Angiotensin-converting-enzyme inhibition counteractsangiotensin II-mediated endothelial cell dysfunctionby modulating the p38/SirT1axis
Francesco Marampona,b,!, Giovanni L. Gravinaa,!, Luca Scarsellaa, Claudio Festucciaa,Francesca Lovatc, Carmela Ciccarellia, Bianca M. Zania, Lorella Polidorob, Davide Grassib,Giovambattista Desiderib, Stefano Evangelistad, and Claudio Ferrib
Objective: Oxidative stress has been linked to endothelialdysfunction and angiotensin II stimulates the reactiveoxygen species production contributing to severalcardiovascular diseases. We have studied the chain ofevents induced by angiotensin-converting-enzyme (ACE)activation in vascular umbilical vein endothelial cells(HUVECs) by using an ACE inhibitor such as zofenoprilat.
Methods: We used specific assay to measure thesuperoxide anion production, tetrazolium bromide (MTT)assay for cell viability, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assayfor cell apoptosis, and western blot for protein analysis inthe study.
Results: Zofenoprilat counteracts the superoxide anionproduction and cell apoptosis induced by angiotensin Itreatment by blocking the extrinsic caspase cascade, NF-kBand p38 activation. p38 inhibitor SB203580 reverted theangiotensin II oxidant effects while the p38 constitutivelyactivation, by MKK6 transfection, abrogated thezofenoprilat effects. Characterizing the zofenoprilatdownstream effector we found that zofenoprilat revertedthe SirT-1 downregulation induced by angiotensin II. p38activation by angiotensin II was strictly correlated withSirT1 protein downregulation; SB203580 significantlyprevented SirT1 downregulation induced by angiotensin IIwhile the p38 constitutive activation abolished SIRT1protein basal levels. p38 directly bound SirT1 sequesteringit in the cytoplasm. SirT1 inhibition by sirtinol annulledzofenoprilat action while SirT1 overexpression reverted thecytotoxic effects of angiotensin II. Finally, zofenoprilatnegatively controlled angiotensin I receptor proteinexpression through SirT1.
Conclusion: The p38-SirT1 axis is found markedly relevantin modulating the cardiovascular benefit deriving fromACE-inhibitors and might represent a novel target forinnovative drugs in cardiovascular prevention.
T he vascular endothelial cell regulates manyprocesses such as coagulation, inflammation andarterial tone and its dysfunction is a key event
of several cardiovascular and noncardiovascular diseases[1–3]. Oxidative stress induces vascular endothelial celldysfunction affecting the function of several organs andsystems [4,5]. Endothelial dysfunction has been observedin patients with atherosclerosis, hyperlipidemia, diabetes,hypertension, aging and obesity [6,7]. The production ofreactive oxygen species (ROS) characterizes the damageinduced by oxidative stress. ROS can be generatedin excessive quantities through different sources suchas superoxide-free radicals, hydrogen peroxide, singletoxygen, nitric oxide (NO), and peroxynitrite [8–10]. Angio-tensin II, the main effector of the renin–angiotensin–aldosterone system (RAAS), is known to be involved invarious pro-inflammatory actions in the vascular wall,including adhesion molecule, chemokine and cytokineexpression, and ROS generation [11,12]. It is also stronglyimplicated in various cardiovascular diseases, such ashypertension, atherosclerosis and heart failure [13–15].Angiotensin II exerts many of its detrimental effectsinteracting with the angiotensin II type 1 receptor(AT1R). AT1R activation results in a myriad of intracellularevents, including the ROS production and the consequentreduction in NO bioavailability, which contribute toreduce insulin signal and stimulate both proliferative andinflammatory responses. Finally, these AT1R-mediated
Journal of Hypertension 2013, 31:000–000aDepartment of Applied Clinical Sciences and Biotechnology, University of L’Aquila,bDepartmentMeSVA, University of L’Aquila, L’Aquila, Italy, cDepartment of MolecularImmunology, Virology, and Medical Genetics, The Ohio State University, Columbus,Ohio, USA and dPreclinical Development Department, Menarini Ricerche, Firenze, Italy
Correspondence to Francesco Marampon, Department of Applied Clinical Sciencesand Biotechnology, University of L’Aquila, Via Vetoio, Coppito 2, L’Aquila, Italy. E-mail: [email protected]!Francesco Marampon and Giovanni L. Gravina contributed equally to the writing ofthis article.
Received 14 February 2013 Revised 24 April 2013 Accepted 23 May 2013
J Hypertens 31:000–000 ! 2013 Wolters Kluwer Health | Lippincott Williams &Wilkins.
effects can induce endothelial dysfunction [16,17]. In thiscontext, the role played by both local and systemic RAAShas been widely documented. Short-term effects on cardio-vascular homeostasis, such as vasoconstriction and hyper-tension, can be ascribed mainly to the systemic RAAS,whereas long-term effects such as inflammatory actionsat the vascular wall level mainly relate to the local RAAS[18]. Angiotensin-converting-enzyme (ACE) has beenidentified locally in the vessel wall [19,20] where it controlsthe local angiotensin II formation. Locally, angiotensin IIactivates angiotensin II receptors on different cell types,leading to progressive lesion formation via proliferationof smooth muscle cells, formation of atheroscleroticplaque and facilitation of thrombosis [21–23]. However,the mechanisms by which angiotensin II contributes to thepathophysiology of endothelium dysfunction is not com-pletely understood as well as the action of ACE inhibitors.The Ras-mitogen activated pathway kinases (MAPKs) play akey role in controlling cell apoptosis after oxidative injury[24,25] but the mechanism in promoting or counteractingendothelium dysfunction remains unclear. The activation ofRas/ERKs signaling protects from oxidative stress-inducedapoptosis [26–28] promoting human umbilical vein endo-thelial cells (HUVECs) survival in response to the oxidativechallenge [29]. On the contrary Ras/p38 [30] and Ras/JNK[31] activation leads to cellular death. Angiotensin IIregulates ERKs and p38 MAPKs activation both in endo-thelial cells [32] and in the vascular smooth muscle cells [33]but their role in controlling cellular response to angiotensinII-mediated oxidative stress remains unclear. MAPKssignaling pathway cascades control cellular responseto the oxidative stress, regulating the expression ofseveral proteins [34] such as SirT1 [35]. Mammalian SirT1NAD"-dependent protein deacetylase, regulates cell cycle,premature senescence, apoptosis and metabolism byinteracting with a number of molecules. SirT1 has beenimplicated in the endothelial homeostasis [36,37] prevent-ing cells senescence and oxidative stress-induced death[38,39] and it has been suggested as a novel potential targetfor treatment of vascular diseases [40]. Herein, we haveinvestigated the molecular mechanism regulated by angio-tensin II in inducing oxidative stress and the antioxidanteffects of the ACE inhibitor zofenoprilat [20]. Zofenoprilat isthe active metabolite of zofenopril that contains a sulfhydrylgroup described to scavenge ROS and to exert antioxidantactivity [41]. To this purpose, we used HUVECs, an import-ant model of the human endothelium that is widely usedin vitro in vascular research [42]. We found that zofenoprilatcounteracts angiotensin II-mediated inhibition of super-oxide anion generation and apoptosis by inhibiting extrin-sic caspase cascade activation. Furthermore zofenoprilatturns off the p38 MAPKs pathway angiotensin II-mediatedactivation restoring the SirT1 protein accumulation thatmediates zofenoprilat antioxidant activity.
MATERIALS ANDMETHODSCells culture and reagentsHUVECs (Clonetics, San Diego, California, USA) werecultured in endothelial cell basal medium (EBM-2;Clonetics) supplemented with 2% of fetal calf serum
(FCS; Clonetics) and endothelial growth medium (EGM-2; Clonetics) at 378C and 5% CO2 in a fully humidifiedincubator. For experiments, HUVECs were subculturedin six-well tissue culture plates (Costar) at the second orthe fourth passage with 5000 cells/cm2. The culturemediumwas removed and replaced by an endothelial growthmedium (EGM-2; Clonetics), whichwas supplementedwith10% FCS after 4 days when cells reached the confluence.Fetal bovine serum and penicillin-streptomycin werepurchased from Clonetics. Zofenoprilat was a kind giftfrom Menarini Italia. Angiotensin I, SB203580 and sirtinolwere obtained from Sigma–Aldrich Chemical (St. Louis,Missouri, USA). Concentrations used for zofenoprilat [42]and angiotensin I [43] were reported by the literature.
Plasmids and transfectionsThe transfections were performed using Lipofectamine Plusreagent (Invitrogen, Italy) according to the manufacturer’sinstructions (GIBCO-BRL, Gaithersburg, Maryland, USA).For SirT1 constitutively-expression or p38-activation,HUVEC cells were transiently co-transfected with thehuman SirT1 expression vectors in pcDNA3-SirT1, kindlygift from Richard Pestell [44], or with the human MKK6expression vectors in pCMV5-MKK6 (Addgene), or withthe empty pCMV vector [45] together with puromycinresistance-expressing vector (pPur; Clontech LaboratoriesGmbh, Heidelberg, Germany) [46] to select transfected cellswith puromycin (1mg/ml). After 24 h of puromycintreatment cells media was changed to puromycin freemedia and 24 h cells were treated as described in the resultsparagraphs. RNA interference experiments were performedwith siRNA for SirT1, p38 or scramble (Santa CruzBiotechnology, Santa Cruz, California, USA) usingLipofectamine 2000 reagent (Invitrogen, Italy), accordingto the manufacturer’s instructions. Briefly, cells were platedat 40–50% confluence and transfected after 24 h with100 nmol/l siRNA, which we ascertained was sufficientto detect maximum fluorescence using fluorescein-conjugated control siRNA.
Measurements of angiotensin II, superoxideanion and nitric oxide levels, determination ofangiotensin-converting enzyme and eNOSactivityChemiluminescence Superoxide Anion Assay Kit (CS1000)from Sigma–Aldrich (St. Louis, Missouri, USA) andNitric Oxide Assay Kit (ab65328) from Abcam (Cambridge,Massachusetts, USA) were used to evaluate the influence ofvitamin D treatment on generation of superoxide anionsand NO by HUVECs treated with angiotensin I. The cellswere treated as described in the results. Finally, catalasewas added to block all activity of extracellular H2O2 [47].The assays were performed according to the manufacture’sguidelines. ACE activity was determined according withthe manufacturer’s instructions (ACE Kinetic assay; ALPO,CANADA). eNOS activity was determined according tothe manufacturer’s instructions (NOS Activity Assay Kit,Cayman Chemical). Angiotensin II levels in growthmedium were assessed using angiotensin II EIA Kit(Cayman Chemical).
Marampon et al.
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MTT and transferase-mediated dUTP nick endlabeling assaysTo evaluate the influence of zofenoprilat on protectingHUVECs by angiotensin I treatment, the cellular viabilityby MTT and the transferase-mediated dUTP nick endlabelling (TUNEL) assays were tested. The cells weretreated as described in the results paragraph. For thetetrazolium dye colorimetric assay, MTT assay, HUVECswere incubated in fresh medium containing 1 g/l MTT
for 3 h at 378C. After removal of unconverted MTT, thepurple formazan product was measured colorimetrically atl$ 570 nm with background subtraction at l$ 690 nm [47].TUNEL assay was conducted following manufacturer’sinstructions. Briefly, after treatment the cells were fixedin 4% (w/v) paraformaldehyde for 16 h at 48C andthen stained using TdT-mediated dUTP nick-end label-ing (TUNEL)-based kit (Roche Molecular Biochemicals)following the manufacturer’s instructions.
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FIGURE 1 Effects of zofenoprilat on superoxide anion generation, endothelial cells survival and apoptosis induced by angiotensin II. quantification of angiotensin-convert-ing-enzyme (ACE) activity (a), angiotensin II production (b), superoxide anion generation (c), cells survival (MTT) (d) and cells apoptosis (TUNEL) (e) assays by HUVECs thatwere 1 h pretreated with zofenopril at different concentration (10, 50, 100, 150 or 200mmol/l) and treated or not with angiotensin I (5mmol/l) for 1 h. Any singletreatment was performed in triplicate and the results are expressed as means of two tests (n$3).
ImmunoprecipitationCells were harvested in phosphate-buffered saline (NaCl137 mmol/l, KCl 2,7 mmol/l, Na2HPO4 # 2 H2O 10 mmol/l,KH2PO4 2 mmol/l pH 7.4, sedimented and lysed in10mmol/l Tris pH 7, 50mmol/l NaCl, 1% NP40, 1mmol/lZnCl2, additioned with protease and phosphatase inhi-bitors. Protein extracts were clarified by centrifugation.Supernatant, normalized as equal amounts of proteins,were incubated with SirT1 (Santa Cruz Biotechnology) at48C for 3 h. Thirty microlitres of protein-G Plus (Santa CruzBiotechnology) were added to collect immunocomplexes.Protein G-bound immunocomplexes were washed sixtimes with extraction buffer and processed for SDS-PAGEand immunoblotting.
Immunoblot analysisWestern blotting was conducted as described previously[48]. Briefly, cells from cultures were lysed in 2% SDScontaining phosphatase and protease inhibitors (Roche)sonicated for 30 s. Proteins of whole-cell lysates wereassessed using the Lowry method [49], and equalamounts were separated on SDS-PAGE. The total proteinlevel balance was confirmed by staining the membraneswith Ponceau S (Sigma). Immunoblottings were done asdescribed previously [50] with the following antibodies:anticaspase 3, -caspase 8, -p65, -phospho-p38, -p38, -phospho-ERKs, -ERKs, -SirT1, AT1-R and antia-tubulin(all from Santa Cruz Biotechnology). Peroxidase-conjugateantimouse or antirabbit IgG (Amersham GE Healthcare)were used for enhanced chemiluminescence detection.
Statistical analysisStatistical analysis was performed using a one-wayanalysis of variance (SPSS software version 12.0.1; Chicago,Illinois, USA). The results were expressed as mean% SDof triplicate determination with P< 0.05 considered asstatistically significant.
RESULTSZofenoprilat blocks oxidative stress induced byangiotensin IIOur first task was to define the zofenoprilat concentrationable to block the angiotensin I conversion into angiotensinII by ACE. HUVECs were 1 h pretreated with zofenoprilat atdifferent concentrations and treated or not with angiotensinI (5mmol/l) for 1 h. Zofenoprilat treatment reduced the ACEactivity (P< 0.05; Fig. 1a) and the angiotensin II production(P< 0.05; Fig. 1b) in a concentration-dependent manner,achieving maximum at 150mmol/l. These results wereparticularly evident in angiotensin I pretreated HUVECs.Zofenoprilat antioxidative effects were analyzed bytesting superoxide anion generation, percentage ofsurviving- (MTT assay) and apoptotic cells (TUNEL assay).Zofenoprilat 150mmol/l pretreatment reduced by 65.6% thesuperoxide anion generation (P< 0.05; Fig. 1c), increasedthe number of surviving endothelial cells (Fig. 1d)and decreased the number of apoptotic cells after 1 hof angiotensin I treatment (Fig. 1e). We selected theconcentration of 150mmol/l, for the further tests.
Zofenoprilat inhibits angiotensin II mediatedapoptosis blocking extrinsic caspase cascadeand NF-kB pathway activationThe antiapoptotic activity controlled by zofenoprilat wasinvestigated analyzing the activation status of the extrinsiccaspase cascade pathway. HUVECs were 1 h pretreatedwith zofenoprilat and treated or not with angiotensin Ifor 1 h. Zofenoprilat did not let the cleavage/activation ofp55/p53 and p42/p41 pro-caspase-8 protein into caspase-8active subunits p18 and p10 induced by angiotensin Itreatment (Fig. 2a, upper panel). The analysis ofprotein levels of pro-caspase-3, terminal downstream ofthe extrinsic caspase cascade, shows that zofenoprilatprevented the cleavage/activation of pro-caspase-3 intop19-caspase-3 active subunit induced by angiotensin Itreatment (Fig. 2a, lower panel). Caspase-8 has been impli-cated in NF-kB activation mediated-apoptosis [51,52].We therefore analyzed the p65 nuclear translocation/activation; nuclei were extracted and stained forp65. Angiotensin I treatment significantly increased p65
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FIGURE 2 Zofenoprilat counteracts angiotensin II-induced apoptosis by blockingthe extrinsic caspase cascade activation and NF-kB p65 nuclear translocation.HUVECs were 1h pretreated with zofenopril (150mmol/l) and treated or not withangiotensin I (5mmol/l) for 1 h. (a) Total cell lysates and (b) nuclear (N) orcytosolic (c) fractions were analyzed by immunoblotting with specific antibodiesfor indicated proteins. a-tubulin expression shows the loading of samples. Thevalues of fold increases over the control, arbitrarily set at 1, were obtained bydensitometric analysis. Any single treatment was performed in triplicate. Similarresults were obtained in three (n$3) different experiments.
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4 www.jhypertension.com Volume 30 # Number 1 # Month 2013
translocation compared with untreated. The angiotensin I-mediated p65 translocation was blocked by zofenoprilatpretreatment (Fig. 2b).
Angiotensin-converting-enzyme inhibition byzofenoprilat induces antioxidant effects byinhibiting p38 MAPK pathwayWe investigated on the role of MAPK pathways inzofenoprilat-mediated effects. Cells were 1 h pretreatedwith zofenoprilat and treated or not with angiotensin Ifor 1 h: the phosphorylation/activation status of extracellu-lar signal-regulated kinase 1/2 (ERK1 and ERK2) and p38MAPK was assessed. Zofenoprilat counteracted the angio-tensin II-induced phosphorylation/activation of ERK1/2and p38 MAPK. To deepen on the role of ERK1/2 and/orp38 MAPK pathway activation, HUVEC were pretreatedwith the ERK1/2 inhibitor U0126 or with the p38 MAPK
inhibitor SB203580 for 1 h and then stimulated for 1 h withangiotensin I. As shown in Fig. 3, SB203580 (10mmol/l)reduced the superoxide anion (P< 0.05; Fig. 3b,SB203580), increased the number of surviving endothelial(Fig. 3c, SB203580) and decreased the number of apoptoticcells (Fig. 3d, SB203580) induced by angiotensin I treat-ment. On the contrary, ERK1/2 inhibition by U0126 did notmodify angiotensin I treatment ability to induce oxidativestress (Fig. 3, U0126). HUVEC cells were transfected withMKK6, the kinase upstream of p38, pretreated for 1 h withzofenoprilat and treated or not with angiotensin I for 1 h.As shown in Fig. 4, zofenoprilat antioxidant effectswere abrogated by p38 constitutively activation. MKK6expression increasing the superoxide anion generation(P< 0.05; Fig. 4a), decreasing the number of survivingendothelial (Fig. 4b) and increasing the number ofapoptotic cells (Fig. 4c) also in presence of zofenoprilattreatment. Furthermore, MKK6 expression alone increased
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FIGURE 3 Zofenoprilat reduces the ERKs and p38phosphorylation/activation mediated by angiotensin II. (a) Cell lysates from HUVECs 1 h pretreated with zofenopril(150mmol/l) and treated or not with angiotensin I (5mmol/l) for 1 h were analyzed by immunoblotting with specific antibodies for indicated proteins. a-tubulin expressionshows the loading of samples. The values of fold increases over the control, arbitrarily set at 1, were obtained by densitometric analysis of phospho-ERKs versus ERKs orphospo-p38 versus p38. Any single treatment was performed in triplicate. Similar results were obtained in two (n$2) different experiments. Quantification of superoxideanion generation (b), cells survival (MTT) (c) and cells apoptosis (TUNEL) (d) assays by HUVECs that were pretreated with the ERK1/2 inhibitor U0126 (10mmol/l) or withthe p38 MAPK inhibitor SB203580 (10mmol/l) for 1 h and then stimulated for 1 h with angiotensin I (5mmol/l). Any single treatment was performed in triplicate and theresults are expressed as means of two tests (n$3).
the basal levels of ROS generation, cell survival andapoptosis. MKK6 protein expression levels and p38 phos-phorylation/activation status were analyzed bywestern blot(Fig. 4d).
Angiotensin I treatment abrogates the SirT1protein expression and nuclear accumulationthrough the p38 activationWe investigated the role of SirT1 on the antioxidanteffects mediated by zofenoprilat. HUVECs were 1 hpretreated with zofenoprilat and treated or not withangiotensin I for 1 h. Zofenoprilat upregulated SirT1protein expression that was substantially abolishedby angiotensin I treatment while pretreatment withzofenoprilat significantly prevented SirT1 downregulationinduced by angiotensin II (Fig. 5a). To investigate the roleof p38MAPK in controlling SirT1 protein expression levels,HUVEC were pretreated with SB203580 for 1 h then stimu-lated for 1 h with angiotensin I. SB203580 significantly
prevented SirT1 downregulation induced by angiotensinI (Fig. 5b) increasing the SirT1 basal levels proteinexpression. p38 silencing by siRNA transfection increased(Fig. 5c) while the p38-up-stream-activator MKK6-over-expression (Fig. 5d) reduced SirT1 protein basal levels. Therole of p38 in SirT1-cellular localization was then analyzedby transfecting MKK6 in HUVECs. p38 activation byMKK6 transfection increased the cytoplasm-localized SirT1levels (Fig. 5e). Furthermore coimmunoprecipitationexperiments revealed that p38 kinase bound to SirT1protein (Fig. 5f).
The SirT1 downregulation mediated theoxidative stress induced by angiotensin ItreatmentWe tested the role of SirT1 in mediating angiotensin IIoxidant effects. HUVECs were incubated with sirtinol(50mmol/l), a specific inhibitor of SirT1, 1 h pretreated withzofenoprilat and treated or not with angiotensin I for 1 h.
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FIGURE 4 Over-expression of the p38 upstream MKK6 blocks antioxidant effects mediated by zofenoprilat. Quantification of superoxide anion generation (a), cells survival(MTT) (b) and cells apoptosis (TUNEL) (c) assays by HUVECs that transfected with MKK6, the kinase upstream of p38, with empty vector CMV or untransfected (UNT),pretreated for 1 h with zofenoprilat (150mmol/l) and treated or not with angiotensin I (5mmol/l) for 1 h. Any single treatment was performed in triplicate and the resultsare expressed as means of two tests (n$3). (d) Total cell lysates were analyzed by immunoblotting with specific antibodies for indicated proteins. a-tubulin expressionshows the loading of samples. The values of fold increases over the control, arbitrarily set at 1, were obtained by densitometric analysis. Any single treatment wasperformed in triplicate. Similar results were obtained in tree (n$3) different experiments.
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Sirtinol annulled zofenoprilat action restoring superoxideanion generation induced by angiotensin I treatment(P< 0.05; Fig. 6a) reducing the cytoprotective effectsof zofenoprilat on surviving fraction (P< 0.05; Fig. 6b)and cellular apoptosis (P< 0.05; Fig. 6c). Furthermoresirtinol counteracted the eNOS activation [Fig. 6d] andNO production [Fig. 6e] induced by zofenoprilat treatment(P< 0.05). We examined whether SirT1 over-expressioncan protect HUVECs from oxidative stress inducedby angiotensin I treatment. Overexpression of SirT1significantly reduced the superoxide anion generation(P< 0.05; Fig. 7a), increased the surviving fraction(P< 0.05; Fig. 7b), decreased the apoptosis (P< 0.05;Fig. 7c), increased the eNOS activity (P< 0.05; Fig. 7d)and NO production [Fig. 7e] induced by angiotensin Itreatment. SirT1 protein expression levels were assessedby western blott [Fig. 7f].
Zofenoprilat negatively controls angiotensin IItype I receptor expression through SirT1We analyzed the effects of SirT1-restored expression byzofenoprilat on the angiotensin II type I receptor proteinlevels. HUVEC were 1 h pretreated with zofenoprilat andtreated or not with angiotensin I for 1 h. Zofenoprilatannulled the angiotensin II type I receptor protein expres-sion up-regulation induced by angiotensin I treatment[Fig. 8a]. Furthermore, SirT1 overexpression downregulated[Fig. 8b] the basal protein expression levels of angiotensin IItype I receptor.
DISCUSSIONROS signaling represents the first step in the development ofseveral cardiovascular disorders and noncardiovascular dis-orders. Aging, obesity, ischemia/reperfusion, hypertension,
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FIGURE 5 Angiotensin I negatively controls SirT1 protein expression through the p38 activation. (a) HUVECs 1 h pretreated with zofenoprilat (150mmol/l) and treated ornot with angiotensin I (5mmol/l) for 1 h. (b) HUVECs pretreated with SB203580 (10mmol/l) for 1 h then stimulated for 1 h with angiotensin I (5mmol/l). Total cell lysateswere analyzed by immunoblotting with specific antibodies for indicated proteins. a-tubulin expression shows the loading of samples. The values of fold increases over thecontrol, arbitrarily set at 1, were obtained by densitometric analysis. Any single treatment was performed in triplicate. Total cell lysates were analysed by immunoblottingwith specific antibodies for indicated proteins. a-tubulin expression shows the loading of samples. The values of fold increases over the control, arbitrarily set at 1, wereobtained by densitometric analysis. Any single treatment was performed in triplicate. (c) and (d) HUVECs transfected with MKK6 vector, the kinase upstream of p38, withempty vector CMV or untransfected (UNT); (c) total cell lysates and (d) nuclear (N) or cytosolic (Cy) fractions. (e) SirT1-p38 complex was immunoprecipitated (IP) with aSirT1 monoclonal antibody from extracts containing equal amounts of total proteins and subsequently analyzed by immunoblotting with a p38 polyclonal antibody. Samefilter was probed with a SirT1 monoclonal antibody. Similar results were obtained in two (n$3) different experiments.
high-fat diet, low-molecular pro-oxidants, metal ions, andso forth can be responsible for ROS overproduction [1–3].The renin–angiotensin system (RAS) plays a central role inthe pathogenesis of cardiovascular diseases through theproduction of angiotensin II, the final product of the RASsystem [52]. In this article, we demonstrate that angiotensinII-mediated stress in HUVEC, was reduced by zofenoprilattreatment in a dose-dependent manner as a result ofthe decrease in anion superoxide generation, numberof apoptotic cells and of the increase of cells survival.
The biochemical basis of apoptosis events is here docu-mented by extrinsic caspase cascade and NF-kB activationthat is dramatically prevented by zofenoprilat treatment.The beneficial action of the ACE inhibitors in protectingendothelial cells from oxidative stress is well documented inthe literature and zofenopril, thanks to its sulphydrylgroup, might possess a noteworthy antioxidant activity[42]. Interestingly, due to the presence of the sulphydrylgroup, zofenopril exerts significant additional antioxidantproperties in comparison with other ACE-inhibitors, acting
ZofenoprilatAngiotensin I
Sirtinol
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•P < 0.05 vs. Ang.I + Zofenoprilat
(a) (b)
(c)
(c)
(d)
FIGURE 6 SirT1 inhibitions by sirtinol reverts antioxidative proprieties of zofenoprilat. Quantification of superoxide anion generation (a), cells survival (MTT) (b), cellsapoptosis (TUNEL) (c), eNOS activity (d) and NO levels (e) on HUVECs incubated with sirtinol (50mmol/l), 1 h pretreated with zofenoprilat and treated or not withangiotensin I for 1 h.
Marampon et al.
8 www.jhypertension.com Volume 30 # Number 1 # Month 2013
as a scavenger of various ROS in vitro as well as in vivo.Concordantly, Zofenopril, but not lisinopril, enalaprilor ramipril had significant antioxidant actions, favoring vaso-dilation in experimental heart failure and in hypertensivepatients [53–58]. Thus, it is conceivable that the sulphydrylgroup, that is able per se to scavenge endogenous oxidants, isable to confer significant antioxidant properties to the zofe-nopril molecule. However, the signal mechanism by whichzofenoprilat antioxidative ability is elicited still needs to becharacterized [59]. In fact, some authors reported a cross-talkbetween oxidative stress stimuli, such as angiotensin II, and
MAP-kinases activation, which mediates cell-specificresponses. For this reason, we sought to better characterizethe relationship between ERKs-, p38-MAP-Kinase, angioten-sin II and zofenoprilat in endothelial cells. We show thatzofenoprilat treatment counteracts the angiotensin-II inducedincreased amount of phospho-active ERKs and p38, which ispersistent also under angiotensin II-mediated stress.We dem-onstrated, through the use of MEK/ERK and p38 inhibitors,that zofenoprilat protects endothelial cells counteractingp38-angiotensin-II meadiated activation. As a matter of factthe decreases of superoxide anion generation and apoptotic
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T1
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T1
Angiotensin I
– – + +– +
(c) (d)
(a) (b)
(e) (f)
FIGURE 7 SirT1 overexpression counteracts oxidative stress induced by angiotensin I treatment. Quantification of superoxide anion generation (a), cells survival (MTT) (b),cells apoptosis (TUNEL) (c), eNOS activity (d) and NO levels (e) and SirT1 protein expression levels (f) on HUVECs untransfected (UNT) or transfected with the empty vector(CMV) or with the expression vector for SirT1. After 24 h of puromycin selection, cells were treated with angiotensin I for 1 h. Any single treatment was performed intriplicate and the results are expressed as means of two tests (n$3).
cells with consequent increases of cells survival, occurredwhen angiotensin II treatments were done in the presence ofphospho-active-p38 inhibitor but not in the presence ofphospho-active-ERKs inhibitor. Several scientific evidencesdraw attention to the fact that p38 activation leads to theregulation of several proteins implicated in controlling ofseveral cellular functions. In order to identify a downstreamtarget of p38 we investigated the possible involvement ofSIRT1, the mammalian ortholog of yeast Sir2, which has beenshown to regulate numerous physiological processes includ-ing glucosemetabolism,DNA repair and apoptosis [60]. SIRT1also regulates severalproteins implied in stress responsessuchas p53, forkhead transcription factors andNFkB [61,62]. A linkhas been also demonstrated between vascular angiogenesis,homeostasis, senescence, atherosclerosis and SIRT1 levelsproviding a molecular basis for cardiovascular therapy basedon SIRT1 targeting [39]. Thus, identify new treatments capableof increasingprotein levels of SirT1 is oneof the future goals ofmodern pharmacology to cure cardiovascular disease. Here,we show that zofenoprilat restores SIRT1 protein expressionand nuclear accumulation, blocked by angiotensin II. Ingeneral, SIRT1 is localized in nuclei but it has been shownthat cytoplasm-localized SIRT1 is associated with apoptosisand led to increased sensitivity to apoptosis [63]. As
angiotensin II induced phospho-active p38 increase and itis known that p38 is able to phosphorylate an array of targets,including SIRT1 [64], known to prevent endothelial cellssenescence and oxidative stress-induced death [38,39], weinvestigated the role of p38 signaling in SIRT1 expression.We found an inverse correlation between SIRT1 expressionand p38. In fact, p38 inhibitor alone increased the expressionprotein basal level of SIRT1 and drastically counteracted theSIRT1 protein abrogation induced by angiotensin II. ThatSIRT1 expression is related to p38 was also demonstratedby the transfection of p38-activator MKK6 which induced amarked decrease in SIRT1 expression. p38 pharmacologicalinhibition restored while MKK6 transfection blocked theSIRT1 nuclear shuttling. Furthermore immunoprecipitationexperiments show that p38 was physically bound to SIRT1.The functional relationship between p38 signaling andSIRT1 expression in endothelial cells is very similar to thatin chondrocytes, in which the p38/SIRT1 interaction plays acentral role in inducing cellular senescence [65]. The role ofSIRT1 in endothelial protection is in our study substantiatedalso by overexpression of SIRT1 and by the use ofresveratrol. Resveratrol treatment blocked the angioten-sin-II-mediated anion superoxide generation along with adecrease of apoptotic cells and an increase of cells survival.
SirT1
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T1
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Zofenoprilat
Angiotensin I
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– +– +
1 1 2 1
(a)
(b)
FIGURE 8 Zofenopril blocks the AT1-R protein expression through SirT1.(a) HUVECs 1 h pretreated with zofenoprilat (150mmol/l) and treated or not withangiotensin I (5mmol/l) for 1 h. (b) HUVECs transfected with SirT1 vector, with theempty vector CMV or untransfected (UNT); total cell lysates. Total cell lysateswere analyzed by immunoblotting with specific antibodies for indicated proteins.a-tubulin expression shows the loading of samples. The values of fold increasesover the control, arbitrarily set at 1, were obtained by densitometric analysis. Anysingle treatment was performed in triplicate.
Angiotensin II
MKK6
RAS
p38
sirt1
Cytoplasm
Nucleus
p38
Sirt1
AT
1-R
AT1-R
Zofenoprilat
ROS ↑↑NO ↓↓
Oxidative stress
FIGURE 9 Diagram of molecular mechanism regulated by angiotensin IIand responsible of oxidative stress. Diagram summarizes the molecular pathwaycontrolled by angiotensin II and responsible of oxidative stress-mediated endo-thelial dysfunction. Angiotensin II, through the AT-R1 binding, activates RAS/p38MAPKs pathway. p38, forming a protein complex, sequestrates SirT1 into thecytoplasm not permitting the SirT1 nuclear shuttling. Furthermore, p38 activationnegatively controls the SirT1 protein expression do not permitting its negativetranscriptional activity on AT1-R gene. Zofenoprilat, blocking angiotensin II syn-thesis, crushes the entire mechanism do not permitting AT1-R expression throughthe SirT1 protein upregulation.
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10 www.jhypertension.com Volume 30 # Number 1 # Month 2013
Finally, we show that zofenoprilat negatively controlsangiotensin II type I receptor expression through SirT1.Therefore zofenopril ensures a cytoprotective effect againstthe angiotensin blocking not only the signal but alsoeliminating its receptor. Figure 9 shows a diagram thatexplain the molecular pathway responsible for oxidativestress induced by angiotensin II and negatively controlledby zofenoprilat. This prevents any signal mediatedby angiotensinmay induce cellular dysfunction. All togetherthese data suggest that angiotensin II controlling p38 acti-vation, negatively regulates SIRT1 functions favoringendothelial dysfunction. The ACE inhibitor zofenoprilatprotects endothelial homeostasis counteracting thismechanism.
ACKNOWLEDGEMENTSThe study has been funded by the Chair of InternalMedicine at the University of L’Aquila.
Conflicts of interestS.E. is an employee of Menarini Ricerche S.p.A., partof Menarini group, owner of the zofenopril rights. Noneof the other authors has conflict of interest to declare.
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Reviewers’ Summary EvaluationsReviewer 1The present paper demonstrates for the first time thatthe ACE-inhibitor zofenoprilat modulates the Ang II-mediated superoxide generation and cell apoptosis byregulation of the p38-SirT1 pathway at the level ofendothelial cells. These findings likely represent anovel and intriguing molecular mechanism explaining aprotective effect against the hypertension-related cellularand vascular damage. Such in-vitro results await futureinvestigation by in-vivo models, which will confirmwhether this pathway is a major mechanism whereby thisACE-inhibitor exerts the previously described cardiovas-cular benefit.
Reviewer 3The message derived from the study is important as itdemonstrates an important relatively new way of endo-thelial protection by ACE inhibition.
The major strength of the study is the very detailedstepwise approach to demonstrate the effect of ACEinhibition by zofenoprilat on the process of AII-inducedsuperoxide anion production and apoptosis in humanumbilical vein endothelial cells.
One limitation is that the study was done in vitrowithoutthe usual in-vivo environment, where other componentsinteract on the endothelial cells. Future studies in vivo willhave to answer this question, and also whether the anti-oxidant effect is unique for zofenoprilat or it is shared withother ACE inhibitors.
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12 www.jhypertension.com Volume 30 # Number 1 # Month 2013
