High glucose facilitated endothelial heparanase transfer to the ...

High glucose facilitated endothelial heparanase

transfer to the cardiomyocyte modifies its cell

death signature

Fulong Wang1 Jocelyn Jia1 Nathaniel Lal1 Dahai Zhang1 Amy Pei-Ling Chiu1

Andrea Wan1 Israel Vlodavsky2 Bahira Hussein1 and Brian Rodrigues1

1Faculty of Pharmaceutical Sciences The University of British Columbia 2405 Wesbrook Mall Vancouver BC Canada V6T 1Z3 and 2Cancer and Vascular Biology Research CenterRappaport Faculty of Medicine Technion Haifa 31096 Israel

Received 15 April 2016 revised 21 September 2016 accepted 16 September 2016

Time of primary review 32 days

Aims The secretion of enzymatically active heparanase (HepA) has been implicated as an essential metabolic adaptationin the heart following diabetes However the regulation and function of the enzymatically inactive heparanase(HepL) remain poorly understood We hypothesized that in response to high glucose (HG) and secretion of HepL

from the endothelial cell (EC) HepL uptake and function can protect the cardiomyocyte by modifying its cell deathsignature

Methods andresults

HG promoted both HepL and HepA secretion from microvascular (rat heart micro vessel endothelial cellsRHMEC) and macrovascular (rat aortic endothelial cells RAOEC) EC However only RAOEC were capable ofHepL reuptake This occurred through a low-density lipoprotein receptor-related protein 1 (LRP1) dependentmechanism as LRP1 inhibition using small interfering RNA (siRNA) receptor-associated protein or an LRP1 neu-tralizing antibody significantly reduced uptake In cardiomyocytes which have a negligible amount of heparanasegene expression LRP1 also participated in the uptake of HepL Exogenous addition of HepL to rat cardiomyocytesproduced a dramatically altered expression of apoptosis-related genes and protection against HG and H2O2

induced cell death Cardiomyocytes from acutely diabetic rats demonstrated a robust increase in LRP1 expressionand levels of heparanase a pro-survival gene signature and limited evidence of cell death observations that werenot apparent following chronic and progressive diabetes

Conclusion Our results highlight EC-to-cardiomyocyte transfer of heparanase to modulate the cardiomyocyte cell death signa-

ture This mechanism was observed in the acutely diabetic heart and its interruption following chronic diabetesmay contribute towards the development of diabetic cardiomyopathy

Keywords Hyperglycemia bull Diabetes bull LRP1 bull Endothelial cell bull Cardiomyocyte bull Cell death

1 Introduction

In the heart where contracting cardiomyocytes are incapable of regen-eration intrinsic mechanisms are available within the endothelial cell(EC) to protect the cardiomyocyte against cellular demise1ndash4 One con-ceivable cardioprotective protein secreted exclusively from the EC inthe heart is heparanase56 This endoglycosidase is initially synthesized asa latent (enzymatically inactive HepL) 65 kDa proheparanase enzymeHepL undergoes cellular secretion which is followed by reuptake intolysosomes for proteolytic cleavage (removal of a 6 kDa linker peptide)5

Consequently a 50 kDa polypeptide (enzymatically active HepA) isformed that is100-fold more active than HepL

In cancer biology HepA degradation of heparan sulphate proteoglycan(HSPG) is associated with extracellular matrix and basement membranedisruption facilitating tumour cell invasion57ndash9 Following its nuclearentry HepA also influences transcription by cleaving nuclear HSPG miti-gating the suppressive effect of heparan sulphate on histone acetyltrans-ferase10ndash14 More recently we established a novel role for HepA inmodulating cardiac metabolism during diabetes1015 The above studies incancer and diabetes fixated on the effects of HepA incorrectly assuming

Corresponding author Tel 604 822 4758 fax 604 822 3035 E-mail rodrigueinterchangeubcca

Published on behalf of the European Society of Cardiology All rights reserved VC The Author 2016 For Permissions please email journalspermissionsoupcom

Cardiovascular Research (2016) 112 656ndash668doi101093cvrcvw211

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that only the HSPG-hydrolyzing ability of heparanase was of importanceIntriguingly HepL also has some remarkable properties including its abil-ity to activate signalling elements like Erk12 PI3K-AKT RhoA and Srcwhich in turn can contribute to changes in transcription5 Cancer cellsuse secreted HepL to alter gene expression (either through its cell signal-ling properties or by its conversion to HepA) in neighbouring cells pre-venting their cellular demise and promoting tumour growth51617 In theheart a similar paradigm would appear advantageous with endothelialHepL protecting the cardiomyocyte against cell death For this to hap-pen HepL needs to be secreted followed by its subsequent binding anduptake into the cardiomyocyte We hypothesized that following itssecretion from the EC HepL uptake and function in the cardiomyocyteis protective against cell death Results from this study suggest that HGincreases heparanase secretion from EC in addition to augmenting itsuptake into the cardiomyocyte where it has a favourable effect on theexpression of apoptosis-related genes and limits the incidence of celldeath Occurrence of this EC-to-cardiomyocyte transfer of heparanasein the acutely diabetic heart and the interruption of this process follow-ing chronic and progressive diabetes may contribute towards the devel-opment of diabetic cardiomyopathy18ndash20

2 Methods

21 Animal careThis investigation conformed to the Guide for the Care and Use ofLaboratory Animals published by the National Institutes of Health andthe University of British Columbia (Animal Care Certificate A13-0098)

22 Experimental animalsStreptozotocin (STZ) is a b-cell specific toxin used to induce diabetes21

Male Wistar rats (240ndash260 g) were injected intravenously with 55 mgkgSTZ With this dose the animals become hyperglycemic within 24 hThese animals used as a model of poorly controlled Type 1 diabeteswere kept for 4 days (acute) or 6 weeks (chronic) before heart isolation

23 Isolation of cardiomyocytesRats were euthanized using a 100 mgkg intraperitoneal injection ofsodium pentobarbital Once toe pinch and corneal reflexes were lost athoracotomy was performed prior to removal of the heart Rat ventricu-lar calcium-tolerant cardiomyocytes were prepared following previouslydescribed procedures22 Isolated rat cardiomyocytes were plated onlaminin-coated culture dishes and allowed to settle for 3 h Unattachedcells were washed away prior to different treatment protocols

24 EC cultureRepresentative macrovascular (rat aortic endothelial cells RAOEC) andmicrovascular (rat heart micro vessel endothelial cells RHMEC) ECwere cultured at 37 C in a 5 CO2 humidified incubator Cells from thefifth to the eighth passages of three different starting batches for eachcell line were used

25 TreatmentsTo promote the secretion of heparanase EC were incubated with highglucose (25 mM HG) To test whether exogenous heparanase can betaken up into EC and cardiomyocytes cells were treated with 500 ngmLrecombinant myc-tagged HepL (myc-HepL) for different time intervals Toelucidate the contribution of cell surface lipoprotein receptor-related pro-tein 1 (LRP1) towards heparanase uptake we used receptor-associated

protein (RAP 200ndash400 nM 1 h) or LRP1 neutralizing antibodies (20ndash40 mgmL 1 h) to inhibit LRP1 To inhibit LRP1 expression small interfer-ing RNA (siRNA) specific for LRP1 was used in RAOEC SST0001(125lgmL 4 h) was used to inhibit heparanase activity To induce apop-tosis cardiomyocytes were incubated in HG (30 mM) for 48 h or H2O2

(10lM) for 12 h

26 ImmunofluorescenceTo visualize heparanase uptake into cardiomyocytes cells were treatedfor 4 h with myc-HepL Cells were washed with cold PBS and fixed with4 formaldehyde solution This was followed by permeabilization with02 Triton X-100 for 10 min and incubation with blocking buffer con-taining 5 goat serum for 1 h at room temperature Incubation with pri-mary antibodies was at 4 C overnight and secondary antibodies atroom temperature for 1 h To detect lysosome localization LysoTrackerwas added 30 min before fixation For determination of apoptosisAnnexin V (1200) and propidium iodide (PI 1500) were used

27 Nuclear isolationNuclear and cytosolic fractions were separated using the nuclearcytosolfractionation kit from Thermo Fisher Scientific To validate the purity ofproteins we used cytosolic (GAPDH) and nuclear (histone H3) proteinmarkers to detect their predominance in cytosolic and nuclear fractionsrespectively

28 Western blotWestern blot was done as described previously23 In some experimentsusing EC cell culture media was concentrated with an Amicon centrifugefilter (Millipore) before the detection of heparanase protein

29 Quantitative real-time PCRTotal RNA was isolated from EC whole hearts or cardiomyocytes usingTRIzol (Invitrogen) This was followed by extraction using chloroformand isopropanol washing with ethanol and dissolving in RNase-freewater RNA was reverse transcribed into cDNA using a mixture ofdNTPs oligo-(dT) and SuperScript II Reverse Transcriptase cDNA wasamplified by TaqMan probes (b-actin heparanase lrp1 tnfrsf10b tnfsf10tnfrsf11b cflar bcl-2 tradd tnfsf1b bad caspase 7 and caspase 8) in trip-licate using a StepOnePlus Real-Time (RT) PCR system (AppliedBiosystems) Gene expression was calculated by the comparative cyclethreshold (DDCT) method

210 Apoptosis PCR microarraysFor the apoptosis PCR array (Qiagen) 300ndash1000 ng RNA was isolatedusing an RNeasy Mini Kit and cDNA were transcribed using the RT2 FirstStrand Kit The expression of 84 apoptosis-related genes was deter-mined in control and HepL-treated rat cardiomyocytes

211 MaterialsRAOEC and RHMEC were obtained from cell applications and VEC tech-nologies respectively STZ (S0130) and D-Mannitol (M4125) wereobtained from Sigma-Aldrich Anti-LRP1 antibody (ab92544) was pur-chased from Abcam Purified HepL was prepared as described previ-ously24 LysoTracker (L-7528) was purchased from Life Technologies Forwestern blots that detect only HepL we used the heparanase (N-Term)antibody (ABIN786265) which preferentially recognizes the 65 kDaHepL from Aviva Systems Biology For detection of HepA we initiallyused mAb 130 (ANT-193) which can also detect HepL from InSight

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(Rehovot Israel) However due to discontinuation of this antibody wesubsequently used HP317 (INS-26-0000) also from InSight (RehovotIsrael) RAP (03-62221) and the LRP1 neutralizing antibody (8G1) werefrom American Research Products and Millipore respectively Antibodiesfor TNFRSF10B (sc-19529) CFLAR (sc-5276) that recognizes both thefull length and short isoforms and TRAIL (sc-6079) were obtained fromSanta Cruz Biotechnology TNFRSF11B (PA5-19841) was from ThermoFisher Scientific SST0001 was a kind gift from Sigma-Tau ResearchSwitzerland SA Antibodies for PARP (9542) caspase-3 (9662) andcleaved caspase-3 (9664) were purchased from Cell Signalling

212 Statistical analysisValues are means 6 SE Wherever appropriate a non-parametric MannndashWhitney test (for comparison between two groups) or one-way ortwo-way analysis of variance (ANOVA) followed by the Tukey test (forcomparison between multiple groups) was used to determine differen-ces between group mean values The level of statistical significance wasset at Plt 005 Plt 001 or Plt 0001

3 Results

31 Macrovascular and microvascular ECsecretion and reuptake of heparanase inresponse toHGThe concentration and activity of heparanase are elevated in the plasmaand urine of diabetic patients25 We have also reported that HG can stim-ulate the secretion of both latent and active forms of heparanase fromEC26 As EC behave differently based on their vessel type and environ-ment27 in this study we compared the effects of HG on releasing HepL

and HepA from macrovascular and microvascular EC Incubation ofRAOEC in HG promoted the release of both forms of heparanase intothe incubation medium (Figure 1A) HepA by purinergic receptor activationand lysosomal secretion28 and HepL by activation of the serinethreonineprotein kinase D (PKD) an enzyme involved in the fission of proteins des-tined for the cell surface (see Supplementary material online Figure S1)Similar results were observed when using RHMEC (Figure 1B) The osmo-larity control mannitol had no influence on heparanase release in eithercell type (data not shown) After its cellular release HepL must be takenback up into EC5 for maturation into HepA Hence heparanase reuptakewas also determined in EC subsequent to its release by HG The declinein RAOEC lysate HepA at 30 min was followed by a substantial recoveryat 60 min resulting in an increase in the HepAHepL ratio (Figure 1C leftpanel) Measurement of heparanase in the medium also demonstrated ahigher HepAHepL ratio over time (Figure 1C right panel) confirming thereuptake and processing of HepL into HepA which was eventuallysecreted into the medium Remarkably unlike RAOEC the reuptake andsubsequent processing of HepL into HepA was not evident in RHMEC(Figure 1D) To substantiate that only macrovascular but not microvascu-lar EC can take up HepL we used EC incubated with recombinant myc-tagged latent heparanase (myc-HepL) In RAOEC there was a robusttime-dependent uptake of HepL and conversion into HepA effects thatwere not apparent for RHMEC (Figure 1E) suggesting that microvascularEC have a limited capacity for HepL reuptake

32 LRP1 is important for HepL uptakeMultiple receptors have been implicated in facilitating the uptake ofHepL including the mannose-6-phosphate receptor HSPG and LDL

receptor related protein (LRP1)29 We focused on LRP1 given its pro-miscuous role in the endocytosis of a number of different proteins3031

Of considerable interest was the observation that RAOEC demon-strated a robust expression of LRP1 This expression was not apparentin RHMEC (Figure 2A and B) and could explain the disparate abilities ofthese two cell types to take up HepL Using siRNA we effectivelyreduced LRP1 expression in RAOEC (Figure 2C bottom panel and 2D)As a consequence myc-HepL uptake and conversion to HepA over 24 hwas reduced in these cells compared to control (Figure 2C top panel and2E) validating the contribution of LRP1 in HepL uptake (schematic) Inspite of LRP1 knockdown some HepL was still detected albeit at a levelmuch lower as compared to control and likely as a consequence of non-specific binding of HepL to the EC surface Simple binding to the cell sur-face exterior with limited uptake will fail to increase the amount ofHepA as shown in Figure 2C The essential role of LRP1 was further sub-stantiated using the specific blocker RAP (an LRP1 chaperone) (Figure2F) and an LRP1 neutralizing antibody (Figure 2G) both of whichreduced the uptake of HepL by RAOEC Our data implicate LRP1 as anessential contributor in the endocytosis of HepL in EC

33 Extracellular uptake determinespresence of heparanase in cardiomyocytesIn the heart EC outnumber cardiomyocytes by 312 Intriguingly com-pared to EC there is a negligible amount of heparanase gene expressionin cardiomyocytes (Figure 3A) We reasoned that the absence of a reup-take machinery in microvascular EC would lead to HepL secreted fromthese cells to be taken up into cells that are in close proximity for exam-ple the cardiomyocytes Indeed our results indicate that cardiomyocytescontain a significant amount of heparanase protein (Figure 3B) suggestingthat HepL taken up from neighbouring microvascular EC is converted toHepA in the cardiomyocyte lysosome These results are supported by ourprevious work using EC co-cultured with cardiomyocytes10 The uptakeand lysosomal localization of myc-HepL were further confirmed usingimmunofluorescence (Figure 3C) whereas the nuclear presence of HepA

was established using western blot (Figure 3D) Given the importance ofLRP1 in EC HepL uptake we determined and confirmed its expression incardiomyocytes (Figure 3E and F) In addition and analogous to RAOECadministration of either RAP or an LRP1 neutralizing antibody reducedthe cardiomyocyte uptake of myc-HepL (Figure 3G)

34 HepL modulates expression ofapoptosis-related genes in cardiomyocytesEntry of heparanase into the nucleus to regulate histone acetylation hasbeen suggested as a mechanism modulating gene transcription and pro-tection against apoptosis11 We hypothesized that following its uptakeinto the cardiomyocyte HepL can protect against cell death by influenc-ing apoptosis-related genes Using a rat apoptosis gene array in cardio-myocytes incubated with myc-HepL we found that among the 70 genesthat had well-defined functions and significant levels of expression 15out of 27 anti-apoptotic genes were up-regulated and 29 out of 43 pro-apoptotic genes were down-regulated (Figure 4A and B) Of the 18 genesthat were significantly different (fold changegt15) compared to control15 were in favour of cell survival (six anti-apoptotic genes were up-regu-lated nine pro-apoptotic genes were down-regulated) (Figure 4A and BSupplementary material online Table S1) Further examining selectivepro-apoptotic genes that were down-regulated and anti-apoptoticgenes that were up-regulated results from the microarray were con-firmed by quantitative RTndashPCR (Figure 5A and B) and western blot (Figure

658 F Wang et alD

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β -actin

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HepL

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AHep

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Figure 1 Heparanase secretion and reuptake into ECs RAOEC (passage 5ndash8) and RHMEC (passage 5ndash8) were incubated in either 55 (normal glucosecontrol Con) or 25 mM (HG) glucose for 30 min Incubation medium was collected and used to determine latent (HepL) and active (HepA) heparanasesecretion nfrac14 6ndash7 (A and B) Plt 001 compared to Con RAOEC (C) and RHMEC (D) were incubated in 25 (HG) mM glucose for 30 or 60 min Celllysates and incubation medium were used to determine the intracellular and extracellular content of HepL and HepA nfrac14 5 Plt 001 RAOEC andRHMEC were incubated with normal glucose and 500 ngmL myc-HepL Cell lysates were collected at indicated time points to measure the uptake of myc-HepL and its conversion to HepA nfrac14 6 (E) Plt 001 compared to RHMEC

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5C and D) As SST0001 a specific heparanase inhibitor reversed theeffects of heparanase (Figure 5E) our results suggest that heparanase canprotect against apoptotic cell death

35 Contrasting effects of diabetes oncardiomyocyte cell death signatureRAOEC incubated in HG demonstrate an increase in LRP1 expression(see Supplementary material online Figure 2) emphasizing the

importance of HG in mediating its expression Using a model of acute(4 days) diabetes we assessed the impact of HG on whole heart and car-diomyocyte LRP1 Hearts from acute diabetic animals demonstratedaugmented LRP1 expression (Figure 6A) This effect likely contributed toa higher uptake of HepL and its subsequent conversion into HepA whichresulted in a higher HepAHepL ratio (Figure 6A) Extending this observa-tion cardiomyocytes isolated from animals with acute diabetes alsoexhibited higher LRP1 expression and intracellular heparanase content

ARAOEC

RHMEC

LRP1β -actin

85 kDa42 kDa

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LRP1 protein (AU)

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Lysosome

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Figure 2 LRP1 is a key receptor for heparanase reuptake into ECs RAOEC and RHMEC lysates were used to determine the expression of LRP1nfrac14 7 and nfrac14 4 (A and B) In RAOEC siRNA was used to silence LRP1 followed by determination of myc-HepL uptake and conversion to HepA nfrac14 5(CndashE) RAOEC were pre-treated with or without 200 nM RAP (F) or 20lgmL LRP1 neutralizing antibody (G) for 1 h prior to incubation with 500 ngmLmyc-HepL for 4 h Cell lysates were collected to determine HepL uptake nfrac14 5 20lgmL IgG was used as a control for the LRP1 neutralizing antibodyexperiment Plt 005 Plt 001 Plt 0001

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HepLCardiomyocyte

RHMEC

RAOEC

GAPDH

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50 kDa

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GAPDH

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Cardiomyocyte

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85 kDa

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HepLMyc-HepL 75 kDa

65 kDa

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HeparanasemRNA(AU)

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Luptake

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Myc-Hep

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Histone

75 kDa

50 kDa

37 kDa18 kDa

Figure 3 Cardiomyocytes are also capable of HepL uptake Cell lysates of primary rat cardiomyocytes RAOEC or RHMEC were obtained for determi-nation of heparanase mRNA (A) and protein (B) nfrac14 4ndash8 Cardiomyocytes seeded on coverslips were placed in a 6-well plate and treated with 500 ngmL myc-HepL prior to immunofluorescence staining examined under a confocal microscope The merged image of heparanase and lysosomes isdescribed in the third (scale bar 10 mm) and fourth (scale bar 5 mm) panels from left (C) and are data from a representative experiment Isolated myo-cytes were also treated with or without myc-HepL for 4 h Following this incubation nuclear and cytosolic fractions were isolated and HepA protein lev-els determined by western blot (D) Cell lysates of primary rat cardiomyocytes RAOEC or RHMEC were obtained for determination of LRP1 mRNA(E) and protein (F) nfrac14 4 and nfrac14 8 In a different experiment in cardiomyocytes incubated with HG cells were pre-treated with or without 400 nM RAPor 40lgmL LRP1 neutralizing antibody for 1 h prior to incubation with 500 ngmL myc-HepL for 4 h Cell lysates were collected to determine HepL

uptake nfrac14 4 (G) 40lgmL IgG was used as a control for the LRP1 neutralizing antibody experiment Plt 005 Plt 001

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(Figure 6B) The latter effect was unrelated to changes in heparanasegene expression (Figure 6C) It should be noted that unlike EC when car-diomyocytes were exposed to HG no change in LRP1 expression wasobserved up to 48 h after incubation (data not shown) Neverthelesswe observed an increased uptake and lysosomal localization of hepara-nase at 4 h in cardiomyocytes incubated in HG (see Supplementary mate

rial online Figure 3A and B) As the inhibition of Src activation by PP2abrogated this effect this proto-oncogene rather than augmentedexpression of LRP1 can be implicated in HG-mediated cardiomyocyteheparanase uptake in vitro (see Supplementary material online Figure 3Cand D) Whether Src activation also has a contributory effect in vivo iscurrently unclear because its activation by HG was detected within

Apoptosis-relatedgenes n = 81

Lower ΔC t value lt 12

n = 70

n = 27 n = 43

Pro-apoptoticAnti-apoptotic

Fold change gt15

n = 7 n = 11

n=12(44 44 )

n=15(55 56 )

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A

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L og 2foldcha ng e

Il10

Birc5

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l2

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Log 2foldchange

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n=1(14 29 )

n=6(85 71 )

n = 7 n = 11

n=9(81 82 )

n=2(18 18 )

Anti-apoptotic Pro-apoptotic

Figure 4 Expression of apoptosis-related genes in cardiomyocytes exposed to exogenous HepL Primary cardiomyocytes isolated from the adult ratheart were treated with or without 500 ngmL myc-HepL for 12 h prior to RNA isolation and subsequent determination of 81 apoptosis-related genesusing a PCR array (Fig 4A and B)

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TNFRSF10B

VinculinCon

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(NC)

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c-FLIPs

Figure 5 Inhibition of HepA abrogates changes in gene expression RTndashPCR and western blot were employed to confirm our results from the genearray using selected pro- and anti- apoptosis genes nfrac14 5ndash8 (Fig 5AndashD) Vinculin was used as a loading control NC-negative control In a separate experi-ment cardiomyocytes were pre-treated with or without 125 lgmL SST0001 for 4 h prior to incubation with 500 ngmL myc-HepL for 12 h and theexpression of selected genes determined nfrac14 4ndash9 (Fig 5E) Plt 005 Plt 001 Plt 0001

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A

GAPDH

Con

HepL

Diabetes

HepA

LRP165 kDa50 kDa37 kDa

85 kDa

B

4 days

6 weeks

GAPDH

HepL

HepA

LRP1 85 kDa

50 kDa

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Figure 6 Acute and chronic effects of diabetes on cardiomyocyte cell death signature In animals made diabetic with STZ hearts were obtained after4 days of hyperglycemia and LRP1 protein and the HepAHepL ratio determined nfrac14 9 (A) Cardiomyocytes from acute (diabetes-4 days) and chronic(diabetes-6 weeks) diabetic animals were isolated for determination of LRP1 and heparanase protein (B) and heparanase gene (C) nfrac14 7ndash12 Selectedpro- and anti-apoptosis genes (D) and protein (E) were also evaluated in acute and chronic diabetic cardiomyocytes nfrac14 5ndash12 Plt 005 Plt 001Plt 0001

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30 min in vitro whereas diabetic animals are euthanized after 4 days ofSTZ Of considerable significance was the observation that these effectson cardiomyocyte LRP1 and heparanase were abolished upon extendingthe duration of diabetes to 6 weeks (Figure 6B) suggesting that cardio-myocyte LRP1 expression and heparanase uptake are affected in anopposite fashion depending on the duration of hyperglycemia Asapoptosis-related gene (Figure 6D) and protein (Figure 6E) expressionand cleaved caspase 3 and PARP (see Supplementary material onlineFigure S4) followed a similar pattern predicated on the duration of diabe-tes our data suggest that chronic diabetes nullifies the favourable effectsof heparanase in cardiomyocytes

36 HG and H2O2 induced cardiomyocytecell death is attenuated by HepL

In HG a greater production of reactive oxygen species (ROS) togetherwith its disrupted detoxification causes cardiomyocyte cell death32

Given the effects of ROS on gene expression in cells undergoing apopto-sis cardiomyocytes were incubated with HG in the presence or absenceof heparanase In HG HepL caused a significant decrease in the BaxBcl-2mRNA ratio a marker of cellular apoptosis (Figure 7A) Cleaved PARPand caspase 3 apoptosis biomarkers that were augmented in cardiomyo-cytes treated with HG were also significantly decreased upon hepara-nase addition (Figure 7B) Importantly the HG-induced decrease in thenumber of viable cardiomyocytes as determined by Annexin VPI stain-ing was improved by HepL (Figure 7C) As these beneficial effects ofHepL on apoptosis were reproduced in H2O2 induced oxidative stress(see Supplementary material online Figure 5) our data suggest that hep-aranase modulates the cell death signature and is protective against car-diomyocyte cell death

4 Discussion

Under physiological conditions the EC is responsible for secreting fac-tors that support cardiomyocyte function1ndash4 Heparanase is one suchexample having a unique responsibility to release cardiomyocyte cellsurface HSPG-bound lipoprotein lipase (LPL) to promote lipoprotein-TG breakdown The resultant fatty acid (FA) generated is then trans-ported to the cardiomyocyte for oxidative energy generation15 In addi-tion to liberating HSPG-bound proteins heparanase either by binding toputative cell-surface receptors or subsequent to its internalization andnuclear entry has also been suggested to affect gene transcription511ndash

143334 In cancer cells this property of secreted heparanase can inducecell signalling and gene expression in both parent and adjacent cellsmaintaining their survival and delaying demise11161735ndash37 Our data sug-gest for the first time that HG promotes both the secretion of hepara-nase from EC as well as its uptake into cardiomyocytes initiating pro-survival mechanisms to temper the consequences of hyperglycemia inthe diabetic heart

In EC HepA resides in lysosomes5 and hyperglycemia a major compli-cation of diabetes is an effective stimulus for its secretion28 We havepreviously described a mechanism for this process which includes puri-nergic receptor activation as well as cortical and stress actin reorganiza-tion28 As EC are not all created equal and exhibit differences dependingon their anatomical sites-such as arterial compared to venous architec-ture or macro compared to their microvascular locations27mdashwe com-pared the secretion of heparanase in RAOEC and RHMEC Here weshow that HG similarly affects the secretion of HepL from both EC celltypes Following its secretion the EC has a capacity to reuptake HepL for

lysosomal conversion to HepA Interestingly although both cell typeshad a similar capacity to secrete HepL in response to HG only macro-vascular EC were competent for its reuptake an observation that wasconfirmed using myc-HepL A receptor that has been implicated in HepL

uptake is LRP138 Consistent with the differential uptake of HepL intothe two cell types only RAOEC showed a robust expression of LRP1We further established that LRP1 is indispensable for HepL uptake intoRAOEC by silencing the receptor using RAP or an LRP1 neutralizingantibody both of which decreased the uptake of HepL Our data implythat the reuptake of HepL by macrovascular EC is dependent on LRP1an uptake mechanism that is missing in microvascular EC At present themechanism behind the differential LRP1 expression observed in macro-vascular and microvascular ECs is unclear but could be related to shearstress a stimulus that is known to change gene expression3940 Theabsence of this reuptake machinery in microvascular EC suggests thatthe HepL secreted from these cells is likely taken up in the heart byproximal cells Given the proximity of cardiomyocytes (which do notexpress the heparanase gene) to microvascular EC it is plausible to envi-sion the exogenous uptake of EC-secreted heparanase into cardiomyo-cytes In support of this theory we detected both the latent and activeforms of heparanase in isolated cardiomyocytes This observationcoupled with the robust expression of LRP1 in cardiomyocytes whoseinhibition abrogates HepL uptake indicates that transfer from exogenoussources determines the presence of heparanase in cardiomyocytes

One implication of cardiomyocytes acquiring HepL is its subsequentintracellular conversion to HepA followed by its nuclear entry to influ-ence gene transcription By cleaving nuclear HSPG HepA mitigates thesuppressive effect of heparan sulphate on histone acetyltransferase toactivate gene expression11 Using an apoptosis PCR array which detectsboth pro- and anti-apoptotic genes we discovered that cardiomyocytesincubated with HepL down-regulated pro-apoptotic genes (egTnfrsf10b Tnfsf10) whereas anti-apoptotic genes (eg Cflar Tnfrsf11b)were up-regulated As cardiomyocytes isolated from heparanase trans-genic mice also showed a similar trend in this gene expression pattern(unpublished data) our data imply that HepL displayed pro-survivaleffects on the cardiomyocyte by initiating a program that protects againstapoptosis This effect of heparanase on gene expression relies on itsactivity as its inhibition by a specific heparanase inhibitor reversed itsbeneficial effects on gene expression Additionally the changes in geneexpression induced by heparanase translated into protection against car-diomyocyte cell death as confirmed by the reduction in the BaxBcl-2mRNA ratio cleaved PARP and caspase 3 and Annexin VPI staining Indiabetes hyperglycemia can provoke cardiomyocyte cell death and con-tribute to cardiomyopathy18ndash2041 However it should be noted that it isthe EC that is exposed to this metabolic alteration before the cardio-myocyte As such through their release of HepL EC as first respondersto hyperglycemia could pre-condition the cardiomyocyte againstimpending metabolic damage For this to work hyperglycemia also needsto increase HepL uptake into the cardiomyocyte Indeed we observedrobustly increased LRP1 expression and levels of HepA as well as a pro-survival gene signature in whole hearts and cardiomyocytes isolatedfrom acutely diabetic animals Hyperglycemia and its associated oxidativestress which resembles hypoxia and its attendant increase in HIF-1acould be one explanation for LRP1 induction in short-term hyperglyce-mia HIF-1a is a known factor that can induce LRP1 expression in cardio-myocytes42 and in other cell types43ndash46 These effects were lostfollowing chronic diabetes and could contribute to the development ofcardiomyopathy in these animals The disappearance of LRP1 with pro-longed duration of diabetes may be related to a further attenuation of

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0 50 100 150

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Figure 7 HepL protects cardiomyocytes from HG induced apoptosis Isolated rat cardiomyocytes were incubated with 30 mM glucose (HG) andor500 ngmL myc-HepL for 12ndash48 h nfrac14 6 After 12 h the BaxBcl2 mRNA ratio was determined (A) PARP and caspase 3 cleavage were evaluated after 48 h(B) nfrac14 7 Annexin VPI staining as markers of apoptosis were also determined after cardiomyocyte incubation with HG andor myc-HepL (C) nfrac14 195ndash317myocytes pooled from four independent experiments The merged image of Annexin VPI staining is described in the fourth panel (scale bar 50 mm) whereasa higher magnification image (scale bar 10 mm) is described in the fifth panel Data are from a representative experiment Plt 001 Plt 0001

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circulating insulin as islets that escaped the initial insult by STZ are laterlost due to the combined features of hyperglycemia and hyperlipidemia(gluco-lipotoxicity) Interestingly several studies have reported thatLRP1 is down-regulated in brains from chronically diabetic mice an effectassociated with sustained hyperglycemia and insulin deficiency in theseanimals4748 Confirmation of the beneficial effects of heparanase in theprevention of diabetic cardiomyopathy requires the induction of diabe-tes in mice that overexpress heparanase experiments that are currentlyunderway in our lab

In summary our data reveal a novel and complex role for EC in pro-viding functional support to subjacent cardiomyocytes by communicatingvia soluble paracrine mediators In this study HG was a common stimu-lus for HepL secretion from the EC in addition to promoting its uptakeinto the cardiomyocyte The presence of heparanase in the cardiomyo-cyte dramatically changed the expression of apoptosis-related genesproviding an acute cardioprotective effect Data obtained from thesestudies suggesting a novel favourable effect of HepL in the cardiomyo-cyte will assist in devising novel therapeutic strategies to prevent ordelay diabetic heart disease

Supplementary material

Supplementary material is available at Cardiovascular Research online

Conflict of interest none declared

FundingThis work was supported by an operating grant from the Canadian Institutesof Health Research to BR (CIHR-MOP-133547) and the Israel ScienceFoundation (SF60114) to IV AP-LC and DZ are the recipients ofDoctoral Student Research Awards from the Canadian Diabetes AssociationFunding to pay the open access publication charges for this article was pro-vided by CIHR

References1 Kuramochi Y Cote GM Guo X Lebrasseur NK Cui L Liao R Sawyer DB Cardiac

endothelial cells regulate reactive oxygen species-induced cardiomyocyte apoptosisthrough neuregulin-1betaerbB4 signaling J Biol Chem 200427951141ndash51147

2 Narmoneva DA Vukmirovic R Davis ME Kamm RD Lee RT Endothelial cells pro-mote cardiac myocyte survival and spatial reorganization implications for cardiacregeneration Circulation 2004110962ndash968

3 Hsieh PC Davis ME Lisowski LK Lee RT Endothelial-cardiomyocyte interactions incardiac development and repair Annu Rev Physiol 20066851ndash66

4 Tirziu D Giordano FJ Simons M Cell communications in the heart Circulation2010122928ndash937

5 Ilan N Elkin M Vlodavsky I Regulation function and clinical significance of hepara-nase in cancer metastasis and angiogenesis Int J Biochem Cell Biol 2006382018ndash2039

6 Ziolkowski AF Popp SK Freeman C Parish CR Simeonovic CJ Heparan sulfate andheparanase play key roles in mouse beta cell survival and autoimmune diabetes J ClinInvest 2012122132ndash141

7 Hao NB Tang B Wang GZ Xie R Hu CJ Wang SM Wu YY Liu E Xie X Yang SMHepatocyte growth factor (HGF) upregulates heparanase expression via the PI3KAktNF-kappaB signaling pathway for gastric cancer metastasis Cancer Lett201536157ndash66

8 Hammond E Khurana A Shridhar V Dredge K The role of heparanase and sulfa-tases in the modification of heparan sulfate proteoglycans within the tumor microen-vironment and opportunities for novel cancer therapeutics Front Oncol 20144195

9 Purushothaman A Uyama T Kobayashi F Yamada S Sugahara K Rapraeger ACSanderson RD Heparanase-enhanced shedding of syndecan-1 by myeloma cells pro-motes endothelial invasion and angiogenesis Blood 20101152449ndash2457

10 Wang Y Pei-Ling Chiu A Neumaier K Wang F Zhang D Hussein B Lal N Wan ALiu G Vlodavsky I Rodrigues B Endothelial cell heparanase taken up by cardiomyo-cytes regulates lipoprotein lipase transfer to the coronary lumen following diabetesDiabetes 2014632643ndash2655

11 Purushothaman A Hurst DR Pisano C Mizumoto S Sugahara K Sanderson RDHeparanase-mediated loss of nuclear syndecan-1 enhances histone acetyltransferase(HAT) activity to promote expression of genes that drive an aggressive tumor phe-notype J Biol Chem 201128630377ndash30383

12 He YQ Sutcliffe EL Bunting KL Li J Goodall KJ Poon IK Hulett MD Freeman CZafar A McInnes RL Taya T Parish CR Rao S The endoglycosidase heparanaseenters the nucleus of T lymphocytes and modulates H3 methylation at actively tran-scribed genes via the interplay with key chromatin modifying enzymes Transcription20123130ndash145

13 Nobuhisa T Naomoto Y Okawa T Takaoka M Gunduz M Motoki T Nagatsuka HTsujigiwa H Shirakawa Y Yamatsuji T Haisa M Matsuoka J Kurebayashi J NakajimaM Taniguchi S Sagara J Dong J Tanaka N Translocation of heparanase into nucleusresults in cell differentiation Cancer Sci 200798535ndash540

14 Wang F Wang Y Zhang D Puthanveetil P Johnson JD Rodrigues B Fatty acid-induced nuclear translocation of heparanase uncouples glucose metabolism in endo-thelial cells Arterioscler Thromb Vasc Biol 201232406ndash414

15 Wang Y Zhang D Chiu AP Wan A Neumaier K Vlodavsky I Rodrigues BEndothelial heparanase regulates heart metabolism by stimulating lipoprotein lipasesecretion from cardiomyocytes Arterioscler Thromb Vasc Biol 201333894ndash902

16 Nadir Y Brenner B Zetser A Ilan N Shafat I Zcharia E Goldshmidt O Vlodavsky IHeparanase induces tissue factor expression in vascular endothelial and cancer cellsJ Thromb Haemost 200642443ndash2451

17 Gingis-Velitski S Zetser A Flugelman MY Vlodavsky I Ilan N Heparanase inducesendothelial cell migration via protein kinase BAkt activation J Biol Chem 200427923536ndash23541

18 Boudina S Abel ED Diabetic cardiomyopathy revisited Circulation 20071153213ndash3223

19 Poornima IG Parikh P Shannon RP Diabetic cardiomyopathy the search for a unify-ing hypothesis Circ Res 200698596ndash605

20 Fang ZY Prins JB Marwick TH Diabetic cardiomyopathy evidence mechanisms andtherapeutic implications Endocr Rev 200425543ndash567

21 Szkudelski T The mechanism of alloxan and streptozotocin action in B cells of therat pancreas Physiol Res 200150537ndash546

22 Sambandam N Chen XS Cam MC Rodrigues B Cardiac lipoprotein lipase in thespontaneously hypertensive rat Cardiovasc Res 199733460ndash468

23 Pulinilkunnil T An D Ghosh S Qi D Kewalramani G Yuen G Virk N Abrahani ARodrigues B Lysophosphatidic acid-mediated augmentation of cardiomyocyte lipo-protein lipase involves actin cytoskeleton reorganization Am J Physiol Heart CircPhysiol 2005288H2802ndashH2810

24 Zetser A Bashenko Y Miao HQ Vlodavsky I Ilan N Heparanase affectsadhesive and tumorigenic potential of human glioma cells Cancer Res 2003637733ndash7741

25 Shafat I Ilan N Zoabi S Vlodavsky I Nakhoul F Heparanase levels are elevated inthe urine and plasma of type 2 diabetes patients and associate with blood glucoselevels PLoS One 20116e17312

26 Zhang D Wan A Chiu AP Wang Y Wang F Neumaier K Lal N Bround MJJohnson JD Vlodavsky I Rodrigues B Hyperglycemia-induced secretion of endothe-lial heparanase stimulates a vascular endothelial growth factor autocrine network incardiomyocytes that promotes recruitment of lipoprotein lipase Arterioscler ThrombVasc Biol 2013332830ndash2838

27 Zetter BR The endothelial cells of large and small blood vessels Diabetes19813024ndash28

28 Wang F Wang Y Kim MS Puthanveetil P Ghosh S Luciani DS Johnson JD AbrahaniA Rodrigues B Glucose-induced endothelial heparanase secretion requires corticaland stress actin reorganization Cardiovasc Res 201087127ndash136

29 Ben-Zaken O Shafat I Gingis-Velitski S Bangio H Kelson IK Alergand T Amor YMaya RB Vlodavsky I Ilan N Low and high affinity receptors mediate cellular uptakeof heparanase Int J Biochem Cell Biol 200840530ndash542

30 Herz J Strickland DK LRP a multifunctional scavenger and signaling receptor J ClinInvest 2001108779ndash784

31 Lillis AP Van Duyn LB Murphy-Ullrich JE Strickland DK LDL receptor-related pro-tein 1 unique tissue-specific functions revealed by selective gene knockout studiesPhysiol Rev 200888887ndash918

32 von Harsdorf R Li PF Dietz R Signaling pathways in reactive oxygen species-inducedcardiomyocyte apoptosis Circulation 1999992934ndash2941

33 Chen L Sanderson RD Heparanase regulates levels of syndecan-1 in the nucleusPLoS One 20094e4947

34 Yang Y Gorzelanny C Bauer AT Halter N Komljenovic D Bauerle T Borsig LRoblek M Schneider SW Nuclear heparanase-1 activity suppresses melanoma pro-gression via its DNA-binding affinity Oncogene 2015345832ndash5842

35 Purushothaman A Babitz SK Sanderson RD Heparanase enhances the insulin recep-tor signaling pathway to activate extracellular signal-regulated kinase in multiple mye-loma J Biol Chem 201228741288ndash41296

36 Boyango I Barash U Naroditsky I Li JP Hammond E Ilan N Vlodavsky IHeparanase co-operates with Ras to drive breast and skin tumorigenesis Cancer Res2014744504ndash4514

37 Zetser A Bashenko Y Edovitsky E Levy-Adam F Vlodavsky I Ilan N Heparanaseinduces vascular endothelial growth factor expression Correlation with p38 phos-phorylation levels and Src activation Cancer Res 2006661455ndash1463

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38 Bhattacharjee PS Huq TS Potter V Young A Davenport IR Graves R Mandal TKClement C McFerrin HE Muniruzzaman S Ireland SK Hill JM High-glucose-inducedendothelial cell injury is inhibited by a peptide derived from human Apolipoprotein EPLoS One 20127

39 Topper JN Gimbrone MA Blood flow and vascular gene expression fluidshear stress as a modulator of endothelial phenotype Mol Med Today1999540ndash46

40 Chen BPC Li YS Zhao YH Chen KD Li S Lao JM Yuan SL Shyy JYJ Chien S DNAmicroarray analysis of gene expression in endothelial cells in response to 24-h shearstress Physiol Genomics 2001755ndash63

41 Cai L Kang YJ Cell death and diabetic cardiomyopathy Cardiovasc Toxicol20033219ndash228

42 Gao QQ Guan LN Huc SS Yao YW Ren XL Zhang ZW Cheng CL Liu Y Zhang CHuang JP Su DM Ma X Study on the mechanism of HIF1a-SOX9 in glucose-inducedcardiomyocyte hypertrophy Biomed Pharmacother 20157457ndash62

43 Chang ML Chiu CJ Shang F Taylor A High glucose activates ChREBP-mediated HIF-1 alpha and VEGF expression in human RPE Cells under Normoxia Retin DegeneratiDis Mech Exp Ther 2014801609ndash621

44 Kawata K Kubota S Eguchi T Aoyama E Moritani NH Kondo S Nishida TTakigawa M Role of LRP1 in transport of CCN2 protein in chondrocytes J Cell Sci20121252965ndash2972

45 Bonello S Zahringer C BelAiba RS Djordjevic T Hess J Michiels C Kietzmann TGorlach A Reactive oxygen species activate the HIF-1 alpha promoter via a func-tional NF kappa B site Arterioscl Throm Vas 200727755ndash761

46 Castellano J Aledo R Sendra J Costales P Juan-Babot O Badimon L Llorente-Cortes V Hypoxia stimulates low-density lipoprotein receptor-related protein-1expression through hypoxia-inducible factor-1 alpha in human vascular smoothmuscle cells Arterioscler Thromb Vas 2011311411ndash1420

47 Liu CC Hu J Tsai CW Yue M Melrose HL Kanekiyo T Bu GJ Neuronal LRP1 regulatesglucose metabolism and insulin signaling in the brain J Neurosci 2015355851ndash5859

48 Hong H Liu LP Liao JM Wang TS Ye FY Wu J Wang YY Wang Y Li YQ Long YXia YZ Downregulation of LPR1 at the blood-brain barrier in streptozotocin-induced diabetic mice Neuropharmacology 2009561054ndash1059

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