ISSN: 1524-4636 Copyright © 2006 American Heart Association. All rights reserved. Print ISSN: 1079-5642. Online

7272 Greenville Avenue, Dallas, TX 72514Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association.

DOI: 10.1161/01.ATV.0000254855.24394.f9 published online Dec 14, 2006; Arterioscler. Thromb. Vasc. Biol.

Kim-Mitsuyama Tokutomi, Zhong-Fang Lai, Yi-Fei Dong, Shinji Matsuba, Hisao Ogawa and Shokei

Eiichiro Yamamoto, Takuro Yamashita, Tomoko Tanaka, Keiichiro Kataoka, Yoshiko Salt-Sensitive Hypertensive Rats, via Pleiotropic Effects

Pravastatin Enhances Beneficial Effects of Olmesartan on Vascular Injury of

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Pravastatin Enhances Beneficial Effects of Olmesartan onVascular Injury of Salt-Sensitive Hypertensive Rats, via

Pleiotropic EffectsEiichiro Yamamoto, Takuro Yamashita, Tomoko Tanaka, Keiichiro Kataoka, Yoshiko Tokutomi,

Zhong-Fang Lai, Yi-Fei Dong, Shinji Matsuba, Hisao Ogawa, Shokei Kim-Mitsuyama

Objective—This work was undertaken to investigate comparative effect of AT1 receptor blocker (ARB), 3-hydroxy-3-methylglutaryl (HMG) coenzymeA (CoA) reductase inhibitor (statin), and their combination on vascular injury ofsalt-sensitive hypertension.

Methods and Results—Salt-loaded Dahl salt-sensitive hypertensive rats (DS rats) were treated with (1) vehicle, (2)hydralazine (5 mg/kg/d), (3) olmesartan (0.5 mg/kg/d), (4) pravastatin (100 mg/kg/d), and (5) combined olmesartan andpravastatin for 4 weeks. Olmesartan or pravastatin significantly and comparably improved vascular endothelium-dependent relaxation to acetylcholine, coronary arterial remodeling, and eNOS activity of DS rats. Olmesartan preventedvascular eNOS dimer disruption or the downregulation of dihydrofolate reductase (DHFR) more than pravastatin,whereas Akt phosphorylation was enhanced by pravastatin but not olmesartan, indicating differential pleiotropic effectsbetween olmesartan and pravastatin. Add-on pravastatin significantly enhanced the improvement of vascular endothelialdysfunction and remodeling by olmesartan in DS rats. Moreover, pravastatin enhanced the increase in eNOS activity byolmesartan, being associated with additive effects of pravastatin on phosphorylation of Akt and eNOS.

Conclusions—Olmesartan and pravastatin exerted beneficial vascular effects in salt-sensitive hypertension, via differentialpleiotropic effects. Pravastatin enhanced vascular protective effects of olmesartan. Thus, the combination of ARB withstatin may be the potential therapeutic strategy for vascular diseases of salt-sensitive hypertension. (ArteriosclerThromb Vasc Biol. 2007;27:000-000.)

Key Words: eNOS dimers � DHFR � oxidative stress � vascular injury � combined ARB and statin

Accumulating evidence indicates that renin–angiotensinsystem (RAS) plays a crucial role in the pathophysiol-

ogy of cardiovascular diseases in hypertension, and that RASblockers, including angiotensin-converting enzyme inhibitorsand AT1 receptor blockers (ARB), are the useful therapeuticagents for hypertensive cardiovascular diseases.1 As hyper-tension is often accompanied by dyslipidemia in the samepatients, their treatment frequently involves the combinationof RAS blockers with 3-hydroxy-3-methylglutaryl coen-zymeA (HMG-CoA) reductase inhibitors (statins), potentinhibitors of cholesterol biosynthesis. Clinical evidence showthat statins improve endothelial dysfunction and reduce theincidence of atherosclerotic events,2–5 and these vascularprotective effects by statins are at least partially attributed totheir pleiotropic vascular effects beyond lowering of plasmacholesterol.4–6 However, the difference in vascular pleiotro-pic effects between RAS blockers and statins remains to befully understood. Moreover, the significance and the advan-tage of their combination therapy in hypertension, particu-larly salt-sensitive hypertension, are not defined.

Clinically, salt-sensitive hypertensive patients are moreprone to cardiovascular diseases than their salt-insensitivecounterparts.7,8 Therefore, it is a key clinical issue to eluci-date the effect of RAS blockers and statins in salt-sensitivehypertension. Moreover, it is not clear at all whether add-onstatin treatment enhances vascular protective effects of RASblockers in salt-sensitive hypertension. Dahl salt-sensitivehypertensive rats (DS rats) constitute a paradigm of salt-sensitive hypertension in humans and therefore are the usefulanimal model to investigate the mechanism underlying car-diovascular injury in salt-sensitive hypertension.

The aim of our present work was to compare the impact ofolmesartan (an ARB), pravastatin (a statin), and their combi-nation on vascular diseases in DS rats and to examine therelative role of reactive oxygen species (ROS) and eNOS intheir pleiotropic effects. We obtained the evidence thatolmesartan and pravastatin improved vascular injury of DSrats, via different pleiotropic effects on ROS and eNOS, andpravastatin significantly enhanced vascular effects of olme-sartan in DS rats.

Original received June 7, 2006; final version accepted November 6, 2006.From the Departments of Pharmacology and Molecular Therapeutics (E.Y., T.Y., T.T., K.K., Y.T., Z.-F.L., Y.-F.D., S.M., S.K.-M.) and Cardiovascular

Medicine (H.O.), Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan.Correspondence to Shokei Kim-Mitsuyama, MD, PhD, Department of Pharmacology and Molecular Therapeutics, Kumamoto University Graduate

School of Medical Sciences, 1-1-1 Honjyo, Kumamoto 860-8556, Japan. E mail kimmitsu@gpo.kumamoto-u.ac.jp© 2006 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000254855.24394.f9

1

MethodsExperimental AnimalsAll procedures were in accordance with institutional guidelines foranimal research. DS rats (Japan SLC Inc, Shizuoka, Japan) wereused in the present study. At 7 weeks of age, the diet of DS rats wasswitched from a 0.3% NaCl (low-salt) to an 8% NaCl (high-salt)diet.9 Control DS rats were fed a 0.3% NaCl diet, throughout theexperiments.

Experiment I: Time Course of Impact ofSodium LoadingThe first experiments were performed to examine time-dependenteffect of high-salt loading on vascular endothelial function, NADPHoxidase activity, superoxide, eNOS activity and phosphorylation,disruption of eNOS dimers, plasma NO2/NO3, Akt phosphorylation,nitrotyrosine, dihydrofolate reductase (DHFR), and coronary arterialthickening in DS rats. DS rats, fed low-salt diet throughout theexperiment, served as control. Blood pressure (BP) was periodicallymeasured by tail-cuff plethysmography (BP-98A; Softron Co).Eight-, 12-, and 16-week-old DS rats, subjected to high-salt diet for1, 5, and 9 weeks, respectively, were anesthetized with ether, arterialblood was immediately collected by cardiac puncture, and plasmawas collected by centrifugation and stored at �80°C until use. Then,the carotid artery and the thoracic aorta were immediately excised toestimate vascular endothelial function and the above mentionedbiochemical parameters, and the heart was also removed to examinecoronary arterial thickening.

Experiment II: Comparative Effect of an ARB, aStatin, and Their CombinationThe second experiments were carried out to compare the effect ofolmesartan (Sankyo Co. Ltd), pravastatin (Sankyo Co. Ltd), theircombination, or hydralazine on DS rats fed high-salt diet. Twelve-week-old DS rats, which had fed a high-salt diet from 7 weeks ofage, were orally given olmesartan (0.5 mg/kg/d), pravastatin (100mg/kg/d), combined olmesartan (0.5 mg/kg/d) and pravastatin (100mg/kg/d), or hydralazine (5 mg/kg/d) for 4 weeks. Olmesartan andpravastatin were suspended in 0.5% carboxymethyl cellulose(CMC), and were given to rats by gastric gavage once a day.Hydralazine was given to rats as the drinking water. It is well knownthat oral administration of 100 mg/kg/d of pravastatin to rats yieldsplasma pravastatin concentration similar to that seen in patientstaking clinical doses of pravastatin10,11 and does not significantlychange plasma cholesterol levels in DS rats. Furthermore, in prelim-inary experiments, we found that olmesartan, pravastatin, theircombination, and hydralazine at the above mentioned dose exertedsimilar hypotensive effects in DS rats. Therefore, this experimentalprotocol allowed us to elucidate the lipid-independent and bloodpressure-independent effect of each therapy on DS rats. BP and heartrate were measured in conscious rats at 3 to 5 hours after oral dosing,every week. After 4 weeks of treatment, DS rats were anesthetizedwith ether, then the carotid artery, the thoracic aorta, and the heartwere immediately excised to compare the effect of each treatment onvascular endothelial function, NADPH oxidase activity, p22phox,superoxide, eNOS activity, eNOS dimers, phosphorylation of eNOSand Akt, DHFR, extracellular signal regulated-kinase (ERK), nitro-tyrosine, phospho-MEK, coronary arterial thickening, perivascularfibrosis, cardiac hypertrophy, and cardiac fibrosis and macrophageinfiltration.

A detailed description of the Methods is available in the onlinedata supplement at http://atvb.ahajournals.org.

ResultsTime Course of BP, Vascular EndothelialFunction, and Coronary Remodeling in DS RatsFed High-Salt DietDS rats were fed high-salt (8% NaCl) diet from 7 weeks ofage. We measured BP, endothelial function, and coronary

arterial thickening in 8-, 12-, and 16-week-old DS rats, fedhigh-salt diet for 1, 5, and 9 weeks, respectively (supplemen-tal Figure I). Compared with control DS rats fed low-salt diet,BP in salt-loaded DS rats progressively increased with time.As shown by vascular relaxing response to acetylcholine(Ach) (supplemental Figure I-B), vascular endothelium-dependent relaxation in salt-loaded DS rats was alreadyslightly but significantly impaired at 8 weeks of age (P�0.05)and was thereafter progressively impaired with time, whichwas followed by the significant coronary arterial thickeningat 16 weeks of age.

Time Course of Vascular NADPH Oxidase,Superoxide, eNOS Activity, and Plasma NO2/NO3

As shown in Figure 1A and 1B, vascular NADPH oxidaseactivity and superoxide of DS rats were already significantlyincreased by 1 week of high-salt loading (P�0.05) (at 8weeks of age) and remained increased throughout the treat-ment of high salt. On the other hand, vascular eNOS activityof salt-loaded DS rats was not altered at 8 or 12 weeks of age,but was significantly reduced by 53% at 16 weeks of age(P�0.01), compared with low-salt diet (Figure 1C). As invascular eNOS activity, plasma NO2/NO3 concentrationswere significantly decreased only at 16 weeks of age (Figure1D).

Time Course of Phospho-Akt, Phospho-eNOS, andthe Disruption of eNOS DimersAs shown in supplemental Figure II, 1, 5, or 9 weeks ofhigh-salt diet did not affect phosphorylation of vascular Aktthroughout the treatment. On the other hand, vascularphospho-eNOS levels were significantly reduced in 16-week-old DS rats subjected to 9 weeks of high-salt intake(P�0.05).

Supplemental Figure III shows the detection of vasculareNOS protein dimers and monomers by low-temperatureSDS-PAGE followed by Western blot analysis. In low-saltfed DS rats, eNOS existed exclusively as the dimers, whereas

Figure 1. Time course of vascular NADPH oxidase (A), superox-ide (B), eNOS activity (C), and plasma NO2/NO3 (D) in DS rats.High Na, 8% NaCl diet; Low Na, 0.3% NaCl diet. Values aremean�SEM (n�5).

2 Arterioscler Thromb Vasc Biol. March 2007

in high-salt fed 16-week-old DS rats, eNOS was presentpredominantly in the monomers and eNOS dimers werenearly absent, indicating the significant disruption of eNOSprotein dimers by 9 weeks of salt loading. No significantdisruption of vascular eNOS dimers was found in 8- and12-week-old salt-loaded DS rats (data not shown).

Effect of Olmesartan, Pravastatin, CombinedOlmesartan and Pravastatin, and Hydralazine onBP and Plasma Lipids of DS Rats FedHigh-Salt DietEach drug was given 12-week-old DS rats which had beenalready fed high-salt diet for 5 weeks, and drug treatment wascarried out for 4 weeks. As shown in supplemental Figure IV,olmesartan, pravastatin, combined olmesartan and pravasta-tin, and hydralazine slightly reduced BP of DS rats to acomparable degree throughout the treatment, except forgreater reduction of BP by hydralazine than the other drugtreatments at 13 weeks of age.

After 4 weeks of treatment, plasma cholesterol concentra-tions in vehicle-, olmesartan-, pravastatin-, combined olme-sartan and pravastatin-, and hydralazine-treated salt-loadedDS rats, and low-salt DS rats were 77�8, 76�6, 78�6,77�8, 74�5, and 71�4 mg/dL, respectively, and plasmatriglyceride were 140�20, 124�18, 144�19, 108�9,119�5, and 136�14 mg/dL, respectively. There was nosignificant difference in plasma cholesterol or triglyceridelevels, among all groups of DS rats.

Comparative Effect on Serum Creatinine andPlasma Thiobarbituric Acid Reactive SubstancesAs shown in supplemental Figure V, serum creatinine andplasma thiobarbituric acid reactive substances (T-BARS; amarker of lipid peroxidation) were significantly higher in16-week-old salt-loaded DS rats compared with control.Olmesartan, pravastatin, and their combination almost nor-malized serum creatinine. Olmesartan and pravastatin alonesignificantly and comparably reduced plasma T-BARS, andtheir combination reduced it more than either monotherapy.

Comparative Effect on Vascular EndothelialFunction and Coronary Remodeling of DS RatsFed High-Salt DietAs shown in Figure 2, either olmesartan or pravastatin, butnot hydralazine, significantly prevented the impairment ofvascular endothelium-dependent relaxation to Ach and alsoprevented the progression of coronary arterial thickening andperivascular fibrosis, to a comparable degree. The combina-tion of olmesartan with pravastatin prevented vascular endo-thelial dysfunction more than monotherapy with either agentalone and prevented coronary arterial thickening and perivas-cular fibrosis more than pravastatin alone, despite no differ-ence in BP and plasma lipids among all drug treatmentgroups.

As shown in supplemental Figure VI, the phosphorylationof vascular MEK and ERK was significantly enhanced in16-week-old salt-loaded DS rats compared with control.Olmesartan and pravastatin alone similarly prevented thephosphorylation of vascular MEK and ERK compared withcontrol, and their combination decreased the phosphorylationof MEK and ERK more than pravastatin alone.

Comparative Effect on Vascular NADPH Oxidase,p22phox, Superoxide, and eNOS Activity of DSRats Fed High-Salt DietAs shown in Figure 3, either olmesartan or pravastatin alone,but not hydralazine, significantly ameliorated the increase invascular NADPH oxidase activity, the increase in p22phoxprotein expression, the increase in vascular superoxide, or thedecrease in vascular eNOS activity of salt-loaded DS rats.The inhibitory effect of olmesartan, regarding NADPH oxi-dase, p22phox, and superoxide, was greater than that ofpravastatin. On the other hand, no significant difference wasnoted in the beneficial effect on vascular eNOS activitybetween olmesartan and pravastatin. Add-on pravastatin sig-nificantly enhanced the improvement of superoxide (P�0.05)and eNOS activity (P�0.01) by olmesartan alone, whereaspravastatin failed to enhance the suppression of NADPHoxidase and p22phox by olmesartan.

Figure 2. Effect of vehicle (V), olmesartan (O), pra-vastatin (P), combined olmesartan and pravastatin(O�P), and hydralazine (H) on acetylcholine-induced vascular relaxation (A), coronary arterialthickening (B), and perivascular fibrosis (C) of DSrats fed high-salt diet. A, Each plot representsmean�SEM (n�8 to 9 per group). Right top panelsshow representative images of light micrographs ofLV coronary arterial thickening and perivascularfibrosis. Original magnification, �200. Each barrepresents mean�SEM (n�4 to 8 per group). Lindicates DS rats fed low-salt diet; NS, notsignificant.

Yamamoto et al ARB Combined With Statin, and Salt-Sensitivity 3

Comparative Effect on Vascular Phospho-Akt,Phospho-eNOS of DS Rats Fed High-Salt DietIn contrast to no significant effect of olmesartan on vascularAkt phosphorylation, pravastatin alone or olmesartan com-bined with pravastatin significantly and comparably in-creased phosphorylation of Akt in DS rats (Figure 4). How-ever, there was no significant difference in vascular total Aktlevels in each group. Either olmesartan or pravastatin alonesignificantly upregulated vascular phospho-eNOS levels andtotal eNOS, and add-on pravastatin significantly enhanced theupregulation of phospho-eNOS and total eNOS by olmesar-tan (P�0.05).

Comparative Effect on Vascular eNOS Dimers andDHFR Protein Expression of DS Rats FedHigh-Salt DietEither olmesartan or pravastatin alone significantly preventedthe disruption of vascular eNOS protein dimers in salt-loadedDS rats, but olmesartan prevented it more than pravastatin(P�0.01; Figure 5A). Add-on pravastatin did not signifi-cantly enhance the suppressive effect of olmesartan on eNOSdimer disruption. As shown in Figure 5B, vascular DHFRprotein levels were significantly downregulated in 16-week-old salt-loaded DS rats compared with control DS rats(P�0.01), although no significant difference in DHFR was

Figure 3. Effect of each treatment onvascular NADPH oxidase (A), p22phox(B), superoxide (C), and eNOS activity (D)of DS rats fed high-salt diet. Top panelsin B and C show representative Westernblot and fluorescence photomicrographs,respectively, in each group. #P�0.05,*P�0.01 vs V. Each bar representsmean�SEM. A: L, n�9; V, n�9; H, n�4;O, n�9; P, n�9; O�P, n�8. B: L, n�9;V, n�9; H, n�5; O, n�9; P, n�9; O�P,n�8. C: n�5 per group. D: n�4 to 5 pergroup. Abbreviations are the same asFigure 2. L indicates DS rats fed low-saltdiet.

Figure 4. Effect of each treatment onphospho-akt (p-akt), total akt, phospho-eNOS (p-eNOS), and total eNOS of DSrats fed high-salt diet. Top panels showrepresentative Western blot in eachgroup. Each bar represents mean�SEM(n�5 per group). Abbreviations are thesame as Figure 2. L indicates DS ratsfed low-salt diet.

4 Arterioscler Thromb Vasc Biol. March 2007

noted between salt-loaded and control DS rats at the age of 8and 12 weeks. Olmesartan, but not pravastatin, significantlyprevented the downregulation of DHFR in salt-loaded DS rats(P�0.01). Add-on pravastatin did not significantly enhancethe upregulation of DHFR by olmesartan.

Comparative Effect on Vascular Nitrotyrosine ofDS Rats Fed High-Salt DietAs shown by Western blot analysis and immunohistochem-istry in Figure 6, vascular nitrotyrosine levels were signifi-cantly increased in 16-week-old salt-loaded DS rats com-pared with control DS rats (P�0.01), although no significantdifference in nitrotyrosine was noted between salt-loaded andcontrol DS rats at the age of 8 and 12 weeks. Olmesartan and

pravastatin significantly reduced vascular nitrotyrosine. Thecombination of olmesartan with pravastatin did not signifi-cantly enhance the suppressive effect of nitrotyrosine byolmesartan, while significantly enhanced the reduction ofnitrotyrosine by pravastatin.

Comparative Effect on Cardiac Hypertrophy,Fibrosis, and Macrophage Infiltration of DS RatsFed High-Salt DietAs shown in supplemental Figure VII, olmesartan and pra-vastatin alone significantly prevented cardiac hypertrophyand fibrosis in salt-loaded DS rats, and their combinationprevented them more than either monotherapy. Cardiac mac-rophage infiltration was significantly and comparably pre-vented by olmesartan and pravastatin, and their combinationprevented it more than pravastatin monotherapy (supplemen-tal Figure VIII). These results suggest that the combination ofpravastatin and olmesartan may be useful for the treatment ofcardiac remodeling in salt-sensitive hypertension.

DiscussionIt is well established that atherosclerosis is closely associatedwith the impairment of vascular endothelial function, whosehallmark is an impairment of endothelium-dependent vasore-laxation induced by reduced bioavailability of NO.12–14 Adecline in NO bioavailability is mainly caused by the reduc-tion of NO synthesis by eNOS and the increased inactivationof NO by superoxide.12–14 Thus, the dysfunction of eNOS andthe activation of NADPH oxidase which generates superox-ide play a major role in vascular endothelial dysfunction andvascular remodeling. Previous works15–17 on salt-loaded DSrats show that vascular NADPH oxidase and superoxide weresignificantly increased whereas eNOS activity was inverselysignificantly decreased in DS rats suffering from severeendothelial dysfunction and vascular hypertrophy. However,in previous works,15–17 time course of these parameters in DSrats has not been examined and therefore the relative rela-tionship among NADPH oxidase, superoxide, and eNOS,regarding the role in vascular injury, is not elucidated. In ourpresent work, we found that slight but significant impairmentof vascular endothelium-dependent relaxation occurred by 1week after start of salt loading (supplemental Figure I), beingassociated with the early increase in NADPH oxidase activityand superoxide (Figure 1). These results, taken together withno change in eNOS activity at this early stage of salt loading,showed that NADPH oxidase–generated superoxide, but noteNOS, was involved in the early onset of vascular endothelialdysfunction in salt-sensitive hypertension. Notably, muchlater than the onset of increase in NADPH oxidase activity,the remarkable reduction of vascular eNOS activity occurredonly in 16-week-old DS rats fed high-salt diet for 9 weeks(Figure 1). At 16 weeks of age, DS rats had much more severeendothelial dysfunction and remarkable coronary arterialremodeling than at the earlier age (supplemental Figure I).Therefore, in contrast to the importance of NADPH oxidaseat the early phase of salt loading, vascular eNOS seems to bemainly involved in the exacerbation of vascular endothelialdysfunction and vascular structural changes of salt-sensitivehypertension, indicating differential role between NADPH

Figure 5. Effect of each treatment on eNOS dimer disruption (A)and DHFR protein levels (B) of DS rats fed high-salt diet. Toppanels in A and B show representative Western blot in eachgroup. Each bar represents mean�SEM (n�5 per group).Abbreviations are the same as Figure 2. L indicates DS rats fedlow-salt diet.

Figure 6. Effect of each treatment on vascular nitrotyrosine ofDS rats fed high-salt diet. Vascular nitrotyrosine was determinedby immunohistochemistry (A) and Western blot analysis (B).Each bar represents mean�SEM (n�5 per group). Abbreviationsare the same as Figure 2. L indicates DS rats fed low-salt diet.

Yamamoto et al ARB Combined With Statin, and Salt-Sensitivity 5

oxidase and eNOS in the progression of vascular injury insalt-sensitive hypertension.

eNOS activity is regulated by not only its phosphorylationbut also multiple factors. In this study, to elucidate theunderlying mechanism of the decreased eNOS activity in16-week-old salt-loaded DS rats, we examined the phosphor-ylation of vascular Akt and eNOS, the disruption of eNOSdimers, peroxynitrite, and DHFR expression in DS rats. Aktappears to be the principle kinase phosphorylating eNOS,leading to endothelium-dependent vasodilatation.18 Previousstudies show that the formation of eNOS protein homodimersis necessary for enzymatic activity, and peroxynitrite candisrupt eNOS protein dimers through oxidation and displace-ment of the zinc metal ion, leading to the reduction of eNOSactivity.19,20 DHFR is a key enzyme synthesizing tetrahydro-biopterin, eNOS cofactor, and very recent in vitro study oncultured vascular endothelial cells21 indicate that downregu-lation of DHFR causes the uncoupling of eNOS, leading tothe decrease in enzymatic activity.21 However, the in vivorole of Akt, peroxynitrite, disruption of eNOS dimers, andDHFR in hypertension remains unknown. In the presentwork, Akt phosphorylation was not altered throughout saltloading, indicating the minor role of Akt in the decreasedeNOS activity in 16-week-old DS rats. On the other hand,eNOS protein dimers were markedly diminished in 16-week-old DS rats (supplemental Figure III), indicating that thedisruption of eNOS dimers significantly occurred in DS rats.Furthermore, this disruption of eNOS dimers was associatedwith the increase in nitrotyrosine (an indicator of peroxyni-trite) and the downregulation of DHFR. Therefore, we ob-tained the first evidence that the disruption of eNOS dimerswas involved in the reduction of eNOS activity in salt-sensitive hypertension, and eNOS dimer disruption might beattributed to the increased peroxynitrite and the downregula-tion of DHFR.

Although previous studies indicate that monotherapy withARB (candesartan)16 or statin (atorvastatin)15 improves vas-cular endothelial dysfunction and hypertrophy, reduces su-peroxide, and increases eNOS activity in salt-loaded DS rats,previous studies did not rule out the possible involvement ofblood pressure lowering or plasma lipid lowering in thesevascular effects. In our present study, we have obtained theevidence that olmesartan and pravastatin improved vascularinjury of salt-sensitive hypertension, independently of bloodpressure or plasma lipid. Notably, despite similar beneficialeffects between olmesartan and pravastatin on vascular en-dothelial function, coronary remodeling, and eNOS activityin DS rats, olmesartan normalized vascular NADPH oxidaseactivity, p22phox, superoxide, nitrotyrosine (peroxynitrite),and the disruption of eNOS dimers to a greater extent thanpravastatin. Olmesartan significantly enhanced DHFR ex-pression, in contrast to no effect of pravastatin on DHFR.Taken together with the fact that the increase in peroxyni-trite19,20 or DHFR downregulation21 leads to the disruption ofeNOS dimers, our present data support the notion that therestoration of eNOS activity by olmesartan was mediated bythe suppression of eNOS dimer disruption, which was at leastpartially mediated by the amelioration of peroxynitrite andDHFR downregulation. On the other hand, pravastatin sig-

nificantly enhanced the phosphorylation of Akt, differingfrom no effect of olmesartan on Akt. Akt phosphorylation iswell known to enhance eNOS activity.20,22,23 Accordingly, therestoration of eNOS activity by pravastatin might be at leastin part attributed to the enhancement of Akt phosphorylation.Thus, the mechanism responsible for the restoration of eNOSactivity in salt-sensitive hypertension seems to differ betweenolmesartan and pravastatin.

The therapeutic significance of the combination therapy ofRAS blocker with statin remains obscure, and previousclinical and experimental reports are rare and contradicto-ry.24–32 Particularly, the significance of addition of statin toRAS blocker is undefined in salt-sensitive hypertension,32

which encouraged us to investigate whether add-on pravasta-tin enhances the beneficial effect of olmesartan in DS rats. Ofnote are the observations that pravastatin significantly en-hanced the improvement of endothelium-dependent vascularrelaxation and coronary remodeling by olmesartan in DS rats,despite no additive effect of pravastatin on blood pressure andplasma lipid. Therefore, our work provided the first evidencethat pravastatin has the potential to enhance vascular protec-tive effect of olmesartan in salt-sensitive hypertension, inde-pendent of blood pressure and lipid. Interestingly, pravastatinsignificantly enhanced the normalization of vascular eNOSactivity by olmesartan. In contrast to no effect of olmesartanalone on Akt phosphorylation, pravastatin significantly in-creased Akt phosphorylation (Figure 4). Therefore, the addi-tion of pravastatin to olmesartan increased the phosphoryla-tion of Akt, as well as pravastatin alone. Moreover, add-onpravastatin significantly enhanced the phosphorylation ofeNOS by olmesartan, and this enhancement by pravastatinmight be at least in part mediated by the enhancement of theincrease in total eNOS protein levels by olmesartan (Figure4). Thus, the increase in Akt phosphorylation and eNOSphosphorylation seems to be responsible for the increase ineNOS activity by add-on pravastatin.

In the present study, we found no statistically significantadditive effect of pravastatin on olmesartan-treated DS rats,with regard to eNOS dimers, DHFR, or ERK. As describedabove, the formation of eNOS protein homodimers is neces-sary for eNOS enzymatic activity, and is known to bedisrupted mainly by the increase in peroxynitrite19,20 and thereduction of DHFR.21 In this study, we found that vascularperoxynitrite was significantly increased in salt-sensitivehypertension, and that olmesartan and pravastatin alonesignificantly reduced vascular peroxynitrite. The combinationof olmesartan with pravastatin did not significantly enhancethe reduction of peroxynitrite by olmesartan. On the otherhand, very importantly, the combination of olmesartan withpravastatin more potently inhibited vascular peroxynitriteproduction more than pravastatin monotherapy, indicatingthat inhibitory effect of pravastatin on peroxynitrite produc-tion was weaker than that of olmesartan. These findings canaccount for no significant effect of add-on pravastatin onperoxynitrite. Taken together with the fact that peroxynitriteand DHFR are key regulators of eNOS dimer formation, nosignificant effect of add-on pravastatin on eNOS dimers canbe explained by no significant effect of add-on pravastatin onperoxynitrite and DHFR.

6 Arterioscler Thromb Vasc Biol. March 2007

Recent in vitro study using cultured vascular endothelialcells indicated that angiotensin II downregulates DHFR.21

However, it remains to be determined whether or not thedownregulation of DHFR by angiotensin II can apply to invivo situation. We have obtained the first evidence that thedownregulation of DHFR occurred in salt-sensitive hyperten-sion and olmesartan, but not pravastatin, significantly pre-vented the downregulation of DHFR in salt-sensitive hyper-tension. Taken together with the findings that renin-angiotensin system is involved in vascular diseases of salt-sensitive hypertension, our present work demonstrated thatangiotensin II is implicated in the downregulation of vascularDHFR in salt-sensitive hypertension.

As in the case of ERK, add-on pravastatin did not signif-icantly enhance the inhibition of vascular MEK phosphory-lation by olmesartan (supplemental Figure VI). Therefore, noeffect of add-on pravastatin on ERK phosphorylation can beexplained by no effect of add-on pravastatin on MEKphosphorylation, because MEK is the major upstream activa-tor of ERK.

Study limitationPravastatin is a hydrophilic statin, and the direct vasculareffects of pravastatin remain to be fully understood. Takentogether with previous findings10,11 our present results showthe direct vascular pleiotropic effects of pravastatin, and thisnotion is also supported by the findings of MEGA Study33

that a low dose of pravastatin suppresses primary cardiovas-cular events in Japanese hypercholesterolemic patients asmuch as hydrophobic statins. However, our present in vivostudy did not allow us to elucidate the detailed molecularmechanism underlying the effect of pravastatin on vascularinjury in salt-sensitive hypertension, because the study on thedetailed molecular mechanism was out of scope. Furtherstudy is needed to elucidate more detailed molecular mech-anism responsible for the effect of olmesartan, pravastatin,and their combination on vascular injury of salt-sensitivehypertension.

In conclusion, we obtained the evidence that olmesartanand pravastatin exerts beneficial effect on vascular endothe-lial function and remodeling of salt-loaded DS rats, viadifferential multiple pleiotropic effects, and that add-onpravastatin treatment significantly enhanced the improvementof vascular injury by olmesartan via the phosphorylation ofAkt and eNOS. We propose that combination of ARB withstatin may be potentially promising therapeutic strategy ofsalt-sensitive hypertensive patients with hyperlipidemia, be-yond blood pressure and lipid control.

Sources of FundingThis work was supported in part by Grants-in-Aid for ScientificResearch (16590202) from the Ministry of Education, Culture,Sports, Science, and Technology.

DisclosuresNone.

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