Pro-inflammatory effects of early non-enzymatic glycated proteins in human mesothelial cells vary with cell donor s age

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1 British Journl of Phrmcology (26) 149, & 26 Nture Pulishing Group All rights reserved /6 $3. RESEARCH PAPER Pro-inflmmtory effects of erly non-enzymtic glycted proteins in humn mesothelil cells vry with cell donor s ge L Rodríguez-Mñs 1,2,CSánchez-Rodríguez 1, S Vllejo 1, M El-Assr 1, C Peiró 3, V Azcuti 3, N Mtesnz 3,CFSánchez-Ferrer 3 nd J Nevdo 1 1 Unidd de Investigción, Hospitl Universitrio de Getfe, Getfe, Mdrid, Spin; 2 Servicio de Geritrí, Hospitl Universitrio de Getfe, Getfe, Mdrid, Spin nd 3 Deprtmento de Frmcologí y Terpéutic, Fcultd de Medicin, Universidd Autónom de Mdrid, Mdrid, Spin Bckground nd purpose: Dietes mellitus is prevlent in the elderly popultion. It is lso disese cusing tissue dmge through severl different mechnisms. Some of these mechnisms re lso ctivted y geing nd this overlp rises questions out how dietes induces dmge in the elderly. Erly products of non-enzymtic glyction of proteins (Amdori dducts), nd the geing process shre the cpcity to induce oxidtive stress nd inflmmtion in humn peritonel mesothelil cells (HPMCs). We hve evluted the interctions etween the ge of the donor of the HPMCs nd the proinflmmtory effects of Amdori dducts in those cells. Experimentl pproch: HPMCs were isolted from 2 individuls (ge rnge yers) nd grown in culture. Using different experimentl pproches we determined NF-kB dependent trnscriptionl ctivity nd different NF-kB-relted proinflmmtory gene nd protein expressions in sl (or non-stimulted) conditions nd fter stimultion with two Amdori dducts; highly-glycted hemogloin nd glycted ovine serum lumin. Key results: Amdori-induced effects on NF-kB dependent-trnscription nd on the ctivity of NOS, COX nd severl NF-kBrelted pro-inflmmtory genes (inos, COX-2, TNF-, IL-1, nd IL6) diminished s the donor s ge incresed, eing prcticlly sent in cells from donors more thn 65 yers old. Such decresed effects were inversely correlted with n incresed sl expression nd ctivity of these pro-inflmmtory mrkers with ge. Conclusions nd implictions: Pro-inflmmtory effects of Amdori-dducts in HPMCs were strongly dependent on cell donor s ge. This my hve significnt implictions for the mechnisms underlying dietes-induced tissue dmge in ptients of different ges. British Journl of Phrmcology (26) 149, doi:1.138/sj.jp.76864; pulished online 3 Octoer 26 Keywords: Amdori dducts; geing; dietes; glycted humn hemogloin; humn mesothelil cells; inflmmtory response; nitric oxide; nucler fctor-kb Arevitions: AGEs, dvnced glyction end products; CAPD, continuous multory peritonel dilysis; COX, cyclooxygense; gbsa, glycted ovine serum lumin; GAPDH, glycerldehyde-3-phosphte dehydrogense;, highly glycted oxyhemogloin; HPMCs, humn peritonel mesothelil cells; inos, inducile nitric oxide synthse; NF-kB, nucler fctor-kb; NH, oxyhemogloin glycted t norml levels; PBS, phosphte-uffered sline; ROS, rective oxygen species Introduction Among the sustnces involved in tissue dmge induced y dietes mellitus, Amdori dducts hve een shown to ply significnt role (Angulo et l., 1996; Amore et l., 1997; Mndl-Weer et l., 21; Httori et l., 22; Correspondence: Dr J Nevdo, Unidd de Investigción, Hospitl Universitrio de Getfe, Ctr. de Toledo Km 1, Getfe, Mdrid 2895, Spin. E-mil: jnevdo@hug.es Received 1 April 26; revised 26 June 26; ccepted 12 July 26; pulished online 3 Octoer 26 Rodríguez-Mñs et l., 23). Prticulrly, humn hemogloin, when highly glycted () hs een shown to induce functionl nd structurl chnges y mens of rective oxygen species (ROS) nd inflmmtion in severl systems nd tissues (Peiró et l., 1998; Vllejo et l., 2; Peiró et l., 23; Nevdo et l., 25). We hve recently shown tht erly glycted proteins, lso clled Amdori dducts, such s nd glycted ovine serum lumin (gbsa) my fvour proinflmmtory stte in humn peritonel mesothelil cells (HPMCs). Such

2 98 Age-dependence of Amdori-induced effects L Rodríguez-Mñs et l proinflmmtory effect, which involves ROS genertion, is owing to n excerted ctivity of the nitric oxide synthse (NOS) pthwy with enhnced expression of severl nucler fctor-kb (NF-kB)-relted proinflmmtory genes (Nevdo et l., 25). Around 2% of people more thn 65 yers old hve dietes mellitus, with importnt consequences on their totl nd ctive life expectncy (Mggi et l., 24). Different uthors hve reported tht dvncing ge is ccompnied y sl prooxidtive nd proinflmmtory stte (Ferrucci et l., 24), similr to tht induced y dietes or Amdori dducts. Such n oservtion hs een mde in mny systems derived from old nimls, including humn eings (Srkr et l., 24) nd different cell types, including mesothelil cells (Nevdo et l., 26). This fct rises the question of whether sustnces with pro-oxidnt nd proinflmmtory properties, like Amdori dducts, still exert the sme effect in situtions chrcterized y significnt sl proinflmmtory stte, s it hppens during the geing process, or whether the effect of such sustnces is lunted or ttenuted in such sl-enhnced proinflmmtory situtions. The ge of the ptient my thus rise s modulting fctor in the ptho-physiology of the dmge induced y dietes in some tissues, including mong mny others the chnges oserved in the peritoneum during peritonel dilysis, where inflmmtory fctors seem to ply role in trns-differentition of mesothelil cells (Yñez-Mo et l., 23). Therefore, we imed to study the effect of geing on the low-grde inflmmtory response elicited y Amdori dducts in humn cultured omentl mesothelil cells otined from ptients of different ges. Our dt showed tht proinflmmtory effects induced y Amdori dducts in HPMCs depend on the ge of the donor of the HPMCs. Methods Cell culture The study ws pproved y the Clinicl Reserch nd Ethics Committee of Hospitl Universitrio de Getfe (CEIC-Áre1 no. 4/39, 15 June 24). HPMCs were isolted from omentl tissue from 2 persons (see supplementry dt, Tle 1) undergoing non-urgent, non-septic dominl surgery, using previously descried method (Chung-Welch et l., 1997; Nevdo et l., 25, 26). Cell cultures etween pssges two nd five were used. The morphologic nd immunofluorescence-stining fetures (Nevdo et l., 25, 26, nd supplementry dt, Tle 2) of the cells remin stle during these pssges. excess of sodium dithionite. The hemogloin solutions were then extensively dilysed using.25 Å pore dimeter (pproximtely 12 kd mol wt) dilysis memrne (Visking, Serv, Heidelerg, Germny) ginst deionized wter contining 1 mg ml 1 ethylenediminetetrcetic cid (EDTA) nd continuously uled with N 2. Oxyhemogloins were then liquoted nd stored t 71C until used. A5mgml 1 solution of BSA (Sigm Chemicl Co.) ws glycted y incution in phosphte-uffered sline (PBS) (ph 7.4) contining 1 M glucose under sterile nd lightprotected conditions for 6 dys t 371C, s descried previously (Peiró et l., 1998). A control solution ws prepred in prllel using PBS without glucose. Glyction of serum lumin ws verified using the thiorituric cid ssy (Ney et l., 1981). The sence of dvnced glyction end products (AGEs) in our glycted preprtion ws ssessed y mesuring fluorescence in Fluostr fluorometer (BMG Ltechnologies, Offenurg, Germny) t excittion mximum of 37 nm nd emission mximum of 44 nm (Sell nd Monnier, 1989). A stndrd curve (R 2 ¼.99) ws crried out using AGE-modified BSA (.5 5 mgml 1 ). The fluorescence vlues otined in either the (1 nm) or the gbsa solution (.25 mg ml 1 ) were under the fluorescence vlues otined with the mg/ml concentrtion of the AGE BSA in our stndrd curve (dt not shown). In ddition, detection of non-fluorescent AGEs in the glycted oxyhemogloin preprtions ws performed y enzyme-linked immunosorent ssy (ELISA), with the use of rit polyclonl ntiody to AGE (Acm, Cmridge, UK). A stndrd curve (R 2 ¼.98) ws crried out using AGE-modified BSA ( 1 mgml 1 ). The sornce vlues otined in the or norml glycted hemogloin solution (NH) (oth t 1 nm) were not significntly different from the sornce vlues otined with the mg/ml concentrtion of the AGEmodified BSA ( , nd ritrry units for, NH nd AGE-modified BSA, respectively). Finlly, glycted preprtions did not contin significtive cteril endotoxin contmintion (p.5 U endotoxin ml 1 ), s mesured with Pyrogent plus kit (Biowhittker Europe SPRL, Verners, Belgium). Reporter plsmids The reporter plsmids: p5xnf-kb-luc (Strtgene, L Joll, CA, USA), humn inducile NOS (inos) (p7.2 hinos-luc) (Tylor et l., 1998) nd short humn cyclooxygense (COX)-2 (phpes2 327/ þ 59-Luc) (Inoue et l., 1995), were used. Preprtion of Amdori dducts Lyophilized humn hemogloins, non-enzymticlly glycted t either elevted or norml levels, contining 11.1% (ctlogue no. G-112), nd 5.4% (ctlogue no. G-212) HA 1, respectively, were purchsed from Sigm Chemicl Co (St Louis, MO, USA). Before use, hemogloins were prepred s descried previously (Peiró et l., 23; Nevdo et l., 25). Briefly, hemogloins were dissolved in deionized wter nd susequently reduced y incution with n Trnsient trnsfection nd luciferse ssys Trnsient trnsfection experiments were performed s we hve descried previously (Peiró et l., 23). Briefly, HPMC (1 5 cells) were grown in six-well pltes to 8 9% confluence nd the culture medium (M-199) ws then replced y vehicle medium, tht is, serum-free medium supplemented with.1% BSA. The trnsfection mixture ws dded to cell cultures for further 18 2 h. The trnsfection mixture consisted of 2 mg of the ove-mentioned plsmids incuted British Journl of Phrmcology (26)

3 Age-dependence of Amdori-induced effects L Rodríguez-Mñs et l 981 with 75 ml of Dulecco s modified Egle s medium nd 7.5 ml of Superfect (Quigen Gmh, Hilden, Germny) in vehicle medium, following the mnufcturer s instructions. Following tretment with the specified gents, HPMCs were hrvested nd lysed with pssive lysis uffer (1, Promeg, Mdison, WI, USA), followed y one freeze/thw cycle. The extrcts were centrifuged for 3 s t 13 r.p.m. t 41C, nd ssyed with luciferse reporter system (Promeg, Mdison, WI, USA). Luciferse ctivity ws expressed s reltive luciferse units (RLUs, Peiró et l., 23). Determintion of cytokine levels Cytokine levels in confluent cell (1 5 cells) culture superntnts were determined using humn tumour necrosis fctor (TNF), interleukin (IL)-1 nd IL-6 Instnt ELISAs (The Bender Medsystems, Vienn, Austri), y generting stndrd curve provided y the mnufcturer nd normlized to protein content (1 mg). Protein content of whole-cell extrct nd cell culture superntnts were determined using the BCA ssy (Pierce, Rockford, IL, USA). Mesurement of nitrte plus nitrite Nitrite plus nitrte mesurements were performed s we descried previously (Nevdo et l., 25, 26). Briefly, confluent HPMCs (1 5 cells) were grown in six-well pltes nd nitrite plus nitrte production (NO x ), used s n indirect quntifiction of NO, ws mesured in cell superntnts y n ozone-chemiluminescence method (Fries et l., 23), using nitric oxide detector (NOA 28 nlyzer, Sievers, Boulder, CO, USA). A stndrd curve ws generted y injections of known concentrtions of sodium nitrte. The levels of NO x were normlized to protein content (1 mg). Mesurement of COX ctivity Totl COX ctivity ws performed s we descried previously (Nevdo et l., 25, 26). Briefly, confluent HPMCs (1 5 cells) were grown in six-well pltes nd COX ctivity ws mesured y the Cyclooxygense Activity Kit (Stressgen Biotech, Mdison, WI, USA) using specific chemiluminescent sustrte to detect the peroxidtive ctivity of COX enzymes in protein extrct homogentes, s specified y the mnufcturers. Light emission is directly proportionl to COX ctivity in the smple. Results re expressed s RLUs normlized to protein content (1 mg). RNA isoltion nd RT Multiplex PCR ssys Totl RNA from HPMC ( cells) ws otined using RNAquous kit (Amion Inc., Austin, TX, USA), following the mnufcturer s instructions. RT nd Multiplex-PCR (MPCR) were performed with pproprite kits (Mxim Biotech Inc., Sn Frncisco, CA, USA), using one mg of cdna for ech MPCR rection. MPCR kit hs een designed to direct the simultneous mplifiction of specific ORF regions of humn NOS genes nd glycerldehyde-3-phosphte dehydrogense (GAPDH) (hnosg-mpcr). Levels of mrna were normlized to GAPDH trnscript. Sttisticl nlysis Results re expressed s men7s.e.m. s fold induction of the sl (non-stimulted) conditions. Sttisticl significnce (Po.5) using the SttView sttistics progrm (Acus Concepts Inc., Berkeley, CA, USA) ws evluted using the unpired Student t-test or fctoril nlysis of vrince, s required. A simple regression nlysis ws crried out to estlish the potentil correltion etween ge nd ech vrile of interest. The n denotes the numer of experiments performed in triplictes, using cells otined from t lest three different donors for ech group (o65 yers, dult cells () or X65 yers, old cells ()). Mterils M199 medium, L-glutmine nd streptomycin/penicillin solutions were purchsed from Biochrom KG (Berlin, Germny), wheres culture plsticwre ws from Corning- Costr (New York, NY, USA). PBS, foetl clf serum nd trypsin-edta were purchsed from Amresco (Solon, OH, USA), Biologicl Industries (Beit-Hmek, Isrel), nd GIBCO BRM (Pisley, UK), respectively. Humn TNF- nd IL-1 were from Peprotech (London, UK) nd R&D systems (Minnepolis, MN, USA), respectively. Tq DNA polymerse nd deoxynucleoside triphosphtes from ECOGEN (Brcelon, Spin). Unless otherwise stted, ll other regents were purchsed from Sigm Chemicl Co. Results Influence of ge on Amdori-induced NOS nd COX ctivities Age modulted the effects elicited y (1 nm) on two criticl enzymes in the inflmmtory processes, NOS nd COX in HPMCs. Indeed, the effect of tretment (12 h) on levels of nitrte plus nitrite (NO x ) ws nerly three-fold lower in cells from the elderly, over 65 yers (; men ge: yers) thn in those from the younger sujects under 65 yers (; men ge: yers) (Figure 1). Similr results were otined with nother Amdori dduct; gbsa (.25 mg ml 1 ), used to show tht this stimultion ws not n effect unique to (Figure 1). In contrst to the effect of Amdori dducts, cytokine mixture consisting of IL-1 plus TNF- (oth t 1 ng ml 1 ech) nd used s positive control for induction of NOS, showed miniml differences mong the two ge groups (Figure 1). A cler inverse correltion found etween donor s ge nd the effects of on NOS ctivity ws lso shown in Figure 1. Furthermore, Figure 1c shows tht, mesothelil cell donor s ge significntly ffected the effect of (1 nm) nd gbsa (.25 mg ml 1 ) on totl COX ctivity s well. A cler inverse correltion ws lso found etween donor s ge nd the effects of on COX ctivity (Figure 1d). Influence of ge on inos nd COX-2 gene expression, nd NF-kB-dependent trnscription induced y Amdori dducts The rtio of inos/gapdh mrnas in HPMC treted with 1 nm ws significntly lower in s compred with British Journl of Phrmcology (26)

4 982 Age-dependence of Amdori-induced effects L Rodríguez-Mñs et l NOS ctivity NH nbsa gbsa CKm NOS ctivity Y = X; R 2 = c COX ctivity NH nbsa gbsa CKm d COX ctivity Y = X; R 2 = Figure 1 Influence of ge on Amdori-induced NOS nd COX enzymtic ctivities in HPMCs. () Nitrte plus nitrite (NO x ) mesurements in cell culture superntnts or (c) totl COX ctivity (RLUs) in whole protein extrcts were ssessed in HPMC from dult young sujects () nd old sujects () fter 12 h tretment with either NH, (oth t 1 nm); nbsa, gbsa (oth t.25 mg ml 1 ) or the cytokine mixture (CKm; IL-1 þ TNF-, 1ngml 1 ech), n ¼ 4. The correltions etween cell donor ge nd the effects promoted y on either NO x levels is shown in () or on totl COX ctivity, shown in (d). Pp.5 vs sl, Pp.5 vs. (Figure 2). An inverse correltion ws lso found etween donor s ge nd inos gene expression (Figure 2), wheres NH (1 mm) filed to induce significntly inos gene expression in either or (Figure 2). As found for inos, COX-2 gene expression induced y, mesured s the COX-2/GAPDH mrnas rtio ws lso significntly lower in compred to ( vs fold increse over sl in nd, respectively; Pp.1, dt not shown). We next trnsiently trnsfected HPMCs, otined from donors of different ges, with either inos or short COX-2 gene promoter reporter plsmid (p7.2 hinos-luc nd phpes2 327/ þ 59-Luc, respectively) nd then treted with, NH or the cytokine mixture (IL-1 þ TNF-), s descried ove. Figure 2c nd e shows tht the effects of on oth inos nd COX-2 promoter ctivities were higher in compred to. Agin, n inverse correltion ws found etween donor ge nd the effects of on humn inos or humn COX-2 promoter ctivities (Figure 2d nd f). We further monitored the effect of ge on -induced NF-kB-dependent trnscription. HPMC were trnsiently trnsfected with p5xnf-kb-luc reporter plsmid. Agin, NF-kB-induced-dependent trnscriptionl ctivity y ws significntly impired in s compred with (Figure 2g). Additionlly, significnt inverse correltion ws oserved etween donor ge nd NF-kB-dependent trnscriptionl ctivity induced y (Figure 2h). When nd were exposed to gbsa (.25 mg ml 1 ), similr results to those induced y were otined, lthough to lesser extent. Thus, humn inos promoter ctivity ws incresed (Figure 2c), wheres COX-2 promoter ctivity showed comprle increse over sl in nd (Figure 2e). Similrly, NF-kB-dependent trnscription ws incresed (Figure 2g). However, when HPMC ws exposed to the cytokine mixture, no significnt differences were oserved etween nd, regrding humn inos, nd humn COX-2 promoter ctivities or NF-kB-dependent trnscriptionl ctivity (Figure 2c, e nd g, respectively). Age-ssocited effects on -induced proinflmmtory cytokine relese The effect of Amdori dducts on the relese of three proinflmmtory cytokines (IL-1, IL-6 or TNF-) y HPMC ws evluted using our most potent stimuli,. Figure 3 shows tht (1 nm) enhnced IL-1, IL-6 or TNF- levels in HPMC superntnts, this effect eing significntly higher in thn in, where it ws rely detected. The effects elicited y on the levels of these three cytokines decresed ccording to the donor s ge following liner regression curve, especilly for IL-1, ut lso for IL-6 nd TNF- (Figure 3). On the contrry, NH (1 nm) did not ffect the levels of these cytokines, nd ge did not modify this pttern t ll (Figure 3). British Journl of Phrmcology (26)

5 Age-dependence of Amdori-induced effects L Rodríguez-Mñs et l 983 c inos promoter ctivity inos/gapdh mrnas rtio NH -7.2kp inosp LUC NH nbsa gbsa CKm inos/gapdh mrnas rtio : GAPDH inos d inos promoter rtio Y = X; R 2 =.646 Y = X; R 2 = e COX-2 promoter ctivity p COX-2p LUC NH nbsa gbsa CKm f COX-2 promoter rtio Y = X; R 2 = g NF-κB-dependent trnscription x NF-kB sites LUC NH nbsa gbsa CKm h NF-κB-dependent trnscription 5. Y = X; R 2 = Figure 2 Influence of ge on inos nd COX-2 gene expression, nd NF-kB-dependent trnscription induced y Amdori dducts in HPMCs. () Expression of inos mrna fter HPMC (from ; dult sujects, nd ; old sujects) exposure for 12 h to either or NH (oth t 1 nm) ws nlysed y RT MPCR ssy, (n ¼ 3). () A representtive lot of the rtio inos/gadph mrna t different ges ws shown. The effect of Amdori dducts on either humn inos (c) or COX-2 (e) promoter ctivity ws studied using luciferse reporter plsmids (2 mg ech, n ¼ 4, nd n ¼ 5, respectively) in trnsiently trnsfected HPMCs (from nd ), nd then treted for 12 h with either, NH (oth t 1 nm); nbsa, gbsa (oth t.25 mg ml 1 ) or the cytokine mixture (CKm; (IL-1 þ TNF-, 1ngml 1, ech), fter which luciferse ctivity (s RLUs) ws mesured. (g) NF-kB-dependent trnscriptionl ctivity ws ssessed in cells (from nd ) trnsiently trnsfected with p5 NFkB-luc (1 mg) nd then treted for 12 h with the compounds descried ove fter which luciferse ctivity ws mesured (n ¼ 5). Correltions etween cell donor ge nd the effects promoted y on either inos/gapdh mrnas rtio (), or inos promoter ctivity (d) or COX-2 ( 327/ þ 59) promoter ctivity (f) or NF-kB-dependent trnscriptionl ctivity (h). Pp.5 vs sl, Pp.5 vs ). British Journl of Phrmcology (26)

6 984 Age-dependence of Amdori-induced effects L Rodríguez-Mñs et l IL-1β levels IL-1β levels Y = X; R 2 = NH IL-6 levels IL-6 levels Y= 63.17X;R 2 = NH TNFα levels NH TNFα levels Y = X; R 2 = Figure 3 Influence of ge on -induced proinflmmtory cytokine relese in HPMCs. () Levels of IL-1, IL-6 nd TNF- were determined y n ELISA-sed method in cell culture superntnts of HPMCs (from ; dult sujects, nd ; old sujects) treted for 12 h with either NH or (oth t 1 nm), n ¼ 4. () Correltion etween cell donor s ge nd -induced levels of IL-1, IL-6 nd TNF-, respectively. Pp.5 vs sl, Pp.5 vs. Correltion etween sl nd -induced proinflmmtory mrkers over ge We further compred the levels nd ctivities of severl proinflmmtory meditors relesed nd expressed y HPMC, reltive to donor ge, either in sl (non-stimulted conditions) or fter stimultion with (nd normlized over its respective sl). To chieve this, NF-kB-dependent trnscriptionl ctivity nd severl NF-kB-relted proinflmmtory mrkers were mesured in oth sl nd stimulted cells otined from sujects of different ges. Thus, s ge dvnces, cler nd significnt decrese of -induced effects on NF-kB-dependent trnscription (expressed s fold increse over its respective sl) ws oserved, which ws prlleled y n increse in sl NF-kBdependent trnscriptionl ctivity (expressed s RLUs) (Figure 4). The sme responses were lso oserved for severl NF-kBrelted proinflmmtory mrkers, such s inos promoter ctivity, inos mrna levels nd therefore for NO x levels (Figure 4 d, respectively) or other proinflmmtory meditors, such s totl COX ctivity, COX-2 promoter ctivity nd superntnt levels of IL6, IL-1 nd TNF- (Figure 5 e, respectively). Thus, geing induces significnt decrese of -induced effects on ove-descried proinflmmtory British Journl of Phrmcology (26)

7 Age-dependence of Amdori-induced effects L Rodríguez-Mñs et l 985 NF-κB-dependent trsncriptionl ctivity (fold induction y, ) (RLUs, - - -) NF-κB-dependent trnscriptionl ctivity c inos/gapdh mrna rtio (fold induction y HH, ) inos/gapdh mrna (Rtio, ---) inos promoter ctivity (fold induction y, ) Age(yrs) Age(yrs) inos promoter ctivity (RLUs, - - -) d ) NOx ctivity (fold induction y HH, Figure 4 Correltions etween sl levels of severl proinflmmtory mrkers nd HHB-induced effects on the sme proinflmmtory mrkers with ge (prt I). Activities of severl proinflmmtory meditors expressed y HPMCs (otined from donors of different ges) were determined in either sl conditions (non-stimulted) or fter stimultion with (1 mm) nd normlized over its respective sl vlue. In () effects on NF-kB-dependent trnscriptionl ctivity re shown (ssyed y trnsient trnsfection ssy; n ¼ 5), nd in () inos promoter ctivity (n ¼ 4); (c) RT MPCR ssy for inos/gapdh mrna rtio (n ¼ 4); (d) onno x levels (chemiluminescence method; n ¼ 4). Results re expressed s either fold induction of its sl for -treted cells or RLUs (, ), ritrry units (c) nd mm (d) for sl (non-stimulted) conditions. NOx Activity ( M, -- -) mrkers, which ws prlleled y n increse in sl levels of these mrkers, ccording to the previous results in our lortory showing tht ge is ccompnied y the presence of sl inflmmtory stte in HPMC (Nevdo et l., 26). Discussion nd conclusions Amdori dducts or erly non-enzymtic glycted products hve een shown to induce functionl nd structurl chnges in different cell types, including HPMCs (Peiró et l., 1998, 23; Vllejo et l., 2; Rodríguez-Mñs et l., 23; Nevdo et l., 25). Furthermore, through the genertion of ROS, Amdori dducts re le to induce proinflmmtory responses (Nevdo et l., 25). The most outstnding finding of the present study is tht the effect of Amdori dducts s inducers of low-grde inflmmtory response in HPMCs is impired y the ge of the donors of the HPMCs. The most frequently studied non-enzymtic glycted products re the so-clled AGEs (Cermi et l., 1998, Prk et l., 2), which exhiit proinflmmtory properties (Luth et l., 25) nd hve een detected in non-dietic old people t higher levels thn in younger ones (Wu et l., 22; De Groot et l., 24). As the presence of AGEs in our Amdori preprtions ws resonly excluded, the results presented here seem to support tht Amdori dducts my trigger low-grde proinflmmtory response in HPMCs in wy tht is independent of the effects of AGEs. It is worth noting tht erly glycted proteins cn e incorported into mesothelil cells y trnscytosis mechnism (Bodeg et l., 22). Indeed, the existence of non-enzymtic protein glyction hs een lso reported during continuous multory peritonel dilysis (CAPD) (Friedlnder et l., 1996) in the mesothelil lyer of the humn peritoneum (Posthum et l., 21). The proinflmmtory properties of either or gbsa, t concentrtions tht re close to free, physiologicl circulting levels (Tietz, 199), in HPMC cultures, hve een highlighted here in relevnt systems involved in the inflmmtory response, including the enzymes inos nd COX-2, s well s severl proinflmmtory cytokines, ll of them regulted y NF-kB (Go et l., 25, Nevdo et l., 25), proly t trnscriptionl level (Nevdo et l., 25). Interestingly, the effects of Amdori dducts on NF-kB-relted proinflmmtory mrkers were not oserved or were very sutle, in HPMCs otined from donors with ges etween 65 nd 81 yers, where the sl or nonstimulted levels of those inflmmtory mrkers were significntly incresed, s we descried previously (Nevdo et l., 26). As is inferred from dt otined with, negtive correltion etween sl levels of severl proinflmmtory mrkers nd Amdori-induced effects on the sme proinflmmtory sustnces ws found over ge, with steeper decline t erlier ges nd smoother one fter middle ge. Therefore, it ppers tht the response induced y is dependent upon the proinflmmtory stte, tht is, the lower the sl response, the higher the stimulted (induced) one nd vice vers. However, these ge-ssocited effects elicited y re not nonspecific, s in our sme experimentl HPMC model, the proinflmmtory effect of British Journl of Phrmcology (26)

8 986 Age-dependence of Amdori-induced effects L Rodríguez-Mñs et l Totl COX ctivity (fold inductiony, ) Totl COX ctivity (RLUs, - - -) COX-2 promoter ctivity (fold induction y, ) COX-2 promoter ctivity (RLUs, - - -) c IL-1β levels (fold induction y, ) e ) TNF-α levels (fold induction y IL-1β levels (pg/ml, - - -) d IL-6 levels (fold induction y, ) TNF-α levels (pg/ml, ---) Figure 5 Correltions etween sl levels of severl proinflmmtory mrkers nd HHB-induced effects on sme proinflmmtory mrkers over ge (prt II). Activities of severl proinflmmtory meditors expressed y HPMCs (otined from donors of different ges) were determined in either sl conditions (non-stimulted) or fter stimultion with (1 mm) nd normlized over its respective sl vlue. In () effects on totl COX ctivity re shown (luminescence ssy; n ¼ 4); () on COX-2 ( 327/ þ 59) promoter ctivity trnsient trnsfection ssy, n ¼ 5; (c e) An ELISA-sed ssy for IL-1, IL-6, nd TNF- levels, respectively (n ¼ 4). Results re expressed s either fold induction of its sl for -treted cells or RLUs (, ), nd pg ml 1 (c e) for sl (non-stimulted) conditions. IL-6 levels (pg/ml, - - -) mixture of cytokines ws unffected y the ge of the donor. Furthermore, such ge dependency is not oserved for other regultory systems in other cellulr models of geing. Thus, the effect of insulin or insulin-like growth fctor-1 (IGF-1) on the prolifertion nd migrtion cpcity of humn smooth muscle cells is unltered y ge (Ruiz-Torres et l., 23). Finlly, our dt suggest tht, HPMCs from young people seem to e more susceptile to the effects of erly nonenzymtic glycted products s inducers of low-grde proinflmmtory response, thn HPMC from older sujects, which re protected from this compliction. However, findings in clinicl prctice would suggest the opposite. The clinicl urden induced y dietes in the elderly popultion is lredy high, not only in terms of complictions in CAPD (higher rte of filtrtion filure nd infectious complictions), ut lso with n enhnced risk of mjor vsculr complictions. Therefore, fctors others thn Amdori dducts must e plying role in the complictions of dietes with dvncing ge nd it is tempting to suggest tht the physiologicl geing process my e le to modulte different pthwys of cellulr dmge in dietes. In conclusion, our dt suggest tht the proinflmmtory effects induced y Amdori dducts in HPMCs re strongly dependent on the ge of the donor of the cells. Furthermore, the ge dependence of cellulr dmge y glucose-relted mechnisms explored here, rises the possiility tht the mechnisms underlying the involvement of trget orgns in dietes will e different, depending upon the clinicl fetures of the disese, including the ge of the ptient. This possiility opens new opportunities for improving our knowledge out dietes nd its complictions in elderly people, y exploring the iochemicl nd signlling mechnisms y which hyperglycemi-linked events contriute to such complictions in the geing popultion. A more ccurte knowledge will contriute to develop nd implement more specific tht is, ge-trgeted, therpeutic strtegies, leding to more successful tretment nd prevention of the devstting consequences of dietes. British Journl of Phrmcology (26)

9 Age-dependence of Amdori-induced effects L Rodríguez-Mñs et l 987 Acknowledgements This work ws supported y grnts from ISCIII: FIS (1/5, 2/1246, 4/1955, 4/1938) nd RGDM (G3/212), CAM (GR/SAL/899/24) nd SAF (25/145). We thnk Elen Cercs for excellent technicl ssistnce, nd we re grteful to Drs Geller nd Tne for kindly providing plsmids. Dr J Nevdo is recipient of Reserch Contrct from ISC III (FIS 99/377) nd Dr S Vllejo is fellow from CAM. Conflict of interest The uthors stte no conflict of interest. References Amore A, Cirin P, Mitol S, Peruzzi L, Ginoglio B, Rone I et l. (1997). Nonenzymticlly glycted lumin (Amdori dducts) enhnces nitric oxide synthse ctivity nd gene expression in endothelil cells. Kidney Int 51: Angulo J, Sánchez-Ferrer CF, Peiró C, Mrín J, Rodríguez-Mñs L (1996). Impirment of endothelium-dependent relxtions y incresing percentges of glycted humn hemogloin. Hypertension 28: Bodeg F, Zcchi L, Agostini E (22). Alumin trnscytosis in mesothelium. 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