Cerium Oxide Nanoparticles Protect Endothelial Cellsfrom Apoptosis Induced by Oxidative Stress

Shizhu Chen & Yingjian Hou & Gong Cheng &

Cuimiao Zhang & Shuxiang Wang & Jinchao Zhang

Received: 6 February 2013 /Accepted: 17 April 2013 /Published online: 6 June 2013# Springer Science+Business Media New York 2013

Abstract Oxidative stress is well documented to cause injuryto endothelial cells (ECs), which in turn trigger cardiovasculardiseases. Previous studies revealed that cerium oxidenanoparticles (nanoceria) had antioxidant property, but theprotective effect of nanoceria on ROS injury to ECs andcardiovascular diseases has not been reported. In the currentstudy, we investigated the protective effect and underlyingmechanisms of nanoceria on oxidative injury to ECs. The cellviability, lactate dehydrogenase release, cellular uptake, intra-cellular localization and reactive oxygen species (ROS) levels,endocytosis mechanism, cell apoptosis, and mitochondrialmembrane potential were performed. The results indicatedthat nanoceria had no cytotoxicity on ECs but had the abilityto prevent injury by H2O2. Nanoceria could be uptaken intoECs through caveolae- and clathrin-mediated endocytosis anddistributed throughout the cytoplasma. The internalizednanoceria effectively attenuated ROS overproduction inducedby H2O2. Apoptosis was also alleviated greatly by nanoceriapretreatment. These results may be helpful for more rationalapplication of nanoceria in biomedical fields in the future.

Keywords Nanoceria . Antioxidant . Endothelial cells .

Apoptosis

AbbreviationsATPS AminopropyltriethoxysilaneDCFH-DA 2’,7’-Dichlorflourescein diacetateDLS Dynamic light scatteringDMEM Dulbecco’s modified Eagle’s mediumDMF DimethylformamideDMSO DimethylsulfoxideECs Endothelial cellsFITC Fluorescein isothiocyanateLDH Lactate dehydrogenaseMMP Mitochondrial membrane potentialMTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium bromideNBS Neonatal bovine serumOD Optical densityPBS Phosphate-buffered salinePI Propidium iodideRH123 Rhodamine 123ROS Reactive oxygen speciesSD Standard deviationSEM Scanning electron microscopeXRD X-ray diffractometer

Introduction

Vascular endothelial cells (ECs) line the interior surface ofblood vessels, forming an interface between circulatingblood and the rest of the vessel wall. Their injury by oxida-tive stress is a key event in cardiovascular diseases, such ashypertension and atherosclerosis, which are the leadingcauses of mortality and morbidity worldwide. Substantialstudies have shown that chronic or acute reactive oxygenspecies (ROS) overproduction plays the critical role in

S. Chen :G. Cheng : C. Zhang : S. Wang (*) : J. Zhang (*)College of Chemistry and Environmental Science,Chemical Biology Key Laboratory of Hebei Province,Hebei University, Baoding, People’s Republic of Chinae-mail: wsx@hbu.edu.cne-mail: jczhang6970@163.com

Y. HouCollege of Basic Medicine, Hebei University,Baoding, People’s Republic of China

Biol Trace Elem Res (2013) 154:156–166DOI 10.1007/s12011-013-9678-8

endothelial insult and subsequent initiation of vascular disor-ders [1]. Oxidative stress can be elicited by various risk factorsof cardiovascular diseases including smoking, hyperglycemia,angiotensin II, and so on [1, 2]. Excessive exposure to ROSimpairs cellular functions and even triggers apoptosisresulting in vascular barrier disruption. Apoptotic ECs aregenerally found in advanced atherosclerosis [3]. Given thefindings that ROS and subsequent apoptosis correlate closelywith the pathogenesis and progression of cardiovascular dis-eases, antioxidant therapy has been the focus of enormousclinical interest to prevent or ameliorate cardiovascular dis-eases. Antioxidant including probucol, statin, vitamin E, car-otenes, and melatonin are marketed and prescribedextensively. However, many of those antioxidants were foundto have limited success due to their short half-lives, dailydosing requirements, side-effects, etc. [4–6]. Nowadays,much attention has been focused on searching for more pow-erful medicines and therapeutic strategies.

Cerium oxide is a rare earth oxide material from the lan-thanide series of the periodic table. When the cerium oxidereached the nano scales, it can show many special properties.The small size of nanoceria make it have a high surface area tovolume ratio, and the reduction in particle size results in theformation of surface oxygen vacancies, which is endowedwith its ability to exist in either Ce3+ or Ce4+ state on theparticle surface [7]. The coexistence of Ce3+ and Ce4+ ionsproduces redox reactions; Ce3+ is oxidized to Ce4+, and H2O2

is formed when nanoceria reduce superoxide. Ce4+ can alsooxidize H2O2 to O2 and regenerate Ce3+ [8]. This is anadvantageous way of regenerating reduced nanoceria andeliminating the ROS. Now nanoceria have been consideredto possess catalase-mimetic activity [9] and superoxidedismutase-mimetic activity [10, 11]. In addition, several stud-ies suggested that nanoceria had radiation-protective effects[12, 13], anti-inflammatory properties [14], neuroprotectiveeffects [15, 16, and protective effects against ischemic stroke[17]. However, the protective effects of nanoceria on ROSinjury to ECs and cardiovascular diseases are not well-understood. In the current study, we investigated the role ofnanoceria for preventing oxidative stress and apoptosis ofECs. The results demonstrated that nanoceria could protectECs from apoptosis induced by oxidative stress. These resultswill be helpful for more rational application of nanoceria inbiomedical fields in the future.

Materials and Methods

Materials

The endothelial cell line EA.hy926 (human umbilical vein cellline) was gifted kindly by Dr. Yi Zhu at Peking University.Dulbecco’smodified Eagle’smedium (DMEM), neonatal bovine

serum (NBS), phosphate-buffered saline (PBS) and trypsin werepurchased from Gibco (Grand Island, NY, USA). Cerium (IV)oxide nanopowder, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI) and 2’,7’-dichlorflourescein diacetate (DCFH-DA), and rhodamine 123(RH123) were purchased from Sigma-Aldrich (St. Louis, MO,USA). Fluorescein isothiocyanate (FITC), nystatin, andaminopropyltriethoxysilane were obtained from Aladdin(Shanghai, CHN). Wortmannin was purchased from TokyoChemical Industry Co. Ltd. (Shanghai, CHN). Chlorpromazinewas purchased from Santa Cruz Biotechnology Inc. (Dallas,Texas, USA). Lactate dehydrogenase (LDH) release kit andannexin V-FITC apoptosis detection kit were purchased fromBeyotime Institute of Biotechnology (Jiangsu, CHN). All theother chemical reagents were of analytical grade.

Characterization of Nanoceria

The phase of the nanoceria was characterized by X-ray dif-fractometer (XRD) (Bruker, D8 Advance, GER) equippedwith a rotation anode Cu Kα radiation. Phase identificationwas performed using EVA 12.0 software. The morphology ofthe nanoceria was characterized by scanning electron micro-scope (SEM) (JEOL, JSM-7500 F, JPN).

Particle Size Measurement

The protocol described by Horie et al. was followed bysome modifications [18]. Four milligrams of nanoceria weresuspended in 100 μl NBS, sonicated for 10 min at 100 W toprevent agglomeration. Then 900 μl DMEM was added andthen vortexed for another 10 min as a stock solution. Afterthat, the stock solution was diluted into different concentra-tions. The particle size distribution was measured by grainsize analyzer (Beckman Coulter, Delsa Nano C, USA).

Fluorescent Labeling of Nanoceria

The protocol was described by Xia et al. [19]. First, 4 mg ofnanoceria was dispersed in 3 ml of anhydrous DMF; 0.5 μlAPTS mixed in 25 μl DMF was added to the above sus-pension solution. Then the suspension solution was sonicat-ed and stirred under nitrogen at room temperature for 20 h.The modified nanoceria were collected by centrifugation.After being washed two times with DMF, the modifiednanoceria were resuspended in 0.5 ml of DMF and mixedwith a solution of 1 mg of FITC and 0.5 ml of DMF. Thesuspension solution was stirred for 4 h, and the FITC-labeled nanoceria were collected by centrifugation. Afterbeing washed with DMF thoroughly, the particles weredried under vacuum to remove organic solvent and storedas dry powders.

Cerium Oxide Nanoparticles Protect Endothelial Cells 157

Cell Viability Assay

MTT assay was used to determine viability and proliferationof ECs upon treatment with nanoceria as described in detailpreviously [20]. In brief, ECs were seeded in 96-well tissueculture plates at the density of 2×104 cells per well andincubated overnight. Nanoceria solution was added at finalconcentrations of 5, 10, 20, and 40 μg/ml; 24, 48, and 72 hfurther incubationswere performed; 10μl ofMTT (5.0mg/ml)was added and incubated for another 4 h at 37 °C. Then, thesupernatant was removed and 100 μl DMSO was added, andthe OD value at 570 nm was recorded on a microplate spec-trophotometer (Molecular Devices, VersaMax, USA). The cellviability was presented as the fold increase over the controlgroup: [ODsample−ODblank]/[ODcontrol−ODblank].

For protective effect of nanoceria on oxidative injury toECs, 1 mM/l H2O2 was set as a positive control. Cells werecultured as described above. After the cells upon treatmentwith nanoceria for 24-, 48-, and 72-h, the H2O2 solution wasadded, and the cells were incubated for another 6 h. The cellviability was determined by MTT assays.

LDH Assay

LDH assay was used to determine the protective effect ofnanoceria on ROS injury to ECs [21]. The level of LDH in cellculture supernatants was performed as in detail previously [22].In brief, ECs were seeded in 96-well tissue culture plates at thedensity of 2×104 cells per well and cultured overnight. Aftertreatment, the plates were centrifuged at 300×g for 5 min; 60 μlcell-free supernatant was incubated with 30 μl LDH substratesolution for 30 min, and then the absorbance at 490 nm wasrecorded on a microplate spectrophotometer. The LDH releaseactivity was presented as the fold increase over the controlgroup: [ODsample−ODblank]/[ODTriton X-100−ODblank].

Cellular Uptake Assay

ECs were seeded in six-well plates at 3×105 cells/ml andcultured overnight. After the addition of nanoceria at differentconcentrations (final concentration 5, 10, 20, and 40 μg/ml),6-, 12-, 24-, or 48-h further incubations were performed. Thenthe cells were washed with PBS three times, digested bytrypsin, and resuspended in PBS. The uptake of nanoceriainto ECs was analyzed by FACScalibur flow cytometer(Becton Dickinson, FACSCalibur™, USA). The side scatterdata were analyzed using CELLQuest 6.0 software [23].

Endocytosis Assay

The endocytosis studies were performed with flow cytome-try [24]. In brief, ECs were seeded in six-well plates at3×105 cells/ml and cultured overnight. Cells were rinsed

three times with PBS and then treated with inhibitors ofendocytosis (35 μM chlorpromazine hydrochloride, 10 μMnystatin, or 400 nM wortmannin) for 30 min at 37 °C. Afterthat, nanoceria were added to the culture medium (finalconcentration 40 μg/ml), and the cells were incubated foranother 60 min. Alternatively, cells were incubated at 4 °Cwith nanoceria (temperature-related inhibition of endocyto-sis). Subsequently, the cells were washed with PBS threetimes, digested by trypsin, and resuspended in PBS. Theuptake of particles was analyzed by flow cytometry asdescribed above.

Intracellular Localization Assay

ECs were seeded on glass coverslips at 3×105 cells/ml andcultured overnight and then cells were treated with FITC-labeled nanoceria (40 μg/ml) for another 24 h. After that,Cells were rinsed three times with PBS, fixed with parafor-maldehyde for 15 min, and stained with DAPI (5 μg/mL inPBS) for 10 min. The intracellular localization was detectedby fluorescence microscope (Olympus, IX5, JPN) with anexcitation of 350 nm and an emission of 470 nm for DAPI,and with an excitation of 488 nm and an emission of 525 nmfor FITC [19].

Cell Apoptosis Assay

ECs were seeded in six-well plates at 3×105 cells/ml andcultured overnight. After treatment with vehicle, H2O2 ornanoceria+H2O2, cells were fixed and permeabilized with70 % ice-cold methanol for overnight and then washedtwice with PBS. DNA was stained by incubating cells in40 μg/ml PI and 100 μg/ml DNase-free RNase in PBS at37 °C for 20 min. Cells were analyzed by FACScalibur flowcytometer; 20,000 cells per sample were acquired in histo-grams, and data were analyzed with Modfit 3.2 software.Cells falling in the sub-G1 region were considered as apo-ptosis cells [25].

In addition, the apoptosis was further studied by mor-phology. In brief, cells were seeded in glass coverslips at 3×105 cells/ml and cultured overnight. After treatment, cellswere stained with DAPI for 10 min. The apoptosis wasdetected by fluorescence microscope with an excitation of350 nm and an emission of 470 nm for DAPI to observe thenuclei fragment.

Annexin V-FITC/PI Staining Assay

Annexin V, a calcium-dependent phospholipid-binding pro-tein with a high affinity for phosphatidylserine was used todetect apoptosis [26]. Briefly, after treatment as describedabove, cells were harvested and centrifuged at 1,000×g for5 min. Then the cells were washed in PBS and resuspended

158 Chen et al.

in binding buffer solution. Annexin V-FITC was addedat a final concentration of 100 ng/ml, and the cells wereincubated in the dark for 10 min, then washed in PBSand resuspended in 300 μl binding buffer solution. Tenmicroliters PI was added to each sample before flowcytometric analysis. Following gentle vortex, the samplewas analyzed using FACScalibur flow cytometer. Thelower left quadrant contained intact cells; lower rightquadrant, apoptotic; and in the upper right quadrant,necrotic or late-apoptotic cells.

Intracellular ROS Measurement

The DCFH-DA method was used to quantify ROS produc-tion [27]. Briefly, cells were seeded in six-well plates at 3×105 cells/ml and cultured overnight. Then, nanoceria wereadded to culture medium for 24 h, followed by ROS induc-tion with 1 mM H2O2 stimulation for 30 min. Cells wereincubated with DCFH-DA solution at a final concentrationof 6 μM in DMEM without serum for 30 min at 37 °C in5 % CO2 atmosphere. Afterward, cells were washed twicewith PBS, digested with trypsin solution, centrifuged at1,000×g for 5 min, and the supernatant was discarded. Thepellets were suspended with PBS, and 1×104 events wereregistered for each sample by using the FACScalibur flowcytometer; 10,000 cells per sample were acquired in histo-grams. ROS level was expressed as the ratio of mean inten-sity of sample/mean intensity of control cells. The data wereanalyzed by CELLQuest 6.0 software.

Mitochondrial Membrane Potential (MMP) Measurement

The mitochondrial membrane potential (MMP) was measuredusing the RH123 [28]. Briefly, ECs were seeded in six-wellplates at 3×105 cells/ml and cultured overnight. Then cellswere treated as described above. After that, cells were washedtwice with PBS, digested by trypsin solution, and stained byRH123 (10mM) for 30min at 37 °C in the dark. The uptake ofRH123 was analyzed using a FACSCalibur flow cytometerwith the excitation wavelength at 488 nm and the emissionwavelength at 525 nm. Ten thousand cells per sample wereacquired in histograms, and data were analyzed withCELLQuest 6.0 software.

The Oxidation State of Nanoceria Measurement

UV-visible spectroscopy was used to evaluate the change inthe oxidation state of nanoceria in the presence or absence ofthe oxidizing agent H2O2 [10]. Briefly, nanoceria weredissolved in PBS at the concentration of 40 μg/ml, and thenH2O2 were added at different concentrations (final concen-tration 0.2, 0.5, and 1.0 mM). UV-visible spectroscopy of

nanoceria was acquired using the spectrophotometer(SHIMADZU, UV3600, Japan) at room temperature.

Statistical Analysis

Data were collected from at least three separate experimentsand expressed as mean±SD. Statistical significance was ana-lyzed using Student’s ttest. To compare the different treatments,statistical significance was calculated by one-way analysis ofvariance followed by post hoc Tukey’s test. The pvalues lessthan 0.05 were considered to indicate statistical differences.

Results

Characterization of Nanoceria

Figure 1a shows the XRD pattern of the nanoceria. Thediffraction peaks of the nanoceria can be well indexed tothe cubic phase of cerium oxide [JCPDS No. 65–2975,space group: Fm-3 m (255)]. No additional peaks of otherphases can be detected, revealing the formation of puretetragonal phase of CeO2. Moreover, it can be seen thatthe diffraction peaks are sharp and strong, indicating thatthe nanoceria are well crystallized. The SEM image showsthat the nanoceria are composed of uniform and well-dispersed nanoparticles (Fig. 1b). The diameters of thenanoceria are about 20 nm.

Agglomeration is one of the most common phenomenawhen nanoparticles were suspended in solutions, especiallyin culture medium. As shown in Fig. 1c, there was a rise indegree of agglomeration with increase in nanoceria concen-trations. The result indicated that nanoceria were aggregatedwhen they were added to cell culture medium.

Effect of Nanoceria on Viability of ECs

MTT assay was employed to measure the metabolic activityof the mitochondria of cells based on the principle thatliving cells are capable of reducing light color tetrazoliumsalts into an intense color formazan derivative. As shown inFig. 2a, nanoceria have no cytotoxicity on ECs at all testedconcentrations.

In order to validate the ability of nanoceria to protect ECsfrom oxidative damage, H2O2 were added into ECs afterpreincubation with nanoceria. The results showed thatnanoceria dose-dependently decreased the extent of damageinduced by H2O2 (Fig. 2b) (p<0.001).

Effect of Nanoceria on the Level of LDH

LDH is an oxidoreductase which catalyzes the intercon-version of lactate and pyruvate. Cytotoxic compounds

Cerium Oxide Nanoparticles Protect Endothelial Cells 159

often impair cell membrane integrity by inducing cellapoptosis or necrosis. Figure 2c showed that nanoceriacould decrease the level of LDH induced by H2O2 in adose-dependent manner (p<0.001).

Uptake of Nanoceria into ECs

To ascertain whether nanoceria were uptaken by ECs, theuptake of nanoceria was measured by flow cytometer [23].Figure 3a and b showed that nanoceria were uptaken by ECswith obvious dose- or and time-dependent tendency (p<0.001).

Endocytosis Mechanisms of Nanoceria

Three selective inhibitors corresponding to three main path-ways of endocytosis were used to study the endocytosismechanisms of nanoceria. As shown in Fig. 3c, the intensityof side scatter signal had no significant decrease when ECswere treated with a macropinocytosis inhibitor (wortmannin),but the intensity of side scatter signal was significantly de-creased in the presence of a caveolae inhibitor (nystatin) and aclathrin inhibitor(chlorpromazine) (p<0.001). This suggeststhat uptake of nanoceria into ECs is mainly mediated byendocytosis involving caveolae- and clathrin-pathways.

Intracellular Localization of Nanoceria

The intracellular localization of nanoceria in ECs was stud-ied by fluorescence microscope. As shown in Fig. 3d–f, theresults indicated that nanoceria were evenly distributed inthe cytoplasma of ECs, but they cannot enter into the nucleiof ECs. This result is consistent with a previous report aboutintracellular localization of nanoceria in keratinocytes [29].

Nanoceria Reduce the Intracellular ROS Level

ROS is one of crucial detrimental factors leading to EC injuryand cardiovascular diseases. H2O2 was used to induce ROSgeneration in ECs, and DCF signal was analyzed by flowcytometry to detect ROS changes. As shown in Fig. 4, the resultsrevealed that nanoceria could reduce intracellular free radicalsinduced by H2O2 in a dose-dependent manner (p<0.01).

Anti-apoptotic Activity of Nanoceria

Apoptosis is the process of programmed cell death. ROSoverload can cause EC apoptosis, which in turn elicitspathogenesis of several cardiovascular diseases [30].Hence, apoptosis is considered as a main aspect of oxidativeinjury to ECs. As shown in Fig. 5a, the sub-G1 cellsdenoting the proportion of apoptotic cells induced byH2O2 were significantly reduced with the increasing con-centrations of nanoceria (p<0.001). As shown in Fig. 5b,the injury induced by H2O2 were both with early and lateapoptosis. With the addition of nanoceria, the apoptosispercents were significantly reduced. The percents of lowerright and upper right quadrant at the concentrations of 20

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160 Chen et al.

and 40 μg/ml nanoceria were 25.15 % and 19.46 %, respec-tively. The results indicated that nanoceria could effectivelyreduce the apoptotic cells induced by H2O2. The morpho-logical results also exhibited that nanoceria reduced apopto-tic cells induced by H2O2 (Fig. 5c).

Nanoceria Elevated MMP

Mitochondrion is one of the most important compartmentsfor ROS generation in cells, and the mitochondrial disorderis a key step during apoptosis [31]. As shown in Fig. 6, the

MMP dropped dramatically after H2O2 stimulation, butnanoceria could effectively elevate MMP (p<0.001).

Quantification of the Ratio of Ce3+/Ce4+

The UV-visible spectroscopy was performed to evaluate theratio of Ce3+/Ce4+. Absorbance in the ranges of 230–260 nmand 330–380 nm corresponded to Ce3+ and Ce4+, respec-tively [32]. As shown in Fig. 7a and b, the absorbance in the230–260 nm increased after nanoceria treatment with H2O2,whereas the absorbance in the 330–380 nm decreased. The

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Fig. 2 Effect of nanoceria onviability of ECs. a The effect ofnanoceria (5, 10, 20, and 40 μg/ml) on viability of ECs for 24,48, and 72 h. b The protectiveeffect of nanoceria on damageinduced by H2O2 (1 mM/l). (n=6), *p<0.05, ***p<0.001versus H2O2 group. c Effect ofnanoceria on the LDH release.(n=4), ***p<0.001 versusH2O2 group

Cerium Oxide Nanoparticles Protect Endothelial Cells 161

results indicated that the ratio of Ce3+/Ce4+ on the surface ofnanoceria was augmented due to the oxidation-reductionreaction with H2O2.

Discussion

Cardiovascular diseases, including atherosclerosis, hyper-tension, heart failure, and so on, are the major causes ofdeath worldwide. Although the mortality rate has declinedin many industrialized countries over the last two decades,death from cardiovascular has increased at a fast rate indeveloping countries such as China [33]. Forming the inner

lining of blood vessels, endothelium homeostasis alterationsgovern the pathogenesis of cardiovascular diseases [34].Dysfunction and injury of ECs are considered as key char-acteristic pathophysiological features in cardiovascular dis-eases, which are often induced by ROS overproduction andoxidative stress [35]. In response to excessive ROS, endo-thelial functions were impaired, and even apoptosis wastriggered [36, 37]. Apoptosis ECs can be shed from bloodvessel walls, while intact endothelium is indispensable inthe homoestasis of cardiovascular system. Thus, oxidativeinjury and consequent apoptosis of ECs are believed to bemajor causes of cardiovascular diseases. In the present stud-y, MTT assay revealed that nanoceria had no cytotoxicity on

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Fig. 3 Uptake of nanoceriainto ECs. a ECs werepreincubated with differentconcentrations of nanoceria (5,10, 20, and 40 μg/ml) for 24 h.b ECs were preincubated with40 μg/ml nanoceria for 6, 12,24, and 48 h. c Uptake ofnanoceria into ECs in thepresence of differentendocytosis inhibitors (35 μm/L chlorpromazinehydrochloride, 10 Μm/Lnystatin, and 400 nm/Lwortmannin). (n=3), ***p<0.001 versus control. d–f Thefluorescence microscopeimages for intracellularlocalization of nanoceria inECs. Bar=20 μm. d The bluefluorescence of DAPI stainingnuclei. e The greenfluorescence of FITC-conjugated nanoceria. f Themerged image of d and e

162 Chen et al.

ECs at all tested concentrations. Moreover, nanoceria alsocould attenuate endothelial oxidative injury induced byH2O2. Flow cytometry analysis demonstrated the sub-G1

cells denoting the proportion of apoptotic cells induced byH2O2 were reduced with increasing concentrations ofnanoceria; these results were further confirmed by stainingearly and late apoptotic cells with annexin V-FITC and PI.In addition, nanoceria could reduce intracellular free radi-cals induced by H2O2 in a dose-dependent manner.

In previous studies, Clark and colleagues reportednanoceria had a capability of counteracting H2O2 challengeand apoptosis in breast fibrosarcoma cells [38]. Nanoceriawere also reported to exert antioxidant and anti-apoptoticeffects on cardiomyocytes, neuronal cells, macrophages,and mice with autoimmune encephalomyelitis [39, 40].However, the mechanisms underlying anti-apoptotic effectsof nanoceria are still understood poorly. Mitochondrion is akey subcellular compartment where apoptosis is associatedintimately with ROS generation and oxidative stress.Although Pourkhalili et al. found that nanoceria increasedATP/ADP ratio in mitochondrion, they did not provideevidence that nanoceria could alleviate apoptosis throughmitochondrial pathway [41]. Our results showed thatnanoceria have the ability to elevate MMP. This is a solidevidence that nanoceria can prevent the impairment of mi-tochondrial functions induced by proapoptotic factors, be-cause mitochondrial depolarization plays a crucial role incell apoptosis [42, 43]. Several studies documented thatROS induced release of Ca2+ from endoplasmic reticulumand mitochondria, which in turn activated mitochondrialCa2+ uptake [44, 45]. ROS also impairs mitochondrial func-tions, leading to mitochondrial depolarization which is

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Fig. 5 Nanoceria reduce ROS-induced apoptosis. a Fraction of ECswith sub-G1 DNA content (PI staining) after treatment with H2O2 inabsence or presence of nanoceria (n=3), ***p<0.001 versus H2O2

group. b The protective effect of nanoceria on H2O2-induced apoptosisof ECs with annexin V-FITC/PI staining. c Flourescence microscopyimages of DAPI-stained ECs, the white arrows indicate apoptotic cells;bar=20 μm

Cerium Oxide Nanoparticles Protect Endothelial Cells 163

closely associated with change in Ca2+ concentration [46].Thus, ROS, mitochondrial depolarization and Ca2+ are be-lieved to interplay during apoptosis [47]. Lansman et al.reported the cerium had an ability to block the Ca2+ channel[48], which provides a possible way to attenuation of mito-chondrial depolarization and apoptosis by nanoceria. Based

on the previous reports and our experimental results, we deducethat the nanoceria can scavenge ROS through a direct redoxreaction and prevent the Ca2+ influx, which play a major role forprotecting ECs from apoptosis induced by oxidative stress. Fourpossible pathways may be involved in uptake of nanoparticlesinto cells, including caveolae-, clathrin-mediated endocytosis,

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Fig. 7 UV-visible spectroscopyanalysis of H2O2-treatednanoceria. a UV-vis spectra of230 to 260 nm range ofnanoceria treated by H2O2 atvarious concentrations. b UV-vis spectra of 330 to 380 nmrange of nanoceria treated byH2O2 at various concentrations

Fig. 8 The mechanism ofnanoceria protecting ECs fromapoptosis induced by oxidativestress

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and macropinocytosis. In the previous study, Singh et al. havereported that nanoceria can be uptaken into cells and widelydistributed in multiple compartments of the cells [29]. Ourresults indicated that nanoceria can be also uptaken into cellsthrough caveolae- and clathrin-mediated endocytosis. Nanoceriawere distributed throughout the cytoplasma but not into nucleus.So we deduced that the protective effect of nanoceria on theoxidative injury might be attributed to the intracellular nanoceriareduction with ROS.

There are many oxygen vacancy sites on the surface of thenanoceria lattice. These oxygen vacancies are characterizedby Ce3+ in the center of the vacancy surrounded by adjacentCe4+ [10]. The coexistence of Ce3+ and Ce4+ ions producesredox reactions through which ROS are scavenged. It wasreported previously that nanoceria had the ability to mimicboth superoxide dismutase and catalase, depending on changein ratio of Ce3+/Ce4+ [49]. In our study, the absorbance in the230–260 nm increased after nanoceria treatment with H2O2,whereas the absorbance in the 330–380 nm decreased. Theresults indicated that the ratio of Ce3+/Ce4+ on the surface ofnanoceria was augmented due to the redox reaction. Multiplevalence surface redox states make nanoceria be capable ofscavenging ROS. One cerium oxide nanoparticle may offermany sites for reaction, whereas pharmacological agents orenzymes offer only one active site per molecule. Furthermore,another important characteristic of nanoceria is their auto-catalytic ability to regenerate free-radical scavenging surfacesites under a wide range of environmental conditions [8]. Thisfeature makes single-dosage treatments possible. In spite ofthis, there are some obstacles on the way to application anddevelopment of nanoceria. A major concern comes fromagglomeration, a phenomenon that occurs when nanoceriaare suspended in an aqueous culture medium and plasma, asit unavoidably occurs in biological studies. The agglomerationof nanoceria will hamper their mobility in body fluids andaffect absorption by human bodies.

On the basis of the above findings, a schematic model wasproposed to describe the protective effect and underlyingmechanisms of nanoceria on oxidative injury to ECs(Fig. 8). Nanoceria interact with the membrane of ECs andare uptaken through caveolae- and clathrin-mediated endocy-tosis. In cytoplasma, nanoceria scavenge ROS by mimickingthe activities of both superoxide dismutase and catalase. Asnanoceria diminish ROS overproduction, mitochondrial trans-membrane potential is elevated, and then cell apoptosis in-duced by oxidative stress was prevented effectively.

Conclusion

Our study showed that nanoceria were uptaken into ECs andprevented oxidative injury to ECs. Nanoceria were labeledwith FITC, and flow cytometry and fluorescence microscope

were performed. It was found that nanoceria were uptakeninto ECs by caveolae- and clathrin-mediated endocytosis anddistributed throughout the cytoplasma. After being uptakeninto ECs, nanoceria effectively counteract ROS induction byH2O2. Apoptosis, a major cellular injury in response to ROS,is also alleviated greatly, as evidenced by reduction in DNAfraction, reduction in the ratio of necrosis and apoptosis, andelevation ofMMP. The results revealed nanoceria had a strongability to prevent oxidative injury to ECs, which provided aclue that the nanoceria might be developed as a novel ap-proach to control cardiovascular diseases.

Acknowledgments This work was supported in part by Chinese Nat-ural Science Foundation project (no. 21271059 and no. 81200078) andResearch Fund for the Doctoral Program of Higher Education of China(no. 20111301110004).

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