Benjamin Hibbert,1,2 Jessie R. Lavoie,3,4 Xiaoli Ma,1 Tara Seibert,2 Joshua E. Raizman,2 Trevor Simard,1

Yong-Xiang Chen,1 Duncan Stewart,3,4 and Edward R. O’Brien1,5

Glycogen Synthase Kinase-3bInhibition Augments DiabeticEndothelial Progenitor CellAbundance and Functionalityvia Cathepsin B: A NovelTherapeutic Opportunity forArterial Repair

Progenitor cell therapy is hindered in patients withdiabetes mellitus (DM) due to cellular senescence.Glycogen synthase kinase-3b (GSK3b) activity isincreased in DM, potentially exacerbating impairedcell-based therapies. Thus, we aimed to determine ifand how GSK3b inhibitors (GSKi) can improvetherapeutic efficacy of endothelial progenitor cells(EPC) from patients with DM. Patients with DM hadfewer EPCs and increased rates of apoptosis. DMEPCs also exhibited higher levels of GSK3b activityresulting in increased levels of phosphorylatedb-catenin. Proteomic profiling of DM EPCs treatedwith GSKi identified 37 nonredundant, differentiallyregulated proteins. Cathepsin B (cathB) wassubsequently confirmed to be differentiallyregulated and showed 40% less baseline activity inDM EPCs, an effect reversed by GSKi treatment.Finally, in vivo efficacy of cell-based therapy was

assessed in a xenotransplant femoral wire injurymouse model. Administration of DM EPCs reducedthe intima-to-media ratio, an effect that was furtheraugmented when DM EPCs were pretreated withGSKi yet absent when cathB was antagonized. InDM, increased basal GSK3b activity contributes toaccelerated EPC cellular senescence, an effectreversed by small molecule antagonism of GSK3b,which enhanced cell-based therapy after vascularinjury.Diabetes 2014;63:1410–1421 | DOI: 10.2337/db13-0941

Use of endothelial progenitor cell (EPC) populations forcell-based therapies is beneficial in a host of cardiovas-cular conditions, including peripheral vascular disease,pulmonary arterial hypertension, and myocardial in-farction. However, the administration of autologous

1Division of Cardiology, University of Ottawa Heart Institute, Ottawa, Ontario,Canada2Department of Biochemistry, Microbiology and Immunology, University ofOttawa, Ottawa, Ontario, Canada3Ottawa Hospital Research Institute, Sprott Stem Cell Centre and RegenerativeMedicine Program, The Ottawa Hospital, Ottawa, Ontario, Canada4Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa,Ontario, Canada5Division of Cardiology, Libin Cardiovascular Institute of Alberta, Calgary,Alberta, Canada

Corresponding author: Edward R. O’Brien, ed.obrien@albertahealthservices.ca.

Received 23 June 2013 and accepted 21 November 2013.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0941/-/DC1.

© 2014 by the American Diabetes Association. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

See accompanying article, p. 1194.

1410 Diabetes Volume 63, April 2014

PHARMACOLOGYAND

THERAPEUTIC

S

EPCs in patients with established disease is often hin-dered due to attenuated cellular yield and biologic ac-tivity. Accordingly, a myriad of strategies have been usedwith the goal of improving EPC cellular yield, survival,and function, including overexpression of endothelialnitric oxide synthase (1) or administration of smallmolecule inhibitors of glycogen synthase kinase-3b(GSK3b) (2).

GSK3b is a ubiquitously expressed serine/threonineprotein kinase negatively regulated by Wnt signaling.Notably, GSK3b is regulated by various modalities, in-cluding phosphorylation, substrate priming, retention ininhibitory complexes, and subcellular compartmentali-zation (3–8). Accordingly, GSK3b levels do not neces-sarily reflect actual physiologic activity. Under basalconditions, GKS3b phosphorylates b-catenin (b-cat)resulting in proteasomal degradation of this nucleartranscription factor. Pharmacologic GSK3b inhibition orWnt3a stimulation promotes hematopoietic stem cellself-renewal and repopulation of cell lineages in vitro andin vivo (9). In EPCs, GSK3b inhibition by transfection ofa dominant negative mutant or by small molecule in-hibition improves the therapeutic capacity of cells,thereby augmenting angiogenesis in ischemia models andimproving arterial repair after vascular injury (2,10). Thisis of particular interest given that GSK3b expression andactivity are dysregulated in patients with diabetes mel-litus (DM) (11) a population in whom EPC function isattenuated (12).

Hence, we sought to explore the potential for phar-macologic inhibition of GSK3b to improve arterial repairin patients with DM. Herein we demonstrate that in-hibition of GSK3b in DM EPCs abrogates apoptosis andimproves EPC yields in vitro. Moreover, using a proteo-mic approach, we identify and confirm the differentialregulation of candidate proteins for the observed benefitsof GSK3b inhibition. Among the identified proteins, in-creased cathepsin B (cathB) activity is demonstrated tobe essential for reductions in EPC apoptosis and neces-sary for increased efficacy in a model of cell-based ther-apy. These findings suggest that inhibition of GSK3b isan important strategy for improving autologous cell-based therapy in patients with DM and acts througha novel mechanism involving increased activity of cathB.

RESEARCH DESIGN AND METHODS

Cell Isolation

EPCs were isolated as previously described (2,10,13). Cellswere plated on human fibronectin (Sigma-Aldrich) coatedsix-well plates at a density of 5.0 3 106 peripheral bloodmononuclear cells per well in endothelial growth media-2(EGM2; Lonza). After 4 days in culture, nonadherent cellswere removed and plates washed with PBS. All experi-ments performed with day 4–7 cells, with samples fromindividual donors representing a single replicate. Forenumeration, EPCs were incubated with 1,1’-dioctadecyl-3,3,39,39-tetramethlyiodocarbocyanine-acetylated LDL

(acLDL, 2.5 mg/mL; Invitrogen), followed by fluoresceinisothiocyanate-conjugated Ulex europaeus agglutinin-1(5 mg/mL; Sigma-Aldrich) and then counterstainedwith DAPI.

GSK3b Inhibitors

GSK-3b small molecule and peptide inhibitors (GSKi)were assayed for efficacy in increasing cell yield andblocking phosphorylation of b-cat. All inhibitors werediluted in DMSO to a final concentration of 0.1% orsterile PBS if water-soluble. Specifically, AR-A014418(Sigma-Aldrich), CHIR98014 (Cedarlane), (22,3E)-6-bromoindirubin-3-oxime (Calbiochem), GSK peptideinhibitor (Calbiochem), and LiCl (20 nmol/L; Sigma-Aldrich) were tested for in vitro efficacy.

Apoptosis Assay

EPCs were maintained under basal culture conditions orserum-starved for 24 h as indicated. Nonadherent cellswere removed by washing with PBS. Subsequently, ad-herent cells were lifted by gentle agitation with 1 mmol/LEDTA. Cells were counted, and 1 3 105 EPCs werestained with Annexin V-fluorescein isothiocyanate andpropidium iodide, according to the manufacturer’sinstructions (Becton, Dickinson and Company). All flowstudies were performed on a Beckman Coulter CytomicsFC 500 cytometer. Early apoptotic cells were defined asAnnexinV+/propidium iodide–.

Protein Lysate Preparation

Cells were centrifuged at 220g for 5 min at room tem-perature. Cell pellets were put on ice, and cell lysis buffer(7 mol/L urea [w/v], 2 mol/L thiourea [w/v], 4% CHAPS[w/v], and 1% dithiothreitol [DTT; w/v]) was added. Celllysates were vortexed and kept at room temperaturefor 30 min to enable protein solubilization. Cell lysateswere then sonicated, vortexed, and centrifuged at 14,000gfor 15 min at room temperature. The supernatant wastransferred, and protein quantification was realized withthe 2-D Quant Kit (GE Healthcare).

Two-Dimensional PAGE

The total proteins (30 mg) were passively rehydratedovernight and applied to immobilized pH gradient strips(11 cm, pH 4–7; Bio-Rad). Isoelectric focusing was doneusing the Agilent fractionator (Agilent) in the in-gelmode. Each focused strip was subsequently equilibratedin 4.0 mL equilibration buffer I (6 mol/L urea [w/v];50 mmol/L Tris-Cl, pH 8.8; 2% SDS [w/v]; 30% glycerol[v/v]; bromophenol blue [trace]; 1% DTT [w/v]) for15 min with gentle agitation, followed by the equilibra-tion buffer II (equilibration solution I with DTT replacedby 2.5% iodoacetamide [w/v]) for 15 min with gentleagitation. The two-dimensional (2-D) separation wasperformed on a 10% SDS-PAGE gel in an Ettan DALTsixElectrophoresis Unit (GE Healthcare) at 10 mA per gel at25°C for ;18 h. Two technical replicates were doneindependently for each biological sample.

diabetes.diabetesjournals.org Hibbert and Associates 1411

Image Acquisition and 2-D Gel Analyses

The same scanning conditions were used for each gel.The scanned gels were analyzed for gel-to-gel matchingusing PDQuest 2-D analysis software (advanced version8.0; Bio-Rad), according to the protocol provided by thedeveloper. Each matched protein spot was assigned aunique sample spot protein number. For gel comparison,a statistical approach was applied for determining sta-tistically differentially regulated proteins using thePDQuest software. The Student t test was performedwith 95% significance level to determine which proteinswere statistically differentially regulated between thehealthy cells and the patient cells nontreated and thepatient cells nontreated and treated with GSKi. A minimumof 1.5-fold change was considered for the upregulatedproteins and 0.67-fold for downregulated proteins. Proteinspots with differential expression patterns on 2-D gelelectrophoresis maps were excised with the automated spotexcision robot, the EXQuest spot cutter (Bio-Rad).

Protein Identification

Liquid chromatography-mass spectrometry (LC-MS)analysis was performed at the Ottawa Hospital ResearchInstitute Proteomics Core Facility (Ottawa, ON, Canada).Peptides were loaded onto a peptide trap (Agilent) for5 min at 15 mL/min using a Dionex UltiMate 3000 RSLCnano high-performance LC. Peptides were eluted overa 20-min gradient of 3–45% acetonitrile with 0.1% for-mic acid at 0.3 mL/min onto a 10-cm analytical column(New Objective Picofrit self-packed with Zorbax C18) andsprayed directly into a LTQ Orbitrap XL hybrid MS usinga nanospray source (Thermo Scientific, Waltham, MA).MS data were acquired in a data-dependent fashion, withMS scans acquired in the Fourier transform cell whiletandem MS (MS/MS) scans were acquired in the ion trapmodule.

MS/MS spectra were matched against a custom da-tabase (2011_07_human_con) consisting of humansequences from SwissProt (2011_07 version ofuniprot_sprot.fasta.gz from ftp.uniprot.org) concatenatedwith a database of common contaminants (downloadedfrom maxquant.org on 9 June 2011) using MASCOT2.3.01 software (Matrix Science, London, U.K.) with MStolerance of 65 ppm and MS/MS tolerance of 0.6 Da.Oxidation of methionine, carbamidomethylation of cys-teine, deamidation, protein N-terminal acetylation, con-version of peptide N-terminal Glu or Gln to Pyro-Glu,and phosphorylation of serine or threonine were allowedas potential modifications.

Quantitative PCR

Total RNA was isolated using Trizol (Invitrogen) andpurified using RNeasy minikits. Subsequently, RNA wasquantified using a NanoDrop 1000 (Thermo Scientific),and real-time PCR was performed using Omniscript kit asdirected (Qiagen). All real-time PCR experiments wereperformed using the SYBR Green Jumpstart Taq Ready

Mix (Sigma-Aldrich) on a LightCycler 480 (Roche) andanalyzed with accompanying software according to thePfaffl method (14).

Western Blots

Western blots were performed using standard techni-ques. Briefly, protein was isolated in radio-immunoprecipitation assay buffer using a ratio of 50 mL/1million cells. The sample was then allowed to incubate onice for 30 min, followed by centrifugation. The super-natant was assayed using a standard bicinchoninic acidassay (Thermo Scientific). Protein was then separated on10% acrylamide gels and transferred to polyvinylidenefluoride membranes using iBlot as directed (Invitrogen).After transfer, the membrane was blocked for 1 h with5% skim milk in Tris-buffered saline-Tween 20 at roomtemperature. Primary antibodies were incubated over-night at 4ºC. Primary antibodies were plasminogen ac-tivator inhibitor-2 (PAI-2, 8:1000; AP6562c, Abgent),b-actin (1:100000; Sigma-Aldrich), gelsolin (1:1000;ab11081, Abcam), GDP dissociation inhibitor-2 (1:2000;ab49193, Abcam), small calcium-binding mitochondrialcarrier-1 (SCaMC-1) protein (1:500; sc-133987, SantaCruz Biotechnology), and CatB (1:10000; ab58802,Abcam). Membranes were then washed and incubatedwith biotinylated secondary antibodies (Santa Cruz Bio-technology) for 1 h, then visualized using ECL Plus(Amersham Biosciences).

CathB Activity Assay

CathB activity was assayed in day 5 EPCs using a stan-dardized CatB fluorometric assay kit as directed (Abcam).Briefly, EPCs were washed with PBS, lifted with EDTA,and 5 3 106 cells collected by centrifugation. Cells werelysed by incubation with cell lysis buffer, pelleted, and50 mL was transferred to a 96-well plate. Subsequently,2 mL of 10 mmol/L cathB substrate labeled with amino-4-trifluoromethyl coumarin was added, and samples wereincubated for 2 h. Plates were read on the SynergyMxmicroplate reader (BioTek).

Vascular Endothelial Growth Factor Secretion Assay

EPCs were cultured using standard techniques to day 4.Cells were subsequently lifted, counted, and replated in96-well plates at equivalent densities in vascular endo-thelial growth factor (VEGF)-free media with treatmentas indicated. After 24 h, media was removed and assayedfor VEGF levels using a standard VEGF ELISA kit (R&DSystems) according to the manufacturer’s protocol.

Human Umbilical Vein Endothelial Cells AdhesionAssay

Human umbilical vein endothelial cells (HUVEC) werecultured to confluence in 96-well plates, then treatedwith 10 ng/mL tumor necrosis factor-a for 6 h to acti-vate cells. EPCs (106) were cultured in with 5 mmol/Lcalcein acetomethoxy for 30 min, lifted with EDTA, pel-leted, and resuspended in EGM-2. Subsequently, 4 3 104

cells were plated on the activated HUVECs and allowed to

1412 GSK3b and Cell-Based Therapy in DM Diabetes Volume 63, April 2014

adhere for 1 h. Plates were read on the SynergyMxmicroplate reader, washed three times with PBS, andreread. Adherence was expressed as the percentage offluorescence retained after washing.

Cell Invasion Assay

Day 5 EPCs were treated as indicated. A modified Boydenchamber and a 12-mm Nuclepore filter (BectonDickinson) with a matrigel matrix (Becton Dickinson) wasused to assay EPC invasiveness. Briefly, 5 3 106 EPCswere placed in the upper chamber with endothelial basalmedium and with EGM-2 in the lower chamber. Cellswere permitted to migrate for 24 h at 37ºC. Cells werecounterstained with DAPI and counted in six randomhigh-power fields.

CD-1 Nude Femoral Artery Wire Injury Model

CD-1 nude athymic male mice were purchased fromCharles River Laboratories and permitted to acclimatizefor 2 to 6 weeks before surgery. Mice underwent femoralartery wire injury as previously described (15). Sub-sequently, 2 3 105 EPCs from patients with DM were in-fused into the adjacent vein using a blunt needle. None ofthe animals exhibited ischemia in the hind limb. Mice wererecovered and were killed at 14 days for tissue analysis.

Tissue Processing

Mice underwent perfusion fixation with buffered for-malin at time they were killed. Arteries were fixed for24 h in formalin and dehydrated in ethanol. Arterieswere mounted in paraffin blocks and sectioned in 5-mmsections until 100 mm from the branch vessel site. Thesesections were stained with hematoxylin and eosin, andanalysis was performed using a computer-assisted digitalimaging system (Image-Pro Plus; Media Cybernetics).

Ethics and Statistics

All protocols involving human donors were approved bythe Ottawa Heart Institute Research Ethics Committee,with participants providing written informed consent.These studies conformed with the Declaration ofHelsinki for the use of human tissue. Animal experi-mental protocols were approved by the University ofOttawa Animal Care Committee and adhered to theCanadian Council on Animal Care guidelines.

Data are expressed as mean 6 SEM. Statistical sig-nificance was determined for P , 0.05. Pairwise com-parisons were performed using a paired Student t test,with multiple comparisons performed with a one- or two-way ANOVA, with Holm-�Sídák post hoc testing as ap-propriate.

RESULTS

DM Accelerates Apoptosis in EPCs Through IncreasedGSK3b Activity

EPCs isolated from human subjects were cultured for7 days and then characterized using immunolabeling forUlex europeus agglutinin-1 and acetylated-LDL uptake

(Fig. 1A). The baseline characteristics of the humansubjects are presented in Supplementary Table 1. Sam-ples derived from patients with DM yielded fewer EPCsthan those derived from healthy controls (n = 12, 14.9 64.6 vs. 38.5 6 6.7 cells per high-power field, P , 0.01;Fig. 1B). Several GSKis were supplemented in increasingconcentrations to identify the optimal inhibitor andconcentration. CHIR98014 at a concentration of1 mmol/L yielded optimal EPC yields and significantlygreater inhibition of GSK3b (Supplementary Fig. 1B andC). Subsequently, CHIR98014 was used as the preferen-tial GSKi in all experiments.

Notably, supplementation of the culture media withGSKi resulted in ;300% increases in the yields of EPCsin DM and in healthy controls (P , 0.01; Fig. 1C). Underbasal conditions, the apoptosis index at 96 h was higherin patients with DM as measured by annexin V andpropidium iodide double labeling (9.2 6 0.9 vs. 7.3 60.9, P = 0.02; Fig. 1D) an effect attenuated throughGSK3b inhibition. As expected, serum starvation, used toreproduce cell stress after therapeutic transplantation,resulted in marked increases in the apoptosis index inDM and healthy cells, an effect abrogated with GSKitreatment to near basal levels (Fig. 1D). Importantly,higher levels of phosphorylated b-cat), the product ofactive GSK3b activity, in EPCs derived from DM patients(0.55 6 0.08 vs. 0.42 6 0.04, P = 0.04) were markedlyreduced in both cohorts of cells after GSKi treatment(P , 0.01; Fig. 1E). As expected, basal levels of GSK3bwere increased in DM patients, with no change afterGSKi treatment (Supplementary Fig. 2). These findingsdemonstrate that increased basal activity of GSK3b inEPCs from patients with DM results in accelerated apo-ptosis in vitro, an effect abrogated by use of isoform-specific small molecule inhibitors.

Proteomic Profiling of EPCs in DM

To ascertain mechanistic insight into the beneficialeffects of GSKi on EPCs, analyses were performed of theproteome of EPCs from patients with DM, from DMpatients treated with GSKi, and from healthy controlsubjects (n = 3 for each). Isolation protocols were scaledup to yield sufficient cellular yields for the analyses.Differential yields between healthy control subjects andDM patients were maintained in scaled-up protocols, aswere the effects of GSKi (Supplementary Table 2). After2-D gel electrophoresis and digital image analysis, 242unique protein spots were identified. Differentially reg-ulated candidate targets were identified if a 2.0-foldupregulation or 0.5-fold downregulation of spot intensitywas identified between the groups (Supplementary Fig 3).A total of 37 nonredundant proteins met these criteriafor significant differential expression (P , 0.05). Thesespots were excised from the Sypro-Ruby–stained gels andunderwent in-gel trypsinization. The peptide mixtureswere analyzed by LC-MS/MS analysis, and the results ofthe MS identification are presented in Table 1.

diabetes.diabetesjournals.org Hibbert and Associates 1413

Western blot analysis of three target proteins of in-terest was performed. Specifically, cathB upregulation(1.9 6 0.06 vs. 5.8 6 2.0), gelsolin downregulation(5.6 6 1.5 vs. 1.9 6 0.9), and PAI-2 upregulation (2.1 60.7 vs. 5.1 6 0.5) was confirmed in EPCs from patientswith DM (P , 0.05 for all comparisons; Fig. 2B). Becauseb-cat acts as a transcription factor, we hypothesized that

regulation of protein levels seen by GSKi were most likelytranscriptional in nature. Indeed, quantitative PCR ofmRNA isolated from DM EPCs under basal and GSKi-treated conditions revealed a 4.6 6 1.2-fold increase incathB (P , 0.01; Fig. 2C), a 0.5 6 0.4-fold reductionin gelsolin (P , 0.05), and a 10.4 6 5.0-fold increase inPAI-2 (P, 0.01). Neither SCaMC-1 nor GDP dissociation

Figure 1—Increased GSK3b signaling in DM EPC reduces in vitro cell yield. A: EPCs at 7 days labeled with fluorescein isothiocyanate-conjugated Ulex europaeus agglutinin-1 (left panel, green), AcLDL-DiI (red) and DAPI (blue,middle panel), and merged images (right panel).B: Light microscopy images of cells from patients with DM and healthy control patients under basal conditions or treated with GSKi.C: GSKi treatment increases EPC yield in healthy control patients and in patients with DM. HPF, high-power field. D: GSKi treatmentattenuates apoptosis under basal and serum-starved conditions. E: EPCs from patients with DM have higher levels of phosphorylated(phospho)–b-catenin. GSKi treatment in both groups markedly reduced these levels.

1414 GSK3b and Cell-Based Therapy in DM Diabetes Volume 63, April 2014

Table 1—Proteomics analysis

DotNo. Protein identity

SwissProtNo.

MSscore

TheoreticalMW

N statisticallysignificantmatches

Coverage(%)

Ratio(DM-to-healthy)

Ratio(DM+GSKivs. DM)

1 26S protease regulatory subunit 6A P17980 401 49,172 26 46 0.94 1.73

2 Actin, cytoplasmic 1 P60709 1,796 41,710 82 55 1.53 2.99

3 Actin, cytoplasmic 1 P60709 2,638 41,710 115 50 2.90 0.42

4 Actin, cytoplasmic 1 P60709 635 41,710 27 33 2.09 1.79

5 Adenosylhomocysteinase P23526 1,246 47,685 71 31 0.64 1.84

6 a-Actinin-4 O43707 364 104,788 18 35 1.46 0.51

7 Annexin A5 P08758 2,639 35,914 159 52 0.65 1.42

8 ATP-dependent RNA helicaseDDX39A O00148 292 49,098 13 33 0.86 1.54

9 b-Hexosaminidase subunit a P06865 786 60,664 38 29 0.98 2.12

10 b-Hexosaminidase subunit b P07686 83 63,071 8 14 0.55 0.97

11 Calcium-binding mitochondrialcarrier protein SCaMC-1 Q6NUK1 325 53,320 16 34 1.12 1.76

12 CathB P07858 559 37,797 21 33 0.45 4.06

13 CathB P07858 522 37,797 15 30 0.56 3.08

14 Cathepsin D P07339 788 44,524 33 32 0.42 ND

15 Cathepsin D P07339 1,212 44,524 55 31 0.49 ND

16 Coagulation factor XIII A chain P00488 594 83,215 28 25 1.62 0.51

17 Dihydropyrimidinase-relatedprotein 2 Q16555 1,478 62,255 59 62 0.60 0.47

18 Galactokinase P51570 269 42,246 13 26 0.57 1.77

19 Gelsolin P06396 447 85,644 20 30 1.57 0.8

20 Gelsolin P06396 144 85,644 8 10 1.41 1.82

21 Gelsolin P06396 3,025 85,644 108 38 1.3 0.38

22 Gelsolin P06396 2,006 85,644 72 45 1.97 0.34

23 Glycyl-tRNA synthetase P41250 560 83,113 40 32 0.79 1.51

24 Heterogeneous nuclearribonucleoprotein K P61978 603 50,944 44 53 0.77 0.62

25 Lamin-B1 P20700 1,251 66,368 51 49 0.91 2.45

26 Leukocyte elastase inhibitor P30740 1,214 42,715 48 49 0.47 ND

27 Lymphocyte-specific protein 1 P33241 600 37,169 35 44 0.94 0.34

28 Microtubule-associated proteinRP/EB family member 1 Q15691 602 29,980 33 59 0.56 0.33

29 N-acetyl-D-glucosamine kinase Q9UJ70 313 37,352 12 24 0.95 1.56

30 Peroxiredoxin-2 P32119 578 21,878 32 38 1.65 0.88

31 Plasminogen activator inhibitor 2 P05120 2,097 46,566 80 56 0.47 4.43

32 Pyruvate kinase isozymes M1/M2 P14618 3,019 57,900 96 59 2.89 1.9

33 Pyruvate kinase isozymes M1/M2 P14618 1,053 57,900 34 35 0.94 2.62

34 Ras GTPase-activating-like proteinIQGAP1 P46940 1,155 189,134 58 13 1.5 0.86

35 Ribonuclease inhibitor P13489 769 49,941 28 37 1.65 0.94

36 Sorting nexin-6 Q9UNH7 259 46,620 16 27 1.76 1.44

37 Spliceosome RNA helicase DDX39B Q13838 887 48,960 42 37 1.8 0.38

38 Synaptic vesicle membrane proteinVAT-1 homolog Q99536 1,046 41,893 55 50 0.68 1.73

39 Tissue a-L-fucosidase P04066 2,138 53,655 71 43 0.96 5.41

40 Tissue a-L-fucosidase P04066 995 53,655 35 21 0.18 2.59

Continued on next page

diabetes.diabetesjournals.org Hibbert and Associates 1415

inhibitor-2 appeared differentially regulated at themRNA or protein level. These findings confirmed thevalidity of the proteomics results and identified severaltargets known to regulate apoptosis and to be expressedin EPCs.

CathB Is Required for GSKi-Mediated Reductions inApoptosis

To ascertain the role of cathB activity in EPC dysfunctionin patients with DM, we assayed enzyme activity levels.As predicted by mRNA and protein levels, EPCs frompatients with DM had 40% less measurable activitycompared with healthy control cells (5,587.3 6 1,455.1vs. 8,251.2 6 771.9 relative fluorescence units, P = 0.03;Fig. 3A). Treatment with GSKi raised measurable activityfourfold in both groups (P , 0.01). To ascertain whethercathB activity was required for changes in phenotypeassociated with GSKi, we used CA074, a specific inhibitorof cathB (12). Supplementation of the culture media withCA074 abrogated a GSKi-induced dose-dependent in-crease in cathB activity at all levels assayed (Fig. 3B). Inturn, functional assays were performed to ascertain thenecessity of intact cathB activity for phenotypic changesin apoptosis rates, VEGF secretion, and endothelial ad-hesion by EPCs cultured from patients with DM. Notably,the effect of GSKi on apoptosis could be attenuated whencells were cultured in the presence of the cathB inhibitorCA074 (Fig. 3C). Blockade of cathB activity demonstratedno effect on VEGF secretion or binding of EPCs toactivated HUVECs (Fig. 3D and E). Instead, only EPC

invasive capacity, as assessed by matrigel invasion, wasalso cathB-dependent, because this parameter increasedthreefold with GSKi treatment (P , 0.01; Fig. 3F). Thesefindings demonstrate that EPCs derived from patientswith DM have intrinsically lower cathB activity and thatinduction of expression by GSKi attenuates higher levelsof apoptosis and improves invasiveness in vitro.

Improvements in EPC-Based Cell Therapy Mediated byGSK3b Inhibition Require CathB Activity

To investigate the effect of DM on reparative capacity ofEPCs, an increasing number of cells from healthy controlsubjects and patients with DM were administered ina xenotransplant femoral artery wire injury model. Theintima-to-media (IM) ratio was assessed at 14 days. DMand cell dose both significantly altered the therapeuticeffect as assessed by two-way ANOVA, with EPCs frompatients with DM exhibiting lower therapeutic benefit at2.5 3 104 and 1.0 3 105 cells (P , 0.05 for all dosecomparisons; Fig. 4A). To test the therapeutic necessityof cathB upregulation to DM EPC–mediated arterialhealing, we administered cells from DM patients withGSKi and CA074 (Fig. 4B). Administration of cells alonereduced the IM ratio 40% (1.37 6 0.15 vs. 0.81 6 0.12,P , 0.01; Fig. 4C) at 14 days. Pretreatment of cells withGSKi resulted in further reduction in the IM ratio com-pared with untreated EPCs (0.50 6 0.07, P = 0.02), aneffect that was lost when cells were pretreated with GSKiand CA074 (0.77 6 0.11, P = 0.03 vs. EPC + GSKi). Ofnote, effects were observed in each individual subject

Table 1—Continued

DotNo. Protein identity

SwissProtNo.

MSscore

TheoreticalMW

N statisticallysignificantmatches

Coverage(%)

Ratio(DM-to-healthy)

Ratio(DM+GSKivs. DM)

41 Transaldolase P37837 142 37,516 9 19 1.08 1.63

42 Tropomyosin a-1 chain P09493 271 32,689 12 20 2.56 0.47

43 Tropomyosin a-4 chain P67936 2,259 28,504 107 39 1.82 0.43

44 Vimentin P08670 51 53,619 1 6 0.68 1.51

45 Vimentin P08670 735 53,619 34 35 2.03 1.26

46 Vinculin P18206 4,302 123,722 142 58 2.10 0.77

47 Vinculin P18206 2,976 123,722 119 41 2.05 0.4

48 V-type proton ATPase subunitB, brain isoform P21281 1,523 56,465 64 48 0.99 1.95

49 V-type proton ATPasesubunit D 1 P61421 271 40,303 12 25 0.63 1.55

GTPase, guanosine triphosphatase; IQGAP, IQ motif containing GTPase-activating protein; ND, nondetectable; VAT, vesicle aminetransport protein 1. The protein identities for each spot are listed. The other columns depict the Swiss-Prot accession number; the MSscore indicating the significance of protein identification from the peptide mass fingerprint according to MASCOT software application2.3.01 (Matrix Sciences, London, U.K.), score value .50 for P , 0.05; the theoretical molecular weight (MW) of the matching protein;and the number of spectrum-to-peptide matches that were evaluated to be statistically significant (expect ,0.05). Density values werenormalized by the local regression model. Student t test (two-tail, 95% level of confidence) was calculated for pairwise comparisons toidentify proteins that were expressed at significantly different levels. Mean fold-change values are indicated in the final two columns, andsignificant changes are indicated in bold. By computational 2-D gel image comparison, 49 protein spots were differentially expressed,each exhibiting $1.5 fold-change (either increase or decrease) of mean value spot intensity among the three different samples.

1416 GSK3b and Cell-Based Therapy in DM Diabetes Volume 63, April 2014

Figure 2—Proteomic analyses of EPCs from patients with DM with and without GSK3b inhibition. A: Sample 2-D gels used in proteomicsanalysis. Western blot (B) and quantitative analysis (C) of cathB, gelsolin, and PAI-2 (n = 6). D: Quantitative PCR analysis of candidategenes identified by proteomics analysis. Fold change represents GSKi sample compared with control sample in EPCs derived frompatients with DM (n = 6). GDI-2, GDP dissociation inhibitor-2; MW, molecular weight.

diabetes.diabetesjournals.org Hibbert and Associates 1417

Figure 3—CathB is required for the GSKi-mediated reduction in the rate of apoptosis in DM EPCs. A: CathB activity is reduced at baselinein DM EPCs and increased by treatment with GSKi (n = 6). B: Dose-dependent increase in cathB activity is achieved with GSKi, andthe effect is abrogated by treatment with CA074, the cathB inhibitor (n = 12). C: The basal rate of apoptosis is reduced by 60% inEPCs treated with GSKi. This effect is lost with cathB inhibition (n = 12). D: Improvements in VEGF secretion achieved with GSKioccur independently of cathB activity (n = 12). E: Increased EPC adhesion achieved with GSKi occurs independently of cathB activity(n = 12). F: Improvement in EPC invasion achieved with GSKi is dependent of cathB activity (n = 6). RFUs, relative fluorescence units.

1418 GSK3b and Cell-Based Therapy in DM Diabetes Volume 63, April 2014

(Fig. 4D). These findings confirm that upregulation ofcathB by GSK3b inhibition partly explains the thera-peutic enhancement of EPC-based therapy in patientswith DM.

DISCUSSION

Identifying cell-enhancement strategies, be it geneticmodification (1) or small molecule antagonism (2), isessential to improve the therapeutic efficacy of cell-basedtherapies. Indeed, transplanted cells in a wide variety ofmodels demonstrate poor engraftment, with high ratesof cell attrition. Herein, we highlight important differ-ences in GSK3b signaling as a factor for enhanced EPCsenescence in DM resulting in accelerated rates of apo-ptosis. Moreover, using a proteomics approach, weidentified upregulation of cathB as protective for reduc-tions in basal and stress-induced apoptosis. Finally, ina xenotransplant model, we confirm that cathB activity is

required for GSKi-induced improvements in EPC medi-ated arterial repair.

Patients with DM have increased rates of cardiovas-cular disease and markedly higher rates of in-stentrestenosis after revascularization. This, in part, is owingto attenuated EPC function in patients and fewer circu-lating cells (16). Multiple mechanisms of EPC dysfunc-tion have been identified, including endothelial nitricoxide synthase uncoupling (17), increased reactive oxy-gen species, and the effects of advanced glycation endproducts (18). As well, glucose is known to impair theactivity of the phosphatidylinositide 3-kinase/Akt path-way, a regulator of GSK3b signaling, and has been im-plicated in EPC differentiation by forkhead box class O1transcription factors (19). GSK3b is known to be highlyupregulated in a number of tissues in DM, with our datanow confirming increased phosphorylated b-cat, the endproduct of GSK3b, in EPCs derived from patients with

Figure 4—CathB is required for improvement in DM EPC–mediated arterial repair achieved with GSKi. A: Changes in neointima formationusing increasing doses of EPCs from healthy age-matched control subjects and patients with DM (n = 6). B: Representative 14-day cross-sections at low magnification with magnified regions of neointima highlighted. Arrows indicate the internal elastic lamina. C: GSKitreatment of DM EPCs results in important reductions in neointima formation, an effect lost with cathB inhibition (n = 6). D: Changes inneointima formation of individual patients according to treatment group. All patients demonstrated improvement in arterial homeostasiswith GSKi treatment (n = 6). I-to-M, intima-to-media.

diabetes.diabetesjournals.org Hibbert and Associates 1419

DM. Thus, dysregulation of b-cat signaling representsa new target for cell enhancement of EPCs in patientswith DM.

To date, three studies suggest a beneficial effect ofGSK3b antagonism in EPC-based therapy (2,10,20). Thecurrent report is the first to use an unbiased proteomicapproach to identify differentially regulated proteins inEPCs in order to identify a potential mechanism ofbenefit. Using this technique, we identified cathB, a pro-tein with known roles as both a pro- and antiapoptoticfactor. Similar to observations in several cell lines(12,21), we noted cathB downregulation in DM-derivedEPCs resulted in enhanced apoptosis, an effect rescuedwith GSKi therapy. Furthermore, low cathB activity hasbeen identified as being linked to progression of diabeticnephropathy, ostensibly due to insufficient fibronectindegradation (22). Analogous to our model, studies usingbone marrow–derived EPCs have demonstrated pro-motion of angiogenesis in regions of glomerular lesionsrepresenting a potential therapeutic target (23). Our datanow compliment these findings, highlighting the role oflow cathB activity in DM and, in the case of cell-basedtherapy, a mechanism to improve therapeutic effectthrough GSK3b inhibition.

The mechanism by which GSK3b regulates cathBremains to be elucidated. Although others have notedGSK3b-mediated relocation of cathB from lysosomes tocytosol as a means by which it modulates apoptosis(21,24–26), the current study supports transcriptionalmodulation of cathB as the most likely mechanismthrough which increased activity was achieved. Indeed,GSK3b is known to inhibit nuclear factor (NF)-kb inquiescent cells resulting in increased apoptosis, an effectthat depends on b-cat (27). Previous reports have notedan upregulation of cathB by NF-kb, which has a bind-ing site in the cathB promoter (28). Supporting thisconcept are recent reports showing that exogenousrecombinant heat shock protein 27 treatmentimproves EPC function in vivo (29) and increasesNF-kb signaling (30). Nonetheless, although it remainsattractive to hypothesize that GSK3 effects on NF-kbare responsible for cathB regulation, definitive studiesin EPCs are needed.

This study is not without limitations. First, the celltypes being used for therapeutic effect continue tobroaden, and we cannot be certain that the mechanismsdescribed in the current study apply to other cell pop-ulations. However, the current experiments were per-formed in primary cells commonly used in clinical studiesand were derived from patients with DM.

Second, although there are clear improvements inapoptosis and invasiveness, we do not demonstrate in-creased cell retention in our in vivo model. However, it iswell documented in numerous animal models of cell-based therapy that cellular retention is a rare event,whereas the paracrine effect of cell therapy may be animportant early beneficial mechanisms (31).

Finally, although numerous specific inhibitors forGSK3b were tested, we cannot entirely rule out thatnonspecific GSK3a inhibition may have partly contributedto the observed biological effect. However, the relativecontribution of GSK3a in DM-induced EPC dysfunction isunlikely to be significant but remains to be elucidated.

Despite these limitations, our study is the first tohighlight cathB regulation by GSK3b as a potential cell-enhancement strategy for patients with DM, and ourunbiased proteomic approach highlights potential futuretargets, such as PAI-2, for future investigation.

In conclusion, inhibition of GSK3b activity in EPCsfrom patients with DM results in upregulation of cathBexpression and activity. Increased cathB activityimproves EPC invasiveness, reduces apoptosis, andameliorates the therapeutic effect of cell-based therapy.Small molecule antagonism of GSK3b is a cell enhance-ment strategy for patients with DM.

Funding. The Canadian Institute for Health Research and Medtronic col-lectively provide E.R.O. with a peer-reviewed research chair (URC #57093, IGO94418) and an operating grant.

Duality of Interest. No potential conflicts of interest relevant to thisarticle were reported.

Author Contributions. B.H. and J.R.L. planned, designed, and carriedout experiments, analyzed data, and wrote the manuscript. X.M., T.Se., J.E.R.,T.Si., and Y.-X.C. carried out experiments and reviewed the manuscript. D.S.and E.R.O. conceived experiments and reviewed the data and manuscript.E.R.O. is the guarantor of this work and as such had full access to all the datain the study and takes responsibility for the integrity of the data and theaccuracy of the data analysis.

Prior Presentation. This work was presented at the 62nd AnnualScientific Session of the American College of Cardiology, San Francisco, CA,9–11 March 2013.

References1. Lavoie JR, Stewart DJ. Genetically modified endothelial progenitor cells in

the therapy of cardiovascular disease and pulmonary hypertension. CurrVasc Pharmacol 2012;10:289–299

2. Hibbert B, Ma X, Pourdjabbar A, et al. Inhibition of endothelial progenitorcell glycogen synthase kinase-3beta results in attenuated neointima for-mation and enhanced re-endothelialization after arterial injury. CardiovascRes 2009;83:16–23

3. Dajani R, Fraser E, Roe SM, et al. Crystal structure of glycogen synthasekinase 3 beta: structural basis for phosphate-primed substrate specificityand autoinhibition. Cell 2001;105:721–732

4. Diehl JA, Cheng M, Roussel MF, Sherr CJ. Glycogen synthase kinase-3betaregulates cyclin D1 proteolysis and subcellular localization. Genes Dev1998;12:3499–3511

5. Jope RS, Johnson GVW. The glamour and gloom of glycogen synthasekinase-3. Trends Biochem Sci 2004;29:95–102

6. Rubinfeld B, Albert I, Porfiri E, Fiol C, Munemitsu S, Polakis P. Binding ofGSK3beta to the APC-beta-catenin complex and regulation of complexassembly. Science 1996;272:1023–1026

7. Sutherland C, Leighton IA, Cohen P. Inactivation of glycogen synthasekinase-3 beta by phosphorylation: new kinase connections in insulin andgrowth-factor signalling. Biochem J 1993;296:15–19

1420 GSK3b and Cell-Based Therapy in DM Diabetes Volume 63, April 2014

8. Thomas GM, Frame S, Goedert M, Nathke I, Polakis P, Cohen P. A GSK3-binding peptide from FRAT1 selectively inhibits the GSK3-catalysedphosphorylation of axin and beta-catenin. FEBS Lett 1999;458:247–251

9. Trowbridge JJ, Xenocostas A, Moon RT, Bhatia M. Glycogen synthasekinase-3 is an in vivo regulator of hematopoietic stem cell repopulation.Nat Med 2006;12:89–98

10. Ma X, Hibbert B, Dhaliwal B, et al. Delayed re-endothelialization withrapamycin-coated stents is rescued by the addition of a glycogen synthasekinase-3beta inhibitor. Cardiovasc Res 2010;86:338–345

11. Eldar-Finkelman H, Schreyer SA, Shinohara MM, LeBoeuf RC, Krebs EG.Increased glycogen synthase kinase-3 activity in diabetes- and obesity-prone C57BL/6J mice. Diabetes 1999;48:1662–1666

12. Szpaderska AM, Frankfater A. An intracellular form of cathepsin B con-tributes to invasiveness in cancer. Cancer Res 2001;61:3493–3500

13. Hibbert B, Ma X, Pourdjabbar A, et al. Pre-procedural atorvastatin mobilizesendothelial progenitor cells: clues to the salutary effects of statins onhealing of stented human arteries. PLoS ONE 2011;6:e16413

14. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29:e45

15. Sata M, Maejima Y, Adachi F, et al. A mouse model of vascular injury thatinduces rapid onset of medial cell apoptosis followed by reproducibleneointimal hyperplasia. J Mol Cell Cardiol 2000;32:2097–2104

16. Fadini GP, Boscaro E, de Kreutzenberg S, et al. Time course and mech-anisms of circulating progenitor cell reduction in the natural history of type2 diabetes. Diabetes Care 2010;33:1097–1102

17. Thum T, Fraccarollo D, Schultheiss M, et al. Endothelial nitric oxide syn-thase uncoupling impairs endothelial progenitor cell mobilization andfunction in diabetes. Diabetes 2007;56:666–674

18. Jarajapu YP, Grant MB. The promise of cell-based therapies for diabeticcomplications: challenges and solutions. Circ Res 2010;106:854–869

19. Marchetti V, Menghini R, Rizza S, et al. Benfotiamine counteracts glucosetoxicity effects on endothelial progenitor cell differentiation via Akt/FoxOsignaling. Diabetes 2006;55:2231–2237

20. Choi JH, Hur J, Yoon CH, et al. Augmentation of therapeutic angiogenesisusing genetically modified human endothelial progenitor cells with altered

glycogen synthase kinase-3beta activity. J Biol Chem 2004;279:49430–49438

21. Malla R, Gopinath S, Alapati K, et al. Downregulation of uPAR and ca-thepsin B induces apoptosis via regulation of Bcl-2 and Bax and inhibitionof the PI3K/Akt pathway in gliomas. PLoS ONE 2010;5:e13731

22. Wyczalkowska-Tomasik A, Bartlomiejczyk I, Gornicka B, Paczek L. Strongassociation between fibronectin accumulation and lowered cathepsin Bactivity in glomeruli of diabetic rats. J Physiol Pharmacol 2012;63:525–530

23. Ikarashi K, Li B, Suwa M, et al. Bone marrow cells contribute to regen-eration of damaged glomerular endothelial cells. Kidney Int 2005;67:1925–1933

24. Chen G, Bower KA, Ma C, Fang S, Thiele CJ, Luo J. Glycogen synthasekinase 3beta (GSK3beta) mediates 6-hydroxydopamine-induced neuronaldeath. FASEB J 2004;18:1162–1164

25. Hsing CH, Chen YH, Chen CL, et al. Anesthetic propofol causes glycogensynthase kinase-3b-regulated lysosomal/mitochondrial apoptosis in mac-rophages. Anesthesiology 2012;116:868–881

26. Huang WC, Lin YS, Chen CL, Wang CY, Chiu WH, Lin CF. Glycogen syn-thase kinase-3beta mediates endoplasmic reticulum stress-induced lyso-somal apoptosis in leukemia. J Pharmacol Exp Ther 2009;329:524–531

27. Graham JR, Tullai JW, Cooper GM. GSK-3 represses growth factor-inducible genes by inhibiting NF-kappaB in quiescent cells. J Biol Chem2010;285:4472–4480

28. Bien S, Ritter CA, Gratz M, et al. Nuclear factor-kappaB mediates up-regulation of cathepsin B by doxorubicin in tumor cells. Mol Pharmacol2004;65:1092–1102

29. Ma X, Hibbert B, McNulty M, et al. Heat shock protein 27 attenuatesneointima formation and accelerates reendothelialization after arterial in-jury and stent implantation: importance of vascular endothelial growthfactor up-regulation. FASEB J 18 October 2013 [Epub ahead of print]

30. Salari S, Seibert T, Chen YX, et al. Extracellular HSP27 acts as a signalingmolecule to activate NF-kB in macrophages. Cell Stress Chaperones 2013;18:53–63

31. Gnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine mechanisms in adult stemcell signaling and therapy. Circ Res 2008;103:1204–1219

diabetes.diabetesjournals.org Hibbert and Associates 1421