The angiotensin II type 2 receptor agonist Compound 21 is protective in experimental diabetes-associated atherosclerosis

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1 etologi (6) 9:77879 DOI.7/s ARTICLE The ngiotensin II type receptor gonist Compound is protective in experimentl dietes-ssocited therosclerosis Bryn S. M. Chow & Christine Koulis & Pooj Krishnswmy & Ulrike M. Steckelings & Thoms Unger & Mrk E. Cooper & Krin A. Jndeleit-Dhm & Terri J. Allen Received: Jnury 6 /Accepted: April 6 /Pulished online: My 6 # Springer-Verlg Berlin Heidelerg 6 Astrct Aims/hypothesis Angiotensin II is well-recognised to e key meditor in driving the pthologicl events of dietesssocited therosclerosis vi signlling through its ngiotensin II type receptor (AT R) sutype. However, its ctions vi the ngiotensin II type receptor (AT R) sutype re still poorly understood. This study is the first to investigte the role of the novel selective AT R gonist, Compound (C) in n experimentl model of dietes-ssocited therosclerosis (DAA). Methods Streptozotocin-induced dietic Apoe-knockout mice were treted with vehicle (. mol/l citrte uffer), C ( mg/kg per dy), cndesrtn cilexetil ( mg/kg per dy) or C + cndesrtn cilexetil over week period. In vitro models of DAA using humn ortic endothelil cells nd monocyte cultures treted with C were lso performed. At the end of the experiments, ssessment of plque content nd mrkers of oxidtive stress, inflmmtion nd firosis were conducted. Bryn S. M. Chow nd Christine Koulis contriuted eqully to the mnuscript. Electronic supplementry mteril The online version of this rticle (doi:.7/s ) contins peer-reviewed ut unedited supplementry mteril, which is ville to uthorised users. Terri J. Allen terri.llen@keridi.edu.u JDRF Dnielle Alerti Memoril Centre for etic Complictions, etic Complictions Division, Bker IDI Hert nd etes Reserch Institute, 7 Commercil Rod, P. O. Box 69, Melourne, VIC, Austrli IMM-Deprtment of Crdiovsculr nd Renl Reserch, University of Southern Denmrk, Odense, Denmrk School for Crdiovsculr Diseses, Mstricht University, Mstricht, the Netherlnds Results C tretment significntly ttenuted ortic plque deposition in mouse model of DAA in vivo, in ssocition with decresed infiltrtion of mcrophges nd meditors of inflmmtion, oxidtive stress nd firosis. On the other hnd, comintion therpy with C nd cndesrtn (AT Rntgonist) ppered to hve limited dditive effect in ttenuting the pthology of DAA when compred with either tretment lone. Similrly, C ws found to confer profound ntitherosclerotic ctions t the in vitro level, prticulrly in the setting of hyperglycemi. Strikingly, these theroprotective ctions of C were completely locked y the AT Rntgonist PD9. Conclusions/interprettion Tken together, these findings provide novel mechnistic nd potentil therpeutic insights into C s monotherpy gent ginst DAA. Keywords Angiotensin II. AT receptor. Atherosclerosis. Compound. etes Arevitions Ang II Angiotensin II ARB Angiotensin receptor locker AT R Angiotensin II type receptor AT R Angiotensin II type receptor C Compound DAA etes-ssocited therosclerosis HAEC Humn ortic endothelil cells MCP Monocyte-chemottrctnt protein NOX NADPH oxidse RAS Reninngiotensin system STZ Streptozotocin TxR Thromoxne receptor VCAM- Vsculr cell dhesion molecule-

2 etologi (6) 9: Introduction Overctivtion of the reninngiotensin system (RAS) is wellrecognised to e mjor underlying pthologicl mechnism tht drives the development of dietic vsculr disese, with its effector peptide, ngiotensin II (Ang II), eing the min meditor []. Phrmcologicl inhiition of the RAS y trgeting either Ang II production with n ngiotensinconverting enzyme inhiitor or the ctivity of Ang II with n ngiotensin receptor locker (ARB) remins first-line tretment for ptients with dietes nd vsculr disese []. However, it hs een proposed tht the eneficil effects of these tretments re out of proportion with the mgnitude of lood pressure reduction, suggesting more direct effects on end orgns, including the mcrovsculture. Although Ang II inds with similr ffinity to oth the AT nd AT receptor sutypes [], the reltive importnce of these receptors in modulting vsculr injury remins poorly understood. Angiotensin II type receptors (AT Rs) re primrily involved in mediting the pthophysiologicl ctions of Ang II, including its ility to regulte lood pressure, vsoconstriction nd cellulr differentition nd promote hypertrophy []. Strikingly, Ang II negtively regultes its clssicl ctions through the ngiotensin II type receptors (AT Rs) [], with this receptor sutype eing mrkedly upregulted in numerous disesed sttes, including therosclerosis [6, 7], possily to counterlnce the dverse effects medited y the AT R. Tken together, these ctions highlight the potentil of the AT R s key therpeutic trget. With numerous studies focusing on the role of the AT R in the non-dietes setting [8], the vsculr ctions of this receptor in the context of dietes still remins uncler. Given our previous findings tht selective ctivtion of the AT R y novel non-peptide gonist, Compound (C), significntly meliorted the progression of nephropthy in n insulin-deficient model [], we imed to further investigte the protective role of C on mcrovsculr disese in this dietes model. C is highly specific AT R gonist, with K i vlue of. nmol/l nd > nmol/l for the AT RndAT R, respectively []. This study is the first to delinete the vsoprotective role of C in n experimentl model of streptozotocin (STZ)-induced insulin-deficient dietes in vivo, followed y extension of the findings to explore potentil underlying mechnisms in vitro. Methods Animls Mle polipoprotein E-knockout (Apoe / )miceon C7BL6/J ckground (Jckson Lortory, Scrmento, CA, USA) were used throughout the study sed on previous findings tht the mice in this model develop ccelerted DAA upon the induction of dietes nd so constitute n pproprite model of DAA []. Mice were housed nd mintined on h lightdrk cycle in pthogen-free environment nd hd free ccess to wter nd rodent l chow (Specilty Feeds, Glen Forrest, WA, Austrli). Experiments were conducted in ccordnce with the Austrlin code of prctice for the cre nd use of lortory nimls for scientific purposes. In vivo experimentl procedure Seven-week-old Apoe / mice were rendered dietic vi dily intrperitonel injection of STZ (Sigm-Aldrich, St Louis, MO, USA) t dose of mg/kg per dy over period of dys. Animls with lood glucose nd HA c levels greter thn mmol/l nd % (8.8 mmol/mol), respectively, fter dys post-stz-dministrtion, were included in the study s dietic. etic mice were rndomised nd treted with vehicle (. mol/l citrte uffer), C ( mg/kg per dy; Vicore Phrm, Göteorg, Sweden), the AT R ntgonist cndesrtn cilexetil ( mg/kg per dy; AstrZenec Södertälje, Sweden) or C + cndesrtn tretment, vi dily gvging, over week period. These concentrtions of C nd cndesrtn hd previously een shown to successfully meliorte dietic nephropthy [, ]. Non-dietic mice treted in the sence or presence of C were lso studied. At the end of the study, mice were killed nd ortic tissues were excised for nlysis. For ssessment of ody weight, metolic vriles nd lood pressure, mice were plced in individul metolic cges (Iff Credo, L Arresele, Frnce) for period of h nd performed s descried []. Cell culture The therosclerotic properties of the AT Rwere further investigted in humn ortic endothelil cells (HAEC) nd monocyte cultures (THP-) (otined from ATCC, Mnsss, VA, USA). Experiments were performed three or four seprte times in duplicte. Cells were regulrly tested for mycoplsm contmintion y the medi services (Bker IDI Hert nd etes Institute, Austrli). HAECs were cultured in endothelil growth medi supplemented with EGM- Bulletkit (Lonz, Allendle, NJ, USA). In ll experiments, EGM- medi contining.6 mmol/l glucose ws used s the norml-glucose condition, while EGM- medi contining mmol/l glucose (Sigm-Aldrich) ws used s high-glucose condition. THP- cells were mintined in RPMI supplemented with % fetl clf serum, penicillin ( U/ml) nd streptomycin ( μg/ml). To induce monocytemcrophge differentition, cells were treted with phorol--myristte--cette ( μmol/l; Sigm-Aldrich) dys efore sujecting the cells to the vrious tretments. In vitro experimentl procedure To determine the optiml dose of C, HAEC nd THP- cultures (tested negtive for mycoplsm contmintion) were seeded into -well pltes t equl density ( to. cells/well) nd treted with

3 78 etologi (6) 9:77879 C (.. μmol/l) in oth norml- ( mmol/l) nd highglucose ( mmol/l) conditions for 7 h. Additionl studies were lso conducted to determine the following: () whether the optiml dose t which C inhiited high-glucosestimulted inflmmtory nd firotic mrkers ws le to inhiit Ang II-medited ctivity; () whether the ility of C to medite this ctivity occurred specificlly through the AT R, y scertining whether C-induced effects in these cells were locked y the AT R ntgonist PD9 ( μmol/l; Cymn Chemicl, Ann Arour, MI, USA), () whether C s comintion therpy with n AT Rlocker, cndesrtn cilexetil ( μmol/l) hd ny dditive effect in ttenuting DAA nd () whether the cndesrtn cilexetilmedited effects were ltered y PD9. At the end of ech experiment, RNA nd protein were extrcted from the cell lyer with Trizol regent (Life Technologies, Rockville, MD, USA) for susequent nlysis. Evlution of theroscleroticplquesizendcontent Plque re ws quntified in n en fce mnner, s descried previously []. Totl nd segmentl plque res were quntified s percentge re of ort stined (Adoe Photoshop, version 7.; Adoe Systems, Chtswood, NSW, Austrli). Frozen sections of ortic sinus were stined with Oil Red O solution (Sigm-Aldrich) for h followed y counterstining with hemtoxylin. Mesurement of necrotic cores ws susequently quntified s descried previously [6]. Results were expressed s re of necrotic core per lesion. Immunohistochemistry Prffin sections of ort were stined for vsculr cell dhesion molecule- (VCAM-; : dilution; BD Phrmingen, Sn Diego, CA, USA), s descried previously [7]. Positive-stined sections were quntified using Imge-Pro Anlyser 7. (Medi Cyernetics, Bethesd, MD, USA) nd stined res were expressed s either stined re (μm ) or percentge of the totl plque re. Quntittive rel-time PCR nlysis Expression of genes encoding severl mrkers of DAA were nlysed y quntittive rel-time PCR (qpcr) using the Tqmn System on n ABI Prism 7 Sequence Detector (Applied Biosystems, Foster City, CA, USA) s descried previously []. Gene expression ws normlised to 8S mrna nd reported s reltive rtio to the control group (either the non-dietic mice or norml-glucose-treted cells), which ws ssigned n ritrry vlue of. Western lotting Proteins ( μg) were electrophoresed on.% crylmide gels under reducing conditions. Western lot nlyses were performed with primry ntiodies to either AT R (H-, :, dilution; Snt Cruz Biotechnology, Snt Cruz, CA, USA) or AT R (N-, :, dilution; Snt Cruz) nd ssessed with pproprite secondry ntiodies. The housekeeping protein, α-tuulin (Sigm-Aldrich) ws included to demonstrte equl loding of protein smples. Blots were detected using n enhnced chemiluminescent detection kit (Sigm-Aldrich) followed y quntifiction y densitometry using Quntity-One.. softwre (Bio-Rd Lortories, Richmond, CA, USA). Sttisticl nlysis Dt were nlysed y one-wy ANOVA using SPSS. softwre (IBM, St Leonrds, NSW, Austrli). Post hoc comprisons were performed mong the vrious groups using Fisher s lest significnt difference method. Dt re expressed s men ± SEM, with p <. considered s sttisticlly significnt. Results Metolic vriles STZ-treted Apoe / mice developed sustined hyperglycemi over the week experimentl period with elevted lood glucose nd HA c levels (p <. vs non-dietic mice). etic mice showed reduced ody weight nd elevted levels of totl plsm cholesterol nd tricylglycerol (Tle ). Neither tretment with C lone nor cndesrtn lone ffected ny of these vriles in either non-dietic (control) or dietic mice. While C did not hve ny systemic hemodynmic effect in either normoglycemic or hyperglycemic groups, dministrtion of cndesrtn lone nd in comintion with C significntly reduced lood pressure in dietic mice. Atherosclerotic plque re En fce nlysis of the whole ort reveled twofold increse in plque re in dietic mice compred with their non-dietic counterprts (p <.) (Fig. ). Similr findings were lso oserved in the ortic rch nd thorcic nd dominl segments. C-tretment significntly reduced therosclerotic lesion re in dietic mice (p <.vsdieticmice).whilesimilr reduction ws seen with cndesrtn tretment lone, there ws no further decrese seen in dietic mice co-treted with C nd cndesrtn. Importntly, C hd no effect in modulting plque lesion in non-dietic mice. Sinus plque content etic mice demonstrted twofold increse in lipid deposition compred with their non-dietic counterprts (Fig. ; p <.). This ccumultion of lipid within the therosclerotic plque ws meliorted y tretment with either C or cndesrtn lone (oth p <. vs dietic mice) nd y co-tretment with C nd cndesrtn when compred with dietic mice (p <.vsdieticmice). The comintion tretment showed trend towrds further modest reduction in plque content when compred with either tretment lone (p =.9 vs dietic mice treted with

4 etologi (6) 9: Tle Metolic vriles of non-dietic nd dietic Apoe / mice treted in the sence or presence of AT R gonist C nd/or AT Rntgonist cndesrtn Vriles Control mice etic mice Vehicle C Vehicle C Cndesrtn Cndesrtn + C Body weight (g). ±.. ±. 6.9 ±.8.8 ±.8.7 ± ±. Plsm glucose (mmol/l). ±.. ±.9. ±.. ±..9 ±. 9. ±. HA c (%).9 ±.. ±.. ±.7.7 ±.6. ±.. ±.8 HA c (mmol/mol) 9 ± ± ± 8 ± 7 ± ± 9 Totl cholesterol (mmol/l) 9. ±.7. ±.6. ±.. ±.7. ±.7. ±. Tricylglycerol (mmol/l). ±.. ±..7 ±..6 ±..9 ±.. ±. Systolic BP (mmhg) 9 ± 9 ± 96 ± 9 ± 87 ± 87 ± Dt re shown s men ± SEM, n = mice per group p <. nd p <. vs vehicle-treted control (non-dietic) group; p <.nd p<. vs vehicle-treted dietic mice Fig. Atherosclerotic lesions in mouse ort stined with Oil Red O nd counterstined with hemtoxylin () nd totl therosclerotic plque re (s percentge re of ort stined) in the whole ort () ndwithin the rch (c) nd thorcic (d) nd dominl (e) ortic segments. Mgnifiction.. Dt re expressed s the men ± SEM of n =6 mice per group. p <. vs vehicle-treted control (non-dietic) group; p <.nd p <.vs dietic group. Cnd, cndesrtn;, control;, dietic Control Control etic etic etic etic Aortic plque re (%) c Aortic rch plque re (%) d Thorcic plque re (%) 8 6 e Adominl plque re (%)

5 78 etologi (6) 9:77879 Control Control dietic mice). Neither C nor cndesrtn tretment inhiited dietes-induced mcrophge infiltrtion. Atherom re ( µm ) CD68 stining ( µm ) etic Control etic etic Control etic etic +cndesrtn C; p =. vs dietic mice treted with cndesrtn lone). However, C hd no effect in modulting lipid content within the intiml lyer of the ortic sinus in non-dietic mice. Mcrophge infiltrtion Immunohistochemiclly stined ortic sections from dietic mice demonstrted significnt increse in CD68 protein expression (Fig. ; p <.vsnon- etic esrtn etic +cndesrtn etic esrtn Fig. () Lipid content within the therosclerotic plque in mouse ort. () CD68 stining. Mgnifiction.. Dt re expressed s the men ± SEM of mice per group. p <. vs vehicle-treted control (nondietic) group; p <. nd p <. vs dietic group. Cnd, cndesrtn;, control;, dietic Aortic expression of AT RndAT R qpcr nlysis performed on mouse ort confirmed the expression of oth AT Rs nd AT Rs. mrna levels of At r (lso known s Agtr) were significntly upregulted in dietic mice (p <. vs non-dietic mice; Fig. ) ut were not modulted y C, cndesrtn or C + cndesrtn tretment. In contrst, no significnt difference in At r (lso known s Agtr) gene expression ws oserved when compring the non-dietic nd dietic mouse groups (Fig. ). Moreover, At r expression remined unchnged in ll tretment groups. Aortic expression of the dhesion mrker VCAM- With the dhesion of monocytes to endothelil cells considered criticl step in the initition nd development of DAA, VCAM- expression ws exmined t oth the gene nd protein level. qpcr nlysis reveled significnt upregultion in Vcm- (lso known s Vcm) gene expression in dietic mice (p <. vs non-dietic mice, Fig. c). While C tretment ppered to induce trend towrds reduction in Vcm- gene expression in dietic mice, this AT R gonist significntly inhiited dietes-induced VCAM- expression t the protein level (p <. vs dietic mice; Fig. d). Conversely, significnt reduction in Vcm- mrna nd VCAM- protein expression ws oserved in dietic mice treted with cndesrtn lone nd in comintion with C. Nonetheless, oth qpcr nd immunohistochemicl nlyses demonstrted tht comintion therpy with C nd cndesrtn produced no dditive effect in further reducing dietes-induced VCAM- expression, when compred with either tretment lone. Importntly, C hd no effect in modulting VCAM- expression t the gene or protein level in nondietic mice. Aortic expression of mrkers of oxidtive stress, inflmmtion nd firosis Expression of p7phox, NADPH oxidse (NOX)- nd NOX- ws determined s prt of the ssessment of oxidtive stress, while tht of monocytechemottrctnt protein- (MCP-) nd TGF-β ws exmined to ssess inflmmtory nd firotic pthwys. Tretment with either C or cndesrtn significntly meliorted dietes-induced p7phox (lso known s Ncf), Nox, Mcp- (Ccl) ndtgf-β (Tgf) mrnalevels(p <. vs dietic mice; Fig. e, f, h, i); with Nox expression this meliortion ws produced y only C nd not cndesrtn (Fig. g). Co-dministrtion of C nd cndesrtn mrkedly inhiited the expression of ll the nlysed mrkers in dietic mice (p <. vs dietic mice) while gin no synergistic effect ws seen when compred with either therpy lone. Importntly, C did not influence ny of the nlysed mrkers in non-dietic mice.

6 etologi (6) 9: Fig. (e) At r (), At r ()nd Vcm- (c) mrnalevelsin mouse ort; lso shown re the representtive photomicrogrphs (mgnifiction ) of VCAM-- stined ortic sinus (d). (ei) Aortic mrna levels of p7phox (e), Nox (f), Nox (g), Mcp- (h) ndtgf-β (i). Dt re shown s the men ± SEM of mice per group. mrna levels were normlised to 8S mrna nd reported s reltive rtio to the control group. p <.nd p <. vs vehicle-treted control (non-dietic); p <. nd p <. vs dietic group. Cnd, cndesrtn;, control;, dietic Aortic At r c Aortic Vcm- Aortic At r d Control etic.... Control etic etic +cndesrtn etic +cndesrtn e Aortic p7phox VCAM- stining ( μm ) f Aortic Nox g Aortic Nox h Aortic Mcp- i Aortic Tgf- β In vitro effect of C on high-glucose- nd Ang IIstimulted cells Consistent with our in vivo dt, qpcr nlysis of mrna showed tht mrkers of inflmmtion (ICAM- [ICAM]), oxidtive stress (NOX-) nd firosis (TGF-β, CTGF) were upregulted in hyperglycemic conditions in oth HAEC nd THP- cells (p <. vs norml-glucose-treted cells) (Fig. ). Administrtion of C significntly inhiited the upregultion of these high-glucose-induced mrkers dose dependently, eing most effective t concentrtion of μmol/l in oth cell popultions. Moreover, C t this dose significntly inhiited Ang II-induced effects (Figs, 6). Strikingly,

7 78 etologi (6) 9:77879 Fig. ICAM-, NOX, TGF-β nd CTGF mrna levels from HAEC () ndthp-cells () cultured in either norml ( mmol/l) or high ( mmol/l) glucose conditions. Cells were treted with C (.. μmol/l); mnnitol ( mmol/l)-treted cells served s osmotic control. Dt re shown s the men ± SEM; n = or. mrna levels were normlised to 8S mrna nd reported s reltive rtio to the norml glucose group. p <. nd p <.vs norml-glucose-treted cells; p <.nd p <.vshighglucose-treted cells Endothelil ICAM- Endothelil TGFmRNA induction (fold) β β 8 6 C (μmol/l) Mnnitol Monocyte ICAM- Monocyte TGFmRNA induction (fold) Norml glucose... + Endothelil NOX Endothelil CTGF... Monocyte NOX Monocyte CTGF Norml glucose C (μmol/l) Mnnitol Norml glucose Norml glucose these protective effects of C were completely olished y the AT R ntgonist, PD9 in oth cell types (Figs, 6). While C ( μmol/l) hd similr efficcy to tht of cndesrtn ( μmol/l), co-dministrtion of C nd cndesrtn produced no dditive effect in inhiiting high-glucose- nd Ang-II-induced ICAM-, NOX-, TGF-β nd Ctgf expression (Figs, 6).Bsedonthe similr efficcy of cndesrtn nd C in ttenuting the pthology of DAA, further delinetion of the involvement of the AT R with respect to the effects of the ARB were further studied in HAEC. Interestingly, cndesrtnmedited protective effects were locked y the AT R ntgonist, PD9 (Fig. ). In vitro expression of AT RndAT R Western lot nlysis confirmed the expression of oth AT RndAT Rin HAEC nd THP- cells (Fig. 7). Although AT Rprotein expression ws significntly elevted in cells cultured in the high-glucose condition, neither C nor the AT Rnd AT R inhiitors (PD9 nd cndesrtn, respectively) hd ny effect in modulting AT R expression. In contrst, AT R expression remined unchnged in ll conditions (Fig. 7). Importntly, oth AT RndAT Rprotein expression remined unchnged in norml-glucose conditions (dt not shown). Similr results were lso oserved t the gene level (electronic supplementry mteril [ESM] Fig. ). Discussion AT R ctivtion is incresingly recognised to confer protective effects in numerous disese sttes including therosclerosis [8, 9], which is considered to e the mjor contriutor to premture mortlity nd moridity. However, the iologicl effects medited y the AT R with respect to DAA hve not

8 etologi (6) 9: Fig. ICAM-, NOX, TGF-β nd CTGF mrna levels from HAEC cells cultured in either norml ( mmol/l) or high ( mmol/l) glucose conditions stimulted in the sence () or presence () of AngII ( μmol/l) nd treted with either C ( μmol/l), PD9 ( μmol/l), cndesrtn ( μmol/l), cndesrtn9, C+ PD9 or C+cndesrtn. Cells treted with mnnitol ( mmol/l) served s osmotic control. Dt re shown s the men ± SEM; n =. mrna levels were normlised to 8S mrna nd reported s reltive rtio to the norml glucose group. p <. nd p <.vs norml glucose (NG); p <. nd p <.vshighglucose (HG); p <. vs HG; p <.nd p <.vshg+ cndesrtn; p <.vsng+ AngII; Φ p <.nd ΦΦ p <. vs NG+AngII; Ψ p <. nd p <. HG+AngII, p <.nd p <.vshg+ AngII, Ω p <.vshg+ AngII+cndesrtn Endothelil ICAM- Endothelil TGFmRNA induction (fold) β β Endothelil ICAM- Endothelil TGFmRNA induction (fold) Norml glucose Φ ΦΦ Ψ Ω Norml glucose Norml glucose +AngII +AngII9 Ω +AngII +AngII9 +AngII +AngII 9 +Angll Endothelil NOX Endothelil CTGF Endothelil NOX Endothelil CTGF 6 6 Norml glucose ΦΦ ΦΦ +AngII +AngII9 Ω Ψ Ω +AngII +AngII9 +AngII +AngII 9 +Angll een extensively delineted. This study is the first to exploit the novel non-peptide AT R gonist C in investigting the role of AT Rs in n experimentl model of type DAA. At the in vivo level in mice, C ws found to significntly reduce ortic plque deposition nd plque size, in ssocition with its ility to reduce expression of key meditors of inflmmtion, oxidtive stress nd firosis in the ort. Indeed, C t reltively low dose ws found to retrd the progression of DAAwith similr efficcy to tht fforded y the AT R ntgonist cndesrtn, when continuously dministered over week tretment period. Interestingly, co-dministrtion of C nd cndesrtn hd no cler-cut dditive effect in ttenuting DAA when compred with either tretment lone. Nevertheless, these findings support the potentil role for C in monotherpy for providing protection ginst the progression of DAA y inhiiting inflmmtion, oxidtive stress nd firosis, with the AT R gonist ffording similr vsoprotective properties to the AT R ntgonist. Consistent with our findings, in n experimentl model of myocrdil infrction, C ws demonstrted to improve crdic function nd reduce infrct size with similr efficcy to tht oserved with cndesrtn lone []. Our dditionl findings tht C hd no effect in influencing metolic control or lood pressure in dietic mice suggest tht the eneficil effects seen with this gent do not occur vi effects on glycemic control or vi direct systemic hemodynmic ction. These protective effects of C were further explored in more mechnistic mnner t the in vitro level, in two humn cell lines considered to e involved in the development of therosclerosis endothelil cells nd mcrophges [, ].

9 786 etologi (6) 9:77879 Fig. 6 ICAM-, NOX, TGF-β nd CTGF mrna levels from THP- cells cultured in norml ( mmol/l) or high ( mmol/l) glucose conditions stimulted in the sence () orpresence() of AngII ( μmol/l) nd treted with either C ( μmol/l), PD9 ( μmol/l), cndesrtn ( μmol/l), C + PD9 or C+ cndesrtn. Cells treted with mnnitol ( mmol/l) served s osmotic control. Dt re shown s the men ± SEM; n =. mrna levels were normlised to 8S mrna nd reported s reltive rtio to the norml glucose group. p <.vs norml glucose (NG); p <. nd p <.vshighglucose (HG); p <. vs HG; p <.nd p <.vsng+ AngII; ΦΦ p <. vs NG+AngII+ C; p <. HG+AngII; p <. vs HG+AngII β Monocyte TGF- β Monocyte ICAM- Monocyte ICAM- Monocyte TGFmRNA induction (fold) 6 ΦΦ +AngII +AngII +AngII +AngII Norml glucose Norml glucose ΦΦ +AngII +AngII +AngII +AngII +AngII Monocyte NOX Monocyte CTGF Monocyte NOX Monocyte CTGF +AngII +AngII +AngII +AngII +AngII Norml glucose Norml glucose 6 ΦΦ ΦΦ +AngII +AngII +AngII +AngII +AngII C ws found to significntly meliorte mrkers of oxidtive stress, inflmmtion nd firosis in oth high-glucosend Ang II-stimulted HAEC nd monocytes cultures, with similr efficcy to tht of cndesrtn tretment. Strikingly, these protective effects of C were completely olished y the AT R ntgonist PD9, therey confirming the protective ctions of C occur vi the AT R. Nevertheless, with limited understnding nd knowledge of the signlling pthwys of the AT R, it remins to e fully elucidted s to how C confers its enefits in dietes. Our in vitro nd in vivo studies hve demonstrted tht these effects of C re not dependent on cell type or species nd emphsise the centrl role of C s n nti-inflmmtory, ntioxidnt nd ntifirotic gent. This is consistent with previous studies demonstrting the efficcy of C s reno-protective gent in hlting the progression of insulin-deficient dietic nephropthy [, ]. Thus, these oservtions strengthen the notion tht the AT R gonist C my represent novel therpy for the tretment of dietes-ssocited complictions, including oth micro- nd mcrovsculr complictions. Given the wide tissue distriution of the AT R[], AT R ctivtion hs een shown to e protective ginst numerous disese sttes including renl [, 6], neuronl [7] nd pncretic injury [8]. However, the role of this receptor in the crdiovsculr system still remins poorly understood. Previous studies hve vriously demonstrted tht the AT R could exert protective [6, 9], detrimentl [] or neutrl [, 6] role in therosclerosis. These conflicting reports on the therosclerotic role of the AT R my suggest tht its ctions re likely to e dependent on numer of fctors. These

10 etologi (6) 9: Fig. 7 AT RndAT Rprotein levels from HAEC () nd THP- cells () cultured in norml ( mmol/l) nd high ( mmol/l) glucose conditions treted in the sence or presence of C ( μmol/l), C( μmol/l) + PD9 (PD) ( μmol/l), cndesrtn ( μmol/l) or C + cndesrtn. α-tuulin lots were used to demonstrte equivlent loding of protein smples. Dt re the men ± SEM sornce (As) levels of AT RndAT R nd re expressed reltive to tht of the norml glucose (NG)-treted cells; n = or. p <.vsng Reltive As ATR Endothelil AT R ( kd) Endothelil AT R ( kd) α-tuulin ( kd) Norml glucose C PD9 Cndesrtn Mnnitol NG HG HG HG HG+ HG Cnd Reltive As ATR... NG HG HG HG HG+ HG Cnd Monocyte AT R ( kd) Monocyte AT R ( kd) α-tuulin ( kd) Norml glucose C PD9 Cndesrtn Mnnitol Reltive As ATR NG HG HG HG HG+ HG Cnd Reltive As ATR.. NG HG HG HG HG+ HG Cnd include the severity of the disese, where the efficcy t which AT R medites its protective effects my e lost in severe chronic conditions [7], the sence or presence of hyperglycemi [, 8] nd the prticulr niml strin exmined in the study []. More recently, the diverse crdic response ssocited with the AT R hs lso een shown to e dependent on the trnscription fctors with which it intercts. The ility of the AT R to ind nd fcilitte the trnsloction of promyelocytic leukemi zinc finger trnscription fctor to the nucleus hs een shown to e implicted in driving its hypertrophic ctivity in response to Ang II [9]. Nonetheless, findings in this study hve demonstrted tht C-medited AT R ctivtion is eneficil ginst DAA. Consistent with these end-orgn protective effects, leit in the non-dietic setting, C hs een shown previously to reduce myocrdil firosis nd vsculr injury [8], improve ventriculr function [] nd promote vsodilttion [] in experimentl models of crdiovsculr disese. Although oth the AT RndAT Rwerefoundtoe expressed in the humn monocytes nd ortic endothelil cells used in this study, C did not hve ny mrked effects on expression of either receptor. This is consistent with the view tht the gonist most likely medites its ctions y influencing post-receptor signlling pthwys. Indeed, recent studies hve demonstrted tht AT R inhiits AT R- medited effects y trgeting its downstrem effectors, including TGF-β[] nd NOX [9], s well s its ility to ctivte vsoconstriction inhiiting fctor, n endogenous co-fctor of Ang II []. Moreover, with previous studies demonstrting tht AT R forms heterodimer receptor complex with AT R to lock the signlling nd medited functions of the ltter [], it is possile tht some of the

11 788 etologi (6) 9:77879 effects of the AT R gonist involves the complex interction etween the two Ang II receptor sutypes. Bsed on our findings tht C exhiits similr ntitherogenic efficcy s cndesrtn nd tht the effects of cndesrtn could e inhiited in the presence of the AT R ntgonist PD9, this could indicte tht cndesrtnmedited ctions occur t lest in prt s result of the unopposed ction of the AT R. Consistent with these findings re studies, leit in the non-dietic setting, where cndesrtnmedited reduction of infrct size in pigs [] nd inhiition of crdic remodelling in rts [] were found to e dependent on AT R ctivtion. Furthermore, it is possile tht AT R lockde nd AT R gonism re cting through similr signlling pthwys. Indeed, severl studies hve demonstrted tht cndesrtn nd C inhiit the mitogen-ctivted protein kinse pthwy nd signl through nitric oxide-dependent pthwy to medite their crdioprotective effects [, 6, 7]. Pilot studies hve suggested tht C cn ntgonise the thromoxne receptor (TxR) [8]. Since TxR ntgonist hs een shown to reduce therosclerosis in similr dietic polipoprotein E model [9], one cnnot exclude the possiility tht the nti-therosclerotic ctions of C could involve the TxR. It remins to e determined whether this is direct effect of C or involves n indirect ction vi the AT R. Future studies re wrrnted to further elucidte interctions etween AT R nd AT R, prticulrly in cells/orgns tht co-express the two Ang II receptors, nd to identify potentil effectors tht re involved in mediting the ctivity of the AT R. Our dditionl finding tht AT Rs were upregulted during dietes could explin why the gonist did not ffect ortic function under norml physiologicl conditions nd only displyed its protective effects in DAA. Moreover, recent studies demonstrte tht the AT R undergoes desensitistion in response to prolonged exposure to its lignd nd tht the AT R is unle to recruit β-rrestins, therey contriuting to the receptor sutype not eing desensitised in the sme mnner s AT R[]. Hence, this llows C to hve comprle long-term effects (compred with ARB) in exerting its protective effects, presumly vi the AT R. Tken together, these findings further highlight the dvntge of C s potentil lterntive therpy for DAA. In conclusion, this study hs clerly demonstrted tht dietes-relted therosclerosis cn e influenced y pproches tht trget the two ngiotensin receptor sutypes. These findings hve importnt clinicl significnce in providing insight into potentil therpeutic gents trgeting the RAS tht cn e developed to hlt the progression of DAA. Tretment with C s monotherpy hs een shown to retrd the development of ortic therosclerosis, prticulrly since it leds to suppression of the underlying oxidtive stress, inflmmtion nd firosis ssocited with insulin-deficient DAA. Furthermore, C ws found to inhiit DAA-induced pthology to n extent similr to tht produced y n ARB, suggesting tht C my provide n lterntive therpy for retrding DAA. Further elucidtion of the signlling pthwys tht re ctivted y C would represent crucil step in determining the strengths nd limittions of this AT Rgonist s therpeutic gent in treting the vsculr complictions of oth type nd type dietes. Acknowledgements The uthors would like to thnk S. Scc, E. Lstvec nd M. Hilly (Bker IDI Hert nd etes Institute, Austrli) for their technicl support. We re grteful to R. Pickering nd A. Shrm (Bker IDI Hert nd etes Institute, Austrli) for providing the monocyte nd endothelil cell lines nd to A. Ljunggren (Vicore Phrm AB, Göteorg, Sweden) for providing C. Funding This study is supported y Ntionl Helth & Medicl Reserch Council of Austrli (NHMRC) Project Grnt. BSMC is recipient of JDRF postdoctorl fellowship. MEC, KAJD nd TJA hve een supported y NHMRC fellowships. Dulity of interest The uthors declre tht there is no dulity of interest ssocited with this mnuscript. KJD nd TJA received Compound from Vicore Phrm AB, Göteorg,, Sweden, s gift. Contriution sttement All uthors contriuted to the study concept nd design, nd the interprettion of the dt. BSMC, CK, UMS, TU, MEC, KAJD nd TJA conceived nd designed the experiments. BSMC, CK nd PK performed the experiments nd BSMC, CK, PK, MEC, KAJD nd TJA nlysed the dt. BSMC wrote the pper. The mnuscript ws edited nd reviewed y CK, PK, UMS, TU, MEC, KAJD nd TJA. All uthors pproved the finl version of this mnuscript. TJA tkes full responsiility for the work conducted in this pper. References. M TK, Km KK, Yn BP, Lm YY () Renin-ngiotensinldosterone system lockde for crdiovsculr diseses: current sttus. 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