Protection of coronary endothelial cells from cigarette

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VPH-06213; No of Pages 10 Vascular Pharmacology xxx (2015) xxx–xxx

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Protection of coronary endothelial cells from cigarette smoke-induced oxidative stress by a new MnII-containing polyamine-polycarboxilate scavenger of superoxide anion Matteo Becatti a,1, Giulia Boccalini b,1, Alessandro Pini b, Claudia Fiorillo a, Andrea Bencini c, Daniele Bani b, Silvia Nistri b,⁎ a b c

Department of Experimental & Clinical Biomedical Sciences “Mario Serio”, Italy Department of Experimental & Clinical Medicine, Research Unit of Histology & Embryology, Italy Department of Chemistry, University of Florence, Florence, Italy

a r t i c l e

i n f o

Article history: Received 18 March 2015 Received in revised form 26 May 2015 Accepted 18 June 2015 Available online xxxx Keywords: Superoxide anion ROS scavenger Guinea pig coronary endothelial cells Cigarette smoke Oxidative stress

a b s t r a c t Oxidative stress plays a major role in cardiovascular injury and dysfunction induced by cigarette smoke. Smokeborne pro-oxidants impair endothelial function and predispose to thrombosis, inflammation and atherosclerosis. This in vitro study evaluates whether MnII(4,10-dimethyl-1,4,7,10-tetraazacyclododecane-1,7-diacetate).2H2O (MnII(Me2DO2A)), a polyamine–polycarboxilate, MnII-containing O•− 2 scavenger, has a direct protective action on guinea pig coronary endothelial (GPCE) cells exposed to cigarette smoke extracts (CSE). MnII(Me2DO2A) (1–10 μmol/l) was added to the culture medium together with CSE and maintained for 4 h. In parallel experiments, the inactive congener ZnII(Me2DO2A), in which ZnII replaced the functional MnII center in the same organic scaffold, was used as negative control. MnII(Me2DO2A), mostly at the higher doses (5 and 10 μmol/l), significantly increased GPCE cell viability (trypan blue assay), improved mitochondrial activity (MTT test, mitochondrial membrane potential Δψ), reduced cellular apoptosis (mPTP, caspase-3 activity, TUNEL assay), decreased intracellular ROS levels (H2DCFDA), lipoperoxidation (BODIPY 581/591) and decreased protein nitrosylation. Of note, ZnII(Me2DO2A) did not preserve cell viability. These findings suggest that MnII(Me2DO2A) is a promising O•− 2 scavenging compound able to protect from cigarette smoke-induced oxidative cell injury. In perspective, should its efficacy be confirmed in future in vivo studies, this molecule might represent a therapeutic or preventive drug to counteract cigarette smoke toxicity. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Cigarette smoke (CS) is viewed as one of the major cardiovascular risk factors, accounting for 80% increased risk for coronary artery insufficiency in smokers vs. non-smokers [1,2]. Perspective analyses indicate that the trend of CS-related cardiovascular disease is expected to

Abbreviations: CS, cigarette smoke; CSE, cigarette smoke extracts; GPCE, guinea pig coronary endothelial cells; H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; II Mn (Me2DO2A) and ZnII(Me2DO2A), MnII and ZnII complexes with 4,10-dimethyl1,4,7,10-tetraazacyclododecane-1,7-diacetic acid; mPTP, Mitochondrial permeability transition pore opening; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NO, nitric oxide; NT, nitrotyrosine; RNS, reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; TMRM, tetramethylrhodamine methyl ester perchlorate; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. ⁎ Corresponding author at: Dept. Experimental & Clinical Medicine, Sect. Anatomy & Histology, Research Unit of Histology & Embryology, University of Florence, Viale G.Pieraccini, 6, I-50139 Florence, Italy. E-mail address: silvia.nistri@unifi.it (S. Nistri). 1 BM and BG contributed equally to this paper.

increase in parallel with the earlier onset of tobacco abuse. In fact, young smokers have a higher risk to develop vascular functional and histological abnormalities which are early hallmarks of atherosclerosis [3]. For these reasons, the development of new therapeutic strategies that can prevent or reduce the adverse effects of CS on the cardiovascular system is a major public health target. There is a general consensus that oxidative stress plays a major role in CS-induced cardiovascular injury and dysfunction [4–9]. In fact, CS contains harmful components including short-lived oxidants such as reactive oxygen and nitrogen species (ROS and RNS) [10]. Several clinical and experimental studies suggest that these pro-oxidants can directly impair endothelial function, primarily inducing a decrease in nitric oxide (NO) bioavailability [9,11–13]. In turn, oxidative stress causes a rapid reduction of the cellular anti-oxidant systems, such as superoxide dismutases (SODs), sparkling a vicious cycle that further enhances ROS/RNS tissue levels [14,15]. Hence, O•− 2 seems to play a major role in CS-induced oxidative stress and can be a suitable target for new pharmacological interventions. On these basis, we performed a series of in vitro and in vivo

http://dx.doi.org/10.1016/j.vph.2015.06.008 1537-1891/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: M. Becatti, et al., Protection of coronary endothelial cells from cigarette smoke-induced oxidative stress by a new MnIIcontaining polyamine-polycarboxilate scavenger of superoxide anion, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.008

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investigations using a non-peptidic, low molecular weight, polyamine– polycarboxylate MnII complex, termed MnII(Me2DO2A), capable to efficiently catalyze O•− 2 dismutation, as endogenous SODs [16–19]. Its efficacy relies on the well-known chemical property of Mn ion to bind to and react with ROS, in particular O•− 2 . Polyamine–polycarboxylate scaffolds are optimal molecules for the synthesis of highly stable MnII complexes, resistant to oxidizing and reducing agents, soluble in aqueous media and non-toxic, all properties that render them suitable for biological and medicinal applications [20,21]. In this context, we previously demonstrated that MnII(Me2DO2A) effectively restores redox balance in cell culture and animal models of oxidative stress and inflammation [16–19]. The present study was designed to explore whether MnII(Me2DO2A) could counteract CS-induced oxidative stress in vitro. As cell substrate we used primary cultures of coronary endothelial cells, based on the notion that the cardiovascular system is one of the major targets of the noxious effects of CS.

2.2. CSE preparation Kentucky Reference cigarettes, 3R4F, each containing 11 mg of total particulate matter, 9.4 mg of tar, and 0.73 mg of nicotine, were obtained from the Kentucky Tobacco Research Council (Lexington, KY). CSE solution was prepared by bubbling smoke from two cigarettes in 50 ml of PBS at a rate of one cigarette every 30 s, according to the method used by Niu et al. [22], with minor modification. The resulting solution, assumed as 100% CSE, was adjusted to pH 7.4, and filtered through a 0.2-μm filter (Millipore Corporation, Bedford, MA) to remove bacteria and large particles. CSE solution was freshly prepared on the day of the experiment and immediately used. To establish the effective dose of CSE, concentration- and timedependent studies were performed. GPCE cells were exposed to 1–15% CSE for 4–12 h and then cell injury was evaluated by Trypan Blue (data not shown). Accordingly, 10% CSE solution (vol/vol) for 4 h was used in all experiments. 2.3. Isolation and culture of guinea pig coronary endothelial (GPCE) cells

2. Materials and methods 2.1. Reagents MnII(Me2DO2A), namely MnII(4,10-dimethyl-1,4,7,10-tetraazacyclododecane-1,7-diacetate).2H2O (Fig. 1), was synthesized in our laboratory as described [16]. Its chemical characterization has been reported in detail in our previous papers [16,19]. The amount required to perform the present experiments was a kind gift from the patent owner General Project Ltd., Montespertoli (Florence), Italy. As negative control, we used the inactive congener ZnII(Me2DO2A), in which the functional MnII was replaced with a ZnII ion lacking the capability to II catalyze O•− 2 decomposition. Zn (Me2DO2A) was synthesized in our laboratory using the organic scaffold Me2DO2A and following the same procedure as MnII(Me2DO2A). The formation constants of the ZnII complexes have been determined using the same procedure reported for the MnII complexes [16]. Unless otherwise specified, the other reagents used for the experiments were from Sigma-Aldrich (Milan, Italy).

GPCE cells were isolated from 2–3-month old albino guinea pigs hearts (Harlan, Correzzana, Italy) based on the previously described method to isolate rat coronary endothelial cells [23]. Animal handling was done in compliance with the rules for the care and use of laboratory animals of the European Community (86/609/CEE), under supervision of the committee for the care and use of laboratory animals of the University of Florence, Italy. Briefly, after enzymatic digestion of heart tissue fragments with type I collagenase, the recovered cells were stirred for 30 min at 37 °C in the presence of 10 mg/50 ml trypsin, washed and plated until confluence (5–6 days). M199 containing 10% fetal calf serum, 10% newborn calf serum, 250 U/ml penicillin G, and 250 μg/ml streptomycin, was used as culture medium. Characterization of the isolated GPCE cells was performed by immunocytochemistry and cytochemistry, as previously reported [23]. The percentage of immunoreactive cells for endothelial markers, such as vimentin and von Willebrand factor ranged between 96% and 98% (data not shown). For all experiments, cells were used at the first and second passage. GPCE cells were treated or not with MnII(Me2DO2A) at increasing doses (1, 2.5, 5 and 10 μmol/l), added together with CSE 10% for 4 h. These Mn II(Me2 DO2A) doses were chosen on the basis of our previous in vitro studies [16,19]. As control for the specific capability of MnII(Me2DO2A) to suppress oxidative stress by redox reaction, cell viability was assayed by replacing MnII(Me2DO2A) with its inactive congener ZnII(Me2DO2A) at the same concentrations, added together with CSE. 2.4. Trypan blue viability assay The trypan blue exclusion method was used to assess cell viability. GPCE cells (5 × 104/well) were seeded in 24-well plates. At the end of the treatments, the cells were gently detached by trypsin/EDTA, resuspended in culture medium and mixed 1:1 with 0.4% trypan blue solution. The final cell suspensions were counted under a phase contrast inverted microscope using a Burker chamber. Viable cells were expressed as percentage of the total counted cells. 2.5. MTT assay

Fig. 1. Polyamine–polycarboxylate scaffold (a) and 3-D structure (b) of MnII(Me2DO2A).

Cell mitochondrial function was measured using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. GPCE cells (5 × 104/well) were seeded in 24-well plates. At the end of the treatments, the culture medium was removed from each well and replaced with 300 μl of MTT (0.5 mg/ml), followed by 4-h incubation at 37 °C. Then, 350 μl DMSO were added to each well to dissolve the formazan crystals. The plate was gently shaken for 5 min and read at 550 nm on a plate reader. Optical density was assumed as indicator of mitochondrial activity and, indirectly, cell viability.

Please cite this article as: M. Becatti, et al., Protection of coronary endothelial cells from cigarette smoke-induced oxidative stress by a new MnIIcontaining polyamine-polycarboxilate scavenger of superoxide anion, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.008

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2.6. Mitochondrial membrane potential (Δψ) Mitochondrial membrane potential was assessed as previously reported [24] using tetramethylrhodamine methyl ester perchlorate (TMRM), a lipophilic potentiometric fluorescent dye that distributes between the mitochondria and cytosol in proportion to Δψ by virtue of its positive charge. At low concentrations, the fluorescence intensity depends on dye accumulation in mitochondria, which in turn is directly related to mitochondrial potential. For confocal microscope analysis, cells were cultured on glass coverslips and loaded for 20 min at 37 °C with TMRM, dissolved in 0.1% DMSO to a 100 nM final concentration in the culture medium. The cells were fixed in 2% buffered paraformaldehyde for 10 min at room temperature and the TMRM fluorescence analyzed under a confocal Leica TCS SP5 scanning microscope (Mannheim, Germany) equipped with a helium–neon laser source, using a 543-nm excitation wavelength, and with a Leica Plan Apo × 20 objective. Mitochondrial membrane potential was also quantified by flow cytometry. Single-cell suspensions were washed twice with PBS and incubated for 20 min at 37 °C in the dark with TMRM dissolved in M199 medium (100 nM). The cells were then washed, resuspended in PBS and analyzed using a FACSCanto flow cytometer (Becton-Dickinson, San Jose, CA). 2.7. Mitochondrial permeability transition pore opening (mPTP) Mitochondrial permeability, an index of mitochondrial dysfunction and early apoptosis, was measured by calcein fluorescence, as described [25]. The fluorescent probe calcein-AM freely enters the cells and emits fluorescence upon de-esterification. Co-loading of cells with cobalt chloride, which cannot cross the mitochondrial membranes in living cells, quenches the fluorescence in the whole cell except mitochondria. During induction of mPTP, cobalt can enter mitochondria and quenches calcein fluorescence, whose decrease can be taken as a measure of the extent of mPTP induction. GPCE cells grown on glass coverslips were loaded with calcein-AM (3 μM) and cobalt chloride (1 mM) added to the culture medium for 20 min at 37 °C. The cells were then washed in PBS, fixed in 2% buffered paraformaldehyde for 10 min at room temperature and analyzed by a Leica TCS SP5 confocal laser scanning microscope equipped with an argon laser source, using 488-nm excitation wavelength, and with a Leica Plan Apo ×20 objective. 2.8. Assessment of caspase-3 activity GPCE cells seeded on glass coverslips were incubated with FAM FLICA™ Caspase 3&7 assay kit (Immunochemistry Technologies, Bloomington, MN, USA) for 30 min as previously reported [26]. After incubation, the cells were thoroughly washed and fixed in 2% buffered paraformaldehyde for 10 min at room temperature. Fluorescence was detected by a confocal Leica TCS SP5 scanning microscope equipped with an argon laser source, using 488-nm excitation wavelength, and a Leica Plan Apo x20 objective. Caspase-3 activity was also quantified by flow cytometry: single-cell suspensions were incubated with FAM-FLICA™ for 30 min at 37 °C, washed twice with PBS and analyzed using a FACSCanto flow cytometer (Becton-Dickinson).

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a humid atmosphere. Then, the cells were incubated with peroxidaseconjugated streptavidin for 30 min at room temperature and the signal was revealed with 3,3′-diaminobenzidine. Finally, nuclear counterstaining was achieved by methyl green. Apoptotic nuclei were recognized by the presence of dark brown staining, at variance with those of viable cells, which instead appeared pale brown or green. TUNELpositive nuclei were counted in 10 microscopic fields for each cell preparation. TUNEL apoptotic index was then expressed as relative percentage of TUNEL-positive nuclei on the total number of methyl green-stained nuclei. 2.10. Determination of intracellular ROS GPCE cells seeded on glass coverslips were loaded with the ROSsensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen, CA, USA; 2.5 μmol/l) – dissolved in 0.1% DMSO and Pluronic acid F-127 (0.01% w/v) – which were added to cell culture media for 15 min at 37 °C [26]. The cells were fixed in 2% buffered paraformaldehyde for 10 min at room temperature and the H2DCFDA fluorescence analyzed using a Leica TCS SP5 confocal scanning microscope equipped with an argon laser source, using 488-nm excitation wavelength, and a Leica Plan Apo x20 objective. ROS generation was also monitored by flow cytometry: briefly, single-cell suspensions were incubated with H2DCFDA (1 μmol/l) for 15 min at 37 °C and immediately analyzed using a FACSCanto flow cytometer (Becton–Dickinson). Data were analyzed using FACSDiva software (Becton–Dickinson). 2.11. Evaluation of lipid peroxidation Lipid peroxidation was investigated by confocal scanning microscopy using BODIPY, a fluorescent probe that is intrinsically lipophilic and thus mimics the properties of natural lipids [27]. BODIPY 581/591 C11 acts as a fluorescent lipid peroxidation reporter that shifts its fluorescence from red to green in the presence of oxidizing agents. Briefly, cells were cultured on glass cover slips and loaded with dye by adding the fluorescent probe BODIPY, dissolved in 0.1% DMSO (2 μM final concentration), to the cell culture media for 30 min at 37 °C. The cells were fixed in 2.0% buffered paraformaldehyde for 10 min at room temperature and the BODIPY fluorescence analyzed (at an excitation wavelength of 581 nm) using a confocal Leica TCS SP5 scanning microscope (Mannheim, Germany) equipped with an argon laser source for fluorescence measurements. Moreover, lipid peroxidation was quantified by flow cytometry. Single-cell suspensions were washed twice with PBS and incubated, in the dark, for 30 min at 37 °C with BODIPY 581/591 (1 μM) in cell culture medium. After labeling, cells were washed and

2.9. TUNEL assay Cell death was assessed by TUNEL assay for apoptosis, performed using a Klenow-FragEL™ DNA fragmentation detection kit (Calbiochem, San Diego, CA, USA), as reported in the manufacturer's instructions. Briefly, GPCE cells grown on glass coverslips were exposed to CS, treated with MnII(Me2DO2A) (1 and 10 μmol/l) and then incubated with 15 mg/ml proteinase K for 5 min at room temperature. After rinsing in PBS, the cells were immersed in the Klenow Labeling Reaction Mixture containing deoxynucleotidyl transferase and biotin-labeled and unlabeled deoxynucleotides, and incubated at 37 °C for 90 min in

Fig. 2. Evaluation of GPCE cell viability by trypan blue assay. Exposure to CSE causes a marked reduction of the amounts of viable cells. This effect was reduced by MnII(Me2DO2A) 1–10 μmol/l, added together with CSE, while ZnII(Me2DO2A) had no effects. Significance of differences: °°° p b 0.001 vs. control; *p b 0.05, **p b 0.01, ***p b 0.001 vs. CSE.

Please cite this article as: M. Becatti, et al., Protection of coronary endothelial cells from cigarette smoke-induced oxidative stress by a new MnIIcontaining polyamine-polycarboxilate scavenger of superoxide anion, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.008

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Please cite this article as: M. Becatti, et al., Protection of coronary endothelial cells from cigarette smoke-induced oxidative stress by a new MnIIcontaining polyamine-polycarboxilate scavenger of superoxide anion, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.008

M. Becatti et al. / Vascular Pharmacology xxx (2015) xxx–xxx

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Fig. 4. Evaluation of apoptosis in GPCE cells by mPTP assay. Exposure to CSE causes the extinction of calcein-AM fluorescence in mitochondria by increased cobalt ion permeation, while this effect was reduced by 5 and 10 μmol/l MnII(Me2DO2A) added together with CSE. Scale bar: 20 μm.

resuspended in PBS and analyzed using a FACSCanto flow cytometer (Becton-Dickinson, San Jose, CA). 2.12. Immunohistochemical detection of nitrotyrosine Nitrotyrosine (NT), an index of protein nitrosylation by harmful ONOO−, was determined by immunocytochemistry. Briefly, GPCE cells grown on glass coverslips, were exposed to CS and treated with MnII (Me2DO2A) (1 and 10 μmol/l). Cells were fixed with formaldehyde for 10 min and then incubated with rabbit polyclonal anti-NT antibody (Upstate Biotechnology, Buckingham, UK; 1:100) at 4 °C overnight. Immune reaction was revealed by Alexa-fluor 568-labeled goat antirabbit IgG (1:300; Invitrogen, Milan, Italy). Negative controls were carried out by omitting the primary antibodies. 2.13. Statistical analysis The reported data are expressed as the mean ± SEM of at least 3 independent experiments. Statistical comparison of differences between groups was carried out using one-way ANOVA followed by StudentNewman–Keuls multiple comparison test. A p value ≤0.05 was considered significant. GraphPad Prism 2.0 statistical program (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. 3. Results 3.1. MnII(Me2DO2A) preserves GPCE cell viability impaired by CSE The trypan blue assay (Fig. 2) showed that 4-h exposure to CSE caused a marked decrease in the amounts of viable GPCE cells. This

effect was significantly reduced by MnII(Me2DO2A) (1–10 μmol/l) added together with CSE. Of note, ZnII(Me2DO2A), composed of a similar organic scaffold but lacking MnII, showed no cytoprotective effect (Fig. 2).

3.2. MnII(Me2DO2A) preserves GPCE cell mitochondrial function impaired by CSE Parallel experiments to explore mitochondrial integrity and function were carried out with the MTT assay, which indicates the respiratory chain efficiency (Fig. 3 A) and the TMRM assay, which evaluates the mitochondrial membrane potential (Fig. 3 B,C). The results of these experiments have consistently shown that exposure to CSE caused a marked impairment in mitochondrial function in GPCE cells. This noxious effect was significantly reduced by MnII(Me2DO2A) (5 and 10 μmol/l), added together with CSE.

3.3. MnII(Me2DO2A) protects GPCE cells from CSE-induced apoptosis The reduction of CSE-induced oxidative stress by MnII(Me2DO2A) resulted in a significant decrease in apoptotic cell death. Indeed, the occurrence of early apoptotic markers, namely mitochondrial permeability transition pores opening (mPTP) (Fig. 4) and caspase-3 activation (Fig. 5 A,B) were markedly increased in GPCE cells exposed to CSE, while the concurrent addition of MnII(Me2DO2A) (5 and 10 μmol/l) significantly attenuated these changes. Evaluation of late apoptosis by the percentage of TUNEL-positive nuclei confirmed the cytoprotective effect of MnII(Me2DO2A) (1 and 10 μmol/l) (Fig. 6 A,B).

Fig. 3. Evaluation of GPCE cell mitochondrial integrity and function by MTT and TMRM assay. Exposure to CSE causes a marked mitochondrial impairment, which was inhibited by MnII(Me2DO2A) given together with CSE (A,B). FACS analysis confirms the microscopical findings of TMRM fluorescence as it shows that, compared with the control cells, CSE shift the fluorescence peaks towards higher values (right), while this effect is markedly reduced by MnII(Me2DO2A) at the higher doses (5 and 10 μmol/l) (C). Scale bar: 20 μm. Significance of differences: °°°p b 0.001 vs. control; *p b 0.05 vs. CSE.

Please cite this article as: M. Becatti, et al., Protection of coronary endothelial cells from cigarette smoke-induced oxidative stress by a new MnIIcontaining polyamine-polycarboxilate scavenger of superoxide anion, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.008

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Fig. 5. Evaluation of apoptosis in GPCE cells by caspase-3 activity. Cytoplasmic fluorescence related to activated caspase-3 was increased by CSE and markedly decreased by 5 and 10 μmol/l MnII(Me2DO2A) (A). FACS analysis confirms the microscopical findings as it shows that, compared with the control cells, CSE shift the fluorescence peaks towards higher values (right), while this effect is markedly reduced by MnII(Me2DO2A), especially at the higher doses (B). Scale bar: 20 μm.

3.4. MnII(Me2DO2A) protects GPCE cells from oxidative damage induced by CSE MnII(Me2DO2A) also decreased GPCE cell death by reducing the oxidative stress related to endogenous ROS. In fact, intracellular

ROS generation and lipoperoxidation, evaluated by loading the cells with the fluorescent probes H 2 DCFDA and BODIPY respectively, were markedly increased by exposure to CSE, whereas they were significantly reduced by Mn II (Me 2 DO2A) (5 and 10 μmol/l) (Fig. 7 A,B).

Please cite this article as: M. Becatti, et al., Protection of coronary endothelial cells from cigarette smoke-induced oxidative stress by a new MnIIcontaining polyamine-polycarboxilate scavenger of superoxide anion, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.008

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Fig. 6. Evaluation of apoptosis in GPCE cells by TUNEL assay. Exposure to CSE causes a marked increase in the number of TUNEL-positive cell nuclei, which was reduced by 1 and 10 μmol/l MnII(Me2DO2A), added together with CSE, in a dose-related fashion (A,B). Scale bar: 10 μm. Significance of differences: °°°p b 0.001 vs. control; ***p b 0.001 vs. CSE.

The levels of immunoreactive nitrotyrosine (NT), a marker of ONOO−-induced protein nitration, were enhanced upon exposure to CSE and were significantly reduced by the addition of MnII(Me2DO2A) (10 μmol/l) (Fig. 8). 4. Discussion ROS, RNS and the resulting nitroxidative stress are the most invoked effectors of immediate cell and tissue damage induced by CS [4–9] thus justifying the search for new devices and drugs capable of reducing the noxious effects of CS-borne oxidizing substances. The need for a pharmacological intervention is further supported by previous observations that blood vessels of smokers are particularly susceptible to oxidative stress due to deposition of oxidation catalyzing metals contained in CS as well as by an imbalance between oxidant-generating and oxidantreducing cellular systems in favor of the former [28,29].

The used cell culture model was set up to study the possible protection afforded by O•− 2 scavenging on GPCE cells exposed to CSEinduced oxidative stress. The rationale of this working hypothesis is based on the major role played by O•− 2 in the harmful effect of CS on the vascular endothelium, which depends on its direct oxidant effects as well as on its reactivity with bioactive NO, a key regulator of vascular function [9]. Moreover, evidence exists in the literature that CS induces endothelial cell death through ROS-dependent mechanisms II [30–32]. In the present study, the O•− 2 scavenger Mn (Me 2DO2A), added at low, micromolar concentrations together with CSE, effectively reduced oxidative stress-induced apoptotic cell death and significantly preserved cell viability. These cytoprotective action appeared to be related to the ability of MnII(Me2DO2A) to reduce mitochondrial injury, lipoperoxidation, protein nitrosylation and intracellular ROS generation. Increased endothelial integrity can interrupt the vicious cycle activated by CS, thus preventing or reducing impaired

Please cite this article as: M. Becatti, et al., Protection of coronary endothelial cells from cigarette smoke-induced oxidative stress by a new MnIIcontaining polyamine-polycarboxilate scavenger of superoxide anion, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.008

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Fig. 7. Evaluation of intracellular ROS generation and lipoperoxidation by H2DCFDA and BODIPY. Confocal microscopy shows that both these parameters were enhanced upon CSE treatment and significantly reduced by 5 and 10 μmol/l MnII(Me2DO2A) (A). FACS analysis confirms the microscopical findings as it shows that, compared with the control cells, CSE shifts the fluorescence peaks towards higher values (right), while this effect is markedly reduced by MnII(Me2DO2A), especially at the higher doses (B). Scale bar: 20 μm.

vascular motility, thrombogenic events, inflammation and predisposition to atherosclerosis, which are hallmarks of dysfunctional blood vessels in smokers [4,6,9]. In a previous study, we have shown that MnII(Me2DO2A) has lipophilic properties, which allow it to easily cross the cell membranes and attain O•− 2 -neutralizing levels within the cells [16]. Moreover, MnII forms a highly stable complex at physiological pH with the polyamine-polycarboxylate scaffold, which prevents both demetallation upon MnII complexation and trans-metallation reactions, due to complexation of the ligand to other intracellular metal cations, mainly CaII, MgII and KI, which give less stable complexes than MnII. Indeed, MnII(Me2DO2A) does not release MnII even in the presence of large excess of CaII and of broad pH variations (at pH 6, MnII release is less than 5%) [16]. Moreover, MnII(Me2DO2A) is less susceptible to inactivation by oxidative stress conditions than endogenous or exogenously administered SODs [15,33]. The possible molecular mechanism II of O•− 2 scavenging by Mn (Me2DO2A) may consist of a catalytic cycle involving first oxidation of MnII to MnIII by O•− 2 and then reduction of II the resulting MnIII complex by another O•− 2 to form the initial Mn II compound [16,17]. Interestingly, Mn in aqueous solution is oxidized via a single-electron process with redox potential higher than that of natural Mn-SODs. This suggests that, in the cellular environment, MnIII(Me2DO2A) reduction may occur upon reaction with O•− 2 as well as other endogenous reductants. These assumptions were confirmed observing that the cytoprotective effects of MnII(Me2DO2A) from CS-induced oxidative stress were completely abolished when

MnII was replaced with inactive ZnII. Taken together, the physical– chemical features of MnII(Me2DO2A) may account for its efficient II O•− 2 scavenging activity. Of note, Mn pentaazamacrocyclic complexes have been shown to react with ONOO−, albeit at lower rates than II with O•− 2 [34]. Whether Mn (Me2DO2A) could also remove harmful ONOO−, thereby increasing its antioxidant properties, remains to be elucidated. In conclusion, our study suggests that MnII(Me2DO2A) is a promising member of a new class of SOD-mimetic scavenging compounds, exerting protective effects against O•− 2 -mediated cell injury caused by CS. Further studies on animal models of CS-induced diseases are definitely needed to validate the possible therapeutic or preventive potential of MnII(Me2DO2A) in CS toxicity.

Declaration of interest The authors declare that they have no conflict of interest.

Acknowledgments The authors gratefully acknowledge Dr. Eng. Moreno Naldoni, CEO of General Project Ltd., for kind gift of MnII(Me2DO2A). This work was supported by research funds from the University of Florence (NISTATEN11) issued to Silvia Nistri.

Please cite this article as: M. Becatti, et al., Protection of coronary endothelial cells from cigarette smoke-induced oxidative stress by a new MnIIcontaining polyamine-polycarboxilate scavenger of superoxide anion, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.008

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Fig. 8. Evaluation of oxidative stress by nitrotyrosine (NT) detection. Immunoreactive NT, an index of protein nitration, was visually enhanced upon CSE treatment and reduced by 10 μmol/l MnII(Me2DO2A), added together with CSE. Scale bar: 10 μm.

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Please cite this article as: M. Becatti, et al., Protection of coronary endothelial cells from cigarette smoke-induced oxidative stress by a new MnIIcontaining polyamine-polycarboxilate scavenger of superoxide anion, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.06.008