Inflammation adversely affects arterial wall biology. 1,2 Much

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1 Ftty cids Regulte Endothelil Lipse nd Inflmmtory Mrkers in Mcrophges nd in Mouse ort Role for PPRγ Un Ju Jung, Cludi Torrejon, Chuchun L. Chng, Hiroko Hmi, Till S. Worgll, Richrd J. Deckelum Ojective Mcrophge endothelil lipse () is ssocited with incresed therosclerosis nd inflmmtion. ecuse of their nti-inflmmtory properties we hypothesized tht n- ftty cids, in contrst to sturted ftty cids, would lower mcrophges nd rteril nd inflmmtory mrkers. Methods nd Results Murine J77 nd peritonel mcrophges were incuted with eicospentenoic cid or plmitic cid in the presence or sence of lipopolysccride (LPS). LPS incresed nd protein. Plmitic cid lone or with LPS dose-dependently incresed nd protein. In contrst, eicospentenoic cid dose-dependently rogted effects of LPS or plmitic cid on incresing expression. expression closely linked to peroxisome prolifertor ctivted receptor (PPR)γ expression. Eicospentenoic cid locked rosiglitzone ( PPRγ gonist)-medited ctivtion nd GW9 ( PPRγ ntgonist)-locked plmitic cid-medited stimultion. Eicospentenoic cid lone or with LPS lunted LPS-medited stimultion of mcrophge proinflmmtory interleukin-, interleukin-p, nd toll-like receptor- mrn nd incresed nti-inflmmtory interleukin- nd mnnose receptor mrn. In vivo studies in low density lipoprotein receptor knockout mice showed tht high sturted ft rich diets, ut not n- diets, incresed rteril, PPRγ, nd proinflmmtory cytokine mrn. Conclusion n- ftty cids, in contrst to sturted ftty cids, decrese in prllel with modulting pro- nd ntiinflmmtory mrkers, nd these effects on link to PPRγ. (rterioscler Throm Vsc iol. ;:99-97.) Key Words: therosclerosis endothelil lipse inflmmtion n- ftty cids PPRγ Downloded from y on Jnury, 9 Inflmmtion dversely ffects rteril wll iology., Much evidence supports protherogenic nd proinflmmtory effect of sturted ftty cids (F)., In contrst, protective ctions with respect to the rteril wll hve een ttriuted to n- F. 7 n- F delivered from dietry fish oil re incorported into therosclerotic plques, enhncing stility, wheres n- F do not hve these effects. Recent reviews indicte tht incresed consumption of long-chin n- F, eicospentenoic cid (EP), nd docoshexenoic cid, ut not of α-linolenic cid (their n- essentil F precursor), reduced the rtes of llcuse mortlity nd crdic nd sudden deth. n- F hve een shown to reduce the mcrophge infiltrtion into the vessel wll nd secretion of protherogenic nd proinflmmtory growth fctors nd cytokines y monocytes nd mcrophges. 7 Mcrophges ply pivotl role in the development nd progression of therosclerosis. Endothelil lipse () is of severl lipses synthesized nd secreted y mcrophges. High levels of expression hve een oserved in mcrophges present in humn therosclerotic plques. 8 deficiency is ssocited with 7% decrese in therosclerotic lesions in poe KO mice. 9 protherogenic effect of in mcrophges hs lso een scried to ridging functions, which plys role in the uptke of lipoproteins or recruitment of monocytes y lood vessel wlls. Moreover, expression is upregulted in mcrophges y proinflmmtory cytokines. Upregultion of mcrophge y toll-like receptor (TLR) nd negtively modultes interleukin (IL)- nd positively modultes IL- production, potentilly influencing therosclerosis. ccordingly, might e considered to e n ttrctive phrmcologicl trget in the prevention of therosclerosis. Peroxisome prolifertor ctivted receptor (PPR)γ lso hs een implicted in therogenesis. PPRγ is highly expressed in mcrophges-derived fom cells in therosclerotic lesions nd ctivtion of PPRγ hs een shown to induce mcrophge lipid ccumultion y incresing the expression of the oxidized low density lipoprotein (LDL) scvenger receptor CD nd lipoprotein lipse (LpL). In contrst to the proposed potentilly protherogenic effects of PPRγ, other limited evidence suggests PPRγ my medite nti-inflmmtory effects y negtively regulting proinflmmtory cytokine expression. lso, mcrophge PPRγ deficiency increses therosclerosis in C7L/ nd LDL receptor knockout (LDL-R KO) mice, 7 Received on: ugust, ; finl version ccepted on: ugust,. From the Institute of Humn Nutrition (U.J.J., C.T., C.L.C., T.S.W., R.J.D.), nd Deprtments of Pthology (H.H., T.S.W.) nd Peditrics (T.S.W., R.J.D.), Columi University, New York, NY. The online-only Dt Supplement is ville with this rticle t Correspondence to Richrd J. Deckelum, Institute of Humn Nutrition, College of Physicins nd Surgeons, Columi University, W 8 th St, PH, New York, NY. E-mil rjd@columi.edu mericn Hert ssocition, Inc. rterioscler Throm Vsc iol is ville t 99 DOI:./TVH..88

2 9 rterioscler Throm Vsc iol Decemer Downloded from y on Jnury, 9 indicting n ntitherogenic role for PPRγ. However, t present, no definitive studies support the premise tht PPRγ is required for nti-inflmmtory effects in mcrophges. 8 In fct, recent clinicl trils hve rised concerns on incresed risk of myocrdil infrction nd crdiovsculr deth in dietic ptients treted with rosiglitzone,, strong PPRγ gonist. We previously demonstrted tht high sturted ft (ST) diets incresed contriutions of LDL selective uptke to totl rteril LDL-cholesteryl ester deposition nd tht incresed selective uptke prllels incresed LpL levels nd distriution in the rteril wll. In contrst, n--rich diets decresed rteril totl LDL delivery nd rogted LDL selective uptke in prllel with chnging rteril wll LpL. We now questioned whether F would regulte expression s different dietry F modulted rteril LpL levels nd distriution s descried in our previous reports. 7 Specificlly, we sked whether n- F, EP, in contrst to sturted F, plmitic cid (P), would decrese expression of nd, if so, would these chnges correlte with chnges in inflmmtory mrkers nd in PPRs, which my lso modulte lipid metolism nd inflmmtory responses.,,8 Our results demonstrte tht P increses expression nd decreses nti-inflmmtory IL- expression in mcrophges. In contrst, EP decreses expression, in prllel with decresing proinflmmtory mrkers nd incresing nti-inflmmtory mrkers. The chnges in mcrophge y F were strongly relted to the regultion of PPRγ. Moreover, LDL-R KO mice fed ST diets, ut not n- diets, showed the increses in, PPRγ, nd proinflmmtory responses in the rteril wll. Mterils nd Methods Methods re descried in more detil in the online-only Dt Supplement. Cell Culture Murine mcrophge-like cells, J77 (), were grown in Dulecco s modified Egle s medium contining % fetl ovine serum (vol/ vol), % glutmine (vol/vol), nd % penicillin/streptomycin (vol/vol). Thioglycollte-elicited mcrophges were otined from C7L/ mice ( weeks old) y peritonel lvge with phosphte-uffered sline t dys fter injection of ml of.8% thioglycollte roth (Sigm-ldrich). Cells were suspended in Dulecco s RPMI supplemented with % fetl ovine serum (vol/vol), % glutmine (vol/vol), nd % penicillin/streptomycin (vol/vol), nd incuted t 7 C for hours. For ll experiments, the cells were wshed twice with phosphte-uffered sline nd the F-contining medi were dded t different doses for hours, wheres the control cells received only ovine serum lumin medium. Then, cells were incuted with lipopolysccride (LPS; μg/ml), PPRγ gonist, or ntgonist for hours (for mrn) or hours (for protein). For experiments requiring PPRγ mrn knock-down, J77 cells ( 7% confluence) were trnsfected with optimized concentrtions of either mouse PPRγ short hirpin RN (shrn) plsmid (Snt Cruz iotechnology, Inc), or control nonsense shrn plsmid using shrn trnsfection regent (sc-98), ccording to the mnufcturer s instructions. Twenty-four hours fter trnsfection, cells were treted with specific F nd processed for rel-time polymerse chin rection nlyses s descried elow. Quntittive Rel-Time Polymerse Chin Rection Totl RN ws isolted with TRIzol regent (Invitrogen) nd quntittive rel-time polymerse chin rection ws crried out on n icycler rel-time mchine (iord) using the SYR Green polymerse chin rection mster kit (pplied iosystems). Vlues were normlized to GPDH levels. Western lot protein expression ws nlyzed y Western lot normlized to β-ctin protein. nimls nd Diets Eight-week-old mle LDL-R KO mice were purchsed from Jckson Lortory. fter week cclimtiztion, mice were fed semipurified, norml chow (totl % ft,.% cholesterol, wt/wt) or high-ft, semipurified diet (totl 9% ft,.% cholesterol, wt/wt) enriched in either n- (9% menhden fish oil nd 9% corn oil; Hrln Tekld; TD. 7) or ST (78% ST from coconut oil, % monounsturted ft from olive oil, nd 9% polyunsturted ft from corn oil; Hrln Tekld; TD. 88) for weeks similr to our previous report. The ort ws dissected nd mesured for mrn of, PPRγ, nd specific inflmmtory mrkers. ll procedures were pproved y the Institutionl niml Cre nd Use Committee of Columi University. Sttisticl nlyses Sttisticl nlyses were determined y -wy NOV (for compring F), -tiled Student s t test, or Person correltion coefficients. Dt re expressed s men±se. Results LPS Increses Expression in Mcrophges We first exmined the effect of LPS, potent endotoxin, on expression in mcrophges. J77 cells were incuted with incresing concentrtion of LPS (. μg/ml). LPS incresed y mximum of.-fold t μg/ml (Figure I in the online-only Dt Supplement). Similrly, LPS dose-dependently incresed protein levels of with mximum increse lso t μg/ml (Figure I in the onlineonly Dt Supplement). We thus chose concentrtion of LPS of μg/ml for the experiments elow. EP Inhiits the Increse of nd Protein Induced y LPS More Thn Other F To determine the effects of different F on mcrophge mrn expression, J77 cells were cultured in the presence or sence of LPS, μg/ml, for hours fter preincution with μmol/l of unsturted F (EP; rchidonic cid; linoleic cid), monounsturted F (oleic cid, O), nd sturted F (P) for hours. Exposure of cells to linoleic cid nd P lone significntly incresed mcrophge levels compred with non-f control y.-fold nd.-fold, respectively (Figure ). P together with LPS induced.- fold higher levels of compred with LPS lone. In contrst, EP, rchidonic cid, nd oleic cid inhiited LPS-induced levels y, 7, nd 9%, respectively. mong F tested, P induced the strongest response on incresing expression, wheres EP ws most potent t inhiiting expression induced y LPS. s EP nd P seemed to show the gretest differences on expression, we chose EP nd P for doseresponse experiments. J77 cells were cultured in the presence or sence of LPS, μg/ml, for to hours fter preincution with,, μmol/l of EP or P for hours. P lone s well s together with LPS dose-dependently incresed (Figure nd C). In contrst, EP lone hd no effect on ut completely rogted effects of LPS

3 Jung et l Ftty cids Regulte Endothelil Lipse 9 (ritrry unit) c c c c c c c S EP O L P LPS(-) LPS(+) Downloded from y on Jnury, 9. C LPS LPS F (µ M) - F (µ M) - - EP P EP P (ritrry unit) D β-ctin LPS F (µm) EP P F (ritrry unit) LPS(-) LPS(+) (ritrry unit) E β-ctin LPS F (µm) EP P on incresing. Consistent with mrn expression, EP hd no significnt effect on protein expression ut inhiited LPS-medited increse of protein (Figure D nd E). P lone s well s with LPS incresed protein levels in dose-dependent mnner. S EP P G mnr (ritry unit) β-ctin LPS(-) LPS(+) S EP P Figure. Effects of different ftty cids (F) on endothelil lipse () expression in mcrophges. J77 ( E) or peritonel mcrophges (F, G) were cultured in the presence or sence of lipopolysccride (LPS), μg/ml, for h (mrn, C, F) or h (protein, D, E, G) fter preincution with μmol/l of F for h. c Mens with unlike letters re significntly different t P<. (-wy NOV). P<., P<. (Student s t test). S indictes ovine serum lumin; EP, eicospentenoic cid;, rchidonic cid; O, oleic cid; L, linoleic cid; P, plmitic cid. mrn y % (Figure F). protein levels were lso incresed y LPS nd P, ut EP significntly rogted effects of LPS on incresing protein (Figure G). Thus, EP cn mitigte the effects of P-medited increses of in different mcrophge lines. P nd LPS Hve Similr Effects on Expression in Peritonel Mcrophges s in J77 Cells nd EP Inhiits LPS-Medited Increse of Expression To determine whether EP nd P lso regulte expression in primry mcrophges, we treted EP nd P with or without LPS in peritonel mcrophges from C7L/ mice. Similr to J77 cells, LPS nd P lone significntly incresed compred with control y.- nd.-fold, respectively, wheres EP significntly inhiited LPS-induced EP Inhiits P-Medited Increses of To determine effects of EP together with P on mcrophge, we incuted cells with different rtios of EP nd P (Figure ). ws lower in J77 cells treted with the comintions of P plus EP compred with the group treted only with P; these differences ecme significnt when EP ccounted for one third or more of totl F (Figure ). protein levels showed similr responses to incutions of P versus P nd EP (Figure ).

4 9 rterioscler Throm Vsc iol Decemer Downloded from y on Jnury, 9 (ritrry unit) P ( M) EP ( M) Rtio - : : : : : : -ctin EP nd P Hve Different Effects on Regultion of PPRγ Expression ecuse PPRs re linked to numer of processes importnt to therogenesis,,8 we exmined potentil ssocitions of PPRγ nd PPRα with expression. PPRγ expression ws more ffected y F thn PPRα expression. In J77 cells, ut not peritonel mcrophges, EP lone or with LPS tended to increse PPRα mrn, wheres P hd no effect (dt not shown). In contrst, P lone s well s with LPS significntly incresed PPRγ mrn expression (Figure nd ). In ddition, PPRγ mrn positively correlted with in oth mcrophges, respectively (r=., P<.; r=., P<.). P ( M) EP ( M) Rtio - : : : : : : Figure. Effects of different rtios of eicospentenoic cid (EP) nd plmitic cid (P) on endothelil lipse (). J77 mcrophges were incuted with the indicted concentrtions of EP nd P for 7 h (mrn, ) or h (protein, ). P<., P<. (Student s t test). To determine how chnges in PPRγ might ffect mcrophge expression, we treted cells with incresing concentrtions of rosiglitzone or GW9 in the presence or sence of EP or P. Rosiglitzone is known PPRγ gonist nd GW9 is PPRγ ntgonist. Rosiglitzone (. µmol/l) dose-dependently incresed protein nd mrn, wheres GW9 mrkedly locked rosiglitzonemedited increses of protein nd mrn in J77 cells (Figure II in the online-only Dt Supplement; Figure nd ). Moreover, GW9 significntly inhiited P-induced nd protein. Of note, EP rogted rosiglitzonemedited increses of nd protein in J77 cells (Figure C). PPRγ mrn expression ws dose-dependently incresed y rosiglitzone, wheres GW9 nd EP rogte effects of rosiglitzone on incresing PPRγ mrn in J77 cells (Figure III in the online-only Dt Supplement). We lso found tht EP inhiited the increse of mrn levels of LpL nd CD, well known PPRγ trget genes,, fter incution with rosiglitzone (Figure IV in the online-only Dt Supplement). To determine whether the PPRγ gonist nd ntgonist lso regulte expression in primry mcrophges, we treted peritonel mcrophge with rosiglitzone nd GW9 in the presence of F. Similr to J77 cells, rosiglitzone incresed nd protein y 7% nd %, respectively, nd EP significntly locked rosiglitzoneinduced expression y 8% (Figure D). lso, GW9 mrkedly locked the P nd rosiglitzone-medited increses of y 9% nd 78%, respectively, nd inhiited the increse in protein induced y P nd rosiglitzone (Figure D nd E). Thus, in cultured cells in vitro closely linked to PPRγ nd ws regulted y F, in prt, through modifying mcrophge PPRγ expression. To further support the ove ssocition etween PPRγ nd expression we performed experiments where we used shrn medited knock-down of PPRγ expression in J77 cells. We chieved knock-down efficiency of >8% using PPRγ shrn s compred with control shrn controls (n=; P<.). We then compred the effects of P on incresing in these PPRγ knock-down J77 cells nd found men % decrese in expression fter incution with P compred with control levels (n=, P=.). Thus, decreses in PPRγ lunt the ility of P to increse expression in mcrophges, indicting tht is modulted, in prt, y PPRγ-dependent pthwys. J77 Peritonel Mcrophges PPR mrn (ritrry unit) LPS(-) LPS(+) S EP P PPR mrn (ritrry unit) LPS(-) LPS(+) S EP P Figure. Effects of ftty cids (F) on mcrophge peroxisome prolifertor ctivted receptor (PPR)γ mrn expression. J77 () or peritonel mcrophges () were incuted with µmol/l of eicospentenoic cid (EP) or plmitic cid (P) s previously descried in Figure. Mens with unlike letters re significntly different t P<. (-wy NOV). LPS indictes lipopolysccride; S, ovine serum lumin.

5 Jung et l Ftty cids Regulte Endothelil Lipse 9 protein -ctin GW9 ( M) - - ROSI ( M) (ritrry unit) 8 GW9 ( M) - - ROSI ( M) Downloded from y on Jnury, 9 D protein m mrn (ritr ry unit) -ctin 8 -ctin EP Decreses Proinflmmtory Mrkers, ut Increses nti-inflmmtory Mrkers in Peritonel Mcrophges LPS is cteril endotoxin tht is commonly used to stimulte inflmmtory responses. Wng et l reported tht induction of mcrophge y LPS cn modulte mcrophge inflmmtory responses. To explore the reltionships of nd inflmmtory mrkers, we compred well-defined pro- nd nti-inflmmtory mrkers in peritonel mcrophges incuted with EP nd P. LPS significntly incresed proinflmmtory cytokines IL- nd IL-p (Figure ). However, EP lone or with LPS lunted the stimulting effects of LPS on IL- nd IL-p mrn y % nd %, respectively. lso, EP mrkedly ttenuted TLR mrn. We lso found tht in J77 cells, LPS incresed IL- nd IL-p mrn y 7- nd -fold, respectively (P<., P<.), nd tht these effects were diminished y EP (dt not shown). P nd EP effects on tumor necrosis fctor (TNF)-α expression were not similr to other proinflmmtory mrkers; P C E protein mr RN (ritr ry unit) -ctin 8 P ( M) EP ( M) - - GW9 ( M) ROSI ( M) ROSI ( M) protein EP ( M) GW9 ( M) ROSI ( M) (ritrry unit) protein (ritrry unit) -ctin P ( M) GW9 ( M) Figure. Interctions of eicospentenoic cid (EP) nd plmitic cid (P) with peroxisome prolifertor ctivted receptor (PPR)γ gonist/ntgonist on endothelil lipse () expression in mcrophges. J77 ( C) or peritonel mcrophges (D E) were pretreted with or without ftty cids for h nd then continuously cultured with PPRγ gonist rosiglitzone (ROSI) nd/or PPRγ ntgonist GW9 for h (mrn) or h (protein). P<., P<. (Student s t test). lone incresed TNF-α mrn level similr to LPS (LPS versus ovine serum lumin, %; P versus ovine serum lumin, %), wheres EP lone or with LPS hd no effect. In contrst, nti-inflmmtory IL- nd mnnose receptor were incresed in EP-treted cells y.- nd.-fold, respectively (Figure ). P hd little effect on proinflmmtory mrkers ut decresed IL- mrn (Figure nd ). Interestingly, showed positive correltions with incresing mrn levels of proinflmmtory mrkers, such s IL-, IL-p, TLR, nd vsculr cell dhesion molecule- (Figure V in the online-only Dt Supplement). In contrst, there were negtive correltions etween nd nti-inflmmtory mrkers, IL- nd mnnose receptor, respectively. PPRγ mrn ws lso positively correlted with proinflmmtory IL-, IL-p, TLR, nd vsculr cell dhesion molecule- mrn, wheres it ws negtively correlted with nti-inflmmtory IL- mrn (Figure V in the online-only Dt Supplement). There were no significnt correltions etween PPRα nd inflmmtory mrkers (dt not shown).

6 9 rterioscler Throm Vsc iol Decemer Downloded from y on Jnury, 9 Dietry Sturted Versus n- Diet Chnges rteril, PPRγ, nd Inflmmtory Mrker Expression in LDL-R KO Mice In Vivo We next investigted the potentil effects of -week feeding of n-- nd ST-rich diets on rteril, PPRγ, nd inflmmtory gene expression in ort of LDL-R KO mice, which re susceptile to therosclerosis. ST diets led to -fold greter rteril compred with chow (Figure ). There ws no significnt difference in rteril levels etween n- nd chow diets. ST diets were ssocited with mrkedly incresed rteril PPRγ mrn compred with chow diets, wheres the n- diets showed % decrese in PPRγ mrn (Figure ). Similr to in vitro dt in mcrophges, ST diets incresed rteril IL- nd IL-p mrn.- nd.8-fold compred with chow, respectively, wheres rteril IL- mrn ws lowered in ST-fed mice compred with chow-fed mice y % (Figure C E). In contrst, n- diets reduced oth proinflmmtory cytokine mrn levels in ort of LDL-R KO mice compred with chow y 7 nd %, respectively, ut incresed IL- mrn compred with ST diet y 9%. Thus, in vivo effects of diets rich in ST versus n- F on rteril expression of nd inflmmtory mrkers prlleled effects oserved in cultured mcrophges in vitro. Discussion Mcrophge-derived in the rteril wll is ssocited with incresed therosclerosis nd rteril inflmmtory mrkers in mice. 9,, ecuse of their nti-inflmmtory properties we hypothesized tht n- F, in contrst to sturted F, would lower expression of in vitro nd in vivo. Our results show tht sturted F, P, increses mcrophge expression nd decreses nti-inflmmtory IL- expression. In contrst, n n- F, EP, inhiits the increse of expression induced y LPS nd P, nd this is ccompnied y decreses in proinflmmtory mrkers nd increses in nti-inflmmtory mrkers in cultured mcrophges. Moreover, regultion of mcrophge in response to F is closely linked to chnges in PPRγ ctivtion. In vivo studies lso show tht ST diets, ut not n- diets, increse, PPRγ, nd proinflmmtory cytokine expression ut decrese nti-inflmmtory cytokine mrn in ort of LDL-R KO mice, suggesting tht chnges in y F hve importnt regultory roles on therosclerosis nd inflmmtion in vivo s well s in vitro. LPS, mjor inflmmtory stimulus, cn ply n importnt role in lipoprotein metolism nd therosclerosis. 9 Ysud et l reported tht expression ws incresed y LPS. In our current study, LPS lso mrkedly incresed protein s well s mrn expression in J77 nd peritonel mcrophges. Interestingly, sturted P lone nd with LPS incresed nd protein levels in oth mcrophges. In contrst, n- EP hd little effect on nd protein ut mrkedly inhiited the increse of mcrophge expression induced y LPS. Furthermore, expression ws significntly lower in cells treted with comintion of P plus EP compred with P lone. sed on these results, EP suppresses the increse of mcrophge expression induced y ctivtors P nd LPS. Figure. Effects of ftty cids on pro- () nd nti-inflmmtory mrkers () mrn expression in murine peritonel mcrophges. Cells were incuted with μmol/l of eicospentenoic cid (EP) or plmitic cid (P) s previously descried in Figure. Mens with unlike letters re significntly different (P<., Student s t test). MR indictes, mnnose receptor; LPS, lipopolysccride; S, ovine serum lumin; IL, interleukin; TNF, tumor necrosis fctor; VCM-, vsculr cell dhesion molecule; TLR, toll-like receptor.

7 Jung et l Ftty cids Regulte Endothelil Lipse 9 Downloded from y on Jnury, 9 (ritrry unit) C IL- mrn (ritr ry unit) CHOW n- ST CHOW n- ST E IL-mRN (ritrry unit) PPR mrn (ritrry unit) mrn ry unit) PPRγ is nucler trnscription fctor tht regultes numerous genes involved in lipoprotein metolism nd is highly expressed in mcrophges, including fom cells of therosclerotic lesions. PPRγ ctivtion incresed mcrophge LpL mrn nd protein expression nd promoted uptke of oxidized LDL through induction of mcrophge CD expression,, which suggest the potentil role of PPRγ in the pthogenesis of therosclerosis. Furthermore, PPRγ gonists, rosiglitzone nd pioglitzone, enhnce mcrophge poptosis vi PPRγ-independent mechnism, nd pioglitzone promotes dvnced plque progression in LDL-R KO mice through enhncement of dvnced lesion mcrophge poptosis. Our dt indicte tht might lso contriute to therogenesis nd tht PPRγ ctivtion is ssocited with this regultion of. In the current study, PPRγ ctivtion using rosiglitzone s well s P ws relted to incresed nd protein, nd GW9, PPRγ ntgonist, EP nd PPRγknock-down cells rogted stimultion y rosiglitzone or P. Rosiglitzone significntly incresed PPRγ mrn in dose-dependent mnner, wheres pioglitzone, n times less potent n ctivtor of PPRγ thn rosiglitzone, hd little effects on PPRγ s well s on in our experiments in mcrophges (dt not shown). lso, GW9 nd EP locked rosiglitzone-induced PPRγ mrn expression. Similr to our results with EP, n- F-docoshexenoic cid suppressed CD expression induced y PPRγ gonist through the inhiition of trnscriptionl ctivity of PPRγ in humn monocytes nd colon tumor cells, nd EP nd docoshexenoic cid reduced the PPRγ response element reporter ctivity in D IL- (ritr CHOW n- ST 8 CHOW n- ST CHOW n- ST Figure. Effects of n-- nd sturted ftty cids-rich diets on rteril endothelil lipse () (), peroxisome prolifertor ctivted receptor (PPR)γ (), nd inflmmtory mrkers (C E) of mrn expression in LDL receptor knockout mice. Mens with unlike letters re significntly different t P<. (-wy NOV). ST, sturted ft; IL, interleukin. colon cncer cells. Edwrds et l proposed tht n- F my directly, or fter eing metolized, ctivte extrcellulr signl-regulted kinse or other pthwys tht countercts PPRγ signling. However, P significntly incresed PPRγ mrn or protein expression in severl cell types, such s crdiomyocytes,, nd high-ft diet enriched in P enhnced PPRγ expression in mcrophges. P lso stimulted the ctivity of the PPRγ response element in primry humn dipocytes, suggesting ctivtion of this nucler signling cscde. 7 Tken together, our findings suggest tht mcrophge expression is prtilly medited y the upregultion of PPRγ nd tht sturted versus n- F ffect the expression of, t lest in prt, y regulting PPRγ. It is possile tht the trnscriptionl induction of gene might e medited through inding PPR-RXR heterodimer, s it is reported tht CD nd the LpL promoter is direct trget of PPR-RXR heterodimer.,8 Others hve lso shown different effects of sturted versus n- F on PPRγ signling nd these differences re lso relted to the specific tissue or cell nlyzed. 7 To clerly understnd whether regultion of mcrophge is most likely medited through PPRγ-dependent mechnism, further experiments using lignd inding ssys re needed to e performed. PPRγ-relted mechnism for regultion of rteril wll y F is supported y our in vivo findings. ST diets, ut not n- diets, incresed rteril nd PPRγ mrn in LDL-R KO mice, nd ws positively correlted with PPRγ (P<.). Ishid et l 9 reported tht protein ws incresed in ort from poe KO mice nd this ws ccentuted y high-ft diet (.% cholesterol, % milk ft). lso, there ws decrese in therosclerotic lesions in nimls lcking oth nd poe compred with poe KO lone. Moreover, protein 9 nd PPRγ mrn were incresed in the ort, especilly the therosclerotic lesions in high-cholesterol diet fed nimls. Herein we show tht n- diets do not shre the stimultory effects of ST diets in vivo. We lso found tht, in prllel with the chnges in mcrophge nd PPRγ, EP lone or plus LPS reduced IL- nd IL-p mrn in mcrophges ut incresed the mrn of IL- nd mnnose receptor which stimultes nti-inflmmtory cytokines production including IL-. Proinflmmtory cytokines, such s IL- nd IL-, promote the development of therosclerotic lesions,, wheres ntiinflmmtory IL- hve ntitherogenic effects. s well s PPRγ positively correlted with proinflmmtory mrkers ut negtively correlted with nti-inflmmtory mrkers. Moreover, similr chnges in rteril inflmmtory mrkers were found in LDL-R KO mice, suggesting tht F-regulted inflmmtory responses could lso occur in vivo. In contrst to other inflmmtory cytokines mrn expression, there ws no significnt effect of EP on TNF-α mrn expression in mcrophges. Preliminry dt (not shown) on LDL-R KO mice lso show tht feeding of n- diets for weeks did not ffect TNF-α mrn expression in ort compred with chow or ST diets. Renier et l reported tht mcrophges derived from mice fed n- diets showed significnt decrese in TNF-α mrn fter weeks, ut not weeks, suggesting tht the mximum effect of n- F might require reltively longer oservtion period thn tht used in our study.

8 9 rterioscler Throm Vsc iol Decemer Downloded from y on Jnury, 9 Severl studies hve consistently reported the proinflmmtory effect of in mcrophges., However, there hve een contrdictory reports on the potency of PPRγ ctivtion on mcrophge inflmmtory responses. PPRγ gonists reduced proinflmmtory cytokine production, including TNF-α nd IL-, in humn monocytes, wheres Thieringer et l 9 filed to otin n inhiitory effect of PPRγ gonists on TNF-α nd IL- production in humn monocytes or mcrophges. Furthermore, PPRγ ctivtion seemed to increse plsm cytokine levels in mice fter LPS dministrtion. 9 Consistent with this finding, rosiglitzone incresed LPS-induced TNF-α production in rt peritonel mcrophges. In fct, PPRγ ctivtor exerted nti-inflmmtory effects in PPRγ-independent mechnism vi inhiition of nucler fctor-κ dependent trnscription, nd PPR gonists inhiited cytokine production in PPRγdeficient mcrophges, indicting tht nti-inflmmtory effects my lso e medited y other iologicl pthwys. nother possile mechnism y which F ffect mcrophgederived my e regultion of TLR nd nucler fctor-κ, trnscription fctor involved in TLR ctivtion. expression ws induced y inflmmtory cytokines nd LPS in endothelil cells nd mcrophges through nucler fctor-κ ctivtion. 9,7 In vitro tretment with n- F, such s EP, diminished TLR nd nucler fctor-κ signling, wheres sturted F enhnced these.,,8 Consistent with these findings, we found tht EP ttenuted TLR mrn, nd this correlted with lower expression. Thus, it seems possile tht F modultion of expression my lso occur through interference with TLR pthwys in mcrophges. Indeed, Rder s group hs shown tht is incresed with incresed TLR expression. Experiments in TLR knock-down cells would e informtive in determining whether TLR is lso directly linked to effects of F on. Our findings descrie mechnisms wherey decreses in ssocited with dietry n- F might e ssocited not only with higher plsm high-density lipoprotein levels, s descried y others, ut lso decreses in inflmmtory pthwys contriutory to therosclerosis. ws ssocited with PPRγ expression, nd the mcrophge-derived lipse could e modified y specific F, in prt, through regulting mcrophge PPRγ expression. We suggest tht similr chnges of the nd inflmmtion could occur in vivo. The ility of to nchor low-density lipoprotein in mcrophges could lso contriute to therogenesis. We hypothesize tht diets rich in n- F (eg, EP), in contrst to sturted F (eg, P) decrese progression of therosclerosis in humns, in prt, y downregulting inflmmtory mrkers nd PPRγ, together with decresing mcrophge in the rteril wll. cknowledgments U.J.J., C.T., C.L.C., nd H.H. performed experiments nd RJD drfted the outline of the mnuscript with U.J.J. U.J.J. wrote the originl rticle nd R.J.D., C.L.C., nd T.S.W. performed sustntil editing. We lso thnk Drs Silke Vogel nd Ir Golderg, Columi University, for their criticl reviews of this rticle. Sources of Funding This work ws supported y Ntionl Institutes of Helth grnt HL (to R.J.D.), T DK77 (to C.L.C.), T HL7 (to C.L.C.), nd K8G8 (to T.S.W.), nd fellowship from the Interntionl Nutrition Foundtion/Ellison Medicl Foundtion (to C.T.). Disclosures Richrd J. 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