Biochemical Pharmacology 178 (2020) 114019 ContentslistsavailableatScienceDirect BiochemicalPharmacology journal homepage: www.elsevier.com/locate/biochempharm Reversalofendothelialdysfunctionbynicotinamidemononucleotide via extracellularconversiontonicotinamideriboside £ukaszMateuszuka,RobertoCampagnaa,d,BarbaraKutryb-Zaj¹cb,KamilKuœa, EwaM.S³ominskab,RyszardT.Smolenskib,StefanChlopickia,c,. aJagiellonianCentreforExperimentalTherapeutics(JCET),JagiellonianUniversity,Krakow,Poland bDepartmentandChairofBiochemistry,MedicalUniversityofGdañsk,Gdañsk,Poland cChairofPharmacology,JagiellonianUniversityMedicalCollege,Krakow,Poland dDepartmentofClinicalSciences,PolytechnicUniversityofMarche,Ancona,Italy ARTICLEINFO Keywords: EndothelialdysfunctionNicotinamideadeninedinucleotide Nicotinemononucleotide Nicotinamideriboside ABSTRACT Background: Nicotinamidemononucleotide(NMN)andnicotinamideriboside(NR) areeffectivesubstratesfor NADsynthesis,whichmayact asvasoprotectiveagents.Here, wecharacterizetheeffectsofNMNandNR on endothelialinflammationanddysfunctionandtesttheinvolvementofCD73intheseeffects.Materialsandmethods: TheeffectofNMNandNRonIL1ß-orTNF.-inducedendothelialinflammation(ICAM1and vWF expression), intracellular NAD concentration and NAD-related enzyme expression (NAMPT, CD38,CD73),werestudiedinHAECs.TheeffectofNMNandNRonangiotensinII-inducedimpairmentofendothelium­dependentvasodilationwasanalyzedinmurineaorticrings.TheinvolvementofCD73inNMNandNReffectswastestedusingCD73inhibitor-AOPCP,orCD73-/-mice. Results: 24h-incubationwith NMN andNRinducedanti-inflammatoryeffectsin HAECstimulated by IL1ß or TNF.,asevidencedby areductioninICAM1andvWFexpression.EffectsofexogenousNMNbutnotNRwasabrogatedinthepresenceofAOPCP,thatefficientlyinhibitedextracellularendothelialconversionofNMNtoNR,withoutasignificanteffectonthemetabolismofNMNtoNA.Surprisingly,intracellularNADconcentrationincreasedinHAECstimulatedbyIL1ßorTNF.andthiseffectwasassociatedwithupregulationofNAMPTandCD73,whereaschangesinCD38expressionwerelesspronounced.NMNandNRfurtherincreasedNADinIL1ß­stimulated HAECs and AOPCP diminished NMN-induced increase in NAD, without an effect on NR-inducedresponse.InexvivoaorticringsstimulatedwithangiotensinIIfor24h,NO-dependentvasorelaxationinducedbyacetylcholinewasimpaired.NMNandNR,bothpreventedAngII-inducedendothelialdysfunctionintheaorta.InaorticringstakenfromCD73-/-miceNMNeffectwaslost,whereasNReffectwaspreserved. Conclusion: NMNandNRmodulateintracellularNADcontentinendothelium,inhibitendothelialinflammation andimproveNO-dependentfunctionbyCD73-dependentandindependentpathways,respectively.Extracellularconversion of NMN to NR by CD73 localized in the luminal surface of endothelial cells represent importantvasoprotectivemechanismstomaintainintracellularNAD. 1. Introduction Nicotinamide riboside (NR) and nicotinamide mononucleotide(NMN) have drawn attention as alternative nicotinamide adenine di­nucleotide (NAD) substrates, devoid of side effects for nicotinic acid(NicA), such as “flushing” orhepatotoxicity andsideeffectsof nicoti­namide(NA),includingsirtuininhibition.BothNADsubstrates,NRandNMN were proposed to be used in sports nutrition as good dietarysupplements [9,8] and display numerous beneficial effects in varioussettings,buttheirbioavailabilityandpathwaysofmetabolismtowards NADdiffers. NR, detectable in cow milk, milk-derived products and in natural products containing yeast [3] has a good bio-availability and in­tracellularly is metabolized via NRK1 and NRK2 to NMN, a majorprecursorofNAD.NRwas showntobeeffectiveinrestoringtheNADpoolbothinmiceandhumans[61].NumerousstudiesonNRshoweda significantimpactofthissubstrateonNADcontent,bioenergetics,andimprovedregenerativecapabilitiesinvariousrodentmodelsofdisease.Forexample,NRtreatmentresultedinincreasedNADconcentrationina mouse model of respiratory chain III complex deficiency [52], . Correspondingauthorat:JagiellonianCentreforExperimentalTherapeutics(JCET),JagiellonianUniversity,Krakow,Poland.E-mailaddress:stefan.chlopicki@jcet.eu(S.Chlopicki). https://doi.org/10.1016/j.bcp.2020.114019 Received5December2019;Accepted4May2020 Available online 08 May 20200006-2952/ © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). improved liver regeneration [46]andrestoredNAD contentin mouse skeletalmusclemyotubes [17].NRtreatmentalsoenhancedoxidative metabolism and prevented weight gain in a mouse model of diet-in­duced obesity [7].Moreover,NR treatment increased NADcontent inthecerebralcortex,thusattenuatedcognitivedeteriorationinamousemodel of Alzheimer’s disease [21]. NR was also effective in heart failure, as NR-supplemented diet administrated to murine models ofdilatedcardiomyopathy orpressureoverload-inducedheartfailure re­storedmyocardialNADlevelsandimprovedimpairedcardiacfunction[15]. IncontrasttoNR,NMNhasaworsebioavailability,asextracellularNMN is unable to pass the endothelial membrane without prior de­phosphorylationbyCD73(ecto-5'-nucleotidase)toNR[24]orpriorto metabolism to nicotinamide by extracellular CD38 [25,28]. Extra­cellular nicotinamide in the presence of phosphoribosyl-1-pyropho­sphate (PRPP) could be also converted NMN by visfatin as reviewedrecently[23,65].Interestingly,incardiomyocytes,itwasdemonstratedthatconnexin43(Cx43)channels arepermeabletoextracellularNAD [4] suggesting that intracellular transport of NAD and NMN may becell-typedependentandreliantonvarioustransporters.Intracellularly,NA could be converted by nicotinamide phosphoribosyltransferase(NAMPT) to endogenous NMN in three-step Preiss-Handler pathway[48,20,31], or methylated by nicotinamide N-methyltransferase(NNMT)to1-methylnicotinamide(MNA)[1].NRisphosphorylatedbynicotinamide riboside kinases (NRK1, NRK2) to endogenous NMN[53,17]. NMN is subsequently transformed to NAD by nicotinamidemononucleotide adenylyltransferases (NMNAT1-3). Apart from in­volvementinredoxreactions,NADisalsosubstrateforsirtuins(SIRT),poly-ADP-ribose polymerases (PARP) and other NAD-dependent en­zymesresultinginreleaseofendogenousNA. Innumerous studies,NMNunequivocallyaffordedNAD-dependentbeneficial effects. For example, NMN improved muscular contractilefunction in mouse age-related models of muscle dysfunction [17,45], restoredcardiacNADcontentin mousemodelofischemia-reperfusion [63], improved metabolic balance in type 2 diabetes mice [66], improvedNADcontentandsurvivalinratmodelsofhemorrhagicshock [55] and had a protective effect in ß-amyloid oligomer-induced ratmodelofAlzheimer’sdisease[62].Insomepreviousreports,theeffects ofNMNandNR werecompared[49,17],butinmostofthesestudies,either NMN or NR was characterized. Still, the role of ecto-enzymesCD73andCD38inNMN-inducedeffectshasnotbeenfullycharacter­ized,soitisnotclearwhetherNMN-triggeredbeneficialeffectsareNR­orNA-dependentandwhatmetabolicenzymesare involved. DespitenumerousstudiesonthebeneficialeffectsofNRandNMNinvariousmodels,thereisstillapaucityofdataasregardstheeffectsofNMNandNRonendothelialfunction.NMNtreatmenthadabeneficial effect in various mouse models of age-related vascular pathologies inlinewiththegradualfallinNADcontentinaging[35,13].Thesestudies demonstrated that NMN restored endothelium-dependent vascularfunctionandmitigatedoxidativestressinage-relatedmodel[51],res­cuedangiogeniccapacityinagedcerebrovascularendothelialcells[35]andrestoredfenestration-likephenotypeofliversinusoidalendothelialcells (LSECs) isolated from old mice [30]. NR was also shown to be effective to improve vascular function. NR improved endothelium-de­pendent relaxation of isolated rat mesenteric arteries in ischaemia-re­perfusion model [60]. Beneficial endothelial effects of NR was also showninamousemodelofendotoxaemia,inwhichmodelNRrestoredNAD contents inlungand heart as well as decreased ROSproduction andapoptosisinisolatedendothelialcells[29]. Inthepresentwork, weaimedtocharacterizetheendothelialpro­file of action of NMN in comparison to NR in cellular and vascularmodels of endothelial inflammation, with particular attention to theinvolvement of extracellular conversion of NMN in these effects. Our researchdemonstrated,thatbothNMNandNRmodulatedintracellularNAD content in the endothelium, inhibited endothelial inflammation and improved NO-dependent function. The important finding of this workwastoshowthatNMNeffectsonendotheliumweremediatedbyCD73-dependentconversionofNMNtoNR. 2. Materialsandmethods 2.1. Cellsandanimals Eahy.926 endothelial hybrid cell line (ATCC® CRL-2922™,Manassas,VA,USA)andHumanAorticEndothelialCells(HAECs,CC­2535, Lonza, Basel, Switzerland) were used to study involvement ofCD73andCD38inNMNmetabolismandtoanalyze,effectsofNMNandNRonNADcontentandonendothelialinflammationinducedbyTNFa or IL1ß.For preliminarystudiesdue toeasier maintenance andfastergrowthrate Eahy.926 line was used,while majorpart of experimentswere performed on primary HAEC line, used as a representative en­dothelialinvitromodeltoexaminetheextracellularmetabolismofNAD substratesinhumanendothelium.Eahy.926andHAEClines wasused up to the fifth passage to avoid the phenotype changes during latepassaging. Cells reaching over ~90% confluence were used for theexperiments, grown on EBM-2 medium (CC-3156, Lonza, Basel,Switzerland)orDMEMmedium(ATCC® 30–2006,Manassas,VA,USA),containingglucose1g/L,L-glutamine1mMand20%FBS. 12–16weeks – old C57Bl/6J control mice (Jackson Laboratories,BarHarbor,ME,USA)andage-matchedCD73-/-mice,developedinHeinrich-Heine-Universität, Düsseldorf, provided by Gdansk MedicalUniversity[36]wereusedforexvivostudiesofvascularfunction.After transportation, animals (only females) were randomly allocated tocontrol and experimental groups and placed in individual cages withindependent ventilation system, hosting up to five animals. Animalswerekeptinquarantinefor1week,havinganunlimitedaccesstowaterandchowdiet.Twoorthreemiceadayweresacrificed(inthemorningand at midday) to isolate thoracic aorta for 24h incubation and sub­sequent wire myograph measurements. All animal procedures con­formed to the ARRIVE standards and EU Directive 2010/63/EU foranimalexperimentsandallexperimentalprocedureswereapprovedbytheFirstLocalEthicalCommitteeonAnimalTestingattheJagiellonianUniversityinKrakow. 2.2. Measurementofextracellularmetabolismofnicotinamide mononucleotide(NMN)inEahy.926cellsandHAECs Eahy.926cells(ATCC,Manassas,VA,USA),culturedinthe24-wellplates,wereincubatedwithsubstratesforCD73(AMPandNMN)orforCD38(onlyNMN)attheconcentrationrange1–5mM.Theenzymaticreactionwascarriedat100%ofconfluenceinincubationmedium(1mlofHankssolution)withorwithout5µMEHNAasadenosinedeaminaseinhibitor, 5µM nucleoside transport inhibitor (NBTI) and ecto-5'nu­cleotidase inhibitor – Adenosine 5'-(.,ß-methylene)diphosphate(AOPCP;50µM),allreagentswerepurchasedfromSigmaAldrich,SaintLouis,MO,USA.MichaelisConstant,Vmaxandreactionkinetics were extractedfromagraphicpresentationofexperimentalpoints.MediumsamplesweretakenforHPLCmeasurementofextracellularadenosine,NA and NR, according to the methodology described by Kutryb-Zajacetal.[38].AfterwashingwithcoldPBS,cellswerefrozenin-80°Cforprotein concentration measurement following solubilization in 0.5MNaOH, using Pierce BCA protein assay kit (Thermo Fisher Scientific,Waltham, MO, USA) and Synergy4 multiplate reader (BioTek, Wi-nooski,VT,USA). To confirm the data from Eahy.926 cells HAEC line (HAECs, CC­2535, Lonza, Basel, Switzerland) were cultured in 24-well plates andtreated with AOPCP (50µM) for 24h. After incubation, cells werewashedandplacedinKrebsbufferfor2h-nicotinamidestarving,thenNMN 100µM was applied for 1h-incubation. The concentration ofNMNwaschosenafterinitialassessmentofKmreactionforadenosine release by CD73, to induce the NR production by this enzyme fromNMN. NBTI was not used since nucleotide uptake was considered a marginalandnotaffectingNMNbio-availabilityduringshort-timein­cubationwiththissubstrate.Effluentsamplesweretakenattimepoints0, 30, 60min. and frozen for further NR and NA measurement, ac­cording to the methodology described previously [43]. After samplecollection,cellswerefrozenforproteinconcentrationmeasurement. 2.3. AssessmentofendothelialinflammationinHAECsbyimmunofluorescentstainingofICAM1and vonWillebrandfactor TostudytheeffectofNRandNMNsupplementationontheICAM1andvWFexpressionofTNF.-andIL1ß-stimulatedHAECline(CC-2535,Lonza,Basel,Switzerland),cellswereplatedin96-wellformatonblackCorning multiplates with clear bottom, then supplemented with NR,NMN,orNA(100µM/24h)andstimulatedwithTNF.(10ng/ml/24h) or IL1ß (Sigma Aldrich,Saint Louis, MO, USA) usedat the same con­centration.After24hincubation,cells werefixedwith a4%formalin solution (10min), washed with PBS, then incubated with a blockingsolution containing 5% normal goat serum (Jackson Immuno,Cambridgeshire,UK)and2%filtereddrymilk(Gostyn,Poland)weretominimalize non-specific binding of antibodies. For indirect im­munohistochemical detection of intercellular adhesion molecule 1 (ICAM1)and vonWillebrandfactor(vWF),cells wereincubatedover­nightwithmouseanti-ICAM1monoclonalIg,(ThermoFisherScientific,Waltham, MO, USA), rabbit anti-vWF polyclonal Ig (Abcam,Cambridge, UK). After rinsing in PBS (Thermo Fisher Scientific,Waltham,MO,USA),HAECcellsweretreatedwithdifferentsecondaryantibodies;AlexaFluor647-conjugatedgoat-anti-mouseforICAM1andAlexaFluor488-conjugatedgoat-anti-rabbitforvWF(JacksonImmuno,Cambridgeshire,UK).Fornucleicounterstaining,Hoechst33258solu­tion (Sigma Aldrich, Saint Louis, MO, USA) was applied. Images ofimmunostainedcellsweretakenusingCQ1confocalquantitativeimagecytometer(Yokogawa,Musashino,Tokio,Japan)andCQ1.04software,thenanalyzedautomaticallybyColumbus2.4.2software(PerkinElmer,Waltham, MA, USA) to assess mean fluorescence specific for im­munostained cells. For data normalization, each immunostaining(n=6)wasperformedusingcellswithsimilarconfluence(.90%),the same primary and secondary antibody concentration and constant in-cubationtimeforeachstainingstep.Asanegativecontrol,cellstreatedonlywithsecondaryantibodies wereusedtoestimatethebackground signal. 2.4. MeasurementsofchangesinNADcontentsinHAECs HAECs (CC-2535, Lonza, Basel, Switzerland) were cultured in 96well format at the density 12–15×104 for 24h. After the requiredconfluence was reached (80–100%), cells were supplemented with100µM of NMN (Sigma Aldrich, Saint Louis, MO, USA) or NR(ChromaDex,Irvine, CA,USA).CD73activity wasinhibitedby50µMAOPCP. For pro-inflammatory activation of HAEC line, IL1ß (SigmaAldrich, Saint Louis, MO, USA) wasused at aconcentration10ng/mlfor24h.TNF.wasalsousedasapro-inflammatorystimulator,butasTNF.–stimulatedcellsdisplayinconsistentresultsofintracellularNADcontent after 24h-incubation, IL1ß was preferably used. Cells werewashed with PBS (pH=7.4), treated with 40µl of cold 0.4M HClO4 (Chempur, Piekary Slaskie, Poland) and frozen at -80°C for at least24h. After thawing on ice, HClO4 cell extracts were centrifuged(14000pm/10min/4°C). The supernatant was collected and neu­tralized to ph=6.5 using 3M K3PO4, kept on ice for 15min, cen­trifuged and frozen for further HPLC-RT analysis as described pre­viously[56].Proteindepositremainingafterremovingthesupernatant wasresuspendedin30µlof0.5NaOH(Chempur,PiekarySlaskie,Po­land)andusedforBCAproteinconcentrationassay. 2.5. AssessmentoftheexpressionofextracellularmetabolismenzymesbyimmunofluorescentstainingandWesternBlotinHAECs ImmunofluorescentstainingofNAMPT,CD73andCD38 wereper­formed in HAECs plated in 96-well format (Corning, NY, USA), sup­plementedwith100µMNMNorNR,afterincubationwithIL1ß(10ng/ml/24h). Cells were fixed with a 4% formalin solution (10min), wa­shedwithPBS,thenincubatedwithablockingsolutioncontaining5%normal goat serum (Jackson Immuno, Cambridgeshire, UK) and 2%filtereddrymilk(Gostyn,Poland).BeforeNAMPTimmunostainingcellswereslightlypermabilizedwithTritonX-100(0,1%inPBSfor5min)toimprove antibody binding. The following primary antibodies wereused; rabbit anti-NAMPT (Thermo Fisher Scientific, Waltham, MO,USA),rabbit-anti-CD38(Abnova,Taipei,Taiwan)andmouse-anti-CD73(MerckMillipore,Burlington,MA,USA).Forvisualization onprimaryantibodybindingsites,AlexaFluor488-conjugatedgoat-anti-rabbitandCy3-conjugated goat-anti-mouse secondary antibodies (JacksonImmuno, Cambridgeshire, UK) were added for 30min. Images weretakenandanalyzedas describedabove. ForWesternBlotanalysisofNAMPT,CD73andCD38expressioninHAECline,cells wereplatedin6-wellformat(Corning,NY,USA),in­cubated or not with 100µM NMN or NR and stimulated with IL1ß(10ng/ml/24h), then collected using Accutase solution for 5min.(ThermoFisherScientific,Waltham,MO,USA),lysedbyM-Perreagent(Thermo FisherScientific,Waltham, MO, USA)and frozenin-80°C.Samples were thawed on ice, spinned down (12000G/10min/4°C) toremove protein clots, then the protein concentration was measured,using BCA protein concentration assay (Thermo Fisher Scientific,Waltham, MO, USA) and Synergy4 multiplate reader (BioTek,Winooski, VT, USA). WB samples were prepared by combining 30µg protein with a proper volume of 4x Laemmli buffer, heating (95°C/5min.) and frozen in -80°C. At the day of the analysis, samples were thawed on ice and placed in a 12% FastCast separating gel (BioRad,Hercules, CA, USA). As a protein standard, 5ul of PrecisionPlusUnstained Protein Ladder was used. The separation was performedunder 100V during 1,5h, then proteins were transferred to PVDFmembrane during 1htransfer, under 400mA,usingPowerPacHCPowerSupply(BioRad,Hercules,CA,USA).Membranes wereblocked with 2% milk solution in TBST (TBS+0,1% Tween 20) for 1h, thenincubated(overnight,4°C)withprimaryantibodies; mouseanti-CD73 (Merck Millipore, Burlington, MA, USA), rabbit anti-NAMPT (NovusBiologicals,Cenntenial,CO,USA)orrabbit-anti-CD38(Abnova,Taipei,Taiwan),usedatdilutions1:1000to1:5000.AfterwashingwithTBST,secondary antibodies were applied for 1h;goat-anti-rabbit-HRP orgoat-anti-mouse-HRP(Santa Cruz Biotechnology, Dallas, Texas, USA),usedatrecommendedconcentration1:5000.As anadditionalcontrol, mouse anti-ß-actin Ig (Santa Cruz Biotechnology, Dallas, Texas, USA)wasusedtodetectß-actinbandsat42kDasite,followedbygoat-anti­mouse-HRP (Santa Cruz Biotechnology, Dallas, Texas,USA). Due to a similar molecular weight of NAMPT and CD38, to avoid band over­lapping,ß-actinIgwasusedonlyonCD73-immunostainedmembranes.NAMPTandCD38,totalproteinloadwasusedasareferenceunstainedcontrolbands.ProteinbandsweredetectedusingClarityMaxWesternECL Substrate and ChemiDoc Imaging Station (BioRad, Hercules, CA,USA). Specific bands representing molecular weights 37kDa, 45kDaand 70kDa were detected for NAMPT, CD38 and CD73, respectively.Western Blot assay was repeated three times for each of analyzedproteins. 2.6. Measurementofendothelium-dependentvasodilationinaorticrings After ketamine/xylazine anesthesia, thoracic aorta was isolated,washedwithcoldPBS,cleanedofperivascularfattytissueanddividedinto three 2-mm-long rings, which was placed in DMEM medium containing a vehicle, NMN, NR (100µM) and/or AOPCP (50µM). Totrigger endothelial dysfunction aortic rings were incubated with an­giotensinIIataconcentrationof100nMfor24h.(SigmaAldrich,SaintLouis, Missouri, USA). Then, each ring was placedseparately in DMT620M multi-wire myograph chamber (Danish Myo Technology A/S,Aarhus,Denmark)containingKrebssolution(370C)withconstantCO2 flow and kept for 15min. under stabilizing conditions. After initialstretchingwith30mMand60mMKCl,theendothelium-dependentandendothelial-independent vasodilatory function were measured ac­cording to a standardized protocol, in phenylephrine-precontractedvessels using increasing concentrations of acetylcholine or sodium ni­troprusside,respectively.DatawereacquiredandanalyzedbyLabChart3.01software(DanishMyoTechnologyA/S,Aarhus,Denmark). 2.7. Statisticalanalysis Data were analyzed by Prism 6.0 software (GraphPad, CA, USA),using the nonparametric Mann-Whitney test and Kruskal-Wallis OnewayANOVA,followedbyposthocmultiplecomparisonsDunntest.Data wereshownasmean+SEM(*.0.05,**.0.01,***.0.001). 3. Results 3.1. EffectofCD73inhibitionbyAOPCPontheextracellularconversionofNMNinEahy.926andHAECcells In Eahy.926 cells exposed to the increasing concentration of exo­genousNMN,thereleaseofNRandNAwasincreasedasmeasuredbyHPLC assay (Fig. 1A and 1B). The Michaelis constant and Vmax forNMN›NR reaction was 1.37mM and 1.138nmol/ml/min, respec­tively, while for NMN›NR reaction: 2.29mM and 0.583nmol/ml/min,respectively.AOPCP(CD73inhibitor)incubatedfor2hat acon­centrationof50µMeffectivelydiminishedadenosineproductionfromAMPbyCD73(Fig.1C)aswellasNRproductionfromNMN,buthadalmost no effect on NA production from NMN (Fig. 1D). In HAECsAOPCP (incubated for 24h) also effectively inhibited conversion ofNMNtoNRasmeasuredbyLC/MS/MSassay(Fig.1F),resultinginverylowconcentrationofextracellularNR,ascomparedwithNMN-treatedgroupnotpretreatedwithAOPCP(33.54nmol/gofprot.vs3355nmol/ gofprot,respectively,p.0.01).NAproduction wasonlymarginallyaffected by AOPCP (1147nmol/g of prot. in NMN/AOPCP-treated groupvs1357nmol/gofprotinNMN-treatedgroup;p.0.05). 3.2. EffectsofNMNandNRonvonWillebrandfactorandICAM1 expressioninIL1ß-andTNF.-stimulatedHAECcells;involvementofCD73 IL1ß-induced the upregulation of vWF and ICAM1 in HAECs, andNMN prevented the pro-inflammatory effects of IL1ß. The anti-in­flammatory effect of NMN was lost in the presenceof CD73 inhibitorAOPCP(50µM).HAECstreatedonlywithAOPCPdisplayedincreasedexpression of vWF, as compared with untreated control HAECs(p.0.05). IL1ß–induced upregulation of vWF and ICAM1 expressionwasalsoreducedinthepresenceofNRbuttheanti-inflammatoryeffectofNRwasnotmodifiedbyAOPCP(Fig.2). Similarly to the effects of IL1ß, 24h-incubation with TNF. alsoresultedintheupregulationofvWFandICAM1inHAECs.BothNMNandNRpreventedTNF.–inducedupregulationofvWFandICAM1andtheseeffectswereabolishedbyCD73inhibitioninNMN-treatedHAECs,while in NR-treated HAECs CD73 inhibition had no effect on vWF­specificandICAM1-specificfluorescence(Fig.3). 3.3. EffectsofNMNandNRonintracellularNADconcentrationinHAECs afterstimulationwithIL1ß;effectsofCD73inhibition NMN or NR raised intracellular NAD content in basal non-stimu­lated HAECs (Fig. 4). NMN-triggered NAD increase was a CD73­dependent response, since, in AOPCP-treated HAECs, NMN supple­mentation did not increase significantly NAD content. In contrast toNMN, NR-induced raise of NAD was not modified by AOPCP. Stimu­lation with IL1ß was not linked to the significant fall in intracellularNAD content, rather an increased was noted. CD73 inhibition byAOPCPpreventedtheriseinNADinducedbyNMNinIL1ß-stimulatedHAECs, however effects of NR on NAD content in this experimentalsetupwasnotmodifiedbyAOPCP. 3.4. ChangesinNAMPT,CD38andCD73expressioninIL1ß-stimulatedHAECs ToexplaintheincreaseofNADcontentinIL1ß-stimulatedHAECsweanalyzedtheexpressionofNAMPTandtwoectoenzymes;CD73andCD38 in this experimental setting by immunocytochemistry andWestern Blot. As shown in Fig. 5A, a specific immunofluorescence ofNAMPT, the main cytosolic enzyme involved in NAD synthesis wasupregulated (p.0.01) in HAECs stimulated by IL1ß. CD73-specificimmunofluorescencewashigherinIL1ß-treatedHAECs, ascompared with untreated HAECs (Fig. 5B). Similarly, CD73 and NAMPT expres­sioninHAECstimulatedbyIL1ßwereincreasedasassessedbyWesternBlot(Fig.5D).SupplementationwithNMNorNRresultedinadecreaseofupregulatedNAMPTandCD73expressioninIL1ß-stimulatedHAECs,whichwasalsoconfirmedbyWBassay.StimulationwithIL1ßhadnosignificanteffectonCD38-specificimmunofluorescence(Fig.5C),whileWBanalysisshownaminorupregulationofCD38inIL1ß-treatedcellsand downregulation after co-incubation with NMN or NR. This datasuggeststhecompensatoryupregulationofNAMPT,CD73inresponsetoinflammatorystimulus,mightcontributetothepreservationofNADpoolinIL1ß-stimulatedHAECs.NMN or NRtreatmentresultedinthe anti-inflammatory effects, and reverted compensatory upregulation ofNAMPTandCD73expressioninIL1ß-stimulatedHAECs. 3.5. EffectsofNMNorNRonangiotensinII-inducedimpairmentofendothelium-dependentvasodilatoryresponseinaorticringsinwildtypeandCD73-/-mice AngiotensinIIimpairedvasodilatoryresponsetoAchinaortatakenfromC57Bl/6mice,withoutaneffectonSNP-inducedvasodilation.Co­incubationwith NMN mitigated theimpairment of Ach-induced vaso­dilationinducedbyAngiotensinII(Fig.6A),andthiseffectofNMNwasabrogated by CD73 inhibitor AOPCP. NMN had no effect on Angio­tensin II-induced impairment of vasodilation of aortic rings to Ach inCD73-/-mice (Fig. 6B). In contrast to NMN, NReffectivelyrestoredresponsetoAchinAngiotensinII-treatedaorticringsisolatedbothfromC57 and CD73-/- mice (Fig. 6C, D). SNP-induced vasodilation wassimilar in magnitude in all experimental groups including NMN-andNR-treated vessels taken from C57Bl/6J and CD73-deficient mice(Fig.6E,F).AOPCPusedalonehadnosignificanteffectonvasodilatoryresponses(datanotshown). 4. Discussion Here we demonstrated that NMN inhibited endothelial inflamma­tionandimprovedNO-dependentfunctionbyextracellularconversionvia ecto-5'-nucleotidase (CD73)-dependent pathway to NR and bymodulationofendothelialintracellularNADpool.Ourresultssuggest,that extracellular conversion of NMN to NR by CD73 localized in theluminalsurfaceofendothelialcellsrepresentsimportantvasoprotectivemechanisms maintaining intracellular NAD and healthy phenotype ofendothelialcells. The salient findings of this work was to demonstrate the anti-in­flammatory(inhibitionofupregulationofICAM1, vWFin responsetoIL1ß and TNF.) and vasoprotective (inhibition of the impairment ofNO-dependentfunctioninresponsetoAngII)effectsofNMN,thatwascomparabletotheeffectsofNRinendothelialcellsinvitroaswellasthe Fig. 1.Effects of CD73 inhibition by AOPCP on extracellular metabolism of NMN in Eahy.926 (A-E) and HAEC cells (F, G). Michaelis constant and Vmax ofextracellular nicotinamide riboside/adenosine (A) and nicotinamideproduction (B) by from exogenous nicotinamide mononucleotide and/or AMP. The rates of adenosineproductionfromadenosinemonophosphate(AMP)(C),nicotinamideribosideproductionfromnicotinamidemonophosphate(NMN)(D)andnicotinamide productionfromNMN (E) onthesurfaceofEA.hy926cells.Theconcentrationofextracellularnicotinamideriboside (F) andnicotinamide (G) releasedbyHAEC cells, measured after 24h-preincubation with CD73 inhibitor AOPCP and 60min-incubation withnicotinamide mononucleotide (100µM). n=6, * p.0.05, ** p.0.01,***p.0.001. intheaorticringsexvivo.Furthermore,NMN-affordedeffects,butnotendothelialcells(Eahy.926,HAECs),buthadalmost noeffectonNA NR-inducedeffectswereabsentinthepresenceofCD73inhibitorproductionfromNMNsuggestingselectiveinhibitionofCD73without AOPCPorinCD73–deficientmice.TosubstantiatethattheinhibitoryaneffectonCD38.TheMichaelisConstantandVmaxforNMN›NR effectofAOPCPonNMNresponseinendothelialcellswasindeedduereactionwasapproximately10xlowerthenAMP›adenosinevalueto the inhibition of CD73, we demonstrated that AOPCP (50µM) ef-that could point out that the major physiological and pathophysiolo­fectively diminished NR production from NMN in two types of gical role of this enzyme might be linked to AMP, not to NMN Fig.2.EffectsofNMNandNRonIL1ß-inducedincreaseinvWFandICAM1expressioninHAECs.ExpressionofvWFinNRandNMN-andNR-treatedHAECcellsafter24h-stimulationwithIL1ßinthepresence orabsenceofCD73inhibitorAOPCP, (A);expressionofICAM1inNR-andNMN-treatedHAECcellsafter 24h­ stimulationwithIL1ßinthepresenceorabsenceofCD73inhibitorAOPCP,(B);n metabolism.However,theextracellularconcentrationofAMPwasde­tected in the nanomolar range [22,10]. On the other hand, although this opinion is not univocally accepted [26], the local extracellularconcentration of NMN may reach micromolar concentration range as suggested by some authors [54]. Therefore, CD73, a well-known en­zyme responsible for the conversion of AMP into adenosine and in­organicphosphate[5]mayrepresentanimportantregulatorypathwayforextracellularNMNmetabolisminendothelialcells. Previously expression of CD73 in endothelial cell membrane waslinked to anti-inflammatory, immunosuppressive, vasoprotective oranti-platelet action of adenosine [69,50,42,12,32,39]. Despite the va­soprotectiveeffectofCD73-derivedadenosine,thispathwayplaysalso animportantroleintumorprogression asapotentsuppressorofanti­cancer immune responses [67,2]. Interestingly, mutation in humanNT5E gene encoding CD73 triggers recently described genetic mal­function (2011) known as ACDC (“arterial calcification due to defi­ciencyofCD73”),whichisreflectedbycomplexphenotypeofvascularcalcification,arteriomegaly,andtortuosity,andsometimescalcificationin small joints [47,33] underscoring the important role of CD73 invascularhomeostasis.MouseCD73-/-modeliscriticizedasitdoesnot reflectthesymptomsofCD73knockoutinhumans[33].However,thisdifferences were ascribed to adenosine-mediated mechanisms; inhumanbloodthehalf-lifeofadenosineis<15s,whileinmicethehalf­life is .2min [58,68,16]. In the present work we used human en­dotheliumtostudyCD73-dependentconversionofNMNtoNRbutnottostudyadenosine-dependentmechanisms.Furtherstudiesareneedtodetermine the relative importance of CD73-dependent regulation ofNADmetabolisminmiceandhumans. Neverthelesstheselimitations,CD73 knockdowninmiceseriously =6,*p.0.05,**p.0.01;whitescalebarrepresents25µm. affectedvascularfunction.Mierzejewskaetal.[44]demonstratedthat CD73-/- mice displayed endothelial dysfunction with enhanced ad­ hesion molecules, activation of pro-inflammatory cytokine and im­ paired L-Arginine metabolism, and these changes progressed with the ageofanimals.CD73wasalsoshowntolimitendothelialpermeability [11], trans-endothelial leukocyte trafficking and immune sequelae of allograftvasculopathy[27].Inallthesestudiesvasoprotectiveandanti­ inflammatory roles of CD73 were uniquely linked to adenosine-medi­ atedmechanisms,forexampleexertedviaA2Breceptors[11,27]. In the present study, we provide evidence suggesting that CD73 represents an important pathway that controls the extracellular con­ version of NMN to NR before it could be used as the intracellular substrateforNMN(byNRK1 orNRK2)andsubsequentlyforNAD(by NMNAT1-3) in endothelial cells. Thus, the vasoprotective effects of CD73 described previously might be not only linked to adenosine­ mediatedpathways,butcouldbealsolinkedtoextracellularconversion ofNMNtoNRbyCD73.However, wecannotexclude,thatin ourex­ perimentalsystemCD73-dependentadenosinesignalinginendothelial cells played a role since HAEC cells incubated with AOPCP only dis­ playedahigherexpressionofvonWillebrandFactor(Fig.2B).Therole ofCD73intheconversionofextracellularsubstrateforendothelialNAD such as NMN converted to NR, seemquite likely not only in aging, a well-knownstateofNADdeficiency [35,13],butalsoinvariousother contextswherevasoprotectiveeffectsofNMNorNRweredemonstrated [51,30,60,29].The keyelement ofthis concept that yethasto be ad­ dressedistheexact sourceandconcentrationofNMNinproximityof endothelial luminal surface. That may not be represented by plasma concentration, but by cell surface concentration that may be vastly differentassuggestedforadeninenucleotides[64]. Fig.3.EffectsofNMNandNRonTNF.–inducedincreaseinvWFandICAM1expressioninHAECs.ExpressionofvWFinNRandNMN-andNR-treatedHAECcellsafter24h-stimulationwithTNF.inthepresence orabsenceofCD73inhibitorAOPCP,(A);expressionofICAM1inNR-andNMN-treatedHAECcellsafter24h­stimulationwithTNF.inthepresenceorabsenceofCD73inhibitorAOPCP,(B);n=6,*p.0.05,**p.0.01;whitescalebarrepresents25µm. Thisnotionsupportedbyexperimentalresultsofthisstudy,staysin[24].TheintrinsicroleofCD73intheNMN-dependenteffectonin­linewiththerecentdiscoveryofNAD-relatedfunctionofCD73afterthetracellularNADconcentration wasconfirmedin numerouscancercell structuralandfunctionalanalysisofHaemophilusinfluenzaeNAD nu-lines(U87,A549,PC3,OVCAR-3,HePG2) aswellasinHEK293cells cleotidase(NADn), anorthologofhumanCD73capableofprocessing [37,57,24].Furthermore,highCD73expressioninthetumortissuehasNMN[19,18].Indeed,CD73waspreviouslyshowntobeinvolvedinthebeenlinkedtopooroverallsurvivalandrecurrence-freesurvivalinNMNdephosphorylationintoextracellularNRtosustainintracellularpatientssufferingfrombreastandovariancancerandthisphenomenonNADinvarioushuman cancercellssubjectedtoinhibitionofNAMPT couldbelinkednotonlytoadenosine-dependentpathway[42,59]but Fig. 4.Effects of NR and NMN on NAD content in HAEC cells, in the presence and absence of CD73 inhibition by AOPCP. Effect of NR (A) and NMN (B) on intracellularNAD after24h-stimulationwithIL1ß-in thepresence or the absenceof CD73inhibitor AOPCP, n=5,*p.0.05, **p.0.01, ns-notstatistically significant. Fig.5.UpregulationofNAMPT,CD73andCD38inHAECafter24h-stimulationwithIL1ß,assessedbyimmunofluorescentstainingandWesternBlotassay;resultsofNAMPT immunofluorescent imaging in IL1ß-stimulated cells in the presence or absence of NMN or NR (A); expression of CD73 (5'ectonucleotidase) in IL1ß­stimulatedcellsintheabsenceorpresenceofNMNor NRassessedbyimmunofluorescentimaging(B);resultsofCD38imagingafterincubationwithIL1ßinthe presenceorabsenceofNMNorNR(C);resultsofWesternBlotanalysisofNAMPT,CD73andCD38expressionafterstimulationwithIL1ß,inapresenceorabsenceof NMNorNR(D);n=6,*p.0.05,**p.0.01;whitescalebarrepresents25µm. possiblyalsotoNAD-dependentmechanisms.In thepresentwork, we demonstrated NAD-related function of CD73 in endothelium and as­cribed beneficial effects of NMN and NR to NAD-dependent mechan­isms described previously in numerous papers in other experimentalsystems[17,15,51]. It was quite a surprising finding of these studies to show that en­dothelial inflammation was not associated with NAD deficiency.However,therewasanupregulationofNAMPTandCD73,asshownbyimmunocytochemistry and Western Blot. These results suggest againthat intracellular NAD synthesis by NAMPT from nicotinamide andextracellular conversion of NMN to NR represents the two major sys­tems maintaining intracellular NAD in endothelial inflammation. WBassay, but not immunofluorescent imaging, showed a slight upregula­tionofCD38afterstimulationwithIL1ß.CD38,identifiedpreviouslyasanmainenzymedegradingNMNinmouse tissuesinvivo[6]seemsto playaminorroleinNMNconversioninhumanendothelialcells.TheseresultssuggestthatpharmacokineticsofNMNinvivoisdependentmoreonNMNuptakebyothertissuesstudiedbyCamacho-Pereiraetal.[6]such as liver, brain skeletal muscle, and spleen, not by endothelialuptakeofNMN. AlthoughinthepresentworkwedemonstratedthatNMNinhibitedendothelial inflammation and improved NO-dependent function byextracellular conversion via ecto-5'-nucleotidase (CD73)-dependentpathway to NR we did not explicitly show that NAD-dependent mechanisms were involved. Obviously, NAD-dependent activation ofsirtuins could play a role, for example endothelial SIRT1 that controlendothelial homeostasis and vascular functionality by modulating en­dothelialnitricoxidesynthase(eNOS)activity,p53,angiotensinII(AngII) type 1 receptor (AT1R), forkhead box O1 (FOXO1) or other me­chanisms[14]. In summary, we demonstrated the nicotinamide mononucleotidereversed endothelial dysfunction and inflammation by extracellularconversion to nicotinamide riboside via CD73, whereas nicotinamideriboside-induced effects were CD73-independent. Beneficial effects ofNMN and NR were comparable and could be both ascribed to NAD-dependent mechanisms, as suggested in previous studies [51,3]. In addition, we demonstrated that endothelial inflammation was asso­ciatedwiththeupregulationofNAMPTandCD73,suggestingthatin­tracellular NAD synthesis by NAMPT from nicotinamide and extra­cellular conversion of NMN toNR, represent the major compensatorysystems activated in endothelial inflammation.Altogether, our results pointtotheextracellularconversionofNMNtoNRbyCD73localizedintheluminalsurfaceofendothelialcells asimportantvasoprotectivemechanisms to maintain intracellular NAD. Thus, the vasoprotectiveroleofendothelialCD73cannotbesolelyattributedtoAMP-adenosinedependent mechanisms and the importance of NAD-dependent me­chanisms in vascular pathologies where CD73 is altered [34,40,41], needstobeelucidatedinfurtherstudies. Fig.6.EffectsofNMNandNRonacetylocholine-inducedendothelium-dependentvasodilationofaorticringstakenfromC57Bl/6J(WT)mice(A,E)andCD73-/- mice (C,G), and incubated with angiotensin II for 24 h. For comparison effects of NMN and NR on endothelium-independent vasodilation induced by sodiumnitroprussidewasassessed(B,D,F,H);n=5,**p.0.01,***p.0.001(AngII-treatedvsAngII/NMN/NR). Authorcontributions S³ominskaandRyszardT.Smolenskiprovidednecessarytools;£ukasz MateuszukandStefanChlopickidraftedandwrittenthefinalversionof £ukaszMateuszukandStefanCh³opickiconcievedanddesignedthethemanuscript;EwaM.S³ominska,RyszardT.SmolenskiandBarbarastudy; £ukasz Mateuszuk, Roberto Campagna, Barbara Kutryb-Zaj¹cKutryb-Zaj¹c revised the manuscript; All authors approved the finalandKamilKuœcollected,analyzedand/orinterpretedthedata;EwaM. versionofthemanuscript. Acknowledgements Nicotinamideriboside waskindlyprovidedbyChromaDex(Irvine, CA,USA). ThisprojectwassupportedbythePolishNationalCentreforScience(OPUS project2015/19/B/NZ3/02302 and partiallyby project2016/23/B/NZ4/03877). 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