Diosgenin Modulates Vascular Smooth Muscle Cell Functionby Regulating Cell Viability, Migration, and CalciumHomeostasis□S

Mitra Esfandiarei, Julia T. N. Lam, Sahar Abdoli Yazdi, Amina Kariminia,Jorge Navarro Dorado, Boris Kuzeljevic, Harley T. Syyong, Kaiji Hu, andCornelis van BreemenDepartments of Anesthesiology, Pharmacology, and Therapeutics (M.E., J.T.N.L., S.A.Y., J.N.D., H.T.S., C.V.B.) and Pediatrics(A.K., K.H.), Child and Family Research Institute, University of British Columbia, Vancouver, Canada

Received July 20, 2010; accepted December 20, 2010

ABSTRACTIn this study, we compared the potencies of diosgenin, a plant-derived sapogenin structurally similar to estrogen and proges-terone, on vascular smooth muscle functions ranging fromcontraction and migration to apoptosis. The effects of diosge-nin on vascular smooth muscle cell viability and migration weremeasured using a primary mouse aortic smooth muscle cellculture. The effects of diosgenin on smooth muscle cell con-traction and calcium signaling were investigated in the isolatedmouse aorta using wire myography and confocal microscopy,respectively. Here, we report that in cultured cells diosgenin(�25 �M) induces apoptosis as measured by the number ofannexin V-positive cells and caspase-3 cleavage, while de-creasing cell viability as indicated by protein kinase B/Akt phos-phorylation. In addition, diosgenin blocks smooth muscle cell

migration in a transwell Boyden chamber in response to serumtreatment and response to injury in a cell culture system. Dios-genin (�25 �M) also significantly blocks receptor-mediatedcalcium signals and smooth muscle contraction in the isolatedaorta. There is no difference in the inhibitory effects of diosge-nin on vascular smooth muscle contraction between the endo-thelium-intact and endothelium-denuded aortic segments, in-dicating that they are caused by altered smooth muscle activity.Our findings suggest that over the concentration range of 10 to15 �M diosgenin may provide overall beneficial effects ondiseased vascular smooth muscle cells by blocking migrationand contraction without any significant cytopathic effects, im-plying a potential therapeutic value for diosgenin in vasculardisorders.

IntroductionVascular smooth muscle cells (SMCs) are key structural

and functional components of vessel walls controlling andmodulating peripheral resistance and regional blood flow.During the progression and development of occlusive vascu-lar diseases such as atherosclerosis and restenosis SMCsundergo phenotypic changes from a stable quiescent contrac-tile state to a more invasive, synthetic, and proliferative state

(Okamoto et al., 1992; Ross, 1999). Thickening and harden-ing of blood vessels is a complex and multifactorial eventassociated mainly with changes in lipid homeostasis anddeposition, medial SMC proliferation, and/or migration inresponse to secreted growth factors and inflammatory cyto-kines, alterations in calcium homeostasis and smooth musclecontraction, and cell apoptosis (Doran et al., 2008; Spragueand Khalil, 2009). Therefore, targeting these smooth muscleaberrations may prove beneficial in the treatment of vasculardisease.

Diosgenin (3�-hydroxy-5-spirostene), a plant-derived sapo-genin structurally similar to estrogen and progesterone, isthe precursor for the industrial large-scale production ofprogesterone and norethisterone (Marker et al., 1940; Au etal., 2004; Dias et al., 2007). Diosgenin can be extracted froma variety of plants such as wild yam root (Dioscorea villosa),fenugreek (Trigonella foenum graecum), and soybean (Gly-

This work was supported by the Canadian Institutes of Health Research[Grant 20R92112]. M.E. was supported by fellowships from the Michael SmithFoundation for Health Research and the Heart and Stroke Foundation ofCanada/Astrazeneca Canada. H.T.S. was supported by a studentship from theMichael Smith Foundation for Health Research.

Article, publication date, and citation information can be found athttp://jpet.aspetjournals.org.

doi:10.1124/jpet.110.172684.□S The online version of this article (available at http://jpet.aspetjournals.org)

contains supplemental material.

ABBREVIATIONS: SMC, smooth muscle cell; Nif, nifedipine; PE, phenylephrine; SR, sarcoplasmic reticulum; GFP, green fluorescent protein; PSS,physiological salt solution; DMSO, dimethyl sulfoxide; EtOH, ethanol; PBS, phosphate-buffered saline; PI, propidium iodide; VGCC, voltage-gatedcalcium channels; AM, acetoxymethyl ester.

0022-3565/11/3363-925–939$20.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 336, No. 3Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics 172684/3671154JPET 336:925–939, 2011 Printed in U.S.A.

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cine max). Extracts from these plants and a few others havebeen traditionally used to treat hypercholesterolemia (Val-ette et al., 1984; Sauvaire et al., 1991), diabetes (Sharma etal., 1990; Gupta et al., 2001; McAnuff et al., 2005), andgastrointestinal complaints (Pandian et al., 2002; Kaviar-asan et al., 2006). Studies have also suggested a lower inci-dence for coronary artery diseases and disorders related toestrogen deficiencies in humans who have a high consump-tion of diet rich in phytoestrogens (e.g., genistein, daidzein,and diosgenin) (Adlercreutz et al., 1992, 1993; Hertog et al.,1995; Figtree et al., 2000; Au et al., 2004).

In previous years, the effects of diosgenin on cellulargrowth and viability have been tested in different types ofcells, but resulted in controversial findings. In human my-eloid KBM-5 cells and the 1547 osteosarcoma cell line, dios-genin induced apoptosis and cell cycle arrest (Moalic et al.,2001; Leger et al., 2004; Yen et al., 2005; Shishodia andAggarwal, 2006), whereas in MC3T3-E1 mouse clonal osteo-genic cells it increased cell proliferation and angiogenic ac-tivity through the up-regulation of vascular endothelialgrowth factor production (Yen et al., 2005). Thus, it seemsthat the effects of diosgenin on cellular homeostasis andfunction depend on the cell type as well as the dose.

Unfortunately, our understanding of how diosgenin exertsits protective effects in the vasculature is very limited. Onestudy reported that diosgenin could cause endothelium-inde-pendent coronary artery relaxation in precontracted porcineleft anterior descending coronary artery via activation ofiberiotoxin-sensitive Ca2�-activated K� channels (Au et al.,2004). Later, it was suggested that diosgenin-induced relax-ation of precontracted rat superior mesenteric artery is me-diated by endothelium-dependent mechanisms that involvenitric oxide and cyclooxygenase derivatives (Dias et al.,2007). However, the direct effects of diosgenin on vascularsmooth muscle function such as proliferation, migration, con-traction, and viability have yet to be determined.

In this study, we sought to assess the potential therapeuticvalue of diosgenin in vascular disease by investigating therole of diosgenin in regulating vascular SMC viability, mi-gration, and apoptosis, all of which contribute to the initia-tion and progression of occlusive and nonocclusive vasculardisease. We also investigated the effects of diosgenin on ag-onist-induced calcium release in vascular SMCs and recep-tor-mediated vasoconstriction and force development inmouse thoracic aorta.

Materials and MethodsExperimental Animal and Tissue Preparation. All animal

experiments and procedures were conducted in accordance with theguidelines of the Animal Ethics Board at the University of BritishColumbia. Male C57BL/6 mice were obtained from The JacksonLaboratory (Bar Harbor, ME) and housed in the institutional animalfacility (University of British Columbia, Child and Family ResearchInstitute) under standard animal room conditions (12-h light/darkcycle at 25°C). Seven-month-old male mice were anesthetized with amixture of ketamine hydrochloride (80 mg/kg) and xylazine hydro-chloride (12 mg/kg) intraperitoneally. The thoracic aorta was iso-lated, gently cleaned of connective tissue and blood, and cut into2-mm segments (five or six aortic rings). Experiments were per-formed using endothelium-intact aortic rings unless otherwiseindicated.

Cell Culture and Transient Transfection. Primary mouse aor-tic SMCs were isolated as described previously (Srinivasan et al.,

2009). In brief, aortic segments were isolated, cleaned of excessadvential tissue, and digested using collagenase II (0.5 mg/ml). Iso-lated cells were pelleted, resuspended, and grown in Dulbecco’s mod-ified Eagle’s medium. Subconfluent primary mouse aortic SMCswere grown in Dulbecco’s modified Eagle’s medium supplementedwith 10% heat-inactivated newborn calf serum (Invitrogen, Burling-ton, ON, Canada) at 37°C in a humidified incubator in 5% CO2.Penicillin G (100 �g/ml) and streptomycin (100 �g/ml) (Invitrogen)were added to all culture media. These cells maintain the phenotypeand characteristics of vascular smooth muscle cells up to passage 15as confirmed by positive staining for smooth muscle cell �-actin (seeSupplemental Fig. S1) and preservation of contractility (data notshown). To assure the consistency of results, passage 8–13 of SMCswas used for all experiments.

Adenoviral constructs encoding the constitutively active (Ad-Ca-Akt) and wild-type (Ad-Wt-Akt) forms of murine Akt tagged with thehemagglutinin epitope and control green fluorescent protein (GFP)(Ad-GFP), kindly provided by Dr. Kenneth Walsh (Whitaker Cardio-vascular Institute, Boston University School of Medicine, Boston,MA) and Dr. Jason Dyck (University of Alberta, Edmonton, Canada),were described previously (Esfandiarei et al., 2007). Smooth musclecells were infected with adenoviral constructs at a multiplicity ofinfection of 100. After overnight incubation at 37°C, cells were re-plenished with fresh medium. Fluorescence and bright-field micros-copy were used to assess transfection efficiency and cellular mor-phology at 48 h after transfection. Western blot analysis wasperformed to confirm the overexpression of phosphorylated Akt-Ser in SMC culture compared with control (nontransfected) andAD-GFP-transfected cells to assure the efficiency of the transfec-tion protocol.

Reagents and Antibodies. HEPES-PSS containing 140 mMNaCl, 10 mM glucose, 5 mM KCl, 5 mM HEPES, 1.5 mMCaCl2, and1 mM MgCl2, pH 7.4 was used for all calcium measurements andconfocal microscopy. High-K� PSS (60 mM extracellular K�) wasidentical in composition to normal PSS with the exception of 85 mMNaCl and 60 mM KCl. Zero-Ca2� PSS was prepared in the same wayas normal PSS, but CaCl2 was replaced with 1 mM EGTA. UTP,cyclopiazonic acid, nifedipine (Nif), R-(�)-phenylephrine hydrochlo-ride, and diosgenin were obtained from Sigma-Aldrich (Oakville, ON,Canada). Stock solutions of nifedipine and diosgenin were preparedin dimethyl sulfoxide (DMSO) and ethanol (EtOH), respectively. Forall experiments with diosgenin and/or nifedipine, vehicle-treated (0�M) groups were incubated with 1 �l of EtOH and/or DMSO, respec-tively (the maximum volume of solvents used with the highest con-centration of drugs). Further dilutions of reagents were made inzero-Ca2� PSS buffer. Fluo-4AM was purchased from Invitrogen. Alldrugs and molecular target nomenclature conform to the BritishJournal of Pharmacology’s Guide to Receptors and Channels (Alex-ander et al., 2008). All primary antibodies used in this study werepurchased from Cell Signaling Technology (Danvers, MA). Second-ary antibodies conjugated with horseradish peroxidase or AlexaFluor 488 were purchased from Santa Cruz Biotechnology Inc.(Santa Cruz, CA).

Western Blot Analysis. Cells either untreated or treated withvarious experimental reagents were washed twice with ice-cold PBSand kept on ice for 15 min in lysis buffer containing 50 mM pyro-phosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 10mM HEPES, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 100 �MNa3VO4, 0.1% Triton X-100, and 10 �g/ml leupeptin. Cell lysateswere collected by scraping, and protein concentration was deter-mined using Bradford (1976) assay. Extracted protein (40–80 �g)was fractionated by electrophoresis in 7 to 9% SDS-polyacrylamidegels, transferred to nitrocellulose membranes, and blocked with PBScontaining 0.1% Tween 20 and 5% nonfat dry milk for 1 h. Afterward,the membrane was incubated with specific primary antibody over-night at 4°C, followed by secondary antibody for 1 h at room tem-perature. Immunoblots were visualized with an enhanced chemilu-minescence detection system according to the protocol of the

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Fig. 1. Apoptotic effects of diosgenin on vascular SMC. A, diosgenin (�25 �M) increased the number of both annexin V-positive cells (indicator ofapoptosis) and PI-positive cells (indicator of necrosis) in a concentration-dependent manner with the EC50 values of 33 and 43 �M, respectively (n �3 experiments; mean � S.D.; � and #, P � 0.05). B, diogenin increases caspase-3 cleavage (an indicator of final stage of apoptosis) in cultured SMCsin a concentration-dependent manner. Total actin protein assures equal protein loading. Immunoblot represents one experiment of three independentexperiments. The bar graph presents the average band intensity measurement (n � 3; mean � S.D.; � and #, P � 0.05). C, at the concentration of 25�M diosgenin causes cytopathic effects and apoptosis in the SMC culture. Apoptotic bodies are marked by black arrows (original magnification, 200).Control groups represent nontreated cells, and the 0 �M group represents vehicle (EtOH)-treated cells.

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manufacturer (Pierce Biotechnology, Rockford, IL). Densitometryanalysis was performed by using the NIH ImageJ software (version1.27z). Density values for proteins were normalized to the level forcontrol groups (arbitrarily set to 1.0-fold).

Cell Viability Assay. Subconfluent SMCs were plated in a 24-well culture plate. After 24 h, cells were incubated overnight inserum-free to arrest cell growth and proliferation and assure syn-chronized growth after the addition of serum. The next day, cellswere treated with increasing concentrations of diosgenin or vehiclecontrol (EtOH) for 24 h in the presence of 10% serum. The CellTiter96 AQueous Nonradioactive Cell Viability Assay (MTS) was used tomeasure cell viability according to the manufacturer’s protocol (Pro-mega, Madison, WI). Cell viability was presented as percentagecompared with the control group (arbitrarily considered as 100% ofviability).

Measuring Cell Death by Cellomics. To determine the effect ofdiosgenin on SMC death, cell apoptosis and necrosis were measuredusing the ArrayScan HCS system (Cellomics; Thermo Fisher Scien-tific, Waltham, MA). In this system, numbers of annexin-V-positivecells (apoptotic cells) and propidium iodide (PI)-positive cells (ne-crotic cells) were determined. In brief, SMCs (5 105) were seededinto a 96-well culture plate and incubated overnight. Cells were then

treated with increasing concentrations of diosgenin or vehicle control(EtOH) for 24 h. As the positive control for apoptosis, one group ofcells was treated with the apoptosis-inducing drug staurosporine(200 nM) for 24 h. To measure the rate of cell death caused bynecrosis or apoptosis, cells were then subjected to PI (1 �g/ml) orphenylephrine (PE)-conjugated annexin V according to the manufac-turer’s protocol (BD Biosciences, Ontario, Canada). To measure cellnecrosis, approximately 30 min before the ending point of incubation,Hoechst 33342 (1 �g/ml) and propidium iodide (1 �g/ml) were addedto all wells. To measure apoptosis, cells were once washed withannexin V binding buffer and then incubated with Hoechst 33342 (1�g/ml) and phycoerythrin-annexin V (5 �l/well) for 10 min in thepresence of annexin V binding buffer. After the proper incubationperiod, cells were scanned live using the ArrayScan HCS (highcontent screening) system (Thermo Fisher Scientific). Ten focusfields in each well were scanned and analyzed, and the percentage ofapoptotic or necrotic cells was calculated based on the proportion ofphycoerythrin-annexin V- or propidium iodide-positive cells over thetotal cell population (Hoechst 33342-positive cells). Data are pre-sented as percentage of cell death where the level of cell death in thestaurosporine-treated group (the positive control) was arbitrarily setto 100%.

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Fig. 2. Effects of diosgenin on vascular SMC viability andAkt phosphorylation. A, diosgenin (�25 �M) decreasedserum-induced cell proliferation (an index of cell viability)in a concentration-dependent manner with an IC50 value of�25.6 �M. Data present three independent experiments(n � 3 experiments; mean � S.D.; �, P � 0.05). B, diosgenindecreased Akt phosphorylation (an indicator of cell viabil-ity) with no effect on total Akt expression in culturedSMCs. Immunoblot represents one experiment of threeindependent experiments. The bar graph presents the av-erage band intensity measurement (n � 3; mean � S.D.; �,P � 0.05). Control groups represent nontreated cells, andthe 0 �M group represents vehicle (EtOH)-treated cells.

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Cell Migration Assay. Cell migration was measured using QCMTranswell Colorimetric Cell Migration Assay according to the man-ufacturer’s protocol (Millipore Bioscience Research Reagents, Te-mecula, CA). In brief, 105 serum-starved SMCs (to induce quiescenceand synchronize cell proliferation) were loaded onto the upper well ofthe chamber in the presence or absence of agonist (diosgenin) andvehicle control (EtOH), whereas lower wells were filled with serum-containing culture medium with various concentrations of the ago-nist (diosgenin). After 12-h incubation, nonmigrating cells on theupper side of membranes were removed by wiping and rinsing, andmigrated cells on the lower side of membranes were counted usingcolorimetric assay. Data are represented as percentage in cell migra-tion where migratory rates for control groups were arbitrarily set to100%.

Wound Healing Assay. Subconfluent SMCs were grown on glasscoverslips. After overnight serum starvation (to induce quiescenceand synchronize cell proliferation), cell cultures were scratched witha sterile pipette tip to form a wound, washed with prewarmed sterilePBS, and incubated with medium (containing 10% serum). Cellswere then treated with the vehicle (EtOH) or increasing concentra-tions of diosgenin (10, 25, and 50 �M) or left untreated (control). At24 h after injury, cells were fixed and subjected to imaging using a

Nikon (Tokyo, Japan) inverted microscope (400 magnification) andSpot (Sterling Heights, MI) digital camera. The wound size wasmeasured using Image Pro Plus imaging software. The migration ofsmooth muscle cells to the site of injury was measured as percentageof wound closure using the following formula: percentage of woundclosure � [(L0h � L24h)/L0h] 100%, where L0h is the distancebetween two edges of the wound measured at 0 h after injury (im-mediately after scratching), and L24h is the distance at 24 h afterinjury. Data are represented as percentage of wound closure wherethe wound size in control (nontreated) group at 24 h after injury wasarbitrarily set to 100%.

Cytoplasmic Measurement of Calcium. To measure cytoplas-mic calcium in the isolated aorta, 2-mm segments of endothelium-intact aortic rings were gently inverted and then loaded with Fluo-4AM (5 �M with 5 �M Pluronic F-127) for 2 h at 37°C. Later, loadedaortic rings were isometrically mounted, followed by 15-min washoutin HEPES-buffered physiological saline solution. For in vitro mea-surement of calcium signals, subconfluent SMCs were grown onMatrigel-coated (BD Sciences, Ontario, Canada) 35-mm glass-bot-tom culture dishes (MatTek, Ashland, MA) 48 h before each experi-ment. Cells were then loaded with Fluo-4AM for 1 h at 37°C, followed

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Fig. 3. Reversal of diosgenin-induced ap-optosis by overexpression of a constitu-tively active form of Akt. A, SMCs weretransfected with the adenoviral vector ex-pressing the constitutively active form ofAkt for 48 h. As shown, transfected SMCsexpress a high level of Akt phosphoryla-tion (2.5-fold increase) as measured byWestern blot analysis. �-Actin protein ex-pression confirmed equal protein expres-sion. The blot represents one of three in-dependent experiments. The bar graph isthe average band intensity of three inde-pendent experiments (n � 3; mean �S.D.; �, P � 0.05). Control group repre-sents nontransfected SMCs. B, overex-pression of an active form of Akt rescuesSMCs from the apoptotic effects of diosge-nin. Diosgenin-induced caspase-3 cleav-age was blocked by overexpression of anactivated form of Akt. Control group rep-resents nontransfected SMCs. C, in thepresence of serum (a potent activator ofAkt), both wild-type and active forms ofAkt rescue SMCs from the cytopathic ef-fects of diosgenin. In the absence of serumonly the active form of Akt improve cellviability in diosgenin-treated SMCs. Thebar graph represents three independentexperiments (n � 3; mean � S.D. (Left, �,��, P � 0.05. Right, �, P � 0.2086; ��, P �0.05). Groups marked as 0 �M representvehicle (EtOH)-treated cells.

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by a 15- to 20-min wash in HEPES-buffered physiological salinesolution.

To investigate the effects of diosgenin on calcium release, culturedSMCs or isolated aortic segments were pretreated with diosgenin orvehicle (EtOH) for 30 min and then stimulated with various phar-macological agents. Images were acquired on an upright Olympus(Tokyo, Japan) BX50WI microscope with a 60 water-dipping objec-tive (numerical aperture, 0.9) and equipped with an Ultraview Con-focal imaging system (PerkinElmer Life and Analytical Sciences,Waltham, MA). All parameters (laser intensity, gain, etc.) weremaintained constant during the experiment. The tissue was illumi-nated using an argon-krypton laser (488 nm), and a high-gain pho-tomultiplier tube collected the emission (505–550 nm). Representa-tive fluorescence traces reflect the averaged fluorescence signalsfrom 6 to 10 SMCs in each region of interest. The measured changesin Fluo-4 fluorescence level are proportional to the relative changesin [Ca2�]cyto. The confocal images were analyzed off-line with Ultra-view 4.0 Software (PerkinElmer Life and Analytical Sciences). Flu-orescence traces were extracted from the movies to exclude nuclearregions, and traces were normalized to initial fluorescence values.

Measurement of Isometric Force. Aortic segments weremounted isometrically in a small vessel wire myograph (DanishMyotechnology A/S, Aarhus, Denmark) for measuring generatedforce. Krebs’ solution containing 130 mM NaCl, 4 mM KCl, 1.2 mMMgSO4, 4 mM NaHCO3, 1.5 mM CaCl2, 10 mM HEPES, 1.18 mMKH2PO4, 6 mM glucose, and 0.03 mM EDTA, pH 7.4, was used for allisometric contraction studies. The chambers and bath solutions werekept at 37°C and bubbled continuously with 95% O2-5% CO2 inKrebs’ solution. Optimal tension (5 mN) was preliminarily deter-mined as described previously (Chung et al., 2008; Syyong et al.,2009). Aortic segments were stretched to the optimal tension for 1 hand then stimulated twice with 60 mM KCl to ensure tissue viabilityand normal contractile response. To study the effects of diosgenin oncontraction, aortic segments were incubated with increasing concen-

trations of diosgenin (2.5, 10, and 25 �M) or vehicle (ethanol) for 30min before stimulation with PE or 60 mM KCl.

For some experiments and to block voltage-gated calcium chan-nels, SMC cultures or aorta rings were pretreated with 1 �M Nif orvehicle (DMSO) for 10 min before PE or 60 mM KCl stimulation. Theconcentration-response curve of PE-induced contraction was pre-pared and the negative logarithm (pD2) of the concentration of PEgiving half-maximum response (EC50) was assessed by linear inter-polation on the semilogarithm concentration-response curve [pD2 ��log (EC50)].

To determine whether diosgenin can modulate smooth musclecontraction, endothelium-intact aortic rings were pretreated withincreasing concentrations of diosgenin (or the vehicle) for 30 beforestimulation with PE. For some experiments, the endothelial layerwas removed by gently rubbing the intimal layer of aorta with thewire before mounting the segments in the myograph chamber. Aorticsegments were then treated with 25 �M diosgenin for 30 min beforePE treatment. This procedure abolished aortic relaxation in responseto acetylcholine (data not shown).

To calculate the percentage of inhibition of contraction by diosge-nin, the force created by 10 �M PE in the presence of 0 �M diosgenin(vehicle only, EtOH) was arbitrarily set as 100% of contraction forcontrol group. Percentage of contraction for diosgenin-treated groupswas calculated in comparison with the vehicle control group. Thepercentage of inhibition was calculated as percentage of inhibition �percentage contraction for vehicle-treated control group � percent-age contraction for diosgenin-treated group.

Statistical Analysis. Statistical analysis and preparation ofconcentration-response curves were performed using Prism 4.0software (GraphPad Software, Inc., San Diego, CA). Data wereanalyzed using one-way analysis of variance. We also used repeat-ed-measures analysis of variance where those different doses weretreated as repeated measures (within factor) and different repli-cates were treated as “between” factor. We expected “within” to be

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Fig. 4. Effects of diosgenin on vascular SMCmigration. A, diosgenin (�10 �M) blocked SMCmigration into the site of injury and wound clo-sure in a concentration-dependent manner withthe IC50 value of 15.3 �M (n � 6 experiments;mean � S.D.; � and ��, P � 0.05). B, in theBoyden chamber migration assay diosgenin (�10�M) caused a marked decrease in serum-stimu-lated migration in a concentration-dependentmanner with the IC50 value of 10.3 �M (n � 3experiments; mean � S.D.; �, P � 0.05). Graphsrepresent the average of three independent ex-periments. Control groups represent nontreatedcells, and the 0 �M group represents vehicle(EtOH)-treated cells.

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significant and both between-group factors (different replicates)not to be significant. Our analyses showed that although therewas significant difference between different doses (within groups),there was no difference between replicates (between groups).Therefore, appropriate post hoc tests (Bonferroni or Dunnet’s T3)were used depending on result of Levine’s test for homogeneity ofvariances (i.e., assumption of equality of variances). Values shownare the mean � S.D., where a value of P � 0.05 was consideredstatistically significant. The exact number of repetition (n) foreach experiment is specified under Results. The number for cellcultures, aortic rings, or animals is also noted when different fromthe number of experiments. For all experiments, the groupsmarked as “control” represent untreated cells or tissue, whereasthe groups marked as “0 �M” represent cells or tissue treated withonly vehicle (DMSO or EtOH).

Results

Apoptotic Effects of Diosgenin on Mouse AorticSMCs. We investigated the apoptotic and necrotic effects ofdiosgenin in cultured SMCs. Diosgenin (�25 �M) increasedthe number of both the annexin V-positive (indicator of apop-tosis) and PI-positive (indicator of necrosis) cells in a concen-tration-dependent manner. At the concentration of 25 �M,diosgenin caused apoptosis in 17% of the SMC population. Asthe concentration of diosgenin increased to 50 �M, apoptosiswas detected in 60% of the SMC population (Fig. 1A; n � 3experiments of three replicates; �, P � 0.05). Western blotanalysis also confirmed the cleavage of caspase-3 protein indiosgenin-treated SMCs in a concentration-dependent man-

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Fig. 5. Effects of diosgenin on �-adrenergic receptor-medi-ated contraction in the isolated mouse aorta. A, at theconcentration of 10 �M PE induces submaximal contractionin the aorta (n � 5 mice; P � 0.05). B, in the presence ofextracellular calcium diosgenin causes a maximum of 50%inhibition of the PE-induced contraction in the aorta. Uponremoval of extracellular calcium diosgenin completelyblocks PE-induced contraction (n � 5 mice; P � 0.05).C, removal of extracellular calcium also decreases PE-in-duced force development in mouse aortic rings by 50% (n �5 mice; �, P � 0.05).

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ner (Fig. 1B; n � 3 experiments; �, P � 0.05). Morphologicalstudy using phase-contrast microscopy also confirmed thatthe increase in caspase-3 cleavage was associated with theappearance of cytotoxic effects and apoptotic bodies in culturedSMCs (Fig. 1C).

Effects of Diosgenin on Vascular SMC Viabilityand Akt Phosphorylation. Diosgenin (�25 �M) de-creased SMC viability in a concentration-dependent man-ner (Fig. 2A; n � 3 experiments of three replicates; �, P �

0.05). At the concentration of 25 �M, diosgenin caused asignificant decrease (40%) in SMC viability. It is well es-tablished that Akt (protein kinase B) phosphorylation is akey event during cell growth and viability. To understandthe cellular mechanism by which diosgenin decreasesSMCs viability, we assessed the expression of the phos-phorylated form of Akt (phosphor-Akt-Ser473) after dios-genin treatment. Our results showed that diosgenin de-creased Akt phosphorylation in SMCs without affecting

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Fig. 6. Effects of diosgenin on Ca2� influx via L-type calcium channels in the isolated mouse aorta. A, Nif (1 �M) significantly inhibits PE-inducedforce development in isolated aortic rings compared with the control and DMSO-treated groups (n � 6 mice; mean � S.D.; �, P � 0.05). B, diosgenindoes not block aortic contraction in response to high K� buffer, indicating that diosgenin has no inhibitory effects on L-type calcium channels in themouse aorta (n � 7 mice; mean � S.D.; �, P � 0.05; #, P � 0.6515).

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the expression of total Akt protein in SMCs (Fig. 2B; n �3 experiments; �, P � 0.05).

To establish a causal relationship between decreasedAkt phosphorylation and increased caspase-3 cleavage andapoptosis in response to diosgenin treatment, SMCs weretransfected with the adenoviral vectors carrying either aconstitutively active form of Akt (Ad-Ca-Akt) or a wild-type of Akt (Ad-Wt-Akt), or the GFP (Ad-GFP) controlconstruct before diosgenin treatment. To assure the effi-ciency of transfection, Akt phosphorylation was measuredin transfected SMCs. As shown in Fig. 3A, phosphorylationof Akt-Ser was significantly increased in SMCs transfectedwith the constitutively active form of Akt compared withcells transfected with the adenoviral vector expressing theGFP protein (n � 3 experiments; �, P � 0.05). Overexpres-sion of an active form of Akt blocked diosgenin-inducedcaspase-3 cleavage (Fig. 3B; n � 3 experiments; �, P �0.05). In the presence of serum (a potent inducer of Aktphosphorylation), both the wild-type and constitutivelyactive forms of Akt could reverse the cytopathic effectscaused by diosgenin. However, in the absence of serumstimulation, only the active form of Akt could rescue thecells and increase SMC viability (Fig. 3C; n � 3 experi-ments of three replicates). (Fig. 3C, left, �, ��, P � 0.05;right, �, P � 0.2086; ��, P � 0.05).

Effects of Diosgenin on Vascular SMC Migration. Di-osgenin (�10 �M) blocked SMC migration in a concentra-tion-dependent manner in both wound healing (Fig. 4A;n � 6 experiments; �, P � 0.05) and Transwell Boydenchamber assays (Fig. 4B; n � 3 experiments of three rep-licates; �, P � 0.05). The low concentration of diosgenin (10�M) caused a considerable decrease (40%) in SMC migra-tion, whereas at a higher concentration (50 �M) diosgeninnearly completely blocked cell migration. It is of impor-tance that at a low concentration (10 �M) diosgenin pro-vided a significant inhibition in cell migration (Fig. 4B)with no noticeable cytotoxic effects on SMCs (Fig. 1A).

Effects of Diosgenin on �-Adrenergic Receptor-Me-diated Contraction in the Isolated Mouse Aorta. Toinduce contraction, aortic rings were stimulated with 10�M PE (with the threshold effective concentration of 0.1�M), a concentration that induces 90% of maximal forcedevelopment (Fig. 5A; n � 5 mice). Diosgenin inhibited thecontractile response to PE (10 �M) in both the presenceand absence of extracellular calcium in a concentration-dependent manner with IC50 values of 25 �M (pD2 � �4.5)and 10 �M (pD2 � �5.0), respectively (Fig. 5B; n � 5 mice).It is noteworthy that, in the absence of diosgenin, removalof extracellular calcium 1 min before PE application re-duced force development by approximately one-half (Fig.5C; n � 5 mice; �, P � 0.05). It is noteworthy that ourtime-response experiment with diosgenin has shown that aminimum of 12-h incubation is required to observe theearly indications of apoptotic and necrotic responses incultured SMCs (data not shown). The same observationwas reported by other groups using different cell lines(Moalic et al., 2001; Leger et al., 2004; Yen et al., 2005;Shishodia and Aggarwal, 2006). Therefore, we do not ex-pect the involvement of apoptosis in diosgenin-inducedinhibition of contraction in the isolated aorta (ex vivo).

Effects of Diosgenin on Ca2� Influx via L-Type Cal-cium Channels in the Isolated Mouse Aorta. We first

investigated the contribution of L-type Ca2� channels dur-ing PE-induced force development in the isolated mouseaorta. Treatment of aortic rings with 1 �M Nif, a selectiveinhibitor of L-type Ca2� channels, reduced PE-inducedcontraction to 69 � 5% of control and vehicle-treatedgroups, indicating a partial involvement of L-type Ca2�

channels (Fig. 6A; n � 6 mice; �, P � 0.05). However, whenthe aorta was exclusively activated by voltage-gated cal-cium channels (VGCC) during high potassium depolariza-tion, the entire contraction was abolished by 1 �M Nif butnot by diosgenin (Fig. 6B; n � 7 mice; �, P � 0.05). Thisobservation shows that diosgenin does not block VGCC inthe mouse aorta. It is noteworthy that pretreatment ofaorta with diosgenin for 30 min before the stimulationwith 60 mM KCl also had no effect on force development inaortic rings (data not shown).

To determine whether the diosgenin-induced relaxationhad an endothelium-dependent component we treated bothendothelium-intact and endothelium-denuded aortic seg-ments with diosgenin before PE treatment. Diosgenin treat-ment (25 �M) in both groups resulted in a 50% decrease inPE-induced force in the presence of extracellular calcium,indicating an absence of endothelial involvement in our prep-aration (Fig. 7; n � 5 mice; �, P � 0.05). To minimize tissuetrauma during the calcium measurements we thus left theaorta intact and focused the confocal microscope on the me-dial smooth muscle layer.

Effects of Diosgenin on �-Adrenergic Receptor-Me-diated Calcium Signals in the Isolated Mouse Aorta.We measured PE-induced calcium signals in the absenceand presence of 25 �M diosgenin. As shown in Fig. 8A,diosgenin caused a significant decrease (50%) in the PE-induced calcium transient in aortic rings (n � 11 mice;mean � S.D.; �, P � 0.05). As shown, the plateau phase ofcalcium signal (required for maintaining the force) wasalso affected by diosgenin. Removal of extracellular cal-cium before stimulation of aortic rings with PE resulted ina decrease in basal [Ca2�]i, shortening of the PE-inducedcalcium transient, and abolition of the plateau phase (Fig.8B; n � 9 mice; mean � S.D.; �, P � 0.05). In the absenceof extracellular calcium diosgenin completely blocked thePE-induced sarcoplasmic reticulum (SR) calcium releasetransient in parallel with blockade of force development(compared with 50% inhibition in the presence of extracel-lular calcium in Fig. 8A).

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Fig. 7. Role of endothelial layer in diosgenin-induced inhibition of aorticcontraction. Similar inhibitory effect for diosgenin was observed in theabsence and presence of endothelial layer. At the concentration of 25 �Mdiosgenin caused a 50% decrease in PE-induced aortic contraction in bothendothelium-denuded and endothelium-intact aortic rings (n � 5 mice;mean � S.D.; �, P � 0.9600).

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Effects of Diosgenin on Purinergic Receptor-Medi-ated Calcium Signals in Mouse Aortic SMC Culture. Weinvestigated whether the effect of diosgenin on SR Ca2�

release and possibly store-operated Ca2� channels was alsopresent in the cultured cells. As shown in Fig. 9A, diosgenin

caused a significant inhibition of the UTP-induced calciumtransient in cultured SMCs (n � 5 experiments; mean �S.D.; �, P � 0.05). It is noteworthy that Nif had no effects onUTP-induced calcium transients in cultured SMCs (Fig. 9B;n � 5 experiments; mean � S.D.; �, P � 0.05). Furthermore,

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Fig. 8. Effects of diosgenin on �-adrenergic receptor-mediated calcium signals in the isolated mouse aorta. A, treatment of the mouse aorta with 25�M diosgenin resulted in a significant inhibition of PE-induced calcium transient compared with vehicle (EtOH)-treated aorta (n � 11 mice; mean �S.D.; �, P � 0.05). B, in the absence of extracellular calcium diosgenin completely blocked PE-induced calcium transient in the mouse aorta (n � 9 mice;mean � S.D.; �, P � 0.05).

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to investigate the effects of diosgenin on UTP-induced forcedevelopment, isolated aortic rings were pretreated with 25�M diosgenin for 30 min before UTP application. Diosgenintreatment reduced UTP-induced force development by 50%(Fig. 10; n � 5 mice; mean � S.D.; �, P � 0.05).

To test for a possible effect of diosgenin on store-oper-ated Ca2� channels in vitro, extracellular calcium wasremoved from SMC cultures, and SR calcium content wasdepleted using 10 �M cyclopiazonic acid, a sarco/endoplas-

mic reticulum Ca2�-ATPase inhibitor. In control groups,reperfusion of cell cultures with HEPES buffer containing1.5 mM (calcium readmission) resulted in a rapid andtransient 3-fold increase in [Ca2�]cyto followed by a lowerplateau phase. In the diosgenin-treated cells the transientincrease in [Ca2�]cyto was significantly reduced and theplateau was abolished, indicating an inhibitory effect onstore-operated calcium channels in aortic SMCs (Fig. 11;n � 6 experiments; �, P � 0.05).

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Fig. 9. Effects of diosgenin on purinergic receptor-medi-ated calcium signals in mouse aortic SMC culture. A, at theconcentration of 25 �M diosgenin caused a marked de-crease in the UTP-induced calcium transient in culturedSMCs compared with the vehicle (EtOH)-treated group(n � 5 experiments; mean � S.D.; �, P � 0.05). B, nifedi-pine (1 �M) did not affect the UTP-induced calcium signal,indicating that the L-type calcium channel was not acti-vated during UTP stimulation. Bar graphs represent theaverage of three independent experiments (n � 5 experi-ments; mean � S.D.; �, P � 0.9369).

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DiscussionIn the present study, we describe various mechanisms by

which diosgenin modulates vascular SMC function and via-bility in the mouse aorta. In cell culture, diosgenin inhibitsaortic SMC proliferation and migration, but also inducesapoptosis. The apoptotic effect of diosgenin in aortic SMCs isassociated with a significant reduction in Akt phosphoryla-tion and a marked increase in caspase-3 cleavage. Overex-pression of an active form of Akt rescued SMCs from theapoptotic effects of diosgenin, establishing a causal relation-ship between the loss of Akt activity and increased apoptosisin diosgenin-treated SMCs. We also studied the effects ofdiosgenin on SMC viability and found that in the presence ofserum (an activator of Akt) overexpression of both the wild-type and activated forms of Akt were able to improve cellviability after diosgenin treatment. This is caused by imme-diate phosphorylation of the exogenous Akt (overexpressedwild-type Akt) by serum. However, in a serum-starved con-dition, only a constitutively active form of Akt was able toimprove SMC viability, suggesting that only the activeform of Akt could reverse the cytopathic effects caused bydiosgenin.

Diosgenin has been reported to inhibit cancer growthcaused by inhibiting Akt signaling and inducing apoptosis invarious in vitro culture systems (Moalic et al., 2001; Leger etal., 2004; Shishodia and Aggarwal, 2006). Our findings haveshown that at the concentration higher than 10 �M diosgenincould cause apoptosis in SMCs. This observation is in agree-ment with previous studies in tumor cells, in which theapoptotic effects of diosgenin were observed within the con-centration range of 25 to 50 �M. Fortunately, the potency forapoptotic and necrotic effects is low in mouse aortic SMCs,such that at the concentration of 10 �M diosgenin induces 40

to 50% inhibition of SMC migration without any significantapoptotic or necrotic effects (Fig. 12).

Hypersensitivity of vascular smooth muscle to physiologi-cal stimuli results in enhanced vasoconstriction in a widevariety of vascular disorders such as diabetic vascular dis-ease, hypertension, pulmonary vasoconstriction, and coro-nary vasospastic angina pectoris (Brondum et al., 2008;Burger, 2009; Eijking et al., 2009; Morrell et al., 2009). Todetermine whether diosgenin could affect vascular smoothmuscle responses to physiological stimuli, we measured thecontractile behavior of the isolated mouse aorta in the ab-sence and presence of diosgenin. In the presence of extracel-lular calcium, diosgenin caused a maximum of 50% inhibitionof contraction in response to PE stimulation. Likewise, in thepresence of extracellular calcium, blocking the L-type cal-cium channels with nifedipine caused an almost 50% declinein PE-induced force, highlighting that in mouse aorta onlyhalf of PE-induced contraction is caused by calcium entrythrough L-type calcium channels, and the remaining halfprobably depends on calcium release from intracellularstores and/or calcium entry through store operated channels.Consistent with this hypothesis, in the absence of extracel-lular calcium, diosgenin treatment caused 100% inhibition ofcontraction in response to PE. The observation that diosge-nin did not block L-type channel-mediated calcium influxfrom the extracellular space in the aorta further confirmedour assumption that the 50% blockade caused by diosgeninwas probably caused by its effects on calcium release fromthe intracellular stores and possibly calcium influx throughstore-operated calcium channels.

Dias et al. (2007) reported that treatment of rat superiormesenteric arteries with diosgenin resulted in endothelium-dependent vasorelaxation that could be significantly blocked

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Fig. 10. Effects of diosgenin on UTP-induced force devel-opment in the mouse aorta. Pretreatment of aortic seg-ments with 25 �M diosgenin resulted in a 50% decrease inUTP-induced force development in the aorta (n � 5 exper-iments; mean � S.D.; �, P � 0.05).

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by the inhibitor of endothelial nitric-oxide synthase. How-ever, in our isolated mouse aorta, the effects of diosgenin onaortic contraction did not seem to depend on the endotheliallayer, because removal of the endothelium in mouse aorta didnot affect vasodilatation in response to diosgenin. This dif-ference could be related to the origin of the blood vessels(mouse aorta versus rat mesenteric artery) or variations indiosgenin concentrations used (25 �M versus 1 mM).

As expected, diosgenin also decreased the PE-induced cal-cium transient in the mouse aorta by almost half. Removal ofthe extracellular calcium in the presence of diosgenin re-sulted in complete blockade of the PE-induced calcium tran-sient, which corresponds with complete inhibition of forcedevelopment in the aorta.

UTP, an effective agonist for induction of calcium releasein cultured SMCs, exerts its effects via purinergic P2Y recep-tors that activate the phospholipase C pathway (Horiuchi etal., 2001). . In our primary aortic SMC culture, application ofUTP (1 mM) induced a rapid but transient increase in[Ca2�]cyto, which was largely blocked by diosgenin.

In these aortic SMC cultures, blocking L-type calciumchannels with nifedipine had no effect on UTP-induced cal-

cium transients, indicating that in these cells the agonist-induced calcium transient does not depend on the opening ofVGCC, but depends on SR calcium release and possibly cal-cium influx via the store-operated calcium channels. Consis-tent with the above, no elevation in calcium concentrationwas observed in cultured SMCs upon application of 60 mMKCl, suggesting that L-type calcium channels are not ex-pressed or are possibly inactivated in our primary SMC cul-tures (data not shown). To test the effects of diosgenin onstore-operated calcium channels in cultured SMCs, we de-pleted the SR by sarco/endoplasmic reticulum Ca2�-ATPaseinhibition in the absence of extracellular calcium and read-mitting calcium in the absence or presence of diosgenin. Ourdata clearly showed blockade of store-operated calcium chan-nels in mouse SMCs by diosgenin. An interesting aspect ofdiosgenin is its close chemical relationship to estrogens,which are also known to target a variety of calcium trans-porters and enzymes (Martin et al., 1978; Miksicek, 1993).Thus for the purpose of widening our understanding of phys-iological smooth muscle regulation and possibly correctingpathological dysregulation, further investigation of the mo-lecular mechanisms of diosgenin is desirable.

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Fig. 11. Effects of diosgenin on store-operated channels in aortic SMCs. In the absence of diosgenin and after SR depletion, reperfusion of cells withHEPES buffer containing 1.5 mM Ca2� resulted in a rapid and transient increase in cytoplasmic calcium that quickly dropped and then wasmaintained at the basal level. However, in the presence of 25 �M diosgenin the peak response (caused by the opening of store-operated calciumchannels) was significantly blocked (n � 6 experiments; mean � S.D.; �, P � 0.05).

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In conclusion the approach taken in this study relies on thenotion that during the progression of vascular diseases thearterial walls undergo structural remodeling that is associ-ated with an increase in SMC migration and proliferationand irregular vasoconstriction (Okamoto et al., 1992; Ross,1999). In occlusive vascular disorders, SMC apoptosis alsosignificantly contributes to disease progression and end-stage plaque rupture, resulting in blockade of the arteriesand thus impaired blood flow to the targeted organ (Doran etal., 2008; Sprague and Khalil, 2009). Therefore, a desirableand effective therapeutic approach would control enhancedSMC migration and contraction, while viability of vascularSMC is maintained. In this study, we have shown that dios-genin inhibits both SMC migration and contraction. By per-forming comprehensive concentration-response experimentswe were able to show that treatment of vascular smoothmuscle with 10 �M diosgenin could provide 45% inhibition ofmigration and 25% inhibition of contraction (Fig. 12). At thisconcentration, diosgenin causes apoptosis in less than 3.5%of the SMC population, suggesting that the use of diosgeninat the concentration range of 10 to 15 �M may be expected toprovide overall beneficial effects on diseased vascular SMCs.In short, comparison of the concentration-response curves forthese various pharmacological actions of diosgenin indicatesa possible separation of “desirable effects” such as inhibitionof migration and contraction from the “harmful effects” ofapoptosis and loss of viability, making it a possible candidatefor the treatment of vascular diseases.

Authorship Contributions

Participated in research design: Esfandiarei.Conducted experiments: Esfandiarei, Lam, Yazdi, Kariminia,

Syyong, Dorado, and Hu.Performed data analysis: Esfandiarei and Kuzeljevic.Wrote or contributed to the writing of the manuscript: Esfandiarei

and van Breemen.

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48.2±4.9

85.4±3.

60.65±2.13

50µM

1.5±0.4

0.1±0.1

2.7±0.15

0µM

10.3246.7±5.023.9±4.76.7±2.8Inhibition of Contraction

10.1969.1±2.345.1±2.027.3±0.1Inhibition of Migration

32.9917.29±1.013.01±0.392.80±0.32Apoptosis

IC50

(µmol.L-1)25µM10µM5µM

Diosgenin

Effect (%)

-6.5 -6.0 -5.5 -5.0 -4.5 -4.00

102030405060708090

100Apoptosis

Inhibition of Migration

Inhibition of Contraction

Log (PE)

% E

ffec

t

Fig. 12. Family of curves representingvarious effects of diosgenin on mouse aor-tic smooth muscle cells. Treatment ofvascular smooth muscle with 10 �M di-osgenin provides 13% inhibition of prolif-eration, 45% inhibition of migration, and24% inhibition of contraction. Note thatno significant apoptotic (less than 3%)effects are apparent in vascular SMCstreated with 10 �M diosgenin. The tablesummarizes the inhibitory effects of dios-genin and the IC50 values for all of theobserved effects.

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Address correspondence to: Dr. Mitra Esfandiarei, Child and Family Re-search Institute, Department of Anesthesiology, Pharmacology, and Therapeu-tics, University of British Columbia, Room 2099, 950 West 28th Ave., Vancou-ver, BC, V5Z 4H4, Canada. E-mail: mesfandiarei@gmail.com

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