stimulation of neural stem cell proliferation by inhibition of phosphodiesterase 5

Research ArticleStimulation of Neural Stem Cell Proliferation byInhibition of Phosphodiesterase 5

Ana I Santos123 Bruno P Carreira1 Rui J Nobre1

Caetana M Carvalho1 and Inecircs M Arauacutejo123

1 Centre for Neuroscience and Cell Biology University of Coimbra 3004-517 Coimbra Portugal2 Department of Biomedical Sciences and Medicine University of Algarve 8005-139 Faro Portugal3 Institute for Biotechnology and Bioengineering (IBB) Centre for Molecular and Structural BiomedicineUniversity of Algarve 8005-139 Faro Portugal

Correspondence should be addressed to Ines M Araujo imaraujoualgpt

Received 4 July 2013 Revised 20 November 2013 Accepted 24 November 2013 Published 12 January 2014

Academic Editor Su-Chun Zhang

Copyright copy 2014 Ana I Santos et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The involvement of nitric oxide (NO) and cyclic GMP (cGMP) in neurogenesis has been progressively unmasked over the lastdecade Phosphodiesterase 5 (PDE5) specifically degrades cGMP and is highly abundant in the mammalian brain Inhibition ofcGMP hydrolysis by blocking PDE5 is a possible strategy to enhance the first step of neurogenesis proliferation of neural stemcells (NSC) In this work we have studied the effect on cell proliferation of 3 inhibitors with different selectivity and potency forPDE5 T0156 sildenafil and zaprinast using subventricular zone-(SVZ-) derived NSC cultures We observed that a short- (6 h) ora long-term (24 h) treatment with PDE5 inhibitors increased SVZ-derived NSC proliferation Cell proliferation induced by PDE5inhibitors was dependent on the activation of the mitogen-activated protein kinase (MAPK) and was abolished by inhibitors ofMAPK signaling soluble guanylyl cyclase and protein kinase G Moreover sildenafil neither activated ERK12 nor altered p27Kip1levels suggesting the involvement of pathways different from those activated by T0156 or zaprinast In agreement with the presentresults PDE5 inhibitors may be an interesting therapeutic approach for enhancing the proliferation stage of adult neurogenesis

1 Introduction

Neurogenesis is the biological process of generating newneurons from progenitor cells or neural stem cells (NSC)NSC proliferate in two main regions of the adult mammalianbrain the subventricular zone (SVZ) of the lateral ventriclesand the subgranular zone (SGZ) of dentate gyrus of thehippocampus Following brain injury such as stroke NSC inthe endogenous niches proliferate andmigrate to the affectedbrain areas where they may differentiate into neurons butsurvival is limited [1ndash3] There is still a lack of knowledgeconcerning the use of effective therapeutic strategies in orderto overcome the limited ability of brain self-repair followingan insult Understanding the signaling pathways involvedin the regulation of neurogenesis is paramount in order toenhance brain repair

Neurogenesis is affected by several factors includingnitric oxide (NO) NO is a free radical of particular interestdue to its cellular function as a second messenger which

includes the regulation of NSC proliferation Several studiesrecently reported the effect of NO on the stimulation ofadult neurogenesis in the dentate gyrus and in the SVZ [4ndash8] Thus the increase of NO levels following brain injurysuch as seizures or ischemia has been shown to promoteproliferation of NSC and the formation of new neurons [45 8] Two distinct pathways seem to be involved in the prolif-erative effect of NO the extracellular signal-regulated kinase(ERK)mitogen-activated protein kinase (MAPK) pathway[5] and the soluble guanylyl cyclase (sGC)protein kinaseG (PKG) pathway [9] The second messenger cyclic GMP(cGMP) is a signaling molecule whose levels are regulatedby an equilibrium between its production and removalcGMP is produced by sGC which can be activated by NOActivation of sGC by NO leads to increased cGMP levels thatactivate downstream targets such as PKG [10 11] PKG is aserinethreonine cGMP-dependent kinase that regulates theactivity of several transcription factors that control important

Hindawi Publishing CorporationStem Cells InternationalVolume 2014 Article ID 878397 13 pageshttpdxdoiorg1011552014878397

2 Stem Cells International

processes like synaptic plasticity (reviewed by [12]) In severalconditions such as aging cGMP levels are decreased andmay be involved in age-related neurodegeneration decreasedneurogenesis and cognitive decline [13] Similar to theincrease of cGMP production by NO the inhibition of cGMPhydrolysis by targeting phosphodiesterases (PDE) could bea strategy to increase the levels of cGMP and consequentlyreverse these effects by stimulating neurogenesis

PDE are ubiquitous enzymes responsible for the degrada-tion of cyclic nucleotides whose activity is dependent on sub-strate kinetic properties and cellular and subcellular distri-bution of the 11 known families PDE5 6 and 9 are consideredcGMP specific PDEwhereas PDE4 7 and 8mainly hydrolyzecyclic AMP (cAMP) and PDE1 2 3 10 and 11 hydrolyzeboth substrates (reviewed in [14]) Phosphodiesterase type5 is specific for cGMP degradation and is present in themammalian brain [15 16] PDE5 inhibitors have been usedfor the treatment of several pathologies in which an increasein cGMP levels can be beneficial such as erectile dysfunctionand pulmonary hypertension (reviewed by [17 18]) The bestcharacterized inhibitor of PDE5 is sildenafil Sildenafil wasfirst used for the treatment of erectile dysfunction Due tothe presence of PDE5 in the lung sildenafil is also used inthe treatment of pulmonary hypertension (reviewed by [18ndash20]) Since PDE5 is also present in the central nervous systemseveral works have assessed the effect of sildenafil on thebrain (reviewed by [21]) Some studies reported that sildenafilimproves learning and memory [22 23] and also inducesneurogenesis in the SVZ and SGZ [24] Neuronal functionrecovery in adult young rats and in aged rats [25] followingstroke has also been described after sildenafil treatment [2627] Zaprinast the first PDE5 inhibitor to be used has beenshown to be a nonpotent and nonselective inhibitor Sildenafilis typically considered a PDE5 inhibitor with an IC

50of

approximately 4 nM and it has been shown to inhibit PDE5 atconcentrations about 200-fold lower than the concentrationof zaprinast required to inhibit PDE5 [28] However it alsoinhibits PDE1 and PDE6 (IC

50values of 281 and 29 nM

resp [28]) indicating that sildenafil is not totally selective forPDE5More recently a new compoundwas developed T0156which potently inhibits PDE5 [29] In fact T0156 has a muchlower IC

50for PDE5 (023 nM) than sildenafil (IC

5036 nM)

Within this scenario in this work we investigated theeffect of three different PDE5 inhibitors T0156 sildenafiland zaprinast on the first step of neurogenesis by evalu-ating proliferation of SVZ-derived NSC and addressed themechanisms underlying these effects Here we show thatT0156 sildenafil and zaprinast increase SVZ-derived NSCproliferationMoreover blockingMAPK sGC or PKG inde-pendently results in loss of the proliferative effect of PDE5inhibitors pointing out to the involvement of sGCPKG andERKMAPK pathways in the stimulation of NSC prolifera-tion by PD5 inhibition

2 Methods

21 Materials Dulbeccorsquos Modified Eaglersquos Medium F-12 nutrient mixture (D-MEMF-12 with GlutaMAX I)

B27 supplement trypsin-ethylenediaminetetraacetic acid(EDTA) solution antibiotics (10000 unitsmL penicillin10mgmL streptomycin) Click-iT EdU Alexa Fluor 488Flow Cytometry Assay kit Click-iT EdU Alexa Fluor 594HCS Assay kit and StemPro Accutase cell dissociationreagent were purchased from Invitrogen (Paisley UK) Epi-dermal growth factor (EGF) and basic fibroblast growthfactor (bFGF) were from PeproTech Inc (London UK)The Matrix used was from Stoelting Co (Wood Dale ILUSA) Bicinchoninic acid (BCA) Protein Assay kit wasfrom Pierce (Rockford IL USA) 8-bromoguanosine 3101584051015840-cyclic monophosphate (8-Br-cGMP) Phenylmethylsulfonylfluoride orthovanadate chymostatin leuptin antipain pep-statin A trypan blue Tween-20 mouse anti-120572-tubulin 2-Propanol chloroform andTRI Reagent were purchased fromSigma Chemical (St Louis MO USA) T0156 hydrochloridesildenafil citrate zaprinast and 1119867-[1 2 4]Oxadiazolo[43-a]quinoxalin-1-one (ODQ) were obtained from Tocris Bio-science (Bristol UK) KT5823 was obtained from AlomoneLabs (Jerusalem Israel) and 3-(51015840-Hydroxymethyl-21015840-furyl)-1-benzylindazole (YC-1) from Santa Cruz Biotechnology(Santa Cruz CA USA) 14-diamino-23-dicyano-14-bis[2-aminophenylthio] butadiene (U0126) rabbit anti-p27Kip1rabbit anti-ERK12 and rabbit anti-phospho-ERK12 werepurchased from Cell Signaling (Danvers MA USA) Poly-merase chain reaction (PCR) primers were obtained fromEurofins MWGOperon (Ebersberg Germany) and FastStartPCR Master Mix and Transcriptor High Fidelity cDNASynthesis kit were from Roche Diagnostics (Mannheim Ger-many) Bovine serum albumin (BSA) was from Calbiochem(San Diego CA USA) and polyvinylidene difluoride mem-branes were purchased from Millipore (Madrid Spain)Amersham cGMP enzyme immunoassay Biotrak Systemenhanced chemifluorescence reagent and anti-rabbit andanti-mouse alkaline phosphatase-conjugated antibodies werefrom GE Healthcare Life Sciences (Buckinghamshire UK)Other reagents used in immunoblotting experiments werepurchased from Bio-Rad (Hercules CA USA)

22 Animals C57BL6J mice were obtained from CharlesRiver (Barcelona Spain) and kept in our animal facilities withfood and water ad libitum in a 12-hour dark light cycle Allexperiments were performed in accordancewith Institutionaland European Guidelines (86609EEC) for the care and useof laboratory animals

23 Subventricular Zone Cell Cultures Neural stem cell cul-tures were obtained from the SVZ of 0ndash3 postnatal dayC57BL6J mice as previously described [30] The fragmentsof SVZ were digested in 0025 trypsin0265mM EDTAfor 15ndash20 minutes at 37∘C and then mechanically dissoci-ated The cells were resuspended in a 37∘C D-MEMF-12medium with 2mM GlutaMAX-I supplemented with 1B27 1 antibiotic (10000 unitsmL penicillin 10mgmLstreptomycin) 10 ngmL EGF and 5 ngmL bFGF and platedon uncoated flasks with filter cap at a density of 100000cellsmL The SVZ stem cells were grown as floating aggre-gates in a 95 air5 CO

2humidified atmosphere at 37∘C

Stem Cells International 3

during 7 days Then the primary neurospheres were har-vested centrifuged and mechanically dissociated as singlecells Cells were replated as above and allowed to grow assecondary neurospheres 6-7 days later the floating neuro-spheres were collected dissociated and plated for 2-3 dayson poly-L-lysine-coated 12-well plates in the same mediumas above In the 24 h preceding the experimental treatmentsthe cells were kept with a similar medium but without growthfactors in order to ensure that the results observed followingcell treatments were not due to the effect of EGF or bFGF

24 Experimental Treatments For cell proliferation analy-sis the times of cell treatment with PDE5 inhibitors werechosen based on the times used previously to evaluate cellproliferation in SVZ-derived NSC using a NO-donor [9]Thus the cells were exposed for 6 h and 24 h to PDE5inhibitors or stimulated for 24 h with the sGC activator YC-1or the cGMP analogue 8-Br-cGMP (20 120583M) To analyze thephosphorylation of ERK12 and the levels of p27Kip1 differenttimes were tested and SVZ-derived NSC were treated for2 h in the following experiments or as indicated in thefigure legends In both experiments the stimuli applied wereas follows PDE inhibitors T0156 (1 120583M) sildenafil (1 120583M)and zaprinast (10120583M) or sGC activator YC-1 (20120583M) wereincubated alone or together with the MEK12 inhibitorU0126 (1 120583M) the PKG inhibitor KT5823 (1 120583M) or thesGC inhibitor ODQ (50 120583M) U0126 KT5823 and ODQwere applied 30min before exposure to PDE5 inhibitorsand YC-1 and kept throughout the incubation period Fordetermination of cGMP levels cells were treated with T0156(1 120583M) sildenafil (1 120583M) or zaprinast (10 120583M) for 6 h Allthe experiments were performed together with the respectivecontrols (untreated cells)

25 Analysis of Cell Proliferation by Flow Cytometry SVZ-derived neural stem cell proliferation was assessed by incor-poration of ethynyl-21015840-deoxyuridine (EdU) using the Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay kit aspreviously described [31] 10 120583M EdU was added to the SVZcultures for 6 h or 4 h before fixation for the cells treatedfor 6 h or 24 h respectively For fixation cells were washedwith sterile 001M PBS and then sterile StemPro Accutasecell dissociation reagent was added for 20min at 37∘C Cellfixation was performed in 70 ethanol overnight at 4∘C asdescribed previously [5 31] Detection of EdU incorporationwas based on click chemistry a copper catalyzed covalentreaction between an azide (conjugated with the Alexa Fluor488 fluorophore) and an alkyne (EdU) Briefly cells incubatedfor 30 minutes with the azide conjugate and copper sulphateat room temperature protected from light Next the cellswere incubated with ribonuclease A and with the nucleardye 7-actinomycin D (7-AAD) widely used as a cell viabilityand cell cycle marker for 30min EdU incorporation and7-AAD signal were detected on a BD FACScalibur FlowCytometer using the Cellquest Pro software version 03efab(Becton Dickinson San Jose CA USA) Thirty thousandevents were acquired per each experiment in the region ofinterest (including apoptotic cells and G0G1 S and G2M

cell cycle phases) Aminimum of 4 independent experimentswas analyzed for each condition Data were analyzed usingthe WinMDI29 software and are presented as means plusmn SEMof the number of nonapoptotic cells that incorporated EdU( of control) as previously described [31]

26 Western Blot Analysis Cells were scraped and lysedin 50mM Tris-HCl 015M NaCl 1mM EDTA 1 Igepaland 10 glycerol supplemented with 200120583M phenylmethyl-sulfonyl fluoride 1 120583gmL chymostatin 1120583gmL leupeptin1 120583gmL antipain 1 120583gmL pepstatin A 1mM sodium ortho-vanadate 1120583M dithiothreitol and 5mM NaF pH 75 4∘CProtein concentration was determined by the BCA method(BCA Protein Assay kit Pierce Rockford IL USA) accord-ing to manufacturerrsquos instructions 6x concentrated samplebuffer was added and samples were denatured at 95∘C for5minNext samples were used for protein separation by elec-trophoresis in sodium dodecyl sulfate (SDS)-polyacrylamidegels using MiniPROTEAN 3 systems (Bio-Rad Laborato-ries) Equal amounts of protein were separated by elec-trophoresis and then electrophoretically transferred to theactivated polyvinylidene difluoride membranes Membraneswere blocked by 1 h incubation at room temperature withTris-buffered saline (137mMNaCl 20mM Tris-HCl pH 76)containing 01 Tween-20 (TBS-T) and 3 BSA Incubationswith the primary antibodies (rabbit phospho-ERK12 orrabbit anti-p27kip1 1 1000) in TBS-T containing 1 blockingsolution were performed overnight at 4∘C Next the incu-bation with the appropriated alkaline phosphatase-linkedsecondary antibodies (anti-rabbit or anti-mouse 1 20000in TBS-T containing 1 blocking solution) was performedat room temperature during 1 h After extensive washing inTBS-T followed by the incubation of the membranes withthe enhanced chemifluorescence reagent immunoreactivebands were visualized in the VersaDoc 3000 imaging system(Bio-Rad Hercules CA USA) Data were analyzed with theQuantity One software version 469 (Bio-Rad Laboratories)Protein control loadings were either performed after mem-branes reactivation (5ndash10 s in 100 methanol and 20minin TBS-T) using primary antibodies against rabbit ERK12(1 1000) or mouse 120572-tubulin (1 10000)

27 Determination of cGMP Levels cGMP levels in culturedSVZ-derived stem cells were determined after exposure todrugs for 6 h using a cGMP Enzyme immunoassay BiotrakSystem This experimental assay is based on competitionbetween unlabelled cGMP from cell lysates and a fixedquantity of peroxidase-labelled cGMP for a limited numberof binding sites on a cGMP specific antibody coated on a 96-well plate Cell lysis and cGMPmeasurement were performedaccording to manufacturerrsquos instructions and as previouslydescribed [32] Optical density was read at 450 nm All theexperiments were carried out in duplicate The results areexpressed as femtomoles per million of cells

28 Data Analysis Data are expressed as means plusmn SEMStatistical significance was determined by using two-tailed 119905-tests or one-factor analysis of variance (ANOVA) followed


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Figure 1 The PDE5 inhibitors T0156 sildenafil and zaprinast stimulated the proliferation of SVZ neural stem cells following a 6 h or 24 hexposure Cells were treatedwith 1 120583MT0156 (a) 1120583M 10 120583Mand 50 120583Msildenafil (b) or 10 120583Mand 50 120583Mzaprinast (c) for 6 h andwith 1 120583MT0156 (d) 1120583M sildenafil (e) or 10120583M zaprinast (f) for 24 h The incorporation of EdU was assessed by flow cytometry Data are expressedas means plusmn SEM of at least 4 independent experiments (a) (d) (e) and (f) two-tailed 119905-test lowast119875 lt 005 and lowastlowast119875 lt 001 significantly differentfrom control and (b) and (c) one-way ANOVA (Dunnettrsquos post-test) lowast119875 lt 005 and lowastlowast119875 lt 001 significantly different from control

by Bonferronirsquos or Dunnettrsquos post-tests as appropriate andindicated in the figure legends Differences were consideredsignificant when 119875 lt 005 The software used was GraphPadPrism 50 (GraphPad Software La Jolla CA USA)

3 Results

31 Characterization of SVZ Primary Cultures To investigatethe expression of phosphodiesterases in SVZ-derived neuralstem cell cultures we assessed the presence of the cGMP-specific PDE5A PDE9A and PDE6C by PCR Despite PDE6presence is only described in retina photoreceptors [33] wealso studied its expression in SVZ-derivedNSC cultures sincesome of the inhibitors for PDE5 also act on PDE6 Theexpression of several PDEs normally found in the brain wasanalyzed in SVZ cultures by reverse-transcription PCR Westudied the presence of the cGMP-specific isoforms PDE5APDE6C and PDE9A We also assessed the expression ofPDE1 (isoforms PDE1A 1B and 1C) and PDE10A whichhydrolyze both cAMP and cGMP as well as the cAMP-specific PDE7 enzyme isoforms PDE7A and PDE7B whosepresence in the brain is well documented [34ndash36] Weobserved the presence of PDE1 PDE5 PDE7 and PDE9 inSVZ cultures while PDE10 is absent (see SupplementaryFigure 1 in Supplementary materials available online athttpdxdoiorg1011552014878397) Moreover expression

of PDE6 catalytic subunit was not detected in SVZ-derivedNSC cultures but it was detected in the retina

32 Inhibition of PDE5 Increases Cell Proliferation Neuralstem cell proliferation was evaluated by assessing the incor-poration of EdU by flow cytometry following exposure toPDE5 inhibitors for 6 h All PDE5 inhibitors increased theproliferation of SVZ-derived NSC Treatment with T0156(1 120583M [37]) significantly increased the incorporation of EdU(1407 plusmn 119 of the control 119875 lt 005 Figure 1(a)) whencompared to control cultures (no treatment) Both sildenafiland zaprinast increased EdU incorporation in comparison tountreated cultures as follows sildenafil (1 120583M 1374 plusmn 70119875 lt 005 10 120583M 1208 plusmn 104 119875 gt 005 50 120583M 1413 plusmn126 119875 lt 001 Figure 1(b)) and zaprinast (10 120583M 1256 plusmn78 119875 lt 001 50120583M 1181 plusmn 56 119875 gt 005 Figure 1(c))According to these data 1120583M sildenafil and 10 120583M zaprinastwere selected for all the subsequent experiments

In order to investigate whether this effect of PDE5 inhib-itors on SVZ-derived NSC proliferation was maintained forlonger times of exposure cells were treated for 24 h withT0156 sildenafil or zaprinast and EdU incorporation wasassessed by flow cytometry The data presented in Figure 1shows that following 24 h of treatment T0156 (1120583M 1356 plusmn73 of the control 119875 lt 001 Figure 1(d)) sildenafil (1 120583M1332 plusmn 82 119875 lt 005 Figure 1(e)) or zaprinast (10120583M1465 plusmn 139 119875 lt 005 Figure 1(f)) increased SVZ-derived


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Figure 2 Inhibition of the ERK12 pathway prevented proliferation of SVZ cells stimulated by PDE5 inhibitors Cell proliferation followingtreatment with 1 120583M U0126 and 1 120583M T0156 (a d) 1120583M sildenafil (b e) or 10120583M zaprinast (c f) was assessed by incorporation of EdU andevaluated by flow cytometry following 6 h and 24 h of treatment Data are expressed as means plusmn SEM of at least 4 independent experimentsOne-way ANOVA (Bonferronirsquos post-test) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 significantly different from control and +119875 lt 005 and+++119875 lt 0001 significantly different from the PDE5 inhibitor

NSC proliferation in comparison to untreated cultures Theincorporation of EdU was further assessed by microscopyanalysis Likewise following 24 h of treatment T0156 (1 120583M1824 plusmn 118 of the control 119875 lt 0001 SupplementaryFigures 2(a) and 2(b)) sildenafil (1 120583M 1576 plusmn 89 119875 lt001 Supplementary Figures 2(a) and 2(b)) or zaprinast(10 120583M 1421 plusmn 141 119875 lt 005 Supplementary Figures2(a) and 2(b)) increased SVZ-derived NSC proliferation incomparison to untreated cultures (100 corresponding to27 plusmn 04 of the total of live cells) Moreover treatmentwith these concentrations of PDE5 inhibitors did not have acytotoxic effect on SVZ cultures (Supplementary Table 2) asevaluated by detection of the nuclear dye signal 7-AAD byflow cytometry

33 PDE5 Inhibition Increases cGMP Levels To investigatewhether PDE5 inhibition resulted in fact in increasedcGMP levels the levels of cGMP were measured followingexposure to different PDE5 inhibitors As shown in Table 1we observed that cGMP levels were significantly increasedfollowing exposure to T0156 (297 plusmn 42 fmol106 cells 119875 lt005) sildenafil (288 plusmn 51 fmol106 cells 119875 lt 005) or zapri-nast (331plusmn 61 fmol106 cells119875 lt 001) as compared to cGMPlevels measured in untreated cultures (74 plusmn 09 fmol106cells)

Table 1 PDE5 inhibitors increased the levels of cGMP in SVZ cells

Treatment cGMP levels (fmol106 cells)Control 74 plusmn 09

T0156 297 plusmn 42lowast

Sildenafil 288 plusmn 51lowast

Zaprinast 331 plusmn 61lowastlowast

Cells were treated for 6 hwith 1 120583MT0156 1 120583Msildenafil or 10 120583Mzaprinastand then lysed to measure the levels of cGMP Data are expressed as means plusmnSEMof 4 independent experiments One-way ANOVA (Dunnettrsquos post-test)lowast119875 lt 005 and lowastlowast119875 lt 001 significantly different from control

34 PDE5 Inhibition Stimulates Cell Proliferation via theERKMAPK Pathway To evaluate the involvement of theERKMAPK pathway on cell proliferation stimulated byPDE5 inhibition SVZ-derived NSC cultures were treatedwith the MEK12 inhibitor U0126 Exposure to U0126 for6 h prevented the increased EdU incorporation triggered byT0156 (1091 plusmn 66 of the control 119875 lt 005 Figure 2(a))sildenafil (795 plusmn 58 119875 lt 0001 Figure 2(b)) or zaprinast(943 plusmn 74 119875 lt 005 Figure 2(c)) keeping cell proliferationsimilar to control cultures in comparison to T0156 (6 h 1407plusmn 119 119875 lt 001) sildenafil (6 h 1374 plusmn 70 119875 lt 0001)or zaprinast (6 h 1256 plusmn 78 119875 lt 005) alone respectivelySimilar results were obtained following 24 h of treatment


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Figure 3 T0156 or zaprinast but not sildenafil increased ERK12 phosphorylation following 2 h of treatment Phospho-ERK12 levelsfollowing treatment with 1 120583M T0156 (a) 1120583M sildenafil (b) and 10 120583M zaprinast (c) for 2 h were assessed by Western blot Representativeimages are shown Data are expressed as means plusmn SEM of at least 5 independent experiments Two-tailed 119905-test lowast119875 lt 005 and lowastlowast119875 lt 001significantly different from control

In the presence of U0126 the increase in the incorporationof EdU by T0156 (1025 plusmn 107 of the control 119875 lt 005Figure 2(d)) sildenafil (967 plusmn 96 119875 lt 005 Figure 2(e))or zaprinast (1076 plusmn 34 119875 lt 005 Figure 2(f)) wasprevented comparatively to T0156 (24 h 1356 plusmn 73 119875 lt001) sildenafil (24 h 1332 plusmn 82 119875 lt 005) or zaprinast(24 h 1465 plusmn 139 119875 lt 001) alone respectively

35 PDE5 Inhibition Increases ERK12 Phosphorylation Ex-posure to NO triggers cell proliferation in SVZ-derived NSCcultures by activation of ERK12 signaling which can bemonitored by evaluating the phosphorylation of ERK12 andthe concomitant decrease in the levels of cyclin-dependentkinase inhibitor 1 p27Kip1 [5] p27Kip1 is regulated by p90 RSKand once phosphorylated is translocated to the cytosol fol-lowed by ubiquitination and degradation by the proteosomeallowing progression to S-phase [38]

In order to choose the best time to study the effect ofPDE5 inhibition or sGC activation on the levels of phospho-ERK12 and p27Kip1 cultures were exposed to T0156 (1120583MSupplementary Figure 3(a)) sildenafil (1 120583M SupplementaryFigure 3(b)) zaprinast (10 120583M Supplementary Figure 3(c))or YC-1 (20 120583M Supplementary Figure 3(d)) for 30min 1 hor 2 h Treatment with T0156 zaprinast and YC-1 increasedERK12 phosphorylation following 2 h of treatment Further-more the cellular levels of p27Kip1 appear to be decreasedmainly following a 2 h treatment with T0156 According tothese results an exposure of 2 h to T0156 sildenafil zaprinastor YC-1 was used in these experiments

Treatment with T0156 (1 120583M 1285 plusmn 100 of the control119875 lt 005 Figure 3(a)) or zaprinast (10120583M 1400 plusmn 110 119875 lt001 Figure 3(c)) significantly increased ERK12 phosphory-lation in comparison to control cultures However treatmentwith sildenafil (1120583M 899 plusmn 98 119875 gt 005 Figure 3(b))did not increase the levels of phospho-ERK12 in relationto untreated cultures Since a short period of exposure tosildenafil (1 120583M for 2 h) did not affect phospho-ERK12levels we decided to test a longer time of exposure (6 h)

Interestingly we did not observe any remarkable differencesin phospho-ERK12 (1068 plusmn 85 119875 gt 005 SupplementaryFigure 4(a)) following exposure to sildenafil for 6 h whencompared to control cultures

36 PDE5 Inhibition Enhances Cell Proliferation via sGCPKGPathway To analyze whether the sGCPKG pathway isinvolved in cell proliferation stimulated by PDE5 inhibitionSVZ-derivedNSCcultureswere exposed to the sGC inhibitorODQ or the PKG inhibitor KT5823 We found that ODQprevented cell proliferation stimulated by T0156 (1055 plusmn 79of the control 119875 lt 005 Figure 4(a)) sildenafil (983 plusmn 90119875 lt 0001 Figure 4(b)) and zaprinast (1011plusmn 80119875 lt 005Figure 4(c)) as compared to T0156 (1407 plusmn 119 119875 lt 001)sildenafil (1374 plusmn 70 119875 lt 0001) or zaprinast (1256 plusmn 78119875 lt 005) alone respectively Moreover following 24 h oftreatment ODQ is also effective in preventing the increaseof EdU-positive cells by T0156 (1091 plusmn 55 119875 lt 005Figure 4(d)) sildenafil (1108 plusmn 44 119875 lt 005 Figure 4(e))and zaprinast (1051 plusmn 108 119875 lt 005 Figure 4(f)) incomparison to T0156 (1356 plusmn 73 119875 lt 001) sildenafil(24 h 1332 plusmn 82 119875 lt 0001) or zaprinast (1465 plusmn 139119875 lt 005) alone respectively

By inhibiting PKG with KT5823 following 6 h of treat-ment with PDE5 inhibitors we observed a prevention ofthe increase in cell proliferation induced by T0156 (1026 plusmn71 of the control 119875 lt 005 Figure 5(a)) sildenafil (1011plusmn 90 119875 lt 001 Figure 5(b)) and zaprinast (995 plusmn 74119875 lt 005 Figure 5(c)) and cell proliferation became similarto basal levels whereas the response to T0156 (6 h 1407 plusmn119 119875 lt 001) sildenafil (6 h 1374 plusmn 70 119875 lt 0001) orzaprinast (6 h 1256 plusmn 78 119875 lt 005) alone respectivelywas maintained Similarly 24 h of treatment with KT5823prevented the increase in cell proliferation induced by T0156(1085 plusmn 79 119875 lt 005 Figure 5(d)) sildenafil (1068 plusmn 53119875 lt 001 Figure 5(e)) and zaprinast (1081 plusmn 49 119875 lt 001Figure 5(f)) in comparison to T0156 (24 h 1356 plusmn 73 119875 lt001) sildenafil (24 h 1332 plusmn 82 119875 lt 0001) or zaprinast(24 h 1465 plusmn 139 119875 lt 001) alone respectively


0

50

100

150

200 6h

lowastlowast

T0156ODQ

+minus minus

minus minus

+

+ +

+

EdU

-pos

itive

cells

( o

f con

trol)

(a)

0

50

100

150

200

lowastlowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

SildenafilODQ

+minus minus

minus minus

+

+ +

+++

(b)

0

50

100

150

200

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

ZaprinastODQ

+minus minus

minus minus

+

+ +

(c)

0

50

100

150

200 24h

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

+

T0156ODQ

+minus minus

minus minus

+

+ +

(d)

0

50

100

150

200Ed

U-p

ositi

ve ce

lls(

of c

ontro

l)

24h

+

lowastlowastlowast

SildenafilODQ

+minus minus

minus minus

+

+ +

(e)

0

50

100

150

200

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowast

+

ZaprinastODQ

+minus minus

minus minus

+

+ +

(f)

Figure 4 Inhibition of sGC prevented the proliferation of SVZ cells stimulated by PDE5 inhibitors Cell proliferation following treatmentwith 50 120583MODQ and 1 120583MT0156 (a d) 1120583Msildenafil (b e) or 10 120583Mzaprinast (c f) was assessed by incorporation of EdU and evaluated byflow cytometry following 6 h or 24 h treatment Data are expressed as means plusmn SEM of at least 5 independent experiments One-way ANOVA(Bonferronirsquos post-test) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 significantly different from control and +119875 lt 005 and +++119875 lt 0001significantly different from the PDE5 inhibitor

37 PKG Is Involved in ERK12 Phosphorylation Induced byPDE5 Inhibition In order to evaluate whether inhibitingPKG with KT5823 would antagonize the effect of PDE5inhibition by T0156 or zaprinast on ERK12 phosphorylationsamples obtained from SVZ-derived NSC cultures were ana-lyzed by Western blot following 2 h of treatment Treatmentwith KT5823 suppressed the increase in phospho-ERK12levels induced by T0156 (1072 plusmn 62 of the control 119875 gt005 Figure 6(a)) or zaprinast (1105 plusmn 79 119875 gt 005Figure 6(c)) in comparison to T0156 (1285plusmn 100119875 lt 005)and zaprinast (1400 plusmn 110 119875 lt 005) alone respectivelyOn the contrary as sildenafil did not stimulate ERK12phosphorylation (Figure 3(b)) treatment with KT5823 didnot change phospho-ERK12 levels (1202 plusmn 110 119875 gt 005Figure 6(b)) when compared to sildenafil alone (899 plusmn 98119875 gt 005)

38 T0156 Treatment Decreases p27Kip1 Levels Next we eval-uated whether p27Kip1 levels were altered following 2 h ofexposure to T0156 sildenafil or zaprinast by Western blotWe observed that following treatment with T0156 p27Kip1levels significantly decreased (800 plusmn 72 of the control119875 lt 005 Figure 7(a)) in comparison to the levels found inuntreated cultures On the contrary treatment with sildenafil(1064 plusmn 67 119875 gt 005 Figure 7(b)) or zaprinast (974 plusmn

48 119875 gt 005 Figure 7(c)) did not change p27Kip1 levelsin comparison to control cultures Moreover similar towhat was observe for ERK12 phosphorylation following alonger exposure to sildenafil for 6 h we did not observe anyremarkable differences in the levels of p27Kip1 (917 plusmn 89119875 gt 005 Supplementary Figure 4(b)) when compared tocontrol

In order to test whether this effect can be reversed byinhibiting the ERK12 pathway NSC cultures were treatedwith U0126 for 2 h By Western blotting we observed thatU0126 alone increased p27Kip1 levels above the baseline (1180plusmn 75 Figure 8) Furthermore the levels of p27Kip1 weresignificantly increased following treatment with U0126 plusT0156 (1235 plusmn 130 of the control 119875 lt 001 Figure 8(a))comparatively to T0156 (800 plusmn 72 119875 gt 005) alone Onthe other hand U0126 did not change the levels of p27Kip1in samples treated with sildenafil (996 plusmn 96 119875 gt 005Figure 8(b)) or zaprinast (993 plusmn 46 119875 gt 005 Figure 8(c))as compared to sildenafil (1064plusmn 67119875 gt 005) or zaprinast(974 plusmn 48 119875 gt 005) alone respectively

We also observed that inhibition of PKG did not appearto affect the levels of p27Kip1 following treatment with T0156(841 plusmn 102 119875 gt 005 Figure 9(a)) sildenafil (963 plusmn 78119875 gt 005 Figure 9(b)) or zaprinast (1051 plusmn 100 119875 gt 005Figure 9(c)) in comparison to T0156 (800 plusmn 72 119875 lt 005)


0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

6h

lowastlowast

+

EdU

-pos

itive

cells

( o

f con

trol)

(a)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

lowastlowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

++

(b)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

(c)

0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

24h

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

+

(d)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowastlowast

++

(e)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowast

++

(f)

Figure 5 Inhibition of PKG prevented the proliferation of SVZ cells stimulated by PDE5 inhibitors Cell proliferation following treatmentwith 1 120583MKT5823 and 1 120583MT0156 (a d) 1120583M sildenafil (b e) or 10120583M zaprinast (c f) was assessed by incorporation of EdU and evaluatedby flow cytometry following 6 h or 24 h of treatment Data are expressed as means plusmn SEM of at least 4 independent experiments One-wayANOVA (Bonferronirsquos post-test) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 significantly different from control and +119875 lt 005 and ++119875 lt 001significantly different from the PDE5 inhibitor

0

50

100

150

200

lowast

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

T0156KT5823

+minus minus

minus minus

+

+ +

p-ERK 12

ERK 12


(a)

0

50

100

150

200

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

SildenafilKT5823

+minus minus

minus minus

+

+ +

p-ERK 12

ERK 12


(b)

0

50

100

150

200

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

ZaprinastKT5823

+minus minus

minus minus

+

+ +

lowast

p-ERK 12

ERK 12


(c)

Figure 6 Inhibition of PKG prevented the phosphorylation of ERK12 by treatment with T0156 or zaprinast Levels of phospho-ERK12following treatment with 1120583MKT5823 and 1 120583MT0156 (a) 1120583M sildenafil (b) and 10 120583M zaprinast (c) for 2 h were assessed byWestern blotRepresentative images are shown Data are expressed as means plusmn SEM of at least 4 independent experiments One-way ANOVA (Bonferronirsquospost-test) lowast119875 lt 005 significantly different from control


0

50

100

150

200

Control T0156

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

lowast

27kDa

55kDa

(a)

0

50

100

150

200

Control Sildenafil

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

Control Zaprinast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)

Figure 7 Treatment with T0156 decreased p27Kip1 levels p27Kip1 levels following treatment 1 120583M T0156 (a) 1120583M sildenafil (b) or 10 120583Mzaprinast (c) for 2 h were assessed by Western blot Representative images are shown Data are expressed as means plusmn SEM of at least 6independent experiments Two-tailed 119905-test lowast119875 lt 005 significantly different from control

0

50

100

150

200

T0156U0126

+minus minus

minus minus

+

+ +

++

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilU0126

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastU0126

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)

Figure 8 Inhibition of MAPK pathway prevented the decrease of p27Kip1 levels mediated by T0156 p27Kip1 levels following treatment with1 120583M U0126 and 1 120583M T0156 (a) 1120583M sildenafil (b) or 10 120583M zaprinast (c) for 2 h were assessed by Western blot Representative images areshown Data are expressed as means plusmn SEM of at least 5 independent experiments One-way ANOVA (Bonferronirsquos post-test) lowast119875 lt 005significantly different from control and ++119875 lt 001 significantly different from T0156

0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)

Figure 9 Inhibition of PKG did not prevent the decrease of p27Kip1 levels by T0156 p27Kip1 levels following treatment with 1 120583M KT5823and 1 120583M T0156 (a) 1120583M sildenafil (b) or 10 120583M zaprinast (c) for 2 h were assessed by Western blot Representative images are shown Dataare expressed as means plusmn SEM of at least 4 independent experiments One-way ANOVA (Bonferronirsquos post-test) lowast119875 lt 005 significantlydifferent from control


0

50

100

150

200

Control YC-1

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

(a)

0

50

100

150

200

Control YC-1Ph

osph

o-ER

K 1

2 le

vels

( o

f con

trol) lowast

p-ERK 12

ERK 12


(b)

0

50

100

150

200

YC-1KT5823

+minus minus

minus minus

+

+ +

+

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

p-ERK 12

ERK 12


(c)

0

50

100

150

200

Control YC-1

p27Ki

p1le

vels

( o

f con

trol)

lowast

p27Kip1

120572-Tubulin

27kDa

55kDa

(d)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1U0126

+minus minus

minus minus

+

+ +

+

p27Kip1

120572-Tubulin

27kDa

55kDa

(e)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1KT5823

+minus minus

minus minus

+

+ +

p27Kip1

120572-Tubulin

27kDa

55kDa

(f)

Figure 10 Activation of sGC stimulates proliferation of NSC increases ERK12 phosphorylation and decreases p27Kip1 levels Cellproliferation following treatment with 20 120583M YC-1 (a) for 24 h was assessed by the incorporation of EdU and analyzed by flow cytometryLevels of phospho-ERK12 following treatment with 20 120583M YC-1 (b) or YC-1 plus 1 120583M KT5823 (c) and p27Kip1 levels following treatmentwith YC-1 (d) or YC-1 plus 1120583M U0126 (e) or KT5823 (f) for 2 h were assessed by Western blot Representative images are shown Data areexpressed as means plusmn SEM of at least 4 independent experiments (a) (b) and (d) two-tailed 119905-test lowast119875 lt 005 and lowastlowast119875 lt 001 significantlydifferent from control and (c) (e) and (f) one-way ANOVA (Bonferronirsquos post-test) +119875 lt 005 significantly different from YC-1

sildenafil (1064 plusmn 67 119875 gt 005) or zaprinast (974 plusmn 48119875 gt 005) alone respectively

39 YC-1 Stimulates Cell Proliferation via the sGCPKG andERKMAPK Pathways To evaluate the effect on the prolif-eration of NSC of increasing the levels of cGMP by directactivation of sGC SVZ-derived NSC cultures were treatedwith the sGC activator YC-1 (20120583M) EdU incorporationwas assessed by flow cytometry and microscopy analysisExposure to YC-1 for 24 h increased the incorporation ofEdU by NSC (1371 plusmn 68 of the control 119875 lt 001Figure 10(a)) when compared to untreated cultures as eval-uated by flow cytometry Similarly evaluating EdU labelingby microscopy the increase in cell proliferation following24 h of treatment with YC-1 was also observed (1458 plusmn126 119875 lt 005 Supplementary Figures 2(a) and 2(c)) incomparison to untreated cultures Treatment with the sameconcentration of YC-1 for 6 h did not alter the percentage ofEdU-positive cells in comparison to control cultures (data not

shown) Furthermore treatmentwith the cGMP-analogue 8-Br-cGMP also increased proliferation ofNSC (20 120583M 1459plusmn119 119875 lt 005 Supplementary Figures 2(a) and 2(d)) whencompared to control cultures

In order to evaluate whether direct activation of sGCwould also affect phosphorylation of ERK12 and p27Kip1levels samples obtained from SVZ-derived NSC cultureswere treated with the sGC activator ERK12 phosphorylationand p27Kip1 levels were analyzed by Western blot following2 h of treatment In fact treatment with YC-1 significantlyincreased ERK12 phosphorylation (1233 plusmn 89 119875 lt 005Figure 10(b)) in comparison to control cultures Moreoverinhibition of PKG with KT5823 prevented the increase inphospho-ERK12 levels due to exposure to YC-1 (856 plusmn104 119875 lt 005 Figure 10(c)) when compared to YC-1 alone(1233 plusmn 89 119875 gt 005)

Furthermore similar to T0156 the levels of p27Kip1 weresignificantly decreased following treatment with YC-1 (775 plusmn90 119875 lt 005 Figure 10(d)) when compared to untreatedcultures This effect can be reversed by inhibiting the ERK12


pathwaywithU0126 as the levels of p27Kip1 were significantlyincreased following treatment with U0126 plus YC-1 (1149 plusmn34 119875 lt 005 Figure 10(e)) comparatively to YC-1 (775plusmn 90 119875 gt 005) alone Inhibition of PKG also appearedto prevent the decrease in p27Kip1 levels induced by YC-1as treatment with KT5823 and YC-1 tended to raise p27Kip1levels close to basal (1045 plusmn 113 119875 gt 005 Figure 10(f))when compared to YC-1 alone (775 plusmn 90 119875 gt 005)

4 Discussion

In this work we show that proliferation of NSC is stim-ulated by cGMP since inhibition of cGMP hydrolysis byPDE5 enhances the proliferation of NSC isolated from SVZMoreover direct activation of sGC by the activator YC-1 hasa similar effect in increasing NSC proliferation following 24 hof treatmentWe also show that ERK12 is activated followingtreatmentwith PDE5 inhibitors (except sildenafil) suggestingthe involvement of this pathway in cell proliferationmediatedby PDE5 inhibition Taken together our data point outto the activation by PDE5 inhibition in SVZ cells of twopathways the sGCPKG and the ERKMAPK pathways thesame pathways responsible for the proliferative effect of NO[9]

Using inhibitors with different selectivity for PDE5 weshow that inhibition of PDE5 either with T0156 sildenafilor zaprinast enhances the proliferation of NSC the firststep of neurogenesis This is the first study comparing theproliferative potential of different PDE5 inhibitors on NSCSeveral studies report the proneurogenic effect of sildenafilon SVZ cultures [39] as in animal models of brain injury [24ndash26] but the effect of sildenafil on neurogenesis was nevercompared to other PDE5 inhibitors We show a significantincrease in the levels of cGMP in NSC treated with PDE5inhibitors which is responsible for the increase in cellproliferation Moreover direct activation of sGC with YC-1 and thus increasing in cGMP production also enhancesNSC proliferation upon 24 h of treatment Likewise a cGMPanalogue 8-Br-cGMP also enhances SVZ cell proliferationas shown in this work and previously by our group [9]

Here we show that inhibiting the MAPK pathway theproliferative effect of PDE5 inhibitors is abolished in bothshort- and long-term exposures which is in agreement withthe established contribution of ERK pathway to the prolif-eration of NSC [5] In addition cell proliferation inducedby the PDE5 inhibitors was also prevented by inhibitors ofsGC (ODQ) or PKG (KT5823) showing the involvement ofsGCPKG pathway We have previously shown that both NOand cGMP analogues enhance SVZ proliferation by a mech-anism that is dependent on activation of PKG concomitantlywith ERK activation [9] The present study suggests that thesame pathways involved in stimulating cell proliferation bythe endogenousmessenger (NO) are also being stimulated byblocking the hydrolysis of cGMP with PDE5 inhibitors

T0156 and zaprinast were able to activate ERK12 aneffect that is prevented by inhibition of PKG suggestingthat ERK12 activation is dependent on the cGMP effectorkinase PKG Several other studies report the regulation of

ERK12 activation by PKG which is involved in multiplecell functions [9 40ndash42] Furthermore treatment with T0156decreases the levels of the cyclin-dependent kinase inhibitor1 p27Kip1 p27Kip1 prevents progression from G1 to S-phasebeing a key regulator of cell division of neural progenitor cells[43 44] The downregulation of p27Kip1 levels by T0156 isconsistent with the increase in cell proliferation and ERK12phosphorylation shown above In agreement with this wefound that blockade of ERKMAPK pathway by inhibitionof MAPK is able to prevent this effect Moreover directactivation of sGC by YC-1 has a similar effect to that ofT0156 in increasing phospho-ERK12 and decreasing p27Kip1levels similar to those previously described with NO orthe cGMP analogue 8-Br-cGMP [9] However althoughzaprinast increases ERK12 phosphorylation it does notappear to affect the levels of p27Kip1 In fact by the lackof selectivity of zaprinast for PDE5 we cannot exclude thepossibility that this inhibitor could interact with other PDEand thus to activate other pathways than the two studiedhere

Interestingly while sildenafil increased NSC prolifera-tion it neither increased ERK12 phosphorylation nor alteredthe levels of p27Kip1 for any of the times tested in thiswork Even though ERK12 is not phosphorylated follow-ing treatment with sildenafil we observed that the MAPKinhibitor U0126 completely blocks sildenafil-enhanced cellproliferation which suggests the involvement of other sig-naling pathways in the proliferative effect of this drug Asimilar lack of effect of sildenafil on ERK12 phosphorylationhas already been described by Wang and colleagues [39]According to that study sildenafil (300 nM) does not increaseERK12 phosphorylation following 1 h of treatment [39]These authors suggest that the increase in cGMP levelsvia inhibition of PDE5 activity enhances neurogenesis inthe SVZ through the PI3-KAkt pathway [39] Within thisscenario sildenafil appears to act in a proliferative pathwaydifferent from the one activated by NO by cGMP analogues[9] and by the other PDE5 inhibitors used in this studyThus understanding the pathways activated by sildenafil isimportant in order to understand its possible value in the fieldof proneurogenic therapies

Therapies involving the specific inhibition of PDE5 arebeing evaluated for the treatment of several neurologicalconditions Regarding the possible enhancement of endoge-nous neurogenesis by this strategy our study helps in betterunderstanding the initial events that promote proliferation ofNSC in the initial stages of neurogenesis by PDE5 inhibition

Conflict of Interests

The authors of this work declare no conflict of interests withany of the commercial entities mentioned in this paper

Acknowledgments

This work was supported by FEDER funds via ProgramaOperacional Factores de Competitividade (COMPETE)and by national funds by the Foundation for Science


and Technology (FCT Portugal) (projects PTDCSAU-NEU1026122008 PTDCSAU-OSD04732012 PEst-CSAULA00012013-2014 and PEst-OEEQBLA00232013-2014) Ana I Santos Bruno P Carreira and Rui J Nobrewere supported by FCT (fellowships SFRHBD779032011SFRHBPD789012011 and SFRHBPD667052009)

References

[1] A Arvidsson T Collin D Kirik Z Kokaia and O LindvallldquoNeuronal replacement from endogenous precursors in theadult brain after strokerdquo Nature Medicine vol 8 no 9 pp 963ndash970 2002

[2] J M Parent Z S Vexler C Gong N Derugin and DM Ferriero ldquoRat forebrain neurogenesis and striatal neuronreplacement after focal strokerdquo Annals of Neurology vol 52 no6 pp 802ndash813 2002

[3] J M Parent V V Valentin and D H Lowenstein ldquoProlongedseizures increase proliferating neuroblasts in the adult ratsubventricullar zone-olfactory bulb pathwayrdquo The Journal ofNeuroscience vol 22 no 8 pp 3174ndash3188 2002

[4] D Y Zhu S H Liu H S Sun and Y M Lu ldquoExpressionof inducible nitric oxide synthase after focal cerebral ischemiastimulates neurogenesis in the adult rodent dentate gyrusrdquoTheJournal of Neuroscience vol 23 no 1 pp 223ndash229 2003

[5] B P Carreira M I Morte A Inacio et al ldquoNitric oxidestimulates the proliferation of neural stem cells bypassing theepidermal growth factor receptorrdquo Stem Cells vol 28 no 7 pp1219ndash1230 2010

[6] C X Luo X J Zhu Q G Zhou et al ldquoReduced neuronal nitricoxide synthase is involved in ischemia-induced hippocampalneurogenesis by up-regulating inducible nitric oxide synthaseexpressionrdquo Journal of Neurochemistry vol 103 no 5 pp 1872ndash1882 2007

[7] J M Parent ldquoAdult neurogenesis in the intact and epilepticdentate gyrusrdquo Progress in Brain Research vol 163 pp 529ndash8172007

[8] W Jiang L Xiao J-C Wang Y-G Huang and X ZhangldquoEffects of nitric oxide on dentate gyrus cell proliferation afterseizures induced by pentylenetrazol in the adult rat brainrdquoNeuroscience Letters vol 367 no 3 pp 344ndash348 2004

[9] B P Carreira M I Morte A S Lourenco et al ldquoDifferentialcontribution of the guanylyl cyclase-cyclic GMP-protein kinaseG pathway to the proliferation of neural stem cells stimulatedby nitric oxiderdquo Neurosignals vol 21 no 1-2 pp 1ndash13 2013

[10] L J Ignarro ldquoSignal transduction mechanisms involving nitricoxiderdquo Biochemical Pharmacology vol 41 no 4 pp 485ndash4901991

[11] F Murad ldquoRegulation of cytosolic guanylyl cyclase by nitricoxide the NO-cyclic GMP signal transduction systemrdquoAdvances in Pharmacology vol 26 pp 19ndash33 1994

[12] K S Madhusoodanan and F Murad ldquoNO-cGMP signalingand regenerative medicine involving stem cellsrdquoNeurochemicalResearch vol 32 no 4-5 pp 681ndash694 2007

[13] M Chalimoniuk and J B Strosznajder ldquoAging modulates nitricoxide synthesis and cGMP levels in hippocampus and cere-bellum effects of amyloid 120573 peptiderdquo Molecular and ChemicalNeuropathology vol 35 no 1ndash3 pp 77ndash95 1998

[14] C Lugnier ldquoCyclic nucleotide phosphodiesterase (PDE) super-family a new target for the development of specific therapeutic

agentsrdquo Pharmacology andTherapeutics vol 109 no 3 pp 366ndash398 2006

[15] K Loughney T R Hill V A Florio et al ldquoIsolation andcharacterization of cDNAs encoding PDE5A a human cGMP-binding cGMP-specific 3101584051015840-cyclic nucleotide phosphodi-esteraserdquo Gene vol 216 no 1 pp 139ndash147 1998

[16] J Kotera K Fujishige and K Omori ldquoImmunohistochemi-cal localization of cGMP-binding cGMP-specific phosphodi-esterase (PDE5) in rat tissuesrdquo Journal of Histochemistry andCytochemistry vol 48 no 5 pp 685ndash693 2000

[17] S H Francis J L Busch and J D Corbin ldquocGMP-dependentprotein kinases and cGMP phosphodiesterases in nitric oxideand cGMP actionrdquo Pharmacological Reviews vol 62 no 3 pp525ndash563 2010

[18] S Uthayathas S S Karuppagounder B M Thrash K Param-eshwaran V Suppiramaniam andM Dhanasekaran ldquoVersatileeffects of sildenafil recent pharmacological applicationsrdquo Phar-macological Reports vol 59 no 2 pp 150ndash163 2007

[19] H A Ghofrani I H Osterloh and F Grimminger ldquoSildenafilfrom angina to erectile dysfunction to pulmonary hypertensionand beyondrdquo Nature Reviews Drug Discovery vol 5 no 8 pp689ndash702 2006

[20] L B Kane and E S Klings ldquoPresent and future treat-ment strategies for pulmonary arterial hypertension focuson phosphodiesterase-5 inhibitorsrdquo Treatments in RespiratoryMedicine vol 5 no 4 pp 271ndash282 2006

[21] M U Farooq B Naravetla P W Moore A Majid R Guptaand M Y Kassab ldquoRole of sildenafil in neurological disordersrdquoClinical Neuropharmacology vol 31 no 6 pp 353ndash362 2008

[22] K Rutten J de Vente A Sik M M Ittersum J Prickaerts andA Blokland ldquoThe selective PDE5 inhibitor sildenafil improvesobject memory in Swiss mice and increases cGMP Levels inhippocampal slicesrdquo Behavioural Brain Research vol 164 no 1pp 11ndash16 2005

[23] L Orejana L Barros-Minones J Jordan E Puerta andN Aguirre ldquoSildenafil ameliorates cognitive deficits and taupathology in a senescence-accelerated mouse modelrdquo Neurobi-ology of Aging vol 33 no 3 pp 625e11ndash625e20 2012

[24] R Zhang Y Wang L Zhang et al ldquoSildenafil (Viagra) inducesneurogenesis and promotes functional recovery after stroke inratsrdquo Stroke vol 33 no 11 pp 2675ndash2680 2002

[25] R L Zhang Z Zhang L Zhang Y Wang C Zhang and MChopp ldquoDelayed treatment with sildenafil enhances neuroge-nesis and improves functional recovery in aged rats after focalcerebral ischemiardquo Journal of Neuroscience Research vol 83 no7 pp 1213ndash1219 2006

[26] L Zhang R L Zhang Y Wang et al ldquoFunctional recoveryin aged and young rats after embolic stroke treatment with aphosphodiesterase type 5 inhibitorrdquo Stroke vol 36 no 4 pp847ndash852 2005

[27] R L Zhang M Chopp C Roberts et al ldquoSildenafil enhancesneurogenesis and oligodendrogenesis in ischemic brain ofmiddle-aged mouserdquo PLoS ONE vol 7 Article ID e48141 2012

[28] S A Ballard C J Gingell K Tang L A TurnerM E Price andA M Naylor ldquoEffects of sildenafil on the relaxation of humancorpus cavernosum tissue in vitro and on the activities of cyclicnucleotide phosphodiesterase isozymesrdquo Journal ofUrology vol159 no 6 pp 2164ndash2171 1998

[29] H Mochida M Takagi H Inoue et al ldquoEnzymological andpharmacological profile of T-0156 a potent and selective phos-phodiesterase type 5 inhibitorrdquo European Journal of Pharmacol-ogy vol 456 no 1ndash3 pp 91ndash98 2002


[30] F Agasse L Bernardino H Kristiansen et al ldquoNeuropeptideY promotes neurogenesis in murine subventricular zonerdquo StemCells vol 26 no 6 pp 1636ndash1645 2008

[31] M I Morte B P Carreira V Machado et al ldquoEvaluation ofproliferation of neural stem cells in vitro and in vivordquo CurrentProtocols in Stem Cell Biology chapter 2unit 2D14 2013

[32] I M Araujo A F Ambrosio E C Leal P F Santos A PCarvalho and C M Carvalho ldquoNeuronal nitric oxide synthaseproteolysis limits the involvement of nitric oxide in kainate-induced neurotoxicity in hippocampal neuronsrdquo Journal ofNeurochemistry vol 85 no 3 pp 791ndash800 2003

[33] R E Taylor K H Shows Y Zhao and S J Pittler ldquoA PDE6Apromoter fragment directs transcription predominantly in thephotoreceptorrdquo Biochemical and Biophysical Research Commu-nications vol 282 no 2 pp 543ndash547 2001

[34] C Gardner N Robas D Cawkill and M Fidock ldquoCloningand characterization of the human and mouse PDE7B a novelcAMP-Specific cyclic nucleotide phosphodiesteraserdquo Biochemi-cal and Biophysical Research Communications vol 272 no 1 pp186ndash192 2000

[35] T Sasaki J Kotera K Yuasa and K Omori ldquoIdentification ofhuman PDE7B a cAMP-specific phosphodiesteraserdquo Biochem-ical and Biophysical Research Communications vol 271 no 3pp 575ndash583 2000

[36] J M Hetman S H Soderling N A Glavas and J A BeavoldquoCloning and characterization of PDE7B a cAMP-specificphosphodiesteraserdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 1 pp 472ndash476 2000

[37] A J Santos-Silva E Cairrao M Morgado E Alvarez and IVerde ldquoPDE4 and PDE5 regulate cyclic nucleotides relaxingeffects in human umbilical arteriesrdquo European Journal of Phar-macology vol 582 no 1ndash3 pp 102ndash109 2008

[38] HToyoshima andTHunter ldquop27 a novel inhibitor ofG1 cyclin-Cdk protein kinase activity is related to p21rdquo Cell vol 78 no 1pp 67ndash74 1994

[39] L Wang Z G Zhang R L Zhang and M Chopp ldquoActi-vation of the PI3-KAkt pathway mediates cGMP enhanced-neurogenesis in the adult progenitor cells derived from thesubventricular zonerdquo Journal of Cerebral Blood Flow andMetabolism vol 25 no 9 pp 1150ndash1158 2005

[40] K T Ota V J Pierre J E Ploski K Queen and G E SchafeldquoThe NO-cGMP-PKG signaling pathway regulates synapticplasticity and fear memory consolidation in the lateral amyg-dala via activation of ERKMAP kinaserdquo Learning andMemoryvol 15 no 10 pp 792ndash805 2008

[41] E F Gallo andC Iadecola ldquoNeuronal nitric oxide contributes toneuroplasticity-associated protein expression through cGMPprotein kinase G and extracellular signal-regulated kinaserdquoTheJournal of Neuroscience vol 31 no 19 pp 6947ndash6955 2011

[42] A Das L Xi and R C Kukreja ldquoProtein kinase G-dependentcardioprotective mechanism of phosphodiesterase-5 inhibitioninvolves phosphorylation of ERK and GSK3120573rdquo The Journal ofBiological Chemistry vol 283 no 43 pp 29572ndash29585 2008

[43] F Doetsch J M-G Verdugo I Caille A Alvarez-Buylla M VChao and P Casaccia-Bonnefil ldquoLack of the cell-cycle inhibitorp27Kip1 results in selective increase of transit-amplifying cellsfor adult neurogenesisrdquoThe Journal of Neuroscience vol 22 no6 pp 2255ndash2264 2002

[44] X Li X Tang B Jablonska A Aguirre V Gallo and MB Luskin ldquoP27KIP1 regulates neurogenesis in the rostral

migratory stream and olfactory bulb of the postnatal mouserdquoThe Journal of Neuroscience vol 29 no 9 pp 2902ndash2914 2009

Impact Factor 173028 Days Fast Track Peer ReviewAll Subject Areas of ScienceSubmit at httpwwwtswjcom

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal


processes like synaptic plasticity (reviewed by [12]) In severalconditions such as aging cGMP levels are decreased andmay be involved in age-related neurodegeneration decreasedneurogenesis and cognitive decline [13] Similar to theincrease of cGMP production by NO the inhibition of cGMPhydrolysis by targeting phosphodiesterases (PDE) could bea strategy to increase the levels of cGMP and consequentlyreverse these effects by stimulating neurogenesis

PDE are ubiquitous enzymes responsible for the degrada-tion of cyclic nucleotides whose activity is dependent on sub-strate kinetic properties and cellular and subcellular distri-bution of the 11 known families PDE5 6 and 9 are consideredcGMP specific PDEwhereas PDE4 7 and 8mainly hydrolyzecyclic AMP (cAMP) and PDE1 2 3 10 and 11 hydrolyzeboth substrates (reviewed in [14]) Phosphodiesterase type5 is specific for cGMP degradation and is present in themammalian brain [15 16] PDE5 inhibitors have been usedfor the treatment of several pathologies in which an increasein cGMP levels can be beneficial such as erectile dysfunctionand pulmonary hypertension (reviewed by [17 18]) The bestcharacterized inhibitor of PDE5 is sildenafil Sildenafil wasfirst used for the treatment of erectile dysfunction Due tothe presence of PDE5 in the lung sildenafil is also used inthe treatment of pulmonary hypertension (reviewed by [18ndash20]) Since PDE5 is also present in the central nervous systemseveral works have assessed the effect of sildenafil on thebrain (reviewed by [21]) Some studies reported that sildenafilimproves learning and memory [22 23] and also inducesneurogenesis in the SVZ and SGZ [24] Neuronal functionrecovery in adult young rats and in aged rats [25] followingstroke has also been described after sildenafil treatment [2627] Zaprinast the first PDE5 inhibitor to be used has beenshown to be a nonpotent and nonselective inhibitor Sildenafilis typically considered a PDE5 inhibitor with an IC

50of

approximately 4 nM and it has been shown to inhibit PDE5 atconcentrations about 200-fold lower than the concentrationof zaprinast required to inhibit PDE5 [28] However it alsoinhibits PDE1 and PDE6 (IC

50values of 281 and 29 nM

resp [28]) indicating that sildenafil is not totally selective forPDE5More recently a new compoundwas developed T0156which potently inhibits PDE5 [29] In fact T0156 has a muchlower IC

50for PDE5 (023 nM) than sildenafil (IC

5036 nM)

Within this scenario in this work we investigated theeffect of three different PDE5 inhibitors T0156 sildenafiland zaprinast on the first step of neurogenesis by evalu-ating proliferation of SVZ-derived NSC and addressed themechanisms underlying these effects Here we show thatT0156 sildenafil and zaprinast increase SVZ-derived NSCproliferationMoreover blockingMAPK sGC or PKG inde-pendently results in loss of the proliferative effect of PDE5inhibitors pointing out to the involvement of sGCPKG andERKMAPK pathways in the stimulation of NSC prolifera-tion by PD5 inhibition

2 Methods

21 Materials Dulbeccorsquos Modified Eaglersquos Medium F-12 nutrient mixture (D-MEMF-12 with GlutaMAX I)

B27 supplement trypsin-ethylenediaminetetraacetic acid(EDTA) solution antibiotics (10000 unitsmL penicillin10mgmL streptomycin) Click-iT EdU Alexa Fluor 488Flow Cytometry Assay kit Click-iT EdU Alexa Fluor 594HCS Assay kit and StemPro Accutase cell dissociationreagent were purchased from Invitrogen (Paisley UK) Epi-dermal growth factor (EGF) and basic fibroblast growthfactor (bFGF) were from PeproTech Inc (London UK)The Matrix used was from Stoelting Co (Wood Dale ILUSA) Bicinchoninic acid (BCA) Protein Assay kit wasfrom Pierce (Rockford IL USA) 8-bromoguanosine 3101584051015840-cyclic monophosphate (8-Br-cGMP) Phenylmethylsulfonylfluoride orthovanadate chymostatin leuptin antipain pep-statin A trypan blue Tween-20 mouse anti-120572-tubulin 2-Propanol chloroform andTRI Reagent were purchased fromSigma Chemical (St Louis MO USA) T0156 hydrochloridesildenafil citrate zaprinast and 1119867-[1 2 4]Oxadiazolo[43-a]quinoxalin-1-one (ODQ) were obtained from Tocris Bio-science (Bristol UK) KT5823 was obtained from AlomoneLabs (Jerusalem Israel) and 3-(51015840-Hydroxymethyl-21015840-furyl)-1-benzylindazole (YC-1) from Santa Cruz Biotechnology(Santa Cruz CA USA) 14-diamino-23-dicyano-14-bis[2-aminophenylthio] butadiene (U0126) rabbit anti-p27Kip1rabbit anti-ERK12 and rabbit anti-phospho-ERK12 werepurchased from Cell Signaling (Danvers MA USA) Poly-merase chain reaction (PCR) primers were obtained fromEurofins MWGOperon (Ebersberg Germany) and FastStartPCR Master Mix and Transcriptor High Fidelity cDNASynthesis kit were from Roche Diagnostics (Mannheim Ger-many) Bovine serum albumin (BSA) was from Calbiochem(San Diego CA USA) and polyvinylidene difluoride mem-branes were purchased from Millipore (Madrid Spain)Amersham cGMP enzyme immunoassay Biotrak Systemenhanced chemifluorescence reagent and anti-rabbit andanti-mouse alkaline phosphatase-conjugated antibodies werefrom GE Healthcare Life Sciences (Buckinghamshire UK)Other reagents used in immunoblotting experiments werepurchased from Bio-Rad (Hercules CA USA)

22 Animals C57BL6J mice were obtained from CharlesRiver (Barcelona Spain) and kept in our animal facilities withfood and water ad libitum in a 12-hour dark light cycle Allexperiments were performed in accordancewith Institutionaland European Guidelines (86609EEC) for the care and useof laboratory animals

23 Subventricular Zone Cell Cultures Neural stem cell cul-tures were obtained from the SVZ of 0ndash3 postnatal dayC57BL6J mice as previously described [30] The fragmentsof SVZ were digested in 0025 trypsin0265mM EDTAfor 15ndash20 minutes at 37∘C and then mechanically dissoci-ated The cells were resuspended in a 37∘C D-MEMF-12medium with 2mM GlutaMAX-I supplemented with 1B27 1 antibiotic (10000 unitsmL penicillin 10mgmLstreptomycin) 10 ngmL EGF and 5 ngmL bFGF and platedon uncoated flasks with filter cap at a density of 100000cellsmL The SVZ stem cells were grown as floating aggre-gates in a 95 air5 CO

2humidified atmosphere at 37∘C










0

50

100

150

200

0 1

EdU

-pos

itive

cells

( o

f con

trol)

6h

T0156 (120583M)

lowast

(a)

0

50

100

150

200

0 1 10 50

EdU

-pos

itive

cells

( o

f con

trol)


lowastlowast

6h

lowast

(b)

0

50

100

150

200

0 10 50

EdU

-pos

itive

cells

( o

f con

trol)

Zaprinast (120583M)

6h

lowastlowast

(c)

0

50

100

150

200

Control T0156

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowast

(d)

0

50

100

150

200

Control Sildenafil

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowast

(e)

0

50

100

150

200

Control Zaprinast

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowast

(f)



3 Results






0

50

100

150

200 6h

lowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

T0156U0126

+minus minus

minus minus

+

+ +

+

(a)

0

50

100

150

200

EdU

-pos

itive

cells

( o

f con

trol)

6h

lowastlowastlowast

SildenafilU0126

+minus minus

minus minus

+

+ +

+++

(b)

0

50

100

150

200

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

ZaprinastU0126

+minus minus

minus minus

+

+ +

(c)

0

50

100

150

200

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowast

+

T0156U0126

+minus minus

minus minus

+

+ +

(d)

0

50

100

150

200Ed

U-p

ositi

ve ce

lls(

of c

ontro

l)

24h

lowast

+

SildenafilU0126

+minus minus

minus minus

+

+ +

(e)

0

50

100

150

200

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowast

+

ZaprinastU0126

+minus minus

minus minus

+

+ +

(f)












0

50

100

150

200

Control T0156

p-ERK 12

ERK 12

lowast

Phos

pho-

ERK

12

leve

ls(

of c

ontro


(a)

0

50

100

150

200

Control Sildenafil

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

p-ERK 12

ERK 12


(b)

0

50

100

150

200

Control Zaprinast

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l) lowastlowast

p-ERK 12

ERK 12


(c)










0

50

100

150

200 6h

lowastlowast

T0156ODQ

+minus minus

minus minus

+

+ +

+

EdU

-pos

itive

cells

( o

f con

trol)

(a)

0

50

100

150

200

lowastlowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

SildenafilODQ

+minus minus

minus minus

+

+ +

+++

(b)

0

50

100

150

200

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

ZaprinastODQ

+minus minus

minus minus

+

+ +

(c)

0

50

100

150

200 24h

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

+

T0156ODQ

+minus minus

minus minus

+

+ +

(d)

0

50

100

150

200Ed

U-p

ositi

ve ce

lls(

of c

ontro

l)

24h

+

lowastlowastlowast

SildenafilODQ

+minus minus

minus minus

+

+ +

(e)

0

50

100

150

200

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowast

+

ZaprinastODQ

+minus minus

minus minus

+

+ +

(f)








0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

6h

lowastlowast

+

EdU

-pos

itive

cells

( o

f con

trol)

(a)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

lowastlowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

++

(b)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

(c)

0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

24h

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

+

(d)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowastlowast

++

(e)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowast

++

(f)


0

50

100

150

200

lowast

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

T0156KT5823

+minus minus

minus minus

+

+ +

p-ERK 12

ERK 12


(a)

0

50

100

150

200

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

SildenafilKT5823

+minus minus

minus minus

+

+ +

p-ERK 12

ERK 12


(b)

0

50

100

150

200

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

ZaprinastKT5823

+minus minus

minus minus

+

+ +

lowast

p-ERK 12

ERK 12


(c)



0

50

100

150

200

Control T0156

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

lowast

27kDa

55kDa

(a)

0

50

100

150

200

Control Sildenafil

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

Control Zaprinast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)


0

50

100

150

200

T0156U0126

+minus minus

minus minus

+

+ +

++

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilU0126

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastU0126

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)


0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)



0

50

100

150

200

Control YC-1

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

(a)

0

50

100

150

200

Control YC-1Ph

osph

o-ER

K 1

2 le

vels

( o

f con

trol) lowast

p-ERK 12

ERK 12


(b)

0

50

100

150

200

YC-1KT5823

+minus minus

minus minus

+

+ +

+

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

p-ERK 12

ERK 12


(c)

0

50

100

150

200

Control YC-1

p27Ki

p1le

vels

( o

f con

trol)

lowast

p27Kip1

120572-Tubulin

27kDa

55kDa

(d)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1U0126

+minus minus

minus minus

+

+ +

+

p27Kip1

120572-Tubulin

27kDa

55kDa

(e)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1KT5823

+minus minus

minus minus

+

+ +

p27Kip1

120572-Tubulin

27kDa

55kDa

(f)









4 Discussion










Acknowledgments




References




























































0

50

100

150

200

0 1

EdU

-pos

itive

cells

( o

f con

trol)

6h

T0156 (120583M)

lowast

(a)

0

50

100

150

200

0 1 10 50

EdU

-pos

itive

cells

( o

f con

trol)


lowastlowast

6h

lowast

(b)

0

50

100

150

200

0 10 50

EdU

-pos

itive

cells

( o

f con

trol)

Zaprinast (120583M)

6h

lowastlowast

(c)

0

50

100

150

200

Control T0156

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowast

(d)

0

50

100

150

200

Control Sildenafil

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowast

(e)

0

50

100

150

200

Control Zaprinast

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowast

(f)



3 Results






0

50

100

150

200 6h

lowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

T0156U0126

+minus minus

minus minus

+

+ +

+

(a)

0

50

100

150

200

EdU

-pos

itive

cells

( o

f con

trol)

6h

lowastlowastlowast

SildenafilU0126

+minus minus

minus minus

+

+ +

+++

(b)

0

50

100

150

200

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

ZaprinastU0126

+minus minus

minus minus

+

+ +

(c)

0

50

100

150

200

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowast

+

T0156U0126

+minus minus

minus minus

+

+ +

(d)

0

50

100

150

200Ed

U-p

ositi

ve ce

lls(

of c

ontro

l)

24h

lowast

+

SildenafilU0126

+minus minus

minus minus

+

+ +

(e)

0

50

100

150

200

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowast

+

ZaprinastU0126

+minus minus

minus minus

+

+ +

(f)












0

50

100

150

200

Control T0156

p-ERK 12

ERK 12

lowast

Phos

pho-

ERK

12

leve

ls(

of c

ontro


(a)

0

50

100

150

200

Control Sildenafil

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

p-ERK 12

ERK 12


(b)

0

50

100

150

200

Control Zaprinast

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l) lowastlowast

p-ERK 12

ERK 12


(c)










0

50

100

150

200 6h

lowastlowast

T0156ODQ

+minus minus

minus minus

+

+ +

+

EdU

-pos

itive

cells

( o

f con

trol)

(a)

0

50

100

150

200

lowastlowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

SildenafilODQ

+minus minus

minus minus

+

+ +

+++

(b)

0

50

100

150

200

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

ZaprinastODQ

+minus minus

minus minus

+

+ +

(c)

0

50

100

150

200 24h

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

+

T0156ODQ

+minus minus

minus minus

+

+ +

(d)

0

50

100

150

200Ed

U-p

ositi

ve ce

lls(

of c

ontro

l)

24h

+

lowastlowastlowast

SildenafilODQ

+minus minus

minus minus

+

+ +

(e)

0

50

100

150

200

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowast

+

ZaprinastODQ

+minus minus

minus minus

+

+ +

(f)








0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

6h

lowastlowast

+

EdU

-pos

itive

cells

( o

f con

trol)

(a)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

lowastlowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

++

(b)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

(c)

0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

24h

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

+

(d)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowastlowast

++

(e)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowast

++

(f)


0

50

100

150

200

lowast

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

T0156KT5823

+minus minus

minus minus

+

+ +

p-ERK 12

ERK 12


(a)

0

50

100

150

200

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

SildenafilKT5823

+minus minus

minus minus

+

+ +

p-ERK 12

ERK 12


(b)

0

50

100

150

200

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

ZaprinastKT5823

+minus minus

minus minus

+

+ +

lowast

p-ERK 12

ERK 12


(c)



0

50

100

150

200

Control T0156

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

lowast

27kDa

55kDa

(a)

0

50

100

150

200

Control Sildenafil

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

Control Zaprinast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)


0

50

100

150

200

T0156U0126

+minus minus

minus minus

+

+ +

++

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilU0126

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastU0126

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)


0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)



0

50

100

150

200

Control YC-1

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

(a)

0

50

100

150

200

Control YC-1Ph

osph

o-ER

K 1

2 le

vels

( o

f con

trol) lowast

p-ERK 12

ERK 12


(b)

0

50

100

150

200

YC-1KT5823

+minus minus

minus minus

+

+ +

+

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

p-ERK 12

ERK 12


(c)

0

50

100

150

200

Control YC-1

p27Ki

p1le

vels

( o

f con

trol)

lowast

p27Kip1

120572-Tubulin

27kDa

55kDa

(d)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1U0126

+minus minus

minus minus

+

+ +

+

p27Kip1

120572-Tubulin

27kDa

55kDa

(e)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1KT5823

+minus minus

minus minus

+

+ +

p27Kip1

120572-Tubulin

27kDa

55kDa

(f)









4 Discussion










Acknowledgments




References




















































0

50

100

150

200 6h

lowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

T0156U0126

+minus minus

minus minus

+

+ +

+

(a)

0

50

100

150

200

EdU

-pos

itive

cells

( o

f con

trol)

6h

lowastlowastlowast

SildenafilU0126

+minus minus

minus minus

+

+ +

+++

(b)

0

50

100

150

200

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

ZaprinastU0126

+minus minus

minus minus

+

+ +

(c)

0

50

100

150

200

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowast

+

T0156U0126

+minus minus

minus minus

+

+ +

(d)

0

50

100

150

200Ed

U-p

ositi

ve ce

lls(

of c

ontro

l)

24h

lowast

+

SildenafilU0126

+minus minus

minus minus

+

+ +

(e)

0

50

100

150

200

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowast

+

ZaprinastU0126

+minus minus

minus minus

+

+ +

(f)












0

50

100

150

200

Control T0156

p-ERK 12

ERK 12

lowast

Phos

pho-

ERK

12

leve

ls(

of c

ontro


(a)

0

50

100

150

200

Control Sildenafil

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

p-ERK 12

ERK 12


(b)

0

50

100

150

200

Control Zaprinast

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l) lowastlowast

p-ERK 12

ERK 12


(c)










0

50

100

150

200 6h

lowastlowast

T0156ODQ

+minus minus

minus minus

+

+ +

+

EdU

-pos

itive

cells

( o

f con

trol)

(a)

0

50

100

150

200

lowastlowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

SildenafilODQ

+minus minus

minus minus

+

+ +

+++

(b)

0

50

100

150

200

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

ZaprinastODQ

+minus minus

minus minus

+

+ +

(c)

0

50

100

150

200 24h

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

+

T0156ODQ

+minus minus

minus minus

+

+ +

(d)

0

50

100

150

200Ed

U-p

ositi

ve ce

lls(

of c

ontro

l)

24h

+

lowastlowastlowast

SildenafilODQ

+minus minus

minus minus

+

+ +

(e)

0

50

100

150

200

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowast

+

ZaprinastODQ

+minus minus

minus minus

+

+ +

(f)








0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

6h

lowastlowast

+

EdU

-pos

itive

cells

( o

f con

trol)

(a)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

lowastlowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

++

(b)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

(c)

0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

24h

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

+

(d)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowastlowast

++

(e)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowast

++

(f)


0

50

100

150

200

lowast

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

T0156KT5823

+minus minus

minus minus

+

+ +

p-ERK 12

ERK 12


(a)

0

50

100

150

200

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

SildenafilKT5823

+minus minus

minus minus

+

+ +

p-ERK 12

ERK 12


(b)

0

50

100

150

200

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

ZaprinastKT5823

+minus minus

minus minus

+

+ +

lowast

p-ERK 12

ERK 12


(c)



0

50

100

150

200

Control T0156

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

lowast

27kDa

55kDa

(a)

0

50

100

150

200

Control Sildenafil

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

Control Zaprinast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)


0

50

100

150

200

T0156U0126

+minus minus

minus minus

+

+ +

++

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilU0126

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastU0126

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)


0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)



0

50

100

150

200

Control YC-1

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

(a)

0

50

100

150

200

Control YC-1Ph

osph

o-ER

K 1

2 le

vels

( o

f con

trol) lowast

p-ERK 12

ERK 12


(b)

0

50

100

150

200

YC-1KT5823

+minus minus

minus minus

+

+ +

+

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

p-ERK 12

ERK 12


(c)

0

50

100

150

200

Control YC-1

p27Ki

p1le

vels

( o

f con

trol)

lowast

p27Kip1

120572-Tubulin

27kDa

55kDa

(d)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1U0126

+minus minus

minus minus

+

+ +

+

p27Kip1

120572-Tubulin

27kDa

55kDa

(e)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1KT5823

+minus minus

minus minus

+

+ +

p27Kip1

120572-Tubulin

27kDa

55kDa

(f)









4 Discussion










Acknowledgments




References




















































0

50

100

150

200

Control T0156

p-ERK 12

ERK 12

lowast

Phos

pho-

ERK

12

leve

ls(

of c

ontro


(a)

0

50

100

150

200

Control Sildenafil

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

p-ERK 12

ERK 12


(b)

0

50

100

150

200

Control Zaprinast

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l) lowastlowast

p-ERK 12

ERK 12


(c)










0

50

100

150

200 6h

lowastlowast

T0156ODQ

+minus minus

minus minus

+

+ +

+

EdU

-pos

itive

cells

( o

f con

trol)

(a)

0

50

100

150

200

lowastlowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

SildenafilODQ

+minus minus

minus minus

+

+ +

+++

(b)

0

50

100

150

200

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

ZaprinastODQ

+minus minus

minus minus

+

+ +

(c)

0

50

100

150

200 24h

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

+

T0156ODQ

+minus minus

minus minus

+

+ +

(d)

0

50

100

150

200Ed

U-p

ositi

ve ce

lls(

of c

ontro

l)

24h

+

lowastlowastlowast

SildenafilODQ

+minus minus

minus minus

+

+ +

(e)

0

50

100

150

200

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowast

+

ZaprinastODQ

+minus minus

minus minus

+

+ +

(f)








0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

6h

lowastlowast

+

EdU

-pos

itive

cells

( o

f con

trol)

(a)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

lowastlowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

++

(b)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

(c)

0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

24h

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

+

(d)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowastlowast

++

(e)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowast

++

(f)


0

50

100

150

200

lowast

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

T0156KT5823

+minus minus

minus minus

+

+ +

p-ERK 12

ERK 12


(a)

0

50

100

150

200

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

SildenafilKT5823

+minus minus

minus minus

+

+ +

p-ERK 12

ERK 12


(b)

0

50

100

150

200

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

ZaprinastKT5823

+minus minus

minus minus

+

+ +

lowast

p-ERK 12

ERK 12


(c)



0

50

100

150

200

Control T0156

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

lowast

27kDa

55kDa

(a)

0

50

100

150

200

Control Sildenafil

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

Control Zaprinast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)


0

50

100

150

200

T0156U0126

+minus minus

minus minus

+

+ +

++

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilU0126

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastU0126

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)


0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)



0

50

100

150

200

Control YC-1

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

(a)

0

50

100

150

200

Control YC-1Ph

osph

o-ER

K 1

2 le

vels

( o

f con

trol) lowast

p-ERK 12

ERK 12


(b)

0

50

100

150

200

YC-1KT5823

+minus minus

minus minus

+

+ +

+

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

p-ERK 12

ERK 12


(c)

0

50

100

150

200

Control YC-1

p27Ki

p1le

vels

( o

f con

trol)

lowast

p27Kip1

120572-Tubulin

27kDa

55kDa

(d)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1U0126

+minus minus

minus minus

+

+ +

+

p27Kip1

120572-Tubulin

27kDa

55kDa

(e)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1KT5823

+minus minus

minus minus

+

+ +

p27Kip1

120572-Tubulin

27kDa

55kDa

(f)









4 Discussion










Acknowledgments




References




















































0

50

100

150

200 6h

lowastlowast

T0156ODQ

+minus minus

minus minus

+

+ +

+

EdU

-pos

itive

cells

( o

f con

trol)

(a)

0

50

100

150

200

lowastlowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

SildenafilODQ

+minus minus

minus minus

+

+ +

+++

(b)

0

50

100

150

200

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

ZaprinastODQ

+minus minus

minus minus

+

+ +

(c)

0

50

100

150

200 24h

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

+

T0156ODQ

+minus minus

minus minus

+

+ +

(d)

0

50

100

150

200Ed

U-p

ositi

ve ce

lls(

of c

ontro

l)

24h

+

lowastlowastlowast

SildenafilODQ

+minus minus

minus minus

+

+ +

(e)

0

50

100

150

200

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowast

+

ZaprinastODQ

+minus minus

minus minus

+

+ +

(f)








0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

6h

lowastlowast

+

EdU

-pos

itive

cells

( o

f con

trol)

(a)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

lowastlowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

++

(b)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

(c)

0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

24h

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

+

(d)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowastlowast

++

(e)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowast

++

(f)


0

50

100

150

200

lowast

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

T0156KT5823

+minus minus

minus minus

+

+ +

p-ERK 12

ERK 12


(a)

0

50

100

150

200

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

SildenafilKT5823

+minus minus

minus minus

+

+ +

p-ERK 12

ERK 12


(b)

0

50

100

150

200

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

ZaprinastKT5823

+minus minus

minus minus

+

+ +

lowast

p-ERK 12

ERK 12


(c)



0

50

100

150

200

Control T0156

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

lowast

27kDa

55kDa

(a)

0

50

100

150

200

Control Sildenafil

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

Control Zaprinast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)


0

50

100

150

200

T0156U0126

+minus minus

minus minus

+

+ +

++

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilU0126

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastU0126

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)


0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)



0

50

100

150

200

Control YC-1

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

(a)

0

50

100

150

200

Control YC-1Ph

osph

o-ER

K 1

2 le

vels

( o

f con

trol) lowast

p-ERK 12

ERK 12


(b)

0

50

100

150

200

YC-1KT5823

+minus minus

minus minus

+

+ +

+

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

p-ERK 12

ERK 12


(c)

0

50

100

150

200

Control YC-1

p27Ki

p1le

vels

( o

f con

trol)

lowast

p27Kip1

120572-Tubulin

27kDa

55kDa

(d)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1U0126

+minus minus

minus minus

+

+ +

+

p27Kip1

120572-Tubulin

27kDa

55kDa

(e)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1KT5823

+minus minus

minus minus

+

+ +

p27Kip1

120572-Tubulin

27kDa

55kDa

(f)









4 Discussion










Acknowledgments




References




















































0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

6h

lowastlowast

+

EdU

-pos

itive

cells

( o

f con

trol)

(a)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

lowastlowastlowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

++

(b)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

lowast

EdU

-pos

itive

cells

( o

f con

trol)

6h

+

(c)

0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

24h

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

+

(d)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowastlowast

++

(e)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

EdU

-pos

itive

cells

( o

f con

trol)

24h

lowastlowast

++

(f)


0

50

100

150

200

lowast

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

T0156KT5823

+minus minus

minus minus

+

+ +

p-ERK 12

ERK 12


(a)

0

50

100

150

200

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

SildenafilKT5823

+minus minus

minus minus

+

+ +

p-ERK 12

ERK 12


(b)

0

50

100

150

200

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

ZaprinastKT5823

+minus minus

minus minus

+

+ +

lowast

p-ERK 12

ERK 12


(c)



0

50

100

150

200

Control T0156

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

lowast

27kDa

55kDa

(a)

0

50

100

150

200

Control Sildenafil

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

Control Zaprinast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)


0

50

100

150

200

T0156U0126

+minus minus

minus minus

+

+ +

++

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilU0126

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastU0126

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)


0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)



0

50

100

150

200

Control YC-1

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

(a)

0

50

100

150

200

Control YC-1Ph

osph

o-ER

K 1

2 le

vels

( o

f con

trol) lowast

p-ERK 12

ERK 12


(b)

0

50

100

150

200

YC-1KT5823

+minus minus

minus minus

+

+ +

+

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

p-ERK 12

ERK 12


(c)

0

50

100

150

200

Control YC-1

p27Ki

p1le

vels

( o

f con

trol)

lowast

p27Kip1

120572-Tubulin

27kDa

55kDa

(d)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1U0126

+minus minus

minus minus

+

+ +

+

p27Kip1

120572-Tubulin

27kDa

55kDa

(e)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1KT5823

+minus minus

minus minus

+

+ +

p27Kip1

120572-Tubulin

27kDa

55kDa

(f)









4 Discussion










Acknowledgments




References




















































0

50

100

150

200

Control T0156

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

lowast

27kDa

55kDa

(a)

0

50

100

150

200

Control Sildenafil

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

Control Zaprinast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)


0

50

100

150

200

T0156U0126

+minus minus

minus minus

+

+ +

++

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilU0126

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastU0126

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)


0

50

100

150

200

T0156KT5823

+minus minus

minus minus

+

+ +

lowast

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(a)

0

50

100

150

200

SildenafilKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(b)

0

50

100

150

200

ZaprinastKT5823

+minus minus

minus minus

+

+ +

p27Ki

p1le

vels

( o

f con

trol)

p27Kip1

120572-Tubulin

27kDa

55kDa

(c)



0

50

100

150

200

Control YC-1

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

(a)

0

50

100

150

200

Control YC-1Ph

osph

o-ER

K 1

2 le

vels

( o

f con

trol) lowast

p-ERK 12

ERK 12


(b)

0

50

100

150

200

YC-1KT5823

+minus minus

minus minus

+

+ +

+

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

p-ERK 12

ERK 12


(c)

0

50

100

150

200

Control YC-1

p27Ki

p1le

vels

( o

f con

trol)

lowast

p27Kip1

120572-Tubulin

27kDa

55kDa

(d)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1U0126

+minus minus

minus minus

+

+ +

+

p27Kip1

120572-Tubulin

27kDa

55kDa

(e)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1KT5823

+minus minus

minus minus

+

+ +

p27Kip1

120572-Tubulin

27kDa

55kDa

(f)









4 Discussion










Acknowledgments




References




















































0

50

100

150

200

Control YC-1

EdU

-pos

itive

cells

( o

f con

trol) lowastlowast

(a)

0

50

100

150

200

Control YC-1Ph

osph

o-ER

K 1

2 le

vels

( o

f con

trol) lowast

p-ERK 12

ERK 12


(b)

0

50

100

150

200

YC-1KT5823

+minus minus

minus minus

+

+ +

+

Phos

pho-

ERK

12

leve

ls(

of c

ontro

l)

p-ERK 12

ERK 12


(c)

0

50

100

150

200

Control YC-1

p27Ki

p1le

vels

( o

f con

trol)

lowast

p27Kip1

120572-Tubulin

27kDa

55kDa

(d)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1U0126

+minus minus

minus minus

+

+ +

+

p27Kip1

120572-Tubulin

27kDa

55kDa

(e)

0

50

100

150

200

p27Ki

p1le

vels

( o

f con

trol)

YC-1KT5823

+minus minus

minus minus

+

+ +

p27Kip1

120572-Tubulin

27kDa

55kDa

(f)









4 Discussion










Acknowledgments




References





















































4 Discussion










Acknowledgments




References





















































References



















































stimulation of neural stem cell proliferation by inhibition of phosphodiesterase 5

Documents