ARTICLE. Stefano Menini & Carla Iacobini & Carlo Ricci & Claudia Blasetti Fantauzzi & Giuseppe Pugliese

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1 etologi (215) 58: DOI 1.17/s ARTICE Protetion from ietes-inue theroslerosis n renl isese y D-rnosine-otylester: effets of erly vs lte inhiition of vne glytion en-prouts in Apoe-null mie Stefno Menini & Crl Ioini & Crlo Rii & Clui Blsetti Fntuzzi & Giuseppe Pugliese Reeive: 8 Otoer 214 /Aepte: 14 Novemer 214 /Pulishe online: 4 Deemer 214 # Springer-Verlg Berlin Heielerg 214 Astrt Aims/hypothesis AGEs re involve in ieti omplitions n might e responsile for the phenomenon of hyperglyemi memory. D-Crnosine-otylester (DCO) hs een shown to ttenute AGE formtion n vsulr n renl injury inue y high-ft iet in Apoe-null mie. This stuy ime to verify the protetive effet of DCO in theroslerosis n renl isese inue y experimentl ietes n to isover whether reution of AGE formtion y erly vs lte DCO tretment provies etter mro n mirovsulr protetion. Methos Apoe-null mie were renere ieti y streptozotoin n were left untrete or were trete with DCO for 2 weeks (ene), from week 1 to 11 () or from week 9 to 19 (DCO-te). Nonieti Apoe-null mie serve s ontrols. Aorti n renl lesions were evlute y morphometry n protein n gene expression of isese mrkers were ssesse y immunohistohemistry n rel-time PCR. Results ene tretment proue more stle plque phenotype y mrkely ttenuting ietes-inue inreses in lesion size, neroti ore re n plque ontent of Nε-roxymethyllysine, levels of poptoti ells n mrkers of inflmmtion n oxitive stress n lso reutions in ollgen n smooth musle ells. DCO tretment for 11 weeks ffore prtil protetion n this ws signifintly etter in mie thn in DCO-te mie. Renl Stefno Menini n Crl Ioini ontriute eqully to this work. Eletroni supplementry mteril The online version of this rtile (oi:1.17/s ) ontins peer-reviewe ut uneite supplementry mteril, whih is ville to uthorise users. S. Menini: C. Ioini : C. Rii : C. B. Fntuzzi : G. Pugliese (*) Deprtment of Clinil n Moleulr Meiine, Spienz University, Vi i Grottross , 189 Rome, Itly e-mil: giuseppe.pugliese@unirom1.it isese ws ttenute in ene mie n to lesser extent in those trete for 11 weeks, with no signifint ifferene etween mie n DCO-te mie. Conlusions/interprettion These t show tht DCO protets mie from ietes-inue vsulr n renl isese n tht protetion ginst theroslerosis is more effetively hieve y erly tretment thn y lte tretment, thus suggesting tht erly inhiition of AGE formtion ttenutes progression of mrongiopthy n fvours evelopment of more stle lesions. Keywors Avne glytion en-prouts. AGE. Atheroslerosis. Crnosine. Retive ronyl speies. Renl isese Arevitions BCA Brhioephli rtery CM N ε -Croxymethyllysine DCCT/EDIC etes rol n Complitions Tril/ Epiemiology of etes Interventions n Complitions DCO D-Crnosine-otylester fma Frtionl mesngil re GPX1 Glutthione peroxise 1 HIF-1α Hypoxi inuile ftor-1α MCP-1 Monoyte hemottrtnt protein-1 mga Men glomerulr re mma Men mesngil re PAS Perioi i Shiff PG Prostglnin qrt-pcr Quntittive rel-time PCR RAGE Reeptor for AGEs RCS Retive ronyl speies α-sma α-smooth musle tin SOD Superoxie ismutse

2 846 etologi (215) 58: UKPDS VSMC Introution UK Prospetive etes Stuy Vsulr smooth musle ell In linil trils intensive gluose-lowering tretment hs een foun to hve ifferent effets on miro- n mrovsulr isese. Strit glyemi ontrol prevents or retrs mirovsulr omplitions in ptients with oth types of ietes [1 3] n hs enefiil effet, espeilly on nephropthy, in iniviuls with long-stning type 2 ietes [4 6]. Conversely, in ptients with type 1 ietes who took prt in the etes rol n Complitions Tril/Epiemiology of etes Interventions n Complitions (DCCT/EDIC), reution of mrovsulr risk rising from erlier glyemi ontrol emerge only fter severl yers, espite loss of within-tril ifferenes in HA 1 levels phenomenon lle hyperglyemi memory [7]. Similr results were otine in ptients with newly-ignose type 2 ietes from the UK Prospetive etes Stuy (UKPDS) [8] wheres strit glyemi ontrol ws ineffetive in reuing riovsulr risk in iniviuls with long-stning ietes [4 6], suggesting tht there is legy effet from erly tretment [9]. The injurious effet of hyperglyemi hs een ttriute to multiple iohemil onsequenes of intrellulr metolism of exess gluose, inluing non-enzymti glytion with formtion of AGEs [1]. AGEs re heterogeneous ompouns tht umulte in ser n tissues of iniviuls suffering from severl isese onitions, inluing ietes n its omplitions [11]. Inrese AGE formtion is ue to genertion of retive ronyl speies (RCS), whih ret with mino i resiues on proteins to generte stle uts or rosslinks olletively referre s to AGEs [11 13]. Sine AGEs re primrily forme on long-live proteins suh s ollgen, thus using sustine ltertions of vsulr struture n funtion, they hve een implite in meiting the phenomenon of hyperglyemi memory rising from prior glyemi ontrol [14]. Therpeuti strtegies ime t reuing RCS- n AGEinue tissue injury, inluing quenhing of RCS y ronyl svengers, hve een propose n teste suessfully in experimentl nimls [13]. -Crnosine is histiineontining ipeptie tht serves s mjor enogenous quenher of RCS, vi intrmoleulr Mihel ition [15]. Unfortuntely, it hs short hlf-life in humns ue to its rpi intivtion y serum n tissue rnosinse [15]. We hve reently shown tht ioville pro-rug of the rnosinse-resistnt D-rnosine, D-rnosine-otylester (DCO) [16], is highly effetive in ttenuting experimentl theroslerosis n renl isese inue y high-ft iet in Apoe-null mie y reuing ronyl stress n inflmmtion [17]. The im of this stuy ws to investigte whether DCO provies protetion ginst vsulr n renl omplitions of experimentl ietes in Apoe-null mie n whether reution of AGE formtion y erly vs lte DCO tretment results in etter mro- n mirovsulr protetion, thus proviing moleulr sis for the role of AGEs in the legy from hyperglyemi. Methos Design The stuy protool ws pprove y the lolly ppointe Ethis Committee. The mie were house n re for oring to the Priniples of ortory Animl Cre protools (NIH pulition No , revise 1985) n ntionl lws n regultions n h free ess to wter n foo. Six-week-ol ult femle Apoe-null mie (Chrles River, Clo, Itly) were renere ieti y streptozotoin (Sigm, St ouis, MO, USA) ministere y intrperitonel injetion t ose of 5 mg/kg oy weight for 5 onseutive ys. Mie injete with itrte uffer serve s non-ieti ontrols (). eti mie were ivie into four groups, eh onsisting of ten mie: untrete (); mie trete with 6 mg/kg oy weight DCO (Flmm, Chignolo Isol, Itly) issolve in the rinking wter for the entire urtion of the stuy (2 weeks, ene); mie trete with DCO for 11 weeks only, either strting immeitely fter ietes inution n ontinuing up to week 11 () or strte t week 9 n ontinuing to week 19 (DCO-te) followe y 1 week of wsh-out to voi potentil interferene of DCO plsm levels on the omprison with DCO- Erly. At the en of the 2 week perio, the mie were ple into metolism ges to ollet urine. The next y, mie were nesthetise y intrperitonel injetion of ketmine (Imlgene, 6 mg/kg, Meril, Miln, Itly) n xylzine (Rompum, 7.5 mg/kg, Byer, Miln, Itly) n the hert (with tthe ort) n oth kineys were smple n weighe. The orti sinus, the rhioephli rtery (BCA) n the right kiney were fixe in 4% (wt/vol.) formlehye. Then, the orti sinus ws emee in Optiml Cutting Temperture emeing meium (Tissue-Tek, Torrne, CA, USA) for frozen setion preprtion n the BCA n the right kiney speimens were emee in prffin. The ominl ort ws opene longituinlly, lyere on ovlumin-ote glss slies, llowe to ry for 1 h n then fixe in formlin for 24 h for en fe preprtion [18]. The left kiney n the thori prt of the esening ort were frozen in liqui nitrogen for susequent mrna n protein extrtion. In selete mie, termintion ws preee y mesurement of

3 etologi (215) 58: BP y the til-uff metho, using the CODA System (Kent Sientifi, Torrington, CT, USA). Bloo n urine mesurements Bloo gluose ws mesure using n utomte olorimetri instrument (Gluor G meter; Menrini, Florene, Itly), n serum holesterol n triylglyerols y enzymti olorimetri methos (Rohe Dignostis, Miln, Itly). Serum levels of isoprostne 8-epiprostglnin (PG) F 2α were ssesse using n EISA kit (Cymn, Ann Aror, MI, USA) n those of AGEs were mesure y ompetitive EISA tehnique [17, 19, 2]. SerumnurineretininelevelsweremesureyHPC n proteinuri n luminuri were ssesse using the Brfor ye-ining protein ssy kit (Piere, Rokfor, I, USA) n the Mouse Alumin EISA kit (Bethyl, Montgomery, TX, USA), respetively, n results were normlise to urine retinine onentrtion [17, 2]. Morphology/morphometry For the ssessment of therosleroti lesions [17, 19], the extent of lipi umultion ws evlute in en fe preprtions fter stining with Oil Re O. esion re ws quntifie t the orti sinus n BCA level fter stining with Oil Re O n the Weigert vn Gieson metho for elsti n ollgen fires [21], respetively. Five ryosetions of orti sinus (1 μm thik, every other setion) n four setions (5 μm thik) t 2, 4, 6 n 8 μm from the rnhing point of the BCA were evlute. The neroti ore re n ollgen ontent were mesure in five setions of the orti sinus (5 μm prt) n in four setions of the BCA (2 μm prt) stine with the Weigert vn Gieson metho. For the ssessment of renl lesions [21], 4 μm setions were stine with perioi i Shiff (PAS) to llow glomerulr n tuule-interstitil mge to e exmine y light mirosopy. Moreover, the res of t lest 6 glomerulr tuft profiles per smple were mesure n the hrmoni men of the profile re (men glomerulr re, mga) ws otine. Then, PAS-positive mteril ws quntifie n expresse s perentge of the glomerulr tuft re (frtionl mesngil re, fma), n the men mesngil re (mma) ws lulte y the formul: (fma mga)/ 1. All mesurements were performe with the i of the Optims 6.5 imge nlysis system (Biosn, Wshington, DC, USA). Immunohistohemistry Immunohistohemil nlysis [17, 19, 2] ws performe to ssess the orti n kiney ontent n istriution of mrkers of murine mrophge tivtion (F4/8), oxitive stress (nitrotyrosine) n ronyl stress (N ε -roxymethyllysine, CM). Nitrotyrosine ws hosen s mrker of free ril mge sine its proution oes not epen on RCS levels n, hene, is not iretly influene y DCO tretment, t vrine with CM n the uts of mlonyllehye or 4-hyroxy-2-nonenl. The expression of tive spse-3, mrker of poptoti ells, α-smooth musle tin (α-sma), mrker of vsulr smooth musle ells (VSMCs) n reeptor for AGEs (RAGE) in the ort, n of ollgen IV in the kiney, were lso ssesse (see eletroni supplementry mteril [ESM] Tle 1 for primry ntioies). Setions were nlyse using the Optims 6.5 imge nlysis system. Quntittive rel-time PCR Totl RNA ws extrte from ort n kineys y the guniine thioynte phenol hloroform metho using TRIzol Regent (Invitrogen Itli, Sn Giulino Milnese, Itly). Quntittive rel-time PCR (qrt- PCR) ws use for ssessing the gene expression level of multiple isese mrkers [17]. The trnsripts for the following proteins were mesure in the ort: the inflmmtory mrkers F4/8, monoyte hemottrtnt protein-1 (MCP- 1) n I-1β, the ntioxint enzymes CuZn-superoxie ismutse (CuZn-SOD), tlse n glutthione peroxise 1 (GPX1) n hypoxi inuile ftor-1α (HIF-1α), the trnsriptionl tivity of whih is inrese y AGEs [22] n inhiite y rnosine [23]. The trnsripts for the following were mesure in the kiney: fironetin, ollgen IV, TGF-β, MCP-1, CuZn-SOD, tlse, GPX1 n HIF-1α. Sttistil nlysis Results re expresse s mens±sd. Sttistil signifine ws evlute y one-wy ANOVA followe y the Stuent Newmn Keuls test for multiple omprisons. A p vlue <.5 ws onsiere signifint. All sttistil tests were performe on rw t. Results Metoli n hemoynmi vriles Boy weight erese n loo gluose, triylglyerol n holesterol levels inrese in mie n were unffete y DCO tretment (Tle 1). Serum AGE n isoprostne 8-epi-PGF 2α levels inrese mrkely ( two- to threefol) in mie ompre with mie. These levels were lmost normlise in DCO- Extene mie, lthough isoprostne levels remine signifintly higher thn in mie, possily s onsequene of resiul oxitive stress iretly ttriutle to hyperglyemi. Vlues in mie were similr to those foun in mie, wheres they were prtilly reue in DCO-te mie (Tle 1). BP i not iffer mong groups (not shown). Atheroslerosis Oil Re O stining of en fe ort preprtions (ESM Fig. 1) showe signifint inrese (5.5 ) in the extent of lipi umultion in vs mie. Tretment with DCO for 2 weeks reue the inrese in lipi ontent

4 848 etologi (215) 58: Tle 1 Metoli n renl struture n funtion vriles in non-ieti Apoe-null ontrol mie n ieti Apoe-null mie either untrete or trete with DCO for the entire 2-week perio or for 11 weeks only, from week 1 to 11 or from week 9 to 19 Vrile ene DCO-te p vlue Boy weight, g 28.8± ± ± ± ±1.2 <.1 Kiney weight, mg 22.± ± ±14.1, 36.±16.7,f,g 295.±12.9,e,g <.1 Kiney/oy weight rtio 7.7± ±.9 1.3±.5, 11.8±.6,,g 11.7±.6,,g <.1 Gluose, mmol/l 6.88± ± ± ± ±3.5 <.1 Cholesterol, mmol/l 6.22± ± ± ± ±.85 <.1 Triylglyerols, mmol/l 1.8± ± ± ±.3 2.4±.21.2 AGEs, U/ml 4.32± ± ± ±1.52,g 7.73±1.17,,i <.1 Isoprostne 8-epi-PGF 2α, pg/ml 7.5± ± ±19.2, 159.5±16.8,g 13.3±1.3,e,g,h <.1 Cretinine, μmol/l 28.6± ± ± ±.7 29.± Protein/retinine rtio 2.2± ± ±.24, 2.93± ±.28 <.1 Alumin/retinine rtio 1.37± ± ±.45,e 2.26± ±.17.2 Dt re expresse s men±sd Kiney weight refers to oth kineys The p vlues were lulte using one-wy ANOVA for omprison mong groups n the Stuent Newmn Keuls test for post ho multiple omprison: p<.1, p<.1 n p<.5 vs ; p<.1, e p<.1 n f p<.5 vs ; g p<.1 vs ; h p<.1 n i p<.1 vs y 62%. A more moest (yet signifint) reution ws oserve in mie trete with DCO for 11 weeks, with more mrke (though not signifintly ifferent) improvement seen in thn in DCO-te mie (33 vs 2% reution). ikewise, lesion res t the orti sinus n the BCA (Fig. 1) were signifintly higher in thn in mie (2.6 n 3.4, respetively). The inrese lesion res in ieti mie were reue y tretment with DCO for 2 weeks (y 55% n 66%, respetively) n, less mrkely, 11 weeks. The extent of reution ws signifintly higher with the DCO- Erly thn with the DCO-te regimen (y 33% n 44% vs 16% n 21%, respetively). etes inrese the omplexity of lesions, s efine y the presene of holesterol lefts, nerosis n/or firous p formtion, wheres mie showe preominntly ftty streks. DCO tretment inrese fetures of plque stility, s shown y the smller neroti ores n the more extensive firosis s ompre with untrete mie. Agin, lesions showe more stle phenotype in mie trete for 2 weeks thn in those trete for 11 weeks n in those on the DCO- Erly vs DCO-te protool (Fig. 1). Immunohistohemil etetion of tive spse-3 in speimens from mie revele the presene of poptoti ells, espeilly roun the neroti ore (Fig. 2). Stining for F4/8 ws lmost uniform, from the lumen to the mei, suggesting tht plques in these mie were ompose minly of fom ells. Conversely, stining for α-sma ws fint or sent. Positivity for RAGE, CM n nitrotyrosine ws similr to tht for F4/8 (Fig. 3). ene tretment ws very effetive in reuing the numer of tive spse-3 positive ells n the ontent of monoyte/mrophges while inresing the frtion of the plque oupie y VSMCs, espeilly t the luminl sie n ll roun the lesions (Fig. 2). ikewise, this intervention signifintly reue the lesion ontent of RAGE, CM n nitrotyrosine (Fig. 3). The 11-week DCO tretment lso ffete these vriles positively, though to ifferent extent oring to the type of protool. In ft, with DCO-te regimen the reutions in tive spse-3, F4/8, RAGE, CM n nitrotyrosine were reltively moest n the inrese in α-sma ws not signifint (Figs 2, 3). Conversely, the protool provie signifint protetion from ietes-inue hnges in these vriles, lso prouing unique plque phenotype. In prtiulr, stining for poptoti ells, F4/8, RAGE, CM n nitrotyrosine ws onfine to the luminl sie of the plque, wheres the istriution of α-sma positivity resemle tht in plques from ene mie, with VSMCs forming firous p surrouning the lesion (Figs 2, 3). qrt-pcr nlysis of mrna levels showe tht F4/8 (lso known s Emr1), Mp-1 (lso known s Cl2), Il-1β, CuZn-So (lso known s So1), tlse (lso known s Ct), Gpx1 n Hif-1α were higher in mie thn in mie n were signifintly reue y ene tretment (ESM Fig. 2). The gene expression of inflmmtory mrkers tene to erese in mie (though the extent of reution ws signifint only for Il-1β) n to inrese in DCO-te mie (to signifint extent only for F4/8). Moreover, the protool reue the mrna levels of genes enoing ntioxint enzymes (exept Gpx1) nhif-1α to the sme extent s oserve with DCO- Extene therpy, wheres the DCO-te regimen erese signifintly only the expression of genes enoing tlse

5 etologi (215) 58: esion re (μm ) Neroti ore (% lesion re) e * f esion re (μm ) g Neroti ore (% lesion re) DCO-te Collgen (% lesion re) * DCO-te h Collgen (% lesion re) Fig. 1 Oil Re O stining of orti roots () n Weigert vn Gieson s stining of BCAs (e; lk, elsti fires; re, ollgen; *neroti ore) from representtive mie n quntifition of lesion re ( n f, respetively), neroti ore ( n g, respetively) n ollgen ontent ( n h, respetively) in non-ieti Apoe-null ontrol mie (, white rs) n ieti Apoe-null mie either untrete (, lk n, prtiulrly, Hif-1α. Finlly, signifintifferenesetween DCO-te n protools were oserve for F4/8, Il-1β, So n tlse. rs) or trete with ene (hthe rs), -Erly (light grey rs) or -te (rk grey rs) protool (men ± SD; n=1 per group). Sle r, 5 μm in() n 2 μm in(e). Post ho multiple omprison: p<.1 vs ; p<.5 n p<.1 vs ; p<.1 n p<.1 for DCO-te vs Renl isese Kiney weights, oth solute n reltive to oy weight, were signifintly inrese in mie n reue y tretment with DCO for 2 weeks n, to lesser e DCO-te DCO-te DCO-te Ative spse-3 (n/mm 2 ) Fig. 2 Immunohistohemil etetion of tive spse-3 (, rrows inite tive spse-3 positive ells), F4/8 () n α-sma (e) in orti speimens from representtive mie n quntifition of stining (, n f, respetively) in ieti Apoe-null mie either untrete (, lk rs) or trete with ene (hthe rs), -Erly F4/8 (% lesion re) f α-sma (% lesion re) (light grey rs) or -te (rk grey rs) protool (men ± SD; n=1per group)., lumen;, tuni mei. Sle r, 8 μm in() n (e) n 5 μm in (). Post ho multiple omprison using the Stuent Newmn Keuls test: p<.1 vs ; p<.1 for DCO-te vs

6 85 etologi (215) 58: Fig. 3 Immunohistohemil etetion of RAGE (), CM () n nitrotyrosine (e) in orti speimens from representtive mie n quntifition of stining (, n f, respetively) in ieti Apoe-null mie either untrete (, lk rs) or trete with ene (hthe rs), -Erly (light grey rs) or -te (rk grey rs) protool (men ± SD; n=1 per group). Sle r, 8 μm in() n 5 μm in() n (e)., lumen;, tuni mei. Post ho multiple omprison: p<.1 vs ; p<.1 for DCO-te vs DCO-te RAGE (% lesion re) DCO-te CM (% lesion re) f e Nitrotyrosine (% lesion re) DCO-te extent, for 11 weeks, irrespetive of protool ( or - te). Serum retinine levels i not iffer mong groups, wheres proteinuri n luminuri inrese signifintly in mie (y 68% n 14%, respetively) ompre with mie. The inreses were signifintly ttenute (y 54% n 68%, respetively) y ene tretment. Slight, non-signifint, reutions were oserve with oth n DCO-te protools (Tle 1). Glomerulopthy ws etete in mie, with signifintly higher mga, fma n mma (+41, 71 n 142%, respetively). All these inreses were signifintly reue y 2 weeks of DCO tretment (y 69%, 79% n 79%, respetively), n to lesser n omprle extent y oth 11 week protools (y 3%, 4% n 4%, respetively) (Fig. 4). Also, the inrese renl ontent of ollgen IV, F4/8, CM n nitrotyrosine use y ietes ws signifintly reue y DCO tretment, gin more mrkely in mie trete for 2 weeks thn in those trete for 11 weeks (Fig. 5). ikewise, ietes-inue inreses in mrna levels of fironetin (lso known s Fn1), ollgen IV (lso known s Col41), Tgf-β (lso known s Tgf1), Mp-1 n Hif-1α were signifintly reue y the 2 week DCO tretment n lso y the 11 week protools, with the exeption of Mp-1 n prtiulrly of Tgf-β, whih ws even inrese in DCO-te mie (ESM Fig. 3). CuZn-So, tlse n Gpx1 geneexpression ws reue, inrese n unffete y ietes, respetively, n ws signifintly reue y ll DCO protools. Disussion In ieti mie, tretment with DCO for 2 weeks signifintly reue the size n extent of therosleroti lesions, whih presente with more stle phenotype, n meliorte normlities in renl funtion n struture. These effets were ssoite with reue irulting n tissue AGE levels, whih, in the sene of hnges in metoli vriles, support the onept tht the effiy of DCO is relte to its RCS-quenhing tivity whih results in erese AGE formtion, s previously shown in iet-inue, non-ieti moel of theroslerosis [17]. In turn, reue AGE umultion prompte ttenute inflmmtion n lunte oxitive stress, well-reognise pthogeni mehnism of ietes-inue theroslerosis n renl isese in Apoe-null mie [24]. The onept tht ttenution of oxitive stress y DCO tretment is relte to its RCSquenhing tivity is supporte y severl lines of eviene. First, n iniret ntioxint effet of DCO vi reution of AGE formtion hs een postulte on the sis of the known effet of these yprouts in promoting genertion of retive oxygen speies vi reeptor-meite mehnisms [25, 26]. Seon, previous t from our group [17] n other investigtors [27, 28] rgue ginst iret ntioxint tion for rnosine. Finlly, DCO i not pper to reue the uren from oxitive stress y upregulting ntioxint enzymes. In ft, in the ort, tretment with DCO erese the mrna levels of genes enoing ntioxint enzymes, whih were inrese in mie proly s prt of n ptive response to enhne oxitive stress. Conversely, ministrtion of DCO i not normlise the reue gene expression of renl CuZn-So, the ownregultion of whih hs een previously reporte to our in experimentl ieti nephropthy [29]. Nor i it signifintly moifie the mrna levels of tlse or Gpx1. In ition, the inrese mrna expression of Hif-1α ws prevente y DCO tretment, proly vi reution of AGEs, whih re known to enhne Hif-1α gene trnsription n trnsriptionl tivity [22]. Both of the 11 week protools provie moest, yet signifint, protetion. However, while DCO-te ws not

7 etologi (215) 58: Fig. 4 PAS stining of kineys from representtive mie () n quntifition of mga (), fma () nmma() in non-ieti Apoe-null ontrol mie (, white rs) n ieti Apoenull mie either untrete (, lk rs) or trete with DCO- Extene (hthe rs), -Erly (light grey rs) or -te (rk grey rs) protool (men ± SD; n=1 per group). Sle r, 8 μm. Post ho multiple omprison: p<.1 vs ; p<.1 n p<.1 vs ; p<.1 vs DCO- Extene DCO-te mga (μm ) fma (%) mma (μm 2 ) * s effetive s tretment in ttenuting theroslerosis, the two protools meliorte renl isese to similr extent. These isornt effets on trget orgn mge re onsistent with the isornt effets of the two tretment regimens on orti n kiney stining for CM, F4/8 n nitrotyrosine n mrna levels for inflmmtory mrkers n ntioxint enzymes. The fining tht, in the ort, the two protools reue Hif-1α mrna levels to similr extent, espite lower ttenution of AGE umultion in DCO-te thn in mie, suggests tht the known iret effet of rnosine on HIF-1α [23] plye n importnt role in DCO-te mie. Moreover, in these mie, t on protein n gene expression levels of F4/8 were ontrsting, sine F4/8 stining ws reue, wheres its mrna expression ws further inrese when ompre with mie. This might suggest tht the omposition of the monoyte/ Fig. 5 Immunohistohemil etetion of ollgen IV (), F4/ 8 (, rrows inite F4/8 positive ells), CM () n nitrotyrosine () inkiney speimens from representtive mie n quntifition of stining (e, f, g n h, respetively) in non-ieti Apoe-null ontrol mie (, white rs) n ieti Apoenull mie either untrete (, lk rs) or trete with DCO- Extene (hthe rs), -Erly (light grey rs) or -te (rk grey rs) protool (men ± SD; n=1 per group). Sle r, 5 μm in(), () n () n 8 μm in(). Post ho multiple omprison: p<.1 vs ; p<.1 vs ; p<.1 vs ene. Glom, glomerulr DCO-te DCO-te DCO-te DCO-te e Collgen IV (% glom re) f F4/8 (n/fiel ortex) g CM (% glom re) h Nitrotyrosine (% glom re)

8 852 etologi (215) 58: mrophge popultion iffers etween lesions in DCO-te n mie, with the former ontining fewer ut more mture ells, whih re known to express more F4/8 [3]. Furthermore, esstion of the DCO-te tretment 1 week efore mie were kille might hve resulte in prompt retivtion of inflmmtion, with sort of reoun effet, s suggeste y the upwr tren oserve for MCP-1 n I- 1β levels. This interprettion is onsistent with the fining tht the mrna expression of inflmmtory mrkers in DCO- Erly mie ws signifintly reue ompre with DCO- te mie, ut not mie, exept for Il-1β, suggesting tht, fter 9 weeks of unontrolle ietes n unrestrine AGE formtion following esstion of tretment, pro-inflmmtory mehnisms re lmost fully retivte t the moleulr level. The histologil orrelte of this fining is the immunohistohemil pttern note for F4/8, ut lso for RAGE, CM n nitrotyrosine stining, whih ws limite to the luminl ege of plques, suggesting new monoyte reruitment n AGE eposition fter the en of tretment. Similr t were otine in kineys from oth n DCO-te mie, in whih reution in the mrna levels for fironetin n ollgen IV were not ompnie y ereses in the gene expression for Tgf-β n Mp-1, thus suggesting tht oth pro-firoti n pro-inflmmtory pthwys were fully retivte fter esstion of DCO tretment, though the extent of firosis n ellulr inflmmtion, s of AGE umultion n oxitive stress, ws still lower thn in mie. Although onlusive explntion for the ifferent outomes of erly vs lte intervention in the ort n kiney nnot e provie, it is tempting to speulte tht they my reflet profoun ifferenes in the histologil nture of therosleroti n renl lesions. On the one hn, AGEs n mrophges tht umulte uring initil therom formtion prtiipte in viious yle of inflmmtion n mge, frustrting ttempts t tissue repir n promoting further AGE formtion n monoyte/mrophge infiltrtion in the vessel wll [31]. Although DCO-te intervention might hve loke eposition of new AGEs, preforme AGEs tht umulte prior to tretment oul hve sustine plque inflmmtion, growth n progression uring susequent ministrtion of DCO, sine this gent hs no effet on preforme AGEs [32]. On the other hn, the inhiition of AGE eposition (n monoyte reruitment) y tretment uring initil therom formtion might hve ttenute susequent plque progression through reue inflmmtory uren, whih woul lso ontriute to erese AGE formtion uring the tretment-free perio. Conversely, in ieti glomerulopthy, oth eposition of AGEs n infiltrtion y monoyte/mrophges re less mrke thn in therosleroti lesions n re not topogrphilly segregte into lipi-rih, pro-oxint environment s ours in therom. Thus, the umultion of AGEs n inflmmtory ells efore initition of DCO-te tretment might hve not een suffiient, per se, to sustin further progression of glomerulopthy uring DCO ministrtion. This interprettion is in greement with the oservtion tht glomerulr lesions, inluing Kimmelstiel Wilson noules, re reversile in iniviuls with type 1 ietes unergoing isolte pnres trnsplnttion [33], wheres fewer n more elusive t exist on the reversiility of the therosleroti proess [34]. Altogether, our t support the hypothesis of min role for AGEs in the hyperglyemi memory or legy effet, s postulte some yers go [35], lthough this hypothesis is not yet proven. In ft, the lower effiy of lte vs erly intervention with the inhiitor of AGE formtion DCO implies tht erly vs lte eposition of AGEs, whih results in tissue umultion of these yprouts for longer vs shorter perios, respetively, is responsile for the ifferent therpeuti effets of the two regimens. Deposition of AGEs uring the initil phse of therogenesis fvours plque instility n further lesion progression even uring susequent intervention, wheres erly inhiition of AGE formtion proues smller n more stle plques espite esstion of tretment. This interprettion is onsistent with the eviene tht AGE levels re ssoite with rupture-prone plque phenotype [36]. In this view, our finings suggest tht inhiition of AGE formtion, hieve with erly institution of strit glyemi ontrol, might e importnt for preventing riovsulr events, s oserve in the post-tril long-term followup of prtiipnts formerly ssigne to the intensive vs onventionl group of the DCCT/EDIC n UKPDS [7, 8]. Conversely, unrestrine genertion of AGEs uring previous poor glyemi ontrol might explin the filure of intensive tretment to provie riovsulr enefit in iniviuls with long-stning ietes [4 6]. In onlusion, these t show tht DCO protets ginst ietes-inue vsulr n renl isese n tht erly tretment is more effetive thn lte tretment ginst theroslerosis. In prtiulr, the t inite tht erly inhiition of AGE formtion ttenutes progression of mrongiopthy n fvours evelopment of more stle lesions, onsistent with the eviene tht erly ggressive therpy provies protetion from riovsulr events. Funing This work ws supporte y the EFSD n Snofi-Aventis Europen Reserh Progrmme in Miro- n Mrovsulr Complitions of etes 211 n y reserh grnt from the Reserh Fountion of the Itlin Soiety of etology (Fo.Ri.SID). Dulity of interest The uthors elre tht there is no ulity of interest ssoite with this mnusript. riution sttement SM n CI ontriute to oneption n esign, quisition of t, nlysis n interprettion of t n ritil revision of the rtile for importnt intelletul ontent. CR n CBF ontriute to quisition of t n ritil revision of the rtile for

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