European Journal of Pharmacology 604 (2009) 117–124

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European Journal of Pharmacology

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Cardiovascular Pharmacology

S 35171 exerts protective effects in spontaneously hypertensive stroke-prone rats bypreserving mitochondrial function

Paolo Gelosa a,1, Cristina Banfi a,b,1, Maura Brioschi a,b, Elena Nobili a, Anita Gianella a, Uliano Guerrini a,Alice Pignieri a, Elena Tremoli a,b, Luigi Sironi a,b,⁎a Department of Pharmacological Sciences, University of Milan, Italyb Centro Cardiologico Monzino IRCCS, Milano, Italy

⁎ Corresponding author. Department of PharmacolMilan, Via Balzaretti 9, 20133Milano, Italy. Tel.: +39 02 50

E-mail address: luigi.sironi@unimi.it (L. Sironi).1 These authors contributed equally to this study.

0014-2999/$ – see front matter © 2009 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.12.027

a b s t r a c t

a r t i c l e i n f o

Article history:

S 35171 is one of a family Received 8 April 2008Received in revised form 1 December 2008Accepted 11 December 2008Available online 24 December 2008

Keywords:Cerebral ischemiaStroke-prone ratsMitochondriaProteome

of compounds that have been designed to protect mitochondrial function. Wetested the hypothesis that S 35171 exerts protective effects in spontaneously hypertensive stroke-prone rats(SHRSPs), an animal model developing spontaneous brain damage preceded by proteinuria and systemicinflammation revealed by the urinary accumulation of acute-phase proteins (APPs) originating in the liver.Male SHRSPs fed a permissive diet received vehicle or S 35171 (10 mg/kg/day) started simultaneously with ahigh-sodium diet (group A) or after the establishment of proteinuria (group B). The drug delayed urinaryAPPs accumulation and the appearance of magnetic resonance imaging (MRI)-monitored brain lesions (after62±3 days in group A, and 51±2 days in controls, Pb0.01). The delay was more pronounced in group B as 30%of the animals survived the entire 90-day experimental period without brain abnormality. Proteomic analysisshowed no significant alteration in the expression pattern of brain mitochondrial proteins, but the livermitochondrial levels of carbamoylphosphate synthase I (CPS-I), an enzyme involved in urea metabolism) andthe antioxidant peroxiredoxin-3 spot were affected by hypertension and S 35171. Stress reduces CPS-I andinduces the peroxiredoxin-3 spot, whereas S 35171 brought about normal CPS-I expression and a 12-foldhigher level of the peroxiredoxin-3 spot. As both enzymes are involved in maintaining mitochondrialfunctions, their increased expression after S 35171 treatment may be responsible for delaying thepathological condition that leads to the development of brain damage in SHRSPs.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Despite advances in critical caremedicine, cerebral ischemia remainsa major clinical problem because of the lack of appropriate pharmaco-logical tools for its prevention and treatment. This is partially due to thecomplexity and variety of its underlying pathophysiological processes atcellular, sub-cellular and molecular levels, many of which are incom-pletely understood. One aspect that deserves in-depth investigation isthe involvementofmitochondrial dysfunction in thepathological eventsthat occur during the development of stroke. Recent data indicate thatabnormal mitochondrial function plays amajor role in the developmentof organ failure, particularly at renal (de Cavanagh et al., 2005), vascular(Fennell et al., 2002), cardiac (Davidson andDuchen, 2007), and cerebrallevels (Muramatsu et al., 2007). Furthermore, the pharmacologicalinhibition of pathological mitochondrial alterations has beneficialeffects in all organs, but especially the brain (Perez-Pinzon, 2004).

ogical Sciences, University of318280; fax: +39 02 50318250.

l rights reserved.

Spontaneously hypertensive stroke-prone rats (SHRSPs) develop aform of cerebrovascular pathology resembling the human disorder, andcan therefore be used to investigate different elements involved in thegenesis of brain injury. Brain damage in this rat strain occurs naturallyduring the first year of life, but its onset can be brought forward byadministering a sodium-rich and potassium- and protein-poor Japanesepermissive diet (JPD) with drinking water containing 1% NaCl (Blezeret al., 1998; Sironi et al., 2001). Before the onset of stroke, the animalsdevelop systemic inflammation characterised by the accumulation inbiologicalfluids of acute-phase proteins secreted by the liver (Sironi et al.,2001) andbyenhancedoxidativeprocesses (Kim-Mitsuyamaet al., 2005).

Accumulatingdata indicate that thesystemic inflammation is directlyinvolved in the development of cerebral damage (Sironi et al., 2001,2004; Wait et al., 2001) as it increases blood/brain barrier permeability,and biochemical pathways. Structural studies have also revealedalterations in mitochondrial energy metabolism that may be involvedin the development of the inflammation and oxidative processes drivingthe tissuedamage.However, this aspecthas so far beenexploredonly toalimited extent (Crouser et al., 2006a; Miller et al., 2006).

The aim of this study was to verify whether a pharmacologicalapproach capable of preserving mitochondrial functionmight also havebeneficial effects on the pathological events occurring in salt-loaded

118 P. Gelosa et al. / European Journal of Pharmacology 604 (2009) 117–124

SHRSPs. We studied the effects of treatment with S 35171, 7-(2,3-dimethoxy-6(N-(N′-benzylpiperazinylmethyl)-phenoxy)heptanoic aciddihydrochloride), a member of a class of compounds that directly orindirectly protect mitochondrial function (Morin et al., 2000). Aproteomic approach allowed us to view the effects of this compoundin a broader context by revealing protein targets at both systemic (urine)and subcellular level (mitochondria) that could not otherwise bedetected. In addition to the brain, the mitochondrial analysis includedthe liver, the organ that is richest in mitochondria but also the principal“source” of the inflammatory proteins that accumulate in body fluidsand are involved in the organ failure affecting salt-loaded SHRSPs.

2. Methods

2.1. Products and reagents

S 35171 dihydrochloride was obtained from Technologie Servier(Orléans, France). The Japanese permissive diet (JPD) was supplied byLaboratorio Dr. Piccioni (Gessate, Italy), and contained 18.7% protein,0.63% potassium and 0.37% sodium. The immobilised pH-gradient(IPG) strips, acrylamide and other reagents for the polyacrylamide gelpreparations came from Bio-Rad (Hercules, CA, USA). All otherreagents for the proteomic studies came from Sigma (St. Louis, MO,USA).

2.2. Animals and treatments

The procedures involving the animals and their care were carriedout at Milan University's Department of Pharmacological Sciences inconformity with the institution's guidelines, which comply withnational (DL No. 116, G.U., suppl. 40, 18 February 1992; Circular No. 8,G.U., 14 July 1994) and international rules and policies (EEC CouncilDirective 86/609, OJL 358,1, 12 December 1987; Guide for the Care andUse of Laboratory Animals, U.S. National Research Council, 1996).

Forty-seven male SHRSPs from different litters born during a periodof 3–4 days and randomly pooled at the age of 4 weeks were obtainedfromCharles River Italy (Calco, Lecco, Italy), and switched to the JPD and1% NaCl drinking water regime when they were 6 weeks old (day 0).Thirty rats were divided into three groups that underwent early once-daily gavage treatmentwith vehicle alone (0.5% sodiumcarboxymethyl-cellulose; group A1, n=10) or 1 mg/kg/day S 35171 (A2, n=10) or 10mg/kg/day S 35171 (A3, n=10); 14 rats started late treatment with vehiclealone (B1, n=7) or 10 mg/kg/day S 35171 (B2, n=7) when proteinuriaexceeded 40 mg/day. The daily drug dose was adjusted to changes inbody weight every week in order to ensure that the same dose wasadministered throughout the study period. Three rats were excludedfrom high-sodium treatment and served as negative controls through-out the study. On day 0 and once aweek thereafter, all the animals wereweighedand then individuallyhoused inmetabolic cages for 24h so thatfood and water intake could be measured and urine collected fordetermination of total urinary protein excretion. Urinary proteinconcentration was measured according to Bradford using bovinealbumin as standard (Bradford, 1976). Systolic arterial blood pressurewas measured in conscious rats by means of tail-cuff plethysmography(PB Recorder 8006, Ugo Basile, Comerio, Italy).

In order to detect the occurrence of brain damage, all animalsunderwent magnetic resonance imaging (MRI) every other day aspreviously described (Guerrini et al., 2002); after the onset of braindamage, the MRI sessions were repeated daily until the spontaneousdeath of the animals.

2.3. Mitochondria isolation

In order to isolate liver and brain mitochondria, three rats in thevehicle-treated group (A1) and three in the 10 mg/kg/day S 35171group (A3) were sacrificed 3 days after the first signs of brain damage;

three rats that had not undergone salt loading were sacrificed at thesame time. One-gram tissue samples were immediately washed withice-cold phosphate buffer saline (PBS), suspended in 10 mL ofhomogenisation buffer containing 10 mM HEPES, pH 7.4, 70 mMsucrose, 200 mM mannitol, 1 mM EDTA, 1 mM EGTA, 0.2% bovineserum albumin, 1 mM PMSF, 0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin,0.2 mM Na2VO3 and 1 mMNaF, and then homogenised using a Teflon/ceramic homogeniser and centrifuged at 1000 ×g for 10min in order toremove the nuclei and undissolved material. The supernatant wascentrifuged at 10,000 ×g for 20 min to pellet the mitochondrialproteins, and the resulting pellet was washed three times in thehomogenisation buffer without bovine serum albumin before beingsuspended in 200 µL of the same buffer. Protein content wasdetermined using Bradford's method (Bradford, 1976).

2.4. Two-dimensional gel electrophoresis

Immediately before use, 400 µg of mitochondria were diluted inbuffer to reach a final concentration of 8 M urea, 2 M thiourea, 4%CHAPS,1% dithiothreitol (DTT), 20 mM Tris and 2% carrier ampholytes.Two-dimensional electrophoresis (2-DE) was performed according tothe manufacturer's protocol (Protean IEF cell, Biorad): 11-cm IPG-ready strips with a non-linear pH 3–10 gradient (Biorad) were activelyrehydrated at 50 V for 24 h, and the sample was loaded on the cathodeusing the cup-loading tray and focused for a total of 20 kVh. Afterfocusing, the strips were equilibrated for 15 min with a solutioncontaining 50 mM Tris–HCl, 6 M urea, 30% v/v glycerol, 2% sodiumdodecyl sulphate (SDS) and 2% DTT, and then with the same buffercontaining 4.5% iodoacetamide instead of DTT. The focused proteinswere fractionated by size using SDS-PAGE and 7–17% polyacrylamidegradients, and stained with Coomassie Brilliant Blue G-250. Theprotein patterns were acquired using a densitometer (GS800, Biorad).

The gel samples were analysed in triplicate in order to evaluatereproducibility and improve the reliability of the qualitative andquantitative changes in protein expression revealed by 2-DE. PDQuestsoftware (v 6.0, Biorad) was used for spot detection, gel matching, andquantification after normalisation for total spot volume in each gel.The cut-off level for a differentially expressed protein was defined asat least a two-fold increase or decrease in spot intensity. Statisticallysignificant between-group differences for each protein were com-puted by ANOVA. The level of significance of the differences was set atPb0.05.

2.5. Urine analysis by one-dimensional gel electrophoresis (1-DE)

The urine proteins were concentrated by means of trichloroaceticacid-acetone precipitation, and then underwent 1-DE on a 4–20%polyacrylamide gradient in a discontinuous buffer system, in thepresence of SDS and without sample reduction. The sample load was3.75 µg per lane. The proteins were stained with 0.3% w/v colloidalCoomassie Brilliant Blue G-250, and their patterns were digitisedusing a scanner.

2.6. Mass spectrometry analysis

After in-gel digestion with trypsin as previously described (Waitet al., 2002), tandem electrospray mass spectra were recorded using aQ-Tof hybrid quadrupole/orthogonal acceleration time-of-flight spec-trometer (Waters, Manchester, UK) interfaced with a Waters CapLCcapillary chromatograph. The samples were dissolved in 0.1% aqueousformic acid, injected onto a Pepmap C18 column (300 µm×5 mm; LCPackings, Amsterdam, NL), and eluted into the electrospray using anacetonitrile/0.1% formic acid gradient (5–70% acetonitrile over20 min). The capillary voltage was set at 3500 V, and data-dependentMS/MS acquisitions were recorded on precursors with charge states of2, 3 or 4 over a survey mass range of 540–1200. The collision gas was

119P. Gelosa et al. / European Journal of Pharmacology 604 (2009) 117–124

argon, and the collision voltage was varied between 18 and 45 Vdepending on the charge-state and mass of the precursor. Knowntrypsin autolysis products and keratin-derived precursor ions wereautomatically excluded. The proteins were identified by correlatingthe uninterpreted tandemmass spectra with the entries in SwissProt/TREMBL, using the ProteinLynx Global Server (V 1.0; Micromass) (Waitet al., 2002). One missed cleavage per peptide was allowed, andfragment ion tolerance was 100 ppm. Carbamidomethylation ofcysteine was assumed, but other potential modifications were notconsidered in the first-pass search. All of the matching spectra werereviewed manually and, in the case of ProteinLynx Global Serverscores of less than 100, additional searches were made against theNCBI nr database using MASCOT, which is based on a robustprobabilistic scoring algorithm (Perkins et al., 1999).

2.7. Western blotting analysis

Equal amounts of plasma proteins were electrophoreticallyseparated by SDS-PAGE, transferred to nitrocellulose membranes,and incubated with the rabbit polyclonal antibody against CPS-1(Abcam, Cambridge, UK) as previously described (Banfi et al., 1999).

2.8. Statistical analysis

Thedatawere statisticallyanalysedusingPRISM IV software. Survivaland the delay in brain damage were analysed using the log-rank test.Survival is shown as Kaplan–Meier plots and the survival curves werecompared using a log-rank test for trend (significance: Pb0.05, chi-squared test N1). The data are expressed as mean values±S.E.M.

3. Results

3.1. Non-salt treatment

The rats fed a standard rodent diet did not develop proteinuria orbrain ischemia. At the end of the 90-day study, the three live animalswere free from stroke.

3.2. Early treatment

The animals that were fed the JPD and 1% NaCl drinking water, andreceived vehicle alone or 1 mg/kg/day S 35171, showed a progressiveincrease in daily urinary protein excretion (24-h proteinuria), whichsharply and linearly increased after 4 weeks of salt loading. Treatmentwith S 3517110mg/kg/day S 35171 delayed this increase (Fig.1B). At thebeginning of the experiment (week 1; Fig. 2A), the most abundantlyexcreted protein was the major urinary protein (MUP) and other low-molecular-weight (LMW) proteins that accounted for about 70% of totalprotein content. The protein composition of urine changed over time inthe salt-loaded SHRSPs; in particular, there was an accumulation ofhigh-molecular-weight (HMW) proteins previously identified as mar-kers of inflammation (Sironi et al., 2001), and a simultaneous decrease inLMW proteins (Fig. 2A). In the vehicle-treated animals, the HMWproteins became the major component of the excreted proteins (up to70% of total protein content) 4–5 weeks from the start of dietarytreatment, as measured by densitometric analysis of 1-DE. Fig. 2 showsthe results of representative 1-DE of urine samples from one vehicle-treated rat after 6 weeks of dietary treatment and from one rat treatedwith 10mg/kg/day S 35171. In the control animals, the increase in HMW

Fig. 1. Effects of early S 35171 treatment on blood pressure (A), proteinuria (B) andsurvival (C) as a function of the duration of salt loading in SHRSPs receiving vehicle (○)or 1 mg/kg/day S 35171 (▪) or 10 mg/kg/day S 35171 (▲). The treatments were startedsimultaneously with the start of salt loading (⁎⁎Pb0.01 vs rats receiving vehicle). PanelD, representative MRI images showing coronal brain slices from S17135-treated andvehicle-treated SHRSP, 3 days after the first appearance of brain damage in the lattergroup (damage appears as a hyperintense area, here pointed out by the arrow).

Fig. 2. Effects of S 35171onurinaryprotein composition in SHRSPs. PanelA:Representativeone-dimensional electrophoresis of urinary proteins collected weekly from an SHRSPreceiving vehicle (C). Gels shown are representative of results obtained in ten rats. PanelB: Effects of S 35171 on urinary protein composition in SHRSPs. Representative one-dimensional electrophoresis of urinary proteins collected weekly from rats treated withvehicle for 6 weeks (lane 2) or 10mg/kg/day S 35171 for 6, 8,10 and 12weeks (lanes 3, 4, 5and 6). The treatments were started simultaneously with the start of salt loading. The gelsare representative of the results obtained in four rats. Lane 1: Molecular weight marker.HMW, high-molecular-weight proteins; LMW, low-molecular-weight proteins.

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proteins occurred within 6 weeks, but was delayed to week 10 in theanimals receiving 10 mg/kg/day S 35171.

The vehicle-treated rats and the animals receiving 1 mg/kg/day S35171 developed severe hypertension within 5 weeks, whereas theblood pressure of the animals receiving 10 mg/kg/day S 35171 rosemore slowly without reaching statistical significance (Fig. 1A).However, neither systolic blood pressure nor proteinuria could bemeasured in this last group after 9 weeks because of their severelydeteriorated general condition, and so it cannot be concluded thatsystolic blood pressure reached a plateau after 8 weeks.

The first MRI-detected brain lesions (representative MRI imagesare reported in Fig. 1D) were observed a median of 51 days (range 42–60) after the start of the high-salt diet in the vehicle-treated rats(group A1), 52 days (43–62) in the rats treated with 1 mg/kg/day S35171 (group A2), and 62 days (45–77) in the rats treated with 10 mg/kg/day S 35171 (group A3). The delay in initial brain damage (event-free survival) in the animals in group A3 was significantly longer thanin those in group A1 or A2 (Pb0.01; data not shown), as was their

Table 1Median survival with brain damage after start of salt loading, and maximum SBP andproteinuria values

Group Dose of S 35171(mg/kg/day)

No. ofrats

Median survivaldays (range)

Max. SBP(mm Hg)

Max. proteinuria(mg/day)

Control A1 10 57 (43–69) 288±9 387±59A2 1 10 57 (43–69) 281±12 387±30A3 10 10 73 (63–90) 279±7 390±72Control B1 7 51 (41–59) 291±1 300±21B2 10 7 85 (77–90) 290 273±15

cumulative survival (see Kaplan–Meier plot in Fig. 1C; Pb0.01).Treatment not only seemed to delay the appearance of brain damagebut also to allow longer survival with brain damage. The animals ingroup A3 survived for 12±3 days after the first appearance of braindamage, whereas those in groups A1 and A2 respectively survived foronly 6±3 and 6±5 days (Table 1).

3.3. Late treatment

Proteinuria in the vehicle-treated rats (group B1) progressivelyincreased to 300±21 mg/day within 6 weeks, but treatment with

Fig. 3. Effects of S 35171 started when proteinuria exceeded 40 mg/day (late treatment)on blood pressure (A), proteinuria (B) and survival (C) as a function of the duration ofsalt loading in SHRSPs receiving vehicle (○) or 10 mg/kg/day S 35171 (▲) (⁎Pb0.05;⁎⁎Pb0.01 vs rats receiving vehicle).

Fig. 4. Two-dimensional electrophoresis of rat livermitochondrial proteins separated on IPG strips, pH 3–10, and 7–17% acrylamide gradient gels. Panel A:Master gel generated by thePDQUEST software package and used to analyse the 2-DE separations. The circles indicate the spots showing significant difference in expression (⁎Pb0.05). Panels B and C: Expandedviews of spots 1 and 2 in control rats (not salt-loaded), and salt-loaded vehicle-treated and drug-treated rats.

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10mg/kg/day S 35171 (group B2), which was started when proteinuriaexceeded 40 mg/day, significantly delayed the progression ofproteinuria during the first 2 weeks (Fig. 3B), which subsequentlyremained below the level observed in the control group. After 8–10 weeks of the high-sodium diet, proteinuria reached a plateau of∼270 mg/day in the surviving animals.

Systolic blood pressure progressively increased in both groups but,after the start of pharmacological treatment (when proteinuria wasN40 mg/day), it remained stable in group B2 for almost 20 days beforeprogressively increasing to values that were similar to those observedin the vehicle-treated group 8 weeks after the start of the study(Fig. 3A). Systolic blood pressure reached a plateau after 8–10weeks ofsalt loading.

MRI-detected brain abnormalities were observed after 44 days ofdietary treatment (range 35–56) in the vehicle-treated rats, and after70 days (67–75) in the rats treated with S 35171. The between-groupdifference in the time of onset of initial brain damage was statisticallysignificant (Pb0.001; data not shown), as was the difference in meansurvival (see Kaplan–Meier plot in Fig. 3C; Pb0.001). The controlanimals survived 51 days (41–59) and the treated animals 85 days(77–90) (Table 1). Interestingly, three group B2 animals did notdevelop any brain damage before theywere sacrificed at the end of the90-day study and survived without any signs of suffering.

Table 2Identification of differentially expressed proteins by mass spectrometry 3 days after the on

Name Fold increase(salt-loadedvs control)

Fold increase(drug treatmentvs control)

Accessioncode

MWkDa/pI

Sequencecoverage %/Mowse score

Se

Carbamoyl-phosphatesynthase

0.27±0.05 0.86±0.04 P07756a 165/6.75

11.5/1696 (K(K(K(K(R

Peroxiredoxin3

6.43±0.34 12.08±1.44 gi:11968132b 28/7.14 22.1/455 (R(K

Mean values±S.E.M.a Accession code in Swiss-Prot.b Accession code in NCBI.

3.4. Proteomic study

Conventional 2-DE of liver and brain mitochondria was used todetermine the global changes in protein levels due to salt loading-induced brain damage and chronic drug treatment. Mitochondrialproteins (400 µg) from the livers and brains of non-salt-loaded rats,vehicle-treated rats (group A1) and drug-treated rats (group A3) wereseparated in the first dimension using broad-range pH 3–10 IPG strips,and in the second dimension using 7–17% gradient acrylamide gels. Theproteins were visualised by means of Colloidal Blue staining. Proteomicanalysis of brain mitochondrial proteins revealed no protein expressionsignificantlyalteredbybraindamageordrug treatment (datanot shown).On the otherhand, image analysis of livermitochondrial proteins showedthat the expression of two proteins was affected by salt-loading andtreatment (Fig. 4). Protein 1, identified as carbamoyl-phosphatesynthetase I (CPS-I) in two close spots, was down-regulated in the salt-loaded rats but remained at almost baseline levels in the rats treatedwithS 35171. Both CPS-I spots showed similar regulation, with only spot 1reaching statistical significance. Salt-loading clearly reduced the secondand more acidic spot (0.40±0.069-fold vs the control group; PN0.05),whereas S 35171 led to approximately normal expression (0.89±0.39 foldvs control group; PN0.05). Immunoblotting analysis of circulating CPS-Ishowed that the levels of the detectable 150 kDa formwere not increased

set of brain damage in the vehicle-treated group (A1)

quence Spotno.

) IALGIPLPEIK(N) (K)SLGQWLQEEK(V) (K)ATGYPLAFIAAK(I) (R)VSQEHPVVLTK(F))GNDVLVIECNLR(A) (K)GLNSESVTEETLR(Q) (R)FLGVAEQLHNEGFK(F))VLGTSVESIMATEDR(Q) (K)AFAISGPFNVQFLVK(G) (K)EPLFGISTGNVQFLVK(G))EPLFGISTGNIITGLAAGAK(S) (R)SIFSAVLDELKVAQAPWK(A))KEPLFGISTGNIITGLAAGAK(S) (K)VAGLLVLNYSHDYNHWLATK(S)

1

)GLFIIDDKJ(G) (K)ADEGISFR(G) (R)GLFIIDDKGILR(Q))LNCQVIGASVDSHFCHLAWINTPK(K)

2

122 P. Gelosa et al. / European Journal of Pharmacology 604 (2009) 117–124

in the plasma compartment of the salt-loaded SHRSPs at the time of theMRI-detected brain lesions in comparison with the non-salt-loaded rats(not shown). As it has been shown that the release of CPS-I into thesystemic circulation coincides with changes in mitochondrial morphol-ogy, including ultrastructural changes and a reducedmitochondrialmass(Crouser et al., 2006b), we can conclude that our experimental model,unlike that of sepsis, presents alterations in mitochondrial proteins thatcan be detected before the appearance of signs of mitochondrialultrastructural alterations (not shown). The expression of the peroxir-edoxin-3 spot increased 6.4 times with salt loading (Fig. 4 and Table 2)and, when the animals were treatedwith S 35171 during salt-loading, itsexpression was 12 times higher than in the controls.

4. Discussion

SHRSPs provide a well-established experimental model of sponta-neous hypertension, local and systemic inflammation, and consequentkidney and brain damage. Published data indicate that all of the drugsthat protect SHRSPs act by preventing or delaying the occurrence ofthe systemic inflammation, which is characterised by the accumula-tion of acute-phase proteins (APPs) in body fluids, and has recentlybeen shown to increase blood brain barrier permeability with theconsequent development of brain abnormalities (Sironi et al., 2004).

It has been hypothesised that mitochondrial dysfunction may beinvolved in the pathological events occurring in SHRSPs, particularlyin the pathogenesis of cardiac hypertrophy and myocardial degenera-tion (Tokoro et al., 1996; Cristofori et al., 1994) and the worseningcerebral tissue damage experienced by SHRSPs that have undergonemiddle cerebral artery occlusion (Onoue et al., 2005).

Moreover, the renal protective effects of candesartan in sponta-neously hypertensive rats (SHR), a strain closely related to SHRSPs, maybedue to an improvement inmitochondrial function (DeCavanagh et al.,2005). However, to the best of our knowledge, ours is the first studyinvestigating whether a drug acting on mitochondria also affects thepathological events that occur spontaneously in SHRSPs.

We used S 35171, a trimetazidine derivative that protects mitochon-drial function in vitro in a way that is similar to the effects of a series ofstructurally related compounds (Morin et al., 2000) and, whenadministered systemically before ischemia, also reduces lesion size inthe ratmiddle cerebral artery occlusionmodel (unpublished data). It hasbeen widely shown that trimetazidine derivatives are effective inprotecting against ischemic insults in a varietyof tissues, and in differentanimal species, by limiting inflammation and the production ofoxidising free radicals (Di Napoli et al., 2005). Moreover, in an animalmodel, trimetazidine counteracts the hepatic injury associated withischemia-reperfusion by preserving mitochondrial function, and actingonmitochondrial membrane potential and the generation of mitochon-drial permeability transition (Onay-Besikci and Ozkan, 2008).

Our new data show that its chronic oral administration delays theappearance of proteinuria, increases the time to the development ofbrain damage, and prolongs the survival of salt-loaded SHRSP.Furthermore, the analyses of the urine collected during treatmentclearly indicate that it delays the accumulation of liver-secreted APPs.Interestingly, when given after proteinuria levels had reached N40 mg/day, it not only slowed the evolution of proteinuria and delayed theincrease in systolic blood pressure, but also significantly postponed theappearance of brain damage and allowed event-free survival for up to90 days in 30% of the animals. S 35171 had a slight and never statisticallysignificant effect on blood pressure in the early-treatment group, butwecannot exclude the possibility that its beneficial effect is partiallyattributable to this. However, in the late-treatment group, the animalsseemed to survive without any observable signs of suffering althoughtheir blood pressure was maximal (290 mm Hg), and so othermechanism have to be involved in the protection elicited by S 35171.

In order to explain the protective effect of S 35171 in salt-loadedSHRSPs,we analysedprotein expression in brain and livermitochondria,

and its drug modulation. Proteomic analysis showed that neither thedevelopment of brain abnormalities nor S 35171 treatment altersmitochondrial protein expression in the brain, which can be explainedon the basis of previous observations indicating that the brainabnormalities in SHRSPs are focal and thus remain restricted to asmall area of the cerebral parenchyma (Guerrini et al., 2002; Sironi et al.,2004). As the mitochondria were obtained from total brain tissuehomogenates, the presence of focal alterations may not be easilydetectable. Moreover, the mitochondria localised in different tissuesrespond differently to pathological stimuli, and brain mitochondria arethe least sensitive (Kozlov et al., 2006; Taylor and Piantadosi, 1995).

Conversely, liver mitochondria are particularly sensitive, especiallyto pathological conditions characterised by the activation of inflam-matory processes such as endotoxic shock (Kozlov et al., 2006; Crouseret al., 2006a; Kozlov et al., 2007) and we found that salt-loading and S35171 treatment modulated the expression of two liver mitochondrialproteins: CPS-I and peroxiredoxin-3.

Salt-loading decreased CPS-I levels, whereas oral treatment with S35171 restored them to basal levels. CPS-I is located in the innermitochondrial matrix, and catalyses the first and rate-limiting step ofurea cycle, the primary system for removing the waste nitrogenproduced by protein metabolism (Holden et al., 1999). CPS-I deficiencyis biochemically characterised by high levels of blood ammonia(associated with deleterious effects on the central nervous system),glutamine and alanine, and low or zero citrulline and arginine levels(Schofield et al., 1999). Various in vivo experimentalmodels have shownthat ammonia is extremely toxic, especially for cerebral tissue, and thathyperammonemia increases the permeability of the blood/brain barrierand thus leads to brain edema (Butterworth, 2002; Ott and Larsen,2004). Furthermore, growing evidence indicates that hyperammonemiaand systemic inflammation cooperate in inducing encephalopathy andliver failure (Wright et al., 2007). We cannot assess whether hyper-ammonemia is present in our experimental model or not. Measuringammonia is fraught with problems regarding the collection, handling,storage and analysis of blood samples, which involves various methods(distillation and aeration/microdiffusion, ion exchange chromatogra-phy) that still need to be standardised in order tominimise spontaneousammonia release (Barsotti, 2001). However, an increase in ammonialevels would be consistent with the presence of the severe renaldysfunction observed in our experimental model (Gianella et al., 2007).It has been demonstrated that ammoniagenesis is increased as aconsequence of renal disease, and this could contribute to increasingplasmaammonia levels as70%of total renal ammonia is released into therenal vein (the remainder being excreted in urine) (Halperin et al.,1992).

In addition to the detoxification of ammonia, CPS-I also representsthe rate-limiting step in L-citrulline production (Morris, 2002) andcycling to L-arginine with subsequent nitric oxide (NO) production. Adecrease in CPS-I might therefore lead to reduced arginine availabilityand reduce NO synthesis, and it has been shown that cultured aorticendothelial cells from SHRSPs release smaller amounts of NO thanWKYcontrols (Grunfeld et al.,1995).Wehave also found that thebioactivity ofNO is decreased in SHRSPs, and inversely correlateswith the serum levelof the acute phase protein thiostatin, thus indicating a link toinflammatory sequelae (Ballerio et al., 2007). However, there is stilllittle information regarding the regulation of CPS-I expression in liver.Inoue et al. (2004) found that the genetic disruption of hepatic CCAAT/enhancer-binding protein α (C/EBPα) leads to hepatic CPS-I deficiency,thus suggesting that CPS1 expression is regulated by C/EBPα in liver, andit is interesting to note that C/EBP has been found to be essential for theacute-phase response to inflammation (Burgess-Beusse and Darlington,1998), a predominant condition in our experimental model. Otherpossible explanations for the decreased CPS-I content in livermitochon-dria include post-transcriptional mechanisms, such as the increasedcleavage of the protein induced by caspases (Huang et al., 2002) oroxidative stress (Kim-Mitsuyama et al., 2005). In our experimentalanimal model, the normalisation of CPS-I by S 35171may therefore help

123P. Gelosa et al. / European Journal of Pharmacology 604 (2009) 117–124

to maintain the integrity of the blood brain barrier and preserve livertissue, thus directly contributing to attenuating APP release.

S 35171 also protects against oxidant stress by increasing theexpression of the peroxiredoxin-3 spot, an effect that may be anadditional or alternative explanation for the preservation of CPS-I inliver mitochondria and the integrity of the blood brain barrier.Peroxiredoxin-3 is one of six isoforms of the family of peroxiredoxinslocated in mitochondria (Rabilloud et al., 2002). Together withmanganese superoxide dismutase, peroxiredoxins are involved inone of the major mitochondrial antioxidant systems as they catalyse areduction in hydrogen peroxide and scavenge peroxynitrite, thusproviding a primary line of defence against the damage induced byreactive oxygen species (Kang et al., 2005). The peroxiredoxin-3 spotis up-regulated in salt-loaded animals, probably because of mildoxidative stress, and treatment with S 35171 doubled its alreadyincreased expression. This might protect the mitochondrial respira-tory chain, thus reducing oxidative stress and consequently inflam-mation, but how it can slow the development of systemicinflammation is not clear and will require further study.

In conclusion, our data show that chronic oral administration of S35171 delays and even prevents spontaneous brain damage in SHRSPs.This protective effect is not achieved by means of a local mechanism,but seems to be due to the protection of systemic processes that are, forexample, involved in damaging the blood brain barrier. S 35171 pre-vents mitochondrial dysfunction in the liver, the organ responsible forsecreting the APPs that may be directly involved in blood brain barrierimpairment and the consequent formation of brain edema.

Our data cannot establish a causal relationship between themitochondrial alterations, hepatic release of APPs and the occurrenceof brain damage, but they do – for the first time – indicate thatimproving (or preserving) mitochondrial function may contribute todelaying/preventing the pathological events recorded in SHRSPs. Theyalso indicate that liver dysfunction may play a role in thesepathological events, particularly the occurrence of systemic inflam-mation. Further studies are needed to clarify this last aspect, whichwould seem to be particularly important in the hepatic release of APPsthat triggers end-organ damage in SHRSPs.

Acknowledgements

This study was sponsored by the Institut de Recherches Inter-nationales SERVIER (IRIS), Neuilly Sur Seine, France. Alice Pignieri wassupported by grants from the Fondo per gli Investimenti della Ricercadi Base (FIRB) CHEM-PROFARMA-NET.

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