimental Neurology 234 (2012) 230–238
cyclin-dependent kinase inhibitor 1A (Cdkn1a),in oxidative responseincluding nitric oxide synthase 1 (Nos1), endothelial cell nitric oxidesynthase (eNos, Nos3), and superoxide dismutase 2 (Sod2). Genes in-volved in immune/inflammatory response including interleukin 10(Il10), interferon regulatory factor 1 (Irf1), suppressor of cytokine sig-naling 1 and 3 (Socs1, Socs3), Janus kinase 2 (Jak2), signal transducerand activator of transcription 1 and 3 (Stat1, Stat3), and chemokine C-C motif ligand 20 (Ccl20) were likewise induced by intrathecal NDP-MSH. Further, NDP-MSH injection increased mRNA for nuclear factorof kappa light polypeptide gene enhancer in B-cells inhibitor alpha(Nfkbia or IκBα) and IκBα protein in the basilar artery (Figs. 1A–B).
NDP-MSH inhibited SAH-induced disorder of the basilar artery phenotype
The transcription profile of the basilar arteries harvested 4 h afterSAH inductionwas deeply altered. Unsupervised hierarchical clusteringanalysis, which groups samples according to gene expression similari-ties, sorted together the samples from the 2 SAH groups (saline- andNDP-treated) and those from the 2 groups that did not receive i.t.blood injection (control and sham) (Fig. 2). Further, within these 2large clusters, unsupervised clustering sorted together specific treat-ment groups: all the samples from i.v. NDP-MSH-treated animals clus-tered together, separately from i.v. saline-treated SAH animals thatclustered together. Therefore, NDP-MSH treatment was associated
Table 1Transcriptional changes induced by NDP-MSH in the rat basilar artery in the absence ofinjury.
Genesymbol
Gene name Fold changevs sham
q-value
Cell survivalTnf Tumor necrosis factor 3.54 0.000Bcl2 B-cell CLL/lymphoma 2 3.28 0.000Fgf2 Fibroblast growth factor 2 (basic) 2.04 0.000Rapgef3 Rap guanine nucleotide exchange
factor (GEF) 31.99 0.000
Response to oxidative stressSod2 Superoxide dismutase 2, mitochondrial 2.27 0.037Nos3 Nitric oxide synthase 3 (endothelial cell) 1.95 0.036Nos1 Nitric oxide synthase 1 (neuronal) 1.59 0.036
Inflammatory/immune responseSocs1 Suppressor of cytokine signaling 1 4.13 0.036Socs3 Suppressor of cytokine signaling 3 3.74 0.000Il10 Interleukin 10 3.62 0.000Il6ra Interleukin 6 receptor, alpha 3.05 0.000Nfkbia Nuclear factor of kappa light polypeptide
gene enhancer in B-cells inhibitor, alpha2.46 0.016
Irf1 Interferon regulatory factor 1 2.31 0.036Ccl3 Chemokine (C-C motif) ligand 3 2.30 0.047Stat3 Signal transducer and activator of
transcription 32.19 0.000
Stat1 Signal transducer and activator oftranscription 1
2.06 0.036
Jak2 Janus kinase 2 2.02 0.047Ccl20 Chemokine (C-C motif) ligand 20 1.96 0.036
Stress responseAtf3 Activating transcription factor 3 3.84 0.036Cdkn1a Cyclin-dependent kinase inhibitor 1A (p21) 2.44 0.000Cebpb CCAAT/enhancer binding protein (C/EBP),
beta2.22 0.000
p53 Tumor protein p53 2.16 0.036Egr1 Early growth response 1 1.89 0.036
Vascular remodeling and BBB integritySerpine1 Serpin peptidase inhibitor 4.21 0.036Vcam1 Vascular cell adhesion molecule 1 2.26 0.036Mmp9 Matrix metallopeptidase 9 1.90 0.036Icam1 Intercellular adhesion molecule 1 1.67 0.047
Fig. 1. Iκbα mRNA and protein expression in the basilar artery after injection of 10 ngNDP-MSH into the cisterna magna in the absence of blood (n=9 rats for each experi-mental condition; 3 arteries were combined in 3 pools). (A) Iκbα mRNA evaluatedusing qRT-PCR is reported as relative expression. One Way ANOVA followed by Bonfer-roni's multiple comparison test, pb0.05. (B) Iκbα protein expression analyzed byWestern Blotting. Expression level is reported as Iκbα/GAPDH ratio. Representativeblots are shown above the respective densitometric analysis. Bars denote mean±SEM. One Way ANOVA followed by Bonferroni's multiple comparison test, pb0.01.GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Iκbα, inhibitor kappa B alpha;NDP-MSH, Nle4,DPhe7-α-melanocyte stimulating hormone.
with a distinct expression pattern for a number of genes sufficient toendow a specific transcriptional signature.
SAH-associated differentially expressed genes were identified usingSAM two class unpaired analysis comparing samples from saline-treated SAH and sham-operated rats. Based on a fold change >1.5 forup-regulation and b0.65 for down-regulation with a FDR b5%, the anal-ysis identified 58 genes with highly significant differential expression(q=0) in saline-treated SAH relative to sham animals (Table 2). Ex-pression was enhanced for 45 and reduced for 13 genes. Transcript al-teration after SAH included molecules involved in apoptosis, vascularremodeling, inflammatory response, response to oxidative stress, signaltransduction, transcription, and vasoactive molecules. Genes repressedby hemorrhage were transcripts involved in cell division and compo-nents of intercellular junctions, such as occludin (Ocln) and tightjunction protein 1 (Tjp1). Thirty-four of these transcripts were signifi-cantly modulated by systemic NDP-MSH treatment (Table 1). The pep-tide prevented induction of genes involved in inflammatory response,including interleukin 1 beta (Il1b), vascular cell adhesion molecule 1(Vcam1), interleukin 6 (Il6), chemokine C-X-C motif ligand 1 (Cxcl1),nitric oxide synthase 2, inducible (Nos2), S100 calcium binding proteinA8 (s100a8), and S100 calcium binding protein A9 (s100a9). Further,there was inhibition of transcripts associated with apoptosis, such asFas and Fas ligand (Faslg), and genes linked to vascular tone regulation
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and remodeling including endothelin 1 (Edn1), vascular endothelialgrowth factor A (Vegfa), nuclear receptor subfamily 4, groupA,member1 (Nr4a1). Other geneswhose expressionwas reduced byNDP-MSH areAtf3, CCAAT/enhancer binding protein (Cebpb), Egr1; Irf1 or transcriptsinvolved in extracellularmatrix remodeling such asmatrix metallopep-tidase 9 and 13 (Mmp9 and Mmp13). Although the general effect ofNDP-MSH was inhibitory, in samples from peptide-treated relative tosaline-treated SAH rats there was increased expression of protein ki-nase, AMP-activated, alpha 1 (Prkaa1), epidermal growth factor (Egf),cyclin D1 (Ccnd1), Pomc, and Ocln.
Western blot analysis showed that SAH enhanced phosphorylationof ERK1/2 and IκBα in the basilar artery (Fig. 3). This SAH-associatedphosphorylation was significantly lesser in NDP-MSH-treated animals(Fig. 3).
NDP-MSH reduced delayed vasospasm of the basilar artery
Morphometric analysis of the basilar arteries was performed 5 daysafter SAH induction. Arteries from saline-treated SAH rats showedmarked reduction in section diameters relative to control arteries(296±13 and 156±37 μm, respectively, pb0.005). Hemorrhage-induced vasoconstriction was significantly attenuated in rats treatedwith NDP-MSH administered as a single i.v. injection at the time ofSAH injury (217±20 μm, pb0.01 vs SAH) (Fig. 4).
Discussion
The data show that the peptide NDP-MSH induces a salutary pheno-type in the basilar artery in the absence of injury and during subarach-noid hemorrhage. The protective effects of this melanocortin in SAH areconsistent with its well established neuroprotective action (Catania,2008) and with the observation that subnormal α-MSH in blood ofSAH patients is associated with a poorer outcome (Magnoni et al.,2003).
In recent research, NDP-MSH caused a tolerant phenotype in theheart that resembled preconditioning brought about by transient ische-mia or general anesthetics (Catania et al., 2010). Therefore, an aim in thisresearch was to determine whether NDP-MSH induces protectivechanges in cerebral arteries as well. Consistent with this idea, the tran-scriptional profile induced by NDP-MSH in the absence of blood injuryshows striking similarities with the tolerant phenotype observed duringpreconditioning in the brain (Dirnagl et al., 2009; Lehotsky et al., 2009;Ravati et al., 2001; Stevens et al., 2011; Tang et al., 2006). Expressionchanges included induction of anti-apoptotic and survival factors andgenes involved in stress, repair, oxidative, immune, and inflammatoryresponses. A novel observationwas thatNDP-MSHenhances productionof the protective factor IκBα. In this regard, it is worth mentioning thatenhanced IκBα synthesis is considered a mechanism through which is-chemic preconditioning confers a protective phenotype (Blondeau et al.,2001; Tang et al., 2006). Further, among themolecules induced by NDP-MSH, eNOS is particularly interesting as eNOS-derived nitric oxide is a
Fig. 2. Unsupervised analysis of gene expression profiles in the rat basilar arteries.Gene expression was evaluated using RT-qPCR performed on Taqman Low-Density Ar-rays. Columns denote individual samples (each sample consists of a randomized poolof 3 arteries); rows correspond to individual genes, with gene symbols listed on theright. Data were analyzed by agglomerative hierarchical clustering (clustering method:average linkage; distance metric: 1 — Spearman's rank correlation) using dCHIP soft-ware. Unsupervised analysis sorted together samples from the SAH groups (saline-and NDP-treated) and samples from animals not subjected to SAH (controls andsham). Within these 2 large clusters, the analysis separated samples from NDP-MSH-treated animals from saline-treated SAH rats. Gene expression level is represented asa color normalized across each row, with brighter red for higher expression andbrighter green for lower expression relative to control. NDP-MSH, Nle4,DPhe7-α-mela-nocyte stimulating hormone; SAH, subarachnoid hemorrhage.
Table 2SAH-induced changes in gene expressiona in the rat basilar artery and effect of NDP-MSH treatment.
Genesymbol
Gene name Fold changevs sham
q-value
SAH SAH+NDP
SAH+NDPvs SAH
ApoptosisFas Fas (TNF receptor superfamily,
member 6)3.33 2.17 0.000
Casp1 Caspase 1 2.70 1.84 nsFaslg Fas ligand (TNF superfamily,
member 6)2.03 1.32 0.000
Vascular remodelingVegfa Vascular endothelial growth
factor A5.13 3.43 ns
Cdkn1a Cyclin-dependent kinase inhibitor 1A 3.32 1.93 0.015Myc Myelocytomatosis oncogene 3.10 3.50 nsCcng1 Cyclin G1 0.58 0.60 nsEgf Epidermal growth factor 0.47 0.86 0.036Ccnd1 Cyclin D1 0.35 0.65 0.036Cdkn1b Cyclin-dependent kinase inhibitor 1B 0.30 0.40 ns
Cell junctionsOcln Occludin 0.06 0.19 0.013Tjp1 Tight junction protein 1 0.55 0.81 nsCtnnb1 Catenin (cadherin associated protein),
beta 10.41 0.61 ns
Extracellular proteasesMmp13 Matrix metallopeptidase 13 82.13 31.66 0.024Serpine1 Serine (or cysteine) peptidase inhibitor,
clade E, member 147.68 20.31 0.000
Mmp9 Matrix metallopeptidase 9 13.59 7.78 0.024
Stress-induced genesAtf3 Activating transcription factor 3 18.40 9.74 0.000Irf1 Interferon regulatory factor 1 7.10 4.06 0.000Nfkb1 Nuclear factor of kappa light polypeptide
gene enhancer in B-cells 15.23 3.81 ns
Egr1 Early growth response 1 4.99 2.82 0.015Nr4a1 Nuclear receptor subfamily 4, group A,
member 13.44 1.71 0.015
Gadd45a Growth arrest and DNA-damage-inducible,alpha
2.19 1.71 ns
Immune regulatorsSocs1 Suppressor of cytokine signaling 1 31.32 13.96 0.000Ccl20 Chemokine (C-C motif) ligand 20 13.23 8.01 nsSocs3 Suppressor of cytokine signaling 3 11.98 7.53 0.024Il10 Interleukin 10 9.79 5.88 nsPomc Proopiomelanocortin 0.11 0.36 0.050
Inflammatory responseNos2 Nitric oxide synthase 2, inducible 468.46 180.17 0.000Il6 Interleukin 6 333.90 110.97 0.000Cxcl1 Chemokine (C-X-C motif) ligand 1
(melanoma growth stimulating activity,alpha)
33.78 16.49 0.000
Il1rn Interleukin 1 receptor antagonist 31.61 14.27 0.000S100a9 S100 calcium binding protein A9 29.87 9.51 0.000S100a8 S100 calcium binding protein A8 25.32 8.18 0.000Il8rb Interleukin 8 receptor, beta 22.70 8.71 0.000Il1b Interleukin 1 beta 16.56 7.66 0.000Ccl2 Chemokine (C-C motif) ligand 2 15.54 11.51 nsVcam1 Vascular cell adhesion molecule 1 11.98 7.14 0.015Sele selectin E 11.24 5.39 0.038Ccl3 Chemokine (C-C motif) ligand 3 8.99 6.32 nsTnf Tumor necrosis factor (TNF superfamily,
member 2)7.30 3.43 0.024
Ifnb1 Interferon beta 1, fibroblast 7.10 2.09 0.000Nfkbia Nuclear factor of kappa light
polypeptide gene enhancer in B-cellsinhibitor, alpha
6.40 4.10 0.015
Ptgs2 Prostaglandin–endoperoxide synthase 2 6.09 3.05 0.000Il6ra Interleukin 6 receptor, alpha 3.88 2.75 ns
(continued on next page)
Table 2 (continued)
Genesymbol
Gene name Fold changevs sham
q-value
SAH SAH+NDP
SAH+NDPvs SAH
Icam1 Intercellular adhesion molecule 1 3.12 2.70 nsPecam1 Platelet/endothelial cell adhesion
molecule 10.31 0.51 ns
Response to oxidative stressSod2 Superoxide dismutase 2, mitochondrial 12.87 10.47 nsHif1a Hypoxia-inducible factor 1, alpha
subunit (basic helix–loop-helixtranscription factor)
2.56 2.08 ns
Hmox1 heme oxygenase (decycling) 1 2.50 4.03 0.050
Signal transductionJak2 Janus kinase 2 6.40 4.64 nsStat3 Signal transducer and activator of
transcription 33.35 2.52 ns
Stat1 Signal transducer and activator oftranscription 1
2.05 1.64 ns
Prkaa1 Protein kinase, AMP-activated, alpha 1catalytic subunit
0.59 0.92 0.013
Cntf Ciliary neurotrophic factor 0.45 0.61 ns
TranscriptionCebpb CCAAT/enhancer binding protein
(C/EBP), beta4.34 2.77 0.000
Hes1 Hairy and enhancer of split 1(Drosophila)
0.59 0.82 ns
Vascular tone regulationEdn1 Endothelin 1 3.99 2.39 0.000Nos1 Nitric oxide synthase 1, neuronal 0.47 0.59 ns
a Genes reported in the table were identified using SAM two class unpaired analysis(fold change >1.5 for up-regulation and b0.65 for down-regulation, q=0) in saline-treated SAH vs sham animals.
235S. Gatti et al. / Experimental Neurology 234 (2012) 230–238
key mediator of neurovascular protection during preconditioning(Vellimana et al., 2011). Because all these molecular changes occurredafter intrathecal administration of very small peptide concentrations, itappears that there was a local action of NDP-MSH on the artery, withoutinvolvement of systemic modulatory circuits.
The single-blood injection model of experimental SAH exploited inthis research caused marked alteration in the expression profile of thebasilar artery. Systemic treatment with NDP-MSH prevented most ofthese detrimental changes. The peptide significantly inhibited induc-tion of inflammatory mediators including Nos2, prostaglandin–endo-peroxide synthase 2 prostaglandin–endoperoxide synthase 2 (Ptgs2 orCOX-2), cytokines, chemokines, adhesion molecules, and calcium bind-ing proteins. This effect is consistent with the well established anti-inflammatory effect of melanocortins in peripheral tissues and withinthe brain (Brzoska et al., 2008; Catania, 2008; Catania et al., 2004;Getting, 2006; Lipton and Catania, 1997). As a consequence of modula-tion of NF-κB-mediated transcription, melanocortins reduce productionof pro-inflammatory agents by leukocytes and other cell types (Catania,2007). Consistently, inhibition of cytokines and other mediators wasobserved in models of neural damage in vitro and in vivo (Catania,2008; Muceniece and Dambrova, 2010). TNF-α, a cytokine of para-mount importance in neurodegeneration, was inhibited by melanocor-tins in several models of brain inflammation (Lipton et al., 1998; Rajoraet al., 1997; Teare et al., 2004). Further, α-MSH reduced inducible nitricoxide synthase and cyclooxygenase-2 gene expression in rat hypothal-amus (Caruso et al., 2004); natural and synthetic melanocortins pre-vented LPS-induced nitric oxide production in the mouse forebrain(Muceniece et al., 2006).
Although the anti-inflammatory effects are certainly crucial duringSAH, the protective action of NDP-MSH was not restricted to controlof inflammation. Indeed, treatment benefits involved other molecules
Fig. 4. NDP-MSH effect on SAH-induced vasospasm in rat basilar artery. Delayed vaso-constriction evaluated on day 5 after SAH was significantly inhibited by treatment withNDP-MSH administered as a single i.v. injection at time 0 (n=6). Representative im-ages of hematoxylin–eosin stained basilar artery sections from (A) SAH+saline and(B) SAH+NDP-MSH rats are shown. Three sections from the median level of each bas-ilar artery were examined and photographed using a Nikon Eclipse TE300. The luminalcross-sectional diameter of the arteries was measured at 40× magnification using animage analysis program (Zeiss Axiovision). Bars denote mean±SEM of the percentvalue relative to control arteries. **Pb0.01. NDP-MSH, Nle4,DPhe7-α-melanocyte stim-ulating hormone; SAH, subarachnoid hemorrhage. Scale bar=50 μm.
Fig. 3. Phosphorylation of ERK1/2 and Iκbα in the basilar artery after SAH.Western Blotanalysis shows enhanced phosphorylation of ERK1/2 and Iκbα in the basilar artery 4 hafter SAH. Injury-associated phosphorylation was significantly lesser in NDP-MSH-treated animals. Tissue lysates (n=9 rats for each experimental condition; 3 arterieswere combined in 3 pools) were subjected to SDS-page electrophoresis, transferredon a nitrocellulose membrane and probed with anti-phospho-Iκbα-(Ser32) or anti-phospho-Erk1/2 (Thr202/Tyr204) antibodies. An anti-GAPDH antibody was used asloading control. Representative blots for each protein are shown above the respectivedensitometric analysis performed using Kodak Molecular Imaging Software version4.0.5. Bars denote mean±SEM. Probability was estimated using One Way ANOVA fol-lowed by Bonferroni's multiple comparison test, pb0.05. ERK1/2, extracellular-signal-regulated kinases; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Iκbα, inhibi-tor kappa B alpha; NDP-MSH, Nle4,DPhe7-α-melanocyte stimulating hormone; SAH,subarachnoid hemorrhage.
236 S. Gatti et al. / Experimental Neurology 234 (2012) 230–238
of major importance for vascular homeostasis and function. Of particu-lar interest, there wasmarked inhibition of endothelin 1, a potent vaso-constrictor produced by leukocytes and endothelial cells activated byblood clots, hemolysis products, and cytokines. Endothelin 1 is consid-ered the main pathogenetic factor in development and propagation ofcerebral vasospasm (Kozniewska et al., 2006); its inhibition is, there-fore, crucial.
Another relevant effect associated with NDP-MSH treatment wasthe increased expression of Hmox1. Heme oxygenase-1 is a stress-inducible protein that catalyzes breakdown of heme. During SAH,heme oxygenase-1 induction in the artery wall is an intrinsic regula-tory mechanism that acts against delayed vasospasm (Suzuki et al.,
1999). Indeed, intracisternal injection of antisense heme oxygenase-1 oligodeoxynucleotide significantly delayed the clearance of oxyhe-moglobin and deoxyhemoglobin from the subarachnoid space andaggravated angiographic vasospasm (Suzuki et al., 1999).
Angiogenesis and extracellular matrix remodeling in cerebral arter-ies likewise appear to promote vasoconstriction after SAH (Borel et al.,2003; Vikman et al., 2006). Consistently, the present study found en-hanced expression of Vegfa, Mmp9, and Mmp13 in untreated rats. In-crease in these mediators was attenuated when SAH animals receivedNDP-MSH.
With regard to the cell signaling pathways induced by SAH, it is clearthat activation of NF-κB has a prominent importance (Zhou et al., 2007).Therefore, inhibition of IκBα phosphorylation by NDP-MSH likely con-tributes to protective effects. However, it appears that SAH-inducedactivation of the MEK/ERK pathway is likewise responsible for tran-scriptional upregulation of inflammatorymolecules including cytokinesand metalloproteinases in cerebral arteries (Chen et al., 2009; Maddahiet al., 2011). Specific blockade of phosphorylation of this pathway abol-ished the enhanced expression of cytokines andMmp9 in large cerebralarteries and in microvessels (Maddahi et al., 2011). Of interest, the pre-sent data show that NDP-MSH treatment, in addition to reduced phos-phorylation of IκBα, also inhibited ERK1/2 phosphorylation. It appears,therefore, that the modulatory influences of NDP-MSH are exertedthrough different signaling pathways.
Though there is no definite explanation for the enhanced arterial con-tractility that occurs after SAH, it is clear that inflammation, angiogenesis,oxidative stress, and vascular remodeling individually and collectivelyplay a role in this dire complication (Lubrano-Berthelier et al., 2006;Vikman et al., 2006). The initial bleed and the complex pathophysiologicalmechanisms that follow it, obviously predispose the brain to the second-ary injury (Cahill et al., 2006). Although recent research indicates thatprevention of vasospasm may not correlate with clinical outcome(Macdonald et al., 2011), cerebral vasospasm contributes to poor progno-sis and is a negative factor in SAH patients (Lubrano-Berthelier et al.,2006; Macdonald et al., 2007). Therefore, the observation that NDP-MSH treatment, administered as a single peripheral injection at the timeof injury, significantly reduced hemorrhage-induced vasoconstriction is
237S. Gatti et al. / Experimental Neurology 234 (2012) 230–238
encouraging in consideration of the potential use of melanocortins intreatment of this severe condition.
It is now clear that the melanocortin system, a highly conservedevolutionary component, participates in control of disparate func-tions. A major contribution to the host physiology resides in the ca-pacity to prevent tissue injury in the presence of a harmfulchallenge. Monocytes (Rajora et al., 1996; Taherzadeh et al., 1999),macrophages (Star et al., 1995), and endothelial cells (Scholzen etal., 1999) produce α-MSH and express melanocortin receptors in au-tocrine/paracrine circuits. Brain cells are a source and a target for mel-anocortins. Microvascular endothelial cells isolated from mousebrains express high affinity MC1R (de Angelis et al., 1995): these re-ceptors might provide a transport system for melanocortins fromblood into the brain tissue. Melanocortin receptors are also expressedin human astrocytes (Caruso et al., 2007) and microglia (Delgado etal., 1998). LPS-induced NF-κB activation was reduced in human glio-ma cells transfected with an α-MSH vector (Ichiyama et al., 1999a).Further, immunoneutralization of endogenous α-MSH within thebrain enhanced and prolonged cytokine-induced fever in rabbits(Shih et al., 1986). These observations are consistent with the ideathat, during brain injury, circulating or locally produced α-MSH ex-erts modulatory actions. Administration of synthetic melanocortinscould take advantage of this potent endogenous circuit. Indeed, thewide distribution of melanocortin receptors within the nervous sys-tem suggests that these receptors could be pharmacological targetsin treatment of brain and spinal cord disorders. Therefore, adminis-tration of α-MSH in subarachnoid hemorrhage likely reproducedand amplified the signaling pathway based on melanocortins. Thisobservation is novel and significant, although further investigationsare needed to clarify the effective magnitude of the response and pos-sible time limits that could hinder therapeutic use.
Finally, the potential salutary effects of melanocortin treatment dur-ing clinical SAH likely involve additional influences that have not beenspecifically investigated in the present research. Notably,α-MSH exertspotent antipyretic action (Lipton and Catania, 1997). Fever after SAH is awell known negative factor and prophylactic fever control could im-prove outcome (Fernandez et al., 2007). Further, patients withSAH show signs of a systemic inflammatory response syndrome thatworsens patient outcome (Stevens and Nyquist, 2007). Peripheralimmunodepression is associated with increased susceptibility to infec-tion (Stevens and Nyquist, 2007). Previous research has shown thatmelanocortins can effectively reduce systemic consequences of braininjury (Catania et al., 2009); the peripheral effects could complementthe beneficial influences exerted on the brain.
In conclusion, the data show that NDP-MSH administration re-duces early alteration of the basilar artery phenotype and attenuatesdelayed vasoconstriction. Melanocortins are very safe compounds al-ready tested in clinical studies (Hadley and Dorr, 2006) and could beeffective therapeutic candidates for SAH-related complications.
Supplementary materials related to this article can be found on-line at doi:10.1016/j.expneurol.2011.12.039
Acknowledgments
This work was supported by Grants of Fondazione IRCCS Ca'Granda-Ospedale Maggiore Policlinico, Fondazione Fiera Milano, andMinistero dell'Università, Italy.
The authors thank Dr. Silvia Cristini at Fondazione IRCCS IstitutoNeurologico Carlo Besta, Milano for histologic examination.
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