Accumulating evidence indicates that vascular wall inflammation

Transcription

1 vsttin, n HMG-CoA Reductse Inhiitor, Exerts enos-independent Protective Actions Aginst Angiotensin II Induced Crdiovsculr Remodeling nd Renl Insufficiency Shusuke Ygi, Ken-ichi Aihr, Ysums Iked, Yuk Sumitomo, Sumiko Yoshid, Tkyuki Ise, Tkshi Iwse, Kzue Ishikw, Hiroyuki Azum, Msshi Akike, Toshio Mtsumoto Downloded from y on Septemer 14, 218 Astrct Angiotensin II () plys pivotl role in crdiovsculr remodeling leding to hypertension, myocrdil infrction, nd stroke. vsttin, n HMG-CoA reductse inihiitor, is known to hve pleiotropic ctions ginst the development of crdiovsculr remodeling. The ojectives of this study were to clrify the eneficil effects s well s the mechnism of ction of pitvsttin ginst induced orgn dmge. C57BL6/J mice t 1 weeks of ge were infused with for 2 weeks nd were simultneously dministered pitvsttin or vehicle. vsttin tretment improved Ang II induced left ventriculr hypertrophy nd distolic dysfunction nd ttenuted enhncement of crdic firosis, crdiomyocyte hypertrophy, coronry perivsculr firosis, nd medil thickening. induced oxidtive stress, crdic TGF -1 expression, nd Smd 2/3 phosphoryltion were ll ttenuted y pitvsttin tretment. vsttin lso reduced Ang II induced crdic remodeling nd distolic dysfunction in enos / mice s in wild-type mice. In enos / mice, the Ang II induced crdic oxidtive stress nd TGF- Smd 2/3 signling pthwy were enhnced, nd pitvsttin tretment ttenuted the enhnced oxidtive stress nd the signling pthwy. Moreover, pitvsttin tretment reduced the high mortlity rte nd improved renl insufficiency in treted enos / mice, with suppression of glomerulr oxidtive stress nd TGF- -Smd 2/3 signling pthwy. In conclusion, pitvsttin exerts enos-independent protective ctions ginst induced crdiovsculr remodeling nd renl insufficiency through inhiition of the TGF- -Smd 2/3 signling pthwy y suppression of oxidtive stress. (Circ Res. 28;12:68-76.) Key Words: ngiotensin II pitvsttin enos crdiorenl insufficiency Accumulting evidence indictes tht vsculr wll inflmmtion plys key role in the pthogenesis of vsculr diseses nd therosclerotic processes, nd ngiotensin II (), key effector of the renin-ngiotensin system (RAS), plys centrl role in the regultion of vsculr tone, lood pressure (BP), nd electrolyte homeostsis. 1 induces inflmmtion through BP-dependent nd -independent mechnisms, nd ccelertes crdiovsculr remodeling through enhnced oxidtive stress, 1 vsculr permeility, 2 leukocyte infiltrtion, 3 5 nd tissue remodeling. 6 Becuse numerous clinicl studies hve shown tht n ngiotensin-converting enzyme (ACE) inhiitor nd n ngiotensin II receptor locker (ARB) prevent crdiovsculr events nd remodeling, 7 1 lockde of the RAS system is one of the mjor strtegies for tretment of crdiovsculr diseses. In ddition to ACE inhiitors nd ARBs, it hs een shown tht sttins (3-hydroxy-3-methylglutryl coenzyme A [HMG- CoA] reductse inhiitors) re effective in preventing crdiovsculr events nd meliorting endothelil dysfunction. 15 These effects of sttins cnnot e explined merely y their lipid-lowering effects In fct, sttins hve een reported to hve pleiotropic ctions, including ctivtion nd upregultion of the enos system, 15 reduction of oxidtive stress nd inflmmtory cytokines, nd regultion of thromogenesis-relted gene expression. 22,23 However, the mechnisms y which sttins exert these ctions remin elusive. Becuse the trnsforming growth fctor (TGF)- Smd signling pthwy prticiptes in the pthogenesis of multiple crdiovsculr nd renl diseses, 24,25 we speculted tht there is n interply etween induced orgn dmge nd pleiotropic effects of sttins nd tht these fctors re linked with regultion of the TGF- Smd signling pthwy. To elucidte the crdiovsculr protective effects s well s the mechnism of ction of sttins ginst the ctivted RAS, Originl received My 2, 27; resumission received Septemer 1, 27; revised resumission received Octoer 2, 27; ccepted Octoer 17, 27. From the Deprtment of Medicine nd Bioregultory Sciences (S.Y., K.A., Y.I., Y.S., S.Y., T.I., T.I., K.I., H.A., M.A., T.M.), The University of Tokushim Grdute School of Helth Biosciences, Jpn; nd the 21st Century Center of Excellence Progrm of the University of Tokushim (Y.I., T.M.), Jpn. The first three uthors contriuted eqully to this study. Correspondence to Ken-ichi Aihr, MD, PhD, Deprtment of Medicine nd Bioregultory Sciences, The University of Tokushim Grdute School of Helth Biosciences, Kurmoto-cho, Tokushim , Jpn. E-mil ihr@clin.med.tokushim-u.c.jp 28 Americn Hert Assocition, Inc. Circultion Reserch is ville t DOI: /CIRCRESAHA

2 Ygi et l vsttin Exerts enos-independent Protection 69 Tle. Body Weight, Blood Pressure, Hert Rte, Plsm Lipid, nd Glucose in Wild-Type C57BL6/J Mice nd enos / Mice C57BL6/J enos / ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) Body weight, g # Systolic BP, mm Hg Distolic BP, mm Hg Hert Rte, ets/min TC, mg/dl TG, mg/dl HDL, mg/dl FPG, mg/dl indictes ngiotensin II;, pitvsttin; BP, lood pressure; TC, totl cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; FPG, fsting plsm glucose. P.5 (vs ( )); P.5 (vs ( )); #P.5 (vs ( )), ech group n 12. Downloded from y on Septemer 14, 218 we performed studies using infused mice with or without pitvsttin dministrtion. The present study provides novel in vivo evidence tht pitvsttin exerts enosindependent protective effects ginst induced crdiovsculr remodeling nd renl insufficiency with suppression of oxidtive stress nd inhiition of the TGF- 1- Smd 2/3 signling pthwy. Mterils nd Methods Animls nd Experimentl Protocol We used 1-week-old C57BL6/J mle mice nd they were shmoperted or sucutneously infused with (WAKO Tokyo, Jpn) t rte of 2. mg/kg/d for 2 weeks y n osmotic mini-pump (Alzet model 12, Alz Corp, Mountin View, Clif). 26 They were divided into 2 groups, d liitum dministrtion of pitvsttin (NK 14 Kow Phrmceuticl Co. Ltd) or vehicle. In the sme wy, we exmined enos / mle mice (The Jckson Lortory, Br Hror, Me) with or without stimultion, nd they were simultneously dministered orl pitvsttin t.3 mg/kg/d or vehicle. vsttin, lipophilic HMG CoA reductse inhiitor, is ctegorized s strong sttin. vsttin ws chosen for this study ecuse it is hrdly metolized y the cytochrome P45 system. Such chrcteristics give pitvsttin considerle dvntges over other sttins tht re metolized y cytochrome P45. These dvntges include extremely wek expected interctions with mny other commonly used drugs s well s its high serum concentrtions fter sorption from the smll intestine, enling its direct ctions on crdiovsculr nd renl systems. 27 vsttin ws dissolved in drinking wter t concentrtion of 2.5 mg/l. Averge dily mounts of drinking wter for wild-type C57BL6/J mice nd enos / mice were out 3. ml nd 2.5 ml, respectively, nd verge ody weights were out 26 to 27 g nd 22 to 24 g, respectively (Tle). Therefore, estimted intke of pitvsttin ws pproximtely.3 mg/kg/d. We lso evluted plsm concentrtions of pitvsttin y HPLC nd confirmed tht plsm pitvsttin concentrtion in mice with pitvsttin tretment ws comprle to nd elow tht oserved in clinicl use (supplementl dt I, ville online t The survivl experiment in enos / mice ws performed with 25 mice in ech group. Twenty-five mice with pitvsttin tretment nd the sme numer of mice without pitvsttin tretment were infused with y 14-dy relese osmotic mini-pumps. The survivl rtes of those mice were estimted for 14 dys during infusion, nd mice tht survived were euthnized for nlyses. All experimentl procedures were performed in ccordnce with the guidelines of the Animl Reserch Committee, The University of Tokushim Grdute School. We performed the following experimentl procedures: mesurements of lood pressure nd hert rte, mesurements of plsm lipid nd glucose levels, echocrdiogrphic nlysis, histologicl nlysis nd immunohistochemistry, nlysis of urinry excretion of 8-hydroxy-2 -deoxygunosine(8-ohdg), Western lot nlysis, superoxide detection in myocrdil nd renl tissues, evlution of urinry lumin excretion nd glomerulr filtrtion rte, nd sttisticl nlysis s detiled in the online dt supplement (supplementl dt II). Results Blood Pressure nd Hert Rte Systolic, distolic, nd men BP levels were elevted y Ang II infusion. vsttin tretment did not ffect BP levels regrdless of infusion. There ws no significnt difference in hert rte mong the groups (Tle). Plsm Lipid Profiles nd Glucose Levels infusion cused significnt elevtion of plsm totl cholesterol levels oth in pitvsttin-treted nd untreted mice (Tle). Plsm totl cholesterol levels tended to e lower in vehicle-treted mice with pitvsttin. However, pitvsttin did not hve sttisticlly significnt influence on ny prmeters of plsm lipid nd glucose levels mong the groups of mice (Tle). vsttin Prevents Induced Crdic Hypertrophy nd Distolic Dysfunction Ultrsonogrphic studies to exmine crdic dimension reveled tht stimultion cused significnt increses in reltive wll thickness (RWT) nd left ventriculr mss index (LVMI) in vehicle-treted mice. However, tretment did not significntly increse RWT or LVMI in pitvsttintreted mice (Figure 1). We lso evluted crdic performnce of these mice using ultrsonogrphy (Figure 1). Although LV frctionl shortening (LVFS), mrker of systolic function, ws not significntly influenced y either or pitvsttin tretment, stimultion significntly ttenuted LV distolic function s indicted y lower E-to-A wve velocity rtio, prolonged decelertion

3 7 Circultion Reserch Jnury 4/18, 28 Downloded from y on Septemer 14, 218 Reltive wll thickness E/Artio time, nd decresed decelertion rte in trnsmitrl Doppler flow in mice. In contrst, pitvsttin tretment meliorted the impirment in ll of these prmeters of LV distolic function cused y. Left ventriculr mss index (mg/g) Decelertion time (msec) Azn stining Frctionl shortening (%) Decelertion rte (m/s 2 ) Figure 1. Results of echocrdiogrphy in vehicle-treted nd pitvsttin-treted mice with or without stimultion. Upper pnels show the results for reltive wll thickness (left), left ventriculr mss index (middle), nd frctionl shortening (right). Lower pnels show the results for E/A rtio (left), decelertion time (middle), nd decelertion rte (right) in trnsmitrl pulse-wve Doppler velocity. Vlues re mens SE. n 16 in ech group. P.5, P.1. vsttin Attenutes Induced Crdiomyocyte Hypertrophy nd Remodeling of the Coronry Artery Cross-sectionl histopthologicl exmintions of the hert reveled tht evoked crdic hypertrophy with incresed crdic firosis. lso incresed perivsculr firosis round the coronry rtery. vsttin tretment ttenuted those induced crdiovsculr firotic chnges (Figure 2). Quntittive nlyses of microscopic findings of left crdic ventriculr tissues reveled tht cused hypertrophic chnges of crdiomyocytes with mrked interstitil firosis, long with ccelerted perivsculr firosis nd medil thickening of the coronry rtery. vsttin tretment prevented the development of ll these dverse phenotypes of induced crdiovsculr remodeling, nd no significnt induced increse in the size of crdiomyocytes, myocrdil firosis, or intim-medil thickening ws oserved in pitvsttin-treted mice (Figure 2). vsttin Reduces Induced Oxidtive Stress Becuse sttins hve een shown to improve endothelil dysfunction prtly through their ntioxidnt potency, we estimted urinry excretion of 8-OHdG s n oxidtive stress mrker. tretment cused notle increse of urinry 8-OHdG excretion in vehicle-treted mice, wheres pitvsttin tretment significntly ttenuted the induced elevtion of urinry 8-OHdG excretion (Figure 3d). In prllel with these oservtions, DHE-positive spots indicting superoxide produc- 1 mm 5 µm 1µm Cross sectionl re of crdiomyocyte (µm 2 ) Perivsculr firosis re to vessel re rtio 2 1 Myocrdil firosis rtio (%) Intim-medi re to totl vsculr re rtio Figure 2. Pthologicl findings of crdic tissues in vehicle-treted nd pitvsttin-treted mice with or without stimultion., Representtive findings of Azn-stined crdic tissues. Upper pnels show mcroscopic findings of trnsverse sections of the left ventriculr ppillry muscle level. Middle pnels show microscopic findings of the left ventriculr myocrdium. Lower pnels show vsculr nd perivsculr res of the coronry rtery., Quntittive results of crdiovsculr remodeling. Vlues re mens SE. n 16 in ech group. P.5, P

4 Ygi et l vsttin Exerts enos-independent Protection 71 myocrdium coronry rtery DHE stining 5 µm 1µm TGF- 1 protein expression TGF- 1 -ctin Downloded from y on Septemer 14, 218 myocrdium coronry rtery TGF- 1 stining tion were mrkedly incresed in the myocrdium nd coronry rtery y stimultion in vehicle-treted mice (Figure 3). In contrst, pitvsttin tretment lunted the increse in DHEpositive spots in infused mice (Figure 3). vsttin Attenutes Induced Crdic Expression of TGF- 1 nd Smd 2/3 Activtion TGF- 1 plys n importnt role in crdic hypertrophy nd firosis, nd is known to enhnce TGF- 1 expression. On inding of TGF- 1 to its receptor, phosphoryltion nd nucler trnsloction of Smd 2/3 constitute criticl step in the TGF- 1 signling pthwy. In n effort to clrify the mechnism of ction of pitvsttin in the prevention of Ang II induced crdic remodeling, we exmined the effects of pitvsttin on crdic TGF- 1 expression nd Smd 2/3 ctivtion in infused mice (Figure 3, 3, 3c). stimultion cused mrked enhncement in crdic TGF- 1 expression nd phosphoryltion of Smd 2/3 in vehicletreted mice. In contrst, pitvsttin tretment significntly ttenuted these chnges cused y, nd no significnt induced chnges in these prmeters were oserved in pitvsttin-treted mice (Figure 3, 3, 3c). 5 µm 1µm c p-smd 2/3 Smd 2/3 Smd 2/3 phosphoryltion d Urinry excretion of 8-OHdG (ng/dy/g) vsttin Ameliortes Left Ventriculr Hypertrophy nd Distolic Dysfunction in Ang II Treted enos / Mice Recent studies hve indicted tht crdiovsculr effects of sttins involve enos ctivtion. 28,29 To clrify whether crdioprotective effects of pitvsttin depend on enos ctivtion, we studied the effect of pitvsttin on crdiovsculr remodeling using treted enos / mice. As shown in the Tle, treted enos / mice showed significnt weight loss, wheres pitvsttin restored the chnge. Crdic ultrsonogrphy showed tht oth RWT nd LVMI were mrkedly incresed y stimultion in enos / mice (Figure 4). vsttin tretment lmost completely rogted those chnges, similr to the results in wild-type C57BL6/J mice. Although there were no significnt chnges in LVFS mong ll groups, distolic function evluted y trns-mitrl Doppler flow ws distured y tretment in enos / mice s oserved in wild-type mice (Figure 4). vsttin gin meliorted the impired LV distolic function y correcting the E-to-A wve rtio, decelertion time, nd decelertion rte in treted enos / mice (Figure 4). vsttin Attenutes Induced Crdiomyocyte Hypertrophy nd Firosis nd Remodeling of the Coronry Artery in enos / Mice Cross-sectionl histopthologicl studies of the myocrdium reveled tht cused hypertrophic chnges of crdiomyocytes with mrked interstitil crdic firosis in enos / mice (Figure 5 nd 5). lso ccelerted perivsculr firosis nd medil thickening of the coronry rtery in Figure 3. Evlution of oxidtive stress nd TGF- 1-Smd 2/3 signling pthwy in vehicle-treted nd pitvsttin-treted mice with or without stimultion., Representtive findings of superoxide detection nd TGF- 1 expression in the myocrdium nd coronry rteries in C57BL6/J mice., Quntifiction of crdic TGF- 1 protein expression. Vlues re mens SE. n 12 in ech group. P.5, P.1. c, Quntifiction of crdic Smd 2/3 phosphoryltion. Vlues re mens SE. n 12 in ech group. P.5, P.1. d, Quntifiction of dily urinry excretion of 8-OHdG. Vlues re mens SE. n 12 in ech group. P.5, P.1.

5 72 Circultion Reserch Jnury 4/18, 28 Downloded from y on Septemer 14, 218 Reltive wll thickness E / A rtio Left ventriculr mss index (mg/g) Decelertion time (msec) enos / mice (Figure 5 nd 5) s well s in wild-type C57BL6/J mice (Figure 2). vsttin tretment ttenuted these phenotypes of induced crdiovsculr remodeling in enos / mice (Figure 5 nd 5). Frctionl shortening (%) Decelertion rte (m/s 2 ) Figure 4. Results of echocrdiogrphy in vehicle-treted nd pitvsttin-treted enos / mice with or without stimultion. Upper pnels show the results for reltive wll thickness (left), left ventriculr mss index (middle) nd frctionl shortening (right). Lower pnels show the results for E/A rtio (left), decelertion time (middle), nd decelertion rte (right) in trnsmitrl pulse-wve Doppler velocity. Vlues re mens SE. n 16 in ech group. P.5, P.1. Azn stining vsttin Attenutes Induced Oxidtive Stress in enos / Mice Dihydroethidium (DHE) stining showed tht incresed superoxide production in crdiomyocytes nd coronry rteries of enos / mice (Figure 6). DHE stining t the myocrdium nd the coronry rtery ws decresed y pitvsttin tretment in treted enos / mice (Figure 6). vsttin Attenutes AngII Induced TGF- 1 Expression nd Smd 2/3 Phosphoryltion in the Myocrdium nd the Coronry Artery in enos / Mice induced TGF- 1 expression in the myocrdium nd the coronry rtery evluted y Western lot nd immunohistochemistry ws enhnced in enos / mice (Figure 6 nd 6). Crdic Smd 2/3 phosphoryltion ws lso enhnced y stimultion in enos / mice (Figure 6c). The incresed TGF- 1 expression nd incresed Smd 2/3 phosphoryltion in the myocrdium of treted enos / mice were ttenuted y pitvsttin tretment (Figure 6, 6, 6c). vsttin Improves Survivl Rte nd Glomerulr Injury in AngII Treted enos / Mice Although survivl rte nd renl phenotypes were not ltered in treted wild-type C57BL6/J mice (The results of renl studies in wild-type C57BL6/J mice re shown s supplementl dt III), stimultion mrkedly de- 1 mm 5 µm 1µm Cross sectionl re of crdiomyocyte (µm 2 ) Perivsculr firosis re to vessel re rtio Myocrdil firosis rtio (%) Intim-medi re to totl vsculr re rtio Figure 5. Ameliortion of crdiovsculr remodeling y pitvsttin in infused enos / mice., Representtive findings of Aznstined crdic tissues. Upper pnels show mcroscopic findings of trnsverse sections of the left ventriculr ppillry muscle level. Middle pnels indicte microscopic findings of the left ventriculr myocrdium. Lower pnels show vsculr nd perivsculr res of the coronry rtery., Quntittive results of crdiovsculr remodeling. Vlues re mens SE. n 16 in ech group. P.5, P.1.

6 Ygi et l vsttin Exerts enos-independent Protection 73 Downloded from y on Septemer 14, 218 myocrdium coronry rtery myocrdium coronry rtery DHE stining TGF-β1 stining cresed survivl rte of enos / mice with decresed dily urinry output nd GFR long with incresed urinry lumin excretion (Figure 7 nd 7). Immunohistochemicl exmintions reveled tht PAS- nd firin(ogen)- positive glomerulr depositions were incresed y tretment nd tht glomerulr TGF- 1 nd DHE stining ws lso incresed in treted enos / mice (Figure 7c). In contrst, pitvsttin tretment improved the survivl rte s well s renl function nd glomerulr remodeling of treted enos / mice (Figure 7 to 7c). The enhncement of renl TGF- 1 expression nd Smd 2/3 phosphryltion y stimultion of enos / mice ws lso ttenuted y pitvsttin tretment (Figure 7d). Discussion The present study demonstrted tht pitvsttin hs protective ctions ginst induced crdiovsculr remodeling through inhiition of the TGF- Smd 2/3 signling pthwy y suppression of oxidtive stress. Furthermore, lthough sttins re reported to ctivte the enos system nd the crdiovsculr effects of sttins my involve ctivtion of enos, pitvsttin lso showed protective ctions ginst crdiovsculr nd renl impirment in enos / mice. In wild-type mice, our results re consistent with the results of recent study showing tht rosuvsttin dministrtion meliorted crdiovsculr remodeling with suppression of ccentuted myocrdil expression of gp91 phox, p4 phox, 5 µm 1µm 5 µm 1µm p22 phox, nd Rc-1 in trnsgenic rts overexpressing the mouse renin gene. 3 Previous studies hve lso shown tht sttins cn prevent induced crdiovsculr remodeling through inhiition of the ctions of smll G proteins, including Rc-1, Rho A, nd p21rs, 31,32 nd reduction of NAD(P)H suunit p22 phox. 33 In ddition, pitvsttin hs een reported to suppress myocrdil endothelin-1 expression nd reduce crdic firosis in Dhl slt-sensitive rts. 34 Thus, the reduction in oxidtive stress cused y pitvsttin my e explined t lest in prt y suppression of induced enhncement in myocrdil expression of NAD(P)H suunits, leding to suppression of superoxide genertion. LV distolic dysfunction hs een recognized s n erlyphse mnifesttion of hert filure. Impired LV distolic function is ssocited with n ccelertion of crdic interstitil firosis leding to incresed LV wll stiffness, nd progression of LV distolic dysfunction leds to the development of LV systolic filure. 35 Becuse pitvsttin reduced firotic chnges surrounding crdiomyocytes nd coronry rteries in treted mice, the improvement of LV distolic dysfunction y pitvsttin cn e explined y n inhiition of firotic chnges y pitvsttin. In the present study, pitvstin tretment suppressed induced enhncement of crdic TGF- 1 expression nd Smd 2/3 signling. TGF- 1 is one of the most importnt firotic fctors, nd Smd proteins re n essentil component of the intrcellulr signling pthwy to induce firosis y TGFc TGF-β1 β-ctin p-smd 2/3 Smd 2/3 TGF-β1 protein expression Smd 2/3 phosphoryltion Figure 6. Suppression of oxidtive stress nd TGF- 1 Smd 2/3 signling pthwy in pitvsttin-treted enos / mice with stimultion., Upper pnels show representtive findings of superoxide detection in the myocrdium nd coronry rteries in enos / mice. Lower pnels show representtive findings of immunohistochemistry using TGF- 1 ntiody in the myocrdium nd coronry rteries in enos / mice., Quntifiction of crdic TGF- 1 protein expression. Vlues re mens SE. n 12 in ech group. P.5, P.1. c, Quntifiction of crdic Smd 2/3 phosphoryltion. Vlues re mens SE. n 12 in ech group. P.5, P.1.

7 74 Circultion Reserch Jnury 4/18, 28 c Survivl rte (%) Dys fter infusion Urine volume (ml/dy) Glomerulr filtrtion rte (ml/min/g weight) Urinry excretion of lumin (g/g Cre) PAS d Downloded from y on Septemer 14, 218 Firin(ogen) TGF- 1 DHE 25 m Overexpression of TGF- 1 in trnsgenic mice results in crdic hypertrophy chrcterized y oth interstitil firosis nd hypertrophic growth of crdic myocytes. In contrst, heterozygous TGF- 1 deficient mice exhiited decresed firosis of the hert with ging, 37 nd complete TGF- 1 knockout completely olished induced increse in left ventriculr mss nd myocyte size, deteriortion in systolic function, nd induction of tril ntriuretic fctor. 38 In ddition, it hs een reported tht induced hypertensive vsculr firosis is ssocited with ctivtion of the TGF- 1 Smd 2/3 signling pthwy 39 nd tht the ACE inhiitor cptopril prevents crdic firosis y locking collgen production in hypertensive rts, with n inhiition of TGF- -Smd 2 signling. 4 Tken together, those results indicte tht the RAS enhnces crdiovsculr remodeling nd firotic chnges y incresing TGF- expression nd therey enhncing TGF- -Smd 2/3 signling. Thus, the present oservtions s well s those previous results re consistent with the notion tht increses TGF- expression nd Smd 2/3 signling vi enhnced superoxide genertion nd tht pitvsttin cn counterct induced crdiovsculr remodeling nd firotic chnges vi the inhiition of TGF- -Smd 2/3 signling ttriutle to suppression of superoxide genertion enhnced y. Accumulting evidence indictes tht reduced NO iovilility plys n importnt role in the development of TGF- 1 protein expression p-smd2/3 Smd2/3 Smd 2/3 phosphoryltion Figure 7. Protective effects of pitvsttin on survivl rte, renl function, nd remodeling in infused enos / mice., Kpln Meier curves indicting survivl rte. P.5, n 25 in ech group., Renl function (left, dily urinry output; middle, glomerulr filtrtion rte; right, urinry excretion of lumin). P.5, P.1, n 12 in ech group. c, Representtive findings of glomerulr remodeling. d, Quntittive results of renl TGF- 1 protein expression (left) nd Smd 2/3 phosphoryltion (right). Vlues re mens SE. n 12 in ech group. P.5, P.1. TGF- 1 -ctin hert filure. 41 Endothelil dysfunction nd decresed NO iovilility re ssocited with the development of crdiovsculr diseses, 42 nd sttins enhnce NO iovilility y promoting NO production nd preventing superoxideinduced NO inctivtion. 43,44 In study using enos / mice, Lndmesser et l demonstrted tht incresed NO iovilility is required for sttin-induced improvement of endothelil progenitor cell moiliztion, myocrdil neovsculriztion, LV dysfunction, interstitil firosis, nd survivl fter myocrdil infrction. 41 It hs lso een reported tht the effect of low dose of simvsttin on survivl fter myocrdil infrction ws rogted in enos / mice nd tht rosuvsttin did not ffect myocrdil infrct size in enos / mice in contrst to wild-type mice. 45,46 Thus, enos ctivtion is required for meliortion of survivl rte nd ttenution of infrct size induced y t lest some sttins including simvsttin nd rosuvsttin. To determine whether the crdiovsculr protective ction of pitvsttin ginst excess requires incresed NO iovilility y enos ctivtion, we exmined the effects of pitvsttin on crdiovsculr phenotypes of treted enos / mice. Unexpectedly, pitvsttin tretment meliorted induced crdic hypertrophy, perivsculr firosis with medil thickening of coronry rtery, nd LV distolic dysfunction even in enos / mice. vsttin tretment lso ttenuted induced increse in superoxide production, TGF- 1 expression, nd Smd 2/3 phoso

8 Ygi et l vsttin Exerts enos-independent Protection 75 Downloded from y on Septemer 14, 218 phoryltion t the myocrdium nd the coronry rtery in enos / mice s well s in wild-type mice. These results indicte tht the protective effects of pitvsttin ginst Ang II induced crdiovsculr remodeling do not require incresed NO iovilility y enos ctivtion. However, these oservtions do not rule out the possiility tht iovilility of NO from other sources my e incresed y pitvsttin. The present study demonstrted for the first time tht Ang II stimultion cuses high mortlity with decresed GFR nd incresed luminuri in enos / mice. Immunohistochemistry nd DHE stining demonstrted ccelerted plsm protein deposition nd firosis with incresed superoxide production t the glomerulus in treted enos / mice. It hs lso een demonstrted tht enos / mice mnifest progressive focl renl normlities, including glomerulr hypoplsi nd tuulr cell deth, 47 suggesting tht enos my ply n importnt role in the protection of renl function. In the present study, we only nlyzed the surviving mice t the end of oservtion period on dy 14, nd ctul induced crdio-renl dmge in enos / mice might e more severe thn the results we otined. Nevertheless, it is noteworthy tht pitvsttin tretment meliorted the decrese in GFR, the increse in luminuri nd the pthologicl glomerulr chnges, nd reduced the high mortlity rte of treted enos / mice. Ameliortion of induced pthologicl glomerulr chnges y pitvsttin ws ssocited with reduced renl oxidtive stress long with suppressed TGF- 1 expression nd Smd 2/3 phosphoryltion in enos / mice. Becuse TGF- plys mjor role in the ccumultion of extrcellulr mtrix during renl firosis, the present results re consistent with the notion tht suppression of the TGF- Smd 2/3 signling pthwy vi inhiition of superoxide production y pitvsttin tretment leds to renl protection ginst RAS ctivtion. Becuse other sttins such s simvsttin, 19 rosvsttin, 3 nd torvsttin 48 lso reduced induced oxidtive stress, there is possiility tht the oserved crdiovsculr nd renl protective effects of pitvsttin re clss effects of sttins. However, the dvntgeous chrcteristics of pitvsttin, ie, the fct tht it is hrdly metolized y heptic cytochrome p45 fter intestinl sorption nd the fct tht it shows high crdiovsculr nd renl tissue distriution, my mke it s one of the most potent sttins to develop pleiotropic effects including crdiovsculr nd renl protection. To clrify this issue, further in vivo exmintions to compre the effects of vrious orlly dministered sttins re needed. In conclusion, the present study demonstrted tht pitvsttin exerts enos-independent protective ctions ginst Ang II induced crdiovsculr remodeling nd renl insufficiency through inhiition of the TGF- -Smd 2/3 signling pthwy y suppression of oxidtive stress. Sources of Funding This work ws supported in prt y Grnts-in-Aid for Scientific Reserch nd Grnt for the 21st Century Center of Excellence Progrm from the Ministry of Eduction, Science, Sports nd Culture of Jpn, Grnt for Study Group on Aseptic Femorl Neck Necrosis from the Ministry of Helth, Lor, nd Welfre of Jpn, nd grnts from The Uehr Memoril Foundtion, nd grnt for Clinicl Vsculr Function from the Kimur Memoril Crdiovsculr Foundtion. None. Disclosures References 1. Griendling KK, Sorescu D, Ushio-Fuki M. NAD(P)H oxidse: role in crdiovsculr iology nd disese. Circ Res. 2;86: Victorino GP, Newton CR, Currn B. Effect of ngiotensin II on microvsculr permeility. J Surg Res. 22;14: Alvrez A, Cerd-Nicols M, Nim Au Nh Y, Mt M, Issekutz AC, Pnes J, Lo RR, Snz MJ. Direct evidence of leukocyte dhesion in rterioles y ngiotensin II. Blood. 24;14: Kim JA, Berliner JA, Ndler JL. Angiotensin II increses monocyte inding to endothelil cells. Biochem Biophys Res Commun. 1996;226: Krejcy K, Eichler HG, Jilm B, Kpiotis S, Wolzt M, Znschk G, Gsic S, Schutz W, Wgner O. Influence of ngiotensin II on circulting dhesion molecules nd lood leukocyte count in vivo. Cn J Physiol Phrmcol. 1996;74: Mervl E, Muller DN, Schmidt F, Prk JK, Gross V, Bder M, Breu V, Gnten D, Hller H, Luft FC. Blood pressure-independent effects in rts with humn renin nd ngiotensinogen genes. Hypertension. 2;35: Hnsson L, Lindholm LH, Ekom T, Dhlof B, Lnke J, Schersten B, Wester PO, Hedner T, de Fire U. Rndomised tril of old nd new ntihypertensive drugs in elderly ptients: crdiovsculr mortlity nd moridity the Swedish Tril in Old Ptients with Hypertension-2 study. Lncet. 1999;354: Effects of rmipril on crdiovsculr nd microvsculr outcomes in people with dietes mellitus: results of the HOPE study nd MICRO-HOPE sustudy. Hert Outcomes Prevention Evlution Study Investigtors. Lncet. 2;355: Cohn JN, Tognoni G. A rndomized tril of the ngiotensin-receptor locker vlsrtn in chronic hert filure. N Engl J Med. 21;345: Brenner BM, Cooper ME, de Zeeuw D, Kene WF, Mitch WE, Prving HH, Remuzzi G, Snpinn SM, Zhng Z, Shhinfr S. Effects of losrtn on renl nd crdiovsculr outcomes in ptients with type 2 dietes nd nephropthy. N Engl J Med. 21;345: Schwrtz GG, Olsson AG, Ezekowitz MD, Gnz P, Oliver MF, Wters D, Zeiher A, Chitmn BR, Leslie S, Stern T. Effects of torvsttin on erly recurrent ischemic events in cute coronry syndromes: the MIRACL study: rndomized controlled tril. JAMA. 21;285: Rndomised tril of cholesterol lowering in 4444 ptients with coronry hert disese: the Scndinvin Simvsttin Survivl Study (4S). Lncet. 1994;344: Influence of prvsttin nd plsm lipids on clinicl events in the West of Scotlnd Coronry Prevention Study (WOSCOPS). Circultion. 1998; 97: Scks FM, Pfeffer MA, Moye LA, Rouleu JL, Rutherford JD, Cole TG, Brown L, Wrnic JW, Arnold JM, Wun CC, Dvis BR, Brunwld E. The effect of prvsttin on coronry events fter myocrdil infrction in ptients with verge cholesterol levels. Cholesterol nd Recurrent Events Tril investigtors. N Engl J Med. 1996;335: Rikitke Y, Lio JK. Rho GTPses, sttins, nd nitric oxide. Circ Res. 25;97: O Driscoll G, Green D, Tylor RR. Simvsttin, n HMG-coenzyme A reductse inhiitor, improves endothelil function within 1 month. Circultion. 1997;95: Tresure CB, Klein JL, Weintru WS, Tlley JD, Stillower ME, Kosinski AS, Zhng J, Boccuzzi SJ, Cedrholm JC, Alexnder RW. Beneficil effects of cholesterol-lowering therpy on the coronry endothelium in ptients with coronry rtery disese. N Engl J Med. 1995; 332: Lndmesser U, Bhlmnn F, Mueller M, Spiekermnn S, Kirchhoff N, Schulz S, Mnes C, Fischer D, de Groot K, Fliser D, Fuler G, Mrz W, Drexler H. Simvsttin versus ezetimie: pleiotropic nd lipid-lowering effects on endothelil function in humns. Circultion. 25;111:

9 76 Circultion Reserch Jnury 4/18, 28 Downloded from y on Septemer 14, Delosc S, Cristol JP, Descomps B, Mimrn A, Jover B. Simvsttin prevents ngiotensin II-induced crdic ltertion nd oxidtive stress. Hypertension. 22;4: Tsuouchi H, Inoguchi T, Sont T, Sto N, Sekiguchi N, Koyshi K, Sumimoto H, Utsumi H, Nwt H. Sttin ttenutes high glucoseinduced nd dietes-induced oxidtive stress in vitro nd in vivo evluted y electron spin resonnce mesurement. Free Rdic Biol Med. 25;39: Node K, Fujit M, Kitkze M, Hori M, Lio JK. Short-term sttin therpy improves crdic function nd symptoms in ptients with idiopthic dilted crdiomyopthy. Circultion. 23;18: Mrkle RA, Hn J, Summers BD, Yokoym T, Hjjr KA, Hjjr DP, Gotto AM Jr, Nicholson AC. vsttin lters the expression of thromotic nd firinolytic proteins in humn vsculr cells. J Cell Biochem. 23;9: Msmur K, Oid K, Knehr H, Suzuki J, Horie S, Ishii H, Miymori I. vsttin-induced thromomodulin expression y endothelil cells cts vi inhiition of smll G proteins of the Rho fmily. Arterioscler Throm Vsc Biol. 23;23: Ruiz-Orteg M, Rodriguez-Vit J, Snchez-Lopez E, Crvjl G, Egido J. TGF-et signling in vsculr firosis. Crdiovsc Res. 27;74: Bottinger EP. TG. F-et in renl injury nd disese. Semin Nephrol. 27;27: Iked Y, Aihr K, Sto T, Akike M, Yoshizumi M, Suzki Y, Izw Y, Fujimur M, Hshizume S, Kto M, Ygi S, Tmki T, Kwno H, Mtsumoto T, Azum H, Kto S. Androgen receptor gene knockout mle mice exhiit impired crdic growth nd excertion of ngiotensin II-induced crdic firosis. J Biol Chem. 25;28: Kjinmi K, Muchi H, Sito Y. NK-14: novel synthetic HMG-CoA reductse inhiitor. Expert Opin Investig Drugs. 2;9: Lufs U, Lio JK. Post-trnscriptionl regultion of endothelil nitric oxide synthse mrna stility y Rho GTPse. J Biol Chem. 1998;273: Kureishi Y, Luo Z, Shiojim I, Bilik A, Fulton D, Lefer DJ, Sess WC, Wlsh K. The HMG-CoA reductse inhiitor simvsttin ctivtes the protein kinse Akt nd promotes ngiogenesis in normocholesterolemic nimls. Nt Med. 2;6: Hii J, Whley-Connell A, Qzi MA, Hyden MR, Cooper SA, Trmontno A, Thyfult J, Stump C, Ferrrio C, Muniypp R, Sowers JR. Rosuvsttin, HMG-CoA Reductse Inhiitor, Decreses Crdic Oxidtive Stress nd Remodeling in Ren2 Trnsgenic Rts. Endocrinology. 27;148: Lufs U, Kilter H, Konkol C, Wssmnn S, Bohm M, Nickenig G. Impct of HMG CoA reductse inhiition on smll GTPses in the hert. Crdiovsc Res. 22;53: Oi S, Hned T, Oski J, Kshiwgi Y, Nkmur Y, Kwe J, Kikuchi K. Lovsttin prevents ngiotensin II-induced crdic hypertrophy in cultured neontl rt hert cells. Eur J Phrmcol. 1999;376: Wssmnn S, Lufs U, Bumer AT, Muller K, Ahlory K, Linz W, Itter G, Rosen R, Bohm M, Nickenig G. HMG-CoA reductse inhiitors improve endothelil dysfunction in normocholesterolemic hypertension vi reduced production of rective oxygen species. Hypertension. 21; 37: Sk M, Ot K, Ichihr S, Cheng XW, Kimt H, Nishizw T, Nod A, Izw H, Ngt K, Murohr T, Yokot M. vsttin improves crdic function nd survivl in ssocition with suppression of the myocrdil endothelin system in rt model of hypertensive hert filure. J Crdiovsc Phrmcol. 26;47: Berk BC, Fujiwr K, Lehoux S. ECM remodeling in hypertensive hert disese. J Clin Invest. 27;117: Mssgue J, Chen YG. Controlling TGF-et signling. Genes Dev. 2;14: Brooks WW, Conrd CH. Myocrdil firosis in trnsforming growth fctor et(1)heterozygous mice. J Mol Cell Crdiol. 2;32: Schultz Jel J, Witt SA, Glscock BJ, Niemn ML, Reiser PJ, Nix SL, Kimll TR, Doetschmn T. TGF-et1 medites the hypertrophic crdiomyocyte growth induced y ngiotensin II. J Clin Invest. 22;19: Wng W, Hung XR, Cnls E, Ok K, Truong LD, Deng C, Bhowmick NA, Ju W, Bottinger EP, Ln HY. Essentil role of Smd3 in ngiotensin II-induced vsculr firosis. Circ Res. 26;98: Peng H, Crretero OA, Vuljj N, Lio TD, Motivl A, Peterson EL, Rhle NE. Angiotensin-converting enzyme inhiitors: new mechnism of ction. Circultion. 25;112: Lndmesser U, Engerding N, Bhlmnn FH, Schefer A, Wiencke A, Heineke A, Spiekermnn S, Hilfiker-Kleiner D, Templin C, Kotlrz D, Mueller M, Fuchs M, Hornig B, Hller H, Drexler H. Sttin-induced improvement of endothelil progenitor cell moiliztion, myocrdil neovsculriztion, left ventriculr function, nd survivl fter experimentl myocrdil infrction requires endothelil nitric oxide synthse. Circultion. 24;11: Lio JK, Bettmnn MA, Sndor T, Tucker JI, Colemn SM, Creger MA. Differentil impirment of vsodiltor responsiveness of peripherl resistnce nd conduit vessels in humns with therosclerosis. Circ Res. 1991;68: Lufs U, L Ft V, Plutzky J, Lio JK. Upregultion of endothelil nitric oxide synthse y HMG CoA reductse inhiitors. Circultion. 1998;97: Klinowski L, Dorucki LW, Brovkovych V, Mlinski T. Incresed nitric oxide iovilility in endothelil cells contriutes to the pleiotropic effect of cerivsttin. Circultion. 22;15: Ymkuchi M, Greer JJ, Cmeron SJ, Mtsushit K, Morrell CN, Tlot-Fox K, Bldwin WM, 3rd, Lefer DJ, Lowenstein CJ. HMG-CoA reductse inhiitors inhiit endothelil exocytosis nd decrese myocrdil infrct size. Circ Res. 25;96: Greer JJ, Kkkr AK, Elrod JW, Wtson LJ, Jones SP, Lefer DJ. Low-dose simvsttin improves survivl nd ventriculr function vi enos in congestive hert filure. Am J Physiol Hert Circ Physiol. 26;291:H2743 H Fores MS, Thornhill BA, Prk MH, Chevlier RL. Lck of endothelil nitric-oxide synthse leds to progressive focl renl injury. Am J Pthol. 27;17: Wssmnn S, Lufs U, Muller K, Konkol C, Ahlory K, Bumer AT, Linz W, Bohm M, Nickenig G. Cellulr ntioxidnt effects of torvsttin in vitro nd in vivo. Arterioscler Throm Vsc Biol. 22; 22:3 35.