ISSN: 1524-4563 Copyright © 2009 American Heart Association. All rights reserved. Print ISSN: 0194-911X. Online

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DOI: 10.1161/HYPERTENSIONAHA.109.133397 published online Jul 13, 2009; Hypertension

Cabo, Rajamma Mathew, Michael S. Wolin and Zoltan Ungvari Wu, Furong Hu, Praveen Ballabh, Andrej Podlutsky, Gyorgy Losonczy, Rafael de

Anna Csiszar, Nazar Labinskyy, Susan Olson, John T. Pinto, Sachin Gupte, Joseph M. Resveratrol Prevents Monocrotaline-Induced Pulmonary Hypertension in Rats

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Resveratrol Prevents Monocrotaline-Induced PulmonaryHypertension in Rats

Anna Csiszar, Nazar Labinskyy, Susan Olson, John T. Pinto, Sachin Gupte, Joseph M. Wu, Furong Hu,Praveen Ballabh, Andrej Podlutsky, Gyorgy Losonczy, Rafael de Cabo, Rajamma Mathew,

Michael S. Wolin, Zoltan Ungvari

Abstract—Proliferation of pulmonary arterial smooth muscle cells, endothelial dysfunction, oxidative stress, andinflammation promotes the development of pulmonary hypertension. Resveratrol is a polyphenolic compound that exertsantioxidant and anti-inflammatory protective effects in the systemic circulation, but its effects on pulmonary arteriesremain poorly defined. The present study was undertaken to investigate the efficacy of resveratrol to prevent pulmonaryhypertension. Rats injected with monocrotaline progressively developed pulmonary hypertension. Resveratrol treatment(25 mg/kg per day, PO, from day 1 postmonocrotaline) attenuated right ventricular systolic pressure and pulmonaryarterial remodeling, decreased expression of inflammatory cytokines (tumor necrosis factor-�, interleukin 1�,interleukin 6, and platelet-derived growth factor-�/�), and limited leukocyte infiltration in the lung. Resveratrol alsoinhibited proliferation of pulmonary arterial smooth muscle cells. Treatment of rats with resveratrol increased expressionof endothelial NO synthase, decreased oxidative stress, and improved endothelial function in small pulmonary arteries.Pulmonary hypertension was associated with an upregulation of NAD(P)H oxidase in small pulmonary arteries, whichwas significantly attenuated by resveratrol treatment. Our studies show that resveratrol exerts anti-inflammatory,antioxidant, and antiproliferative effects in the pulmonary arteries, which may contribute to the prevention ofpulmonary hypertension. (Hypertension. 2009;54:00-00.)

Key Words: pulmonary hypertension � resveratrol � oxidative stress � endothelial dysfunction � inflammation

Pulmonary hypertension is a syndrome that encompassesseveral diseases, all of which have in common increased

pulmonary artery pressures. Idiopathic (“primary”) pulmo-nary hypertension is a rare disease caused by genetic defectsin the bone morphogenetic protein signaling pathways. Com-mon causes of “secondary” forms of pulmonary hypertensioninclude the following: (1) pulmonary hypertension associatedwith chronic obstructive pulmonary disease; (2) pulmonaryembolism; and (3) pressure/volume overload–related pulmo-nary hypertension. In addition, pulmonary hypertension oftendevelops in patients with autoimmune diseases or as a severeadverse effect of anorectic drug treatment. Despite the diverseetiologic differences, many similarities in the pathologicalalterations in pulmonary arteries occur among the variousforms of pulmonary hypertension, ie, vascular remodeling,including cellular proliferation in both the intima and media;endothelial dysfunction/increased vasoconstriction; and acti-vation of inflammatory processes (eg, inflammatory cytokineexpression and monocyte infiltration).

Current therapies for chronic pulmonary hypertension aredesigned to reduce pulmonary arterial resistance by inducingvasodilation (eg, NO inhalation, stimulation of cGMP pro-duction by phosphodiesterase inhibitors, endothelin receptorantagonists, and prostacyclin analogs). These therapeuticapproaches mainly provide symptomatic relief. Novel treat-ments are required to prevent progression of pulmonaryhypertension by interfering with the pathomechanisms of thedisease at multiple levels. For example, in a preclinicalsetting, experimental therapeutics that exert antimitogeniceffects on proliferation of pulmonary arterial smooth musclecell (PASMC),1–3 in addition to promoting vasodilation, showpromise in enhancing overall prognosis.

Resveratrol (3,5,4�-trihydroxystilbene) is a dietary poly-phenolic compound that exerts significant antioxidant, anti-inflammatory, and endothelial protective effects in the sys-temic circulation.4–7 Importantly, our previous study showsthat resveratrol inhibits proliferation of cultured aorticsmooth muscle cells.8 Thus, we hypothesize that resveratrol

Received March 25, 2009; first decision April 21, 2009; revision accepted June 10, 2009.From the Departments of Physiology (A.C., N.L., M.S.W., Z.U.), Anatomy and Cell Biology (F.H., P.B., R.M.), and Pediatrics (F.H., P.B., R.M.), New

York Medical College, Valhalla, NY; Reynolds Oklahoma Center on Aging (A.C., Z.U.), Department of Geriatric Medicine, University of OklahomaHealth Science Center, Oklahoma City, Okla; Department of Biochemistry (S.O., J.T.P., J.M.W.), New York Medical College-Westchester MedicalCenter, Valhalla, NY; Department of Biochemistry and Molecular Biology (S.G.), University of South Alabama, Mobile, Ala; Sam and Ann BarshopInstitute for Longevity and Aging Studies (A.P.), University of Texas Health Science Center, San Antonio, Tex; Department of Pulmonology (G.L., Z.U.),Semmelweis University, Budapest, Hungary; Laboratory of Experimental Gerontology (R.d.C.), National Institute on Aging, Baltimore, Md.

Correspondence to Zoltan Ungvari, Reynolds Oklahoma Center on Aging, Department of Geriatric Medicine, University of Oklahoma Health ScienceCenter, Oklahoma City, OK 73104. E-mail ungzol@yahoo.com

© 2009 American Heart Association, Inc.

Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.109.133397

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exerts antiproliferative, antioxidant, anti-inflammatory, andendothelial protective effects in the pulmonary circulationand prevents the progression of pulmonary hypertension.

To test this hypothesis we investigated the chronic efficacyof oral resveratrol treatment in monocrotaline-treated rats.Rats develop severe pulmonary hypertension after a singleinjection of monocrotaline (MCT),9 and this model mimicsseveral key aspects of both primary and secondary forms ofhuman pulmonary hypertension, including vascular remodel-ing, proliferation of PASMCs, oxidative stress, endothelialdysfunction, and upregulation of inflammatory cytokines andleukocyte infiltration. We particularly addressed the questionof whether resveratrol exerts beneficial effects on prolifera-tion of PASMCs, pulmonary arterial endothelial function, andinflammation.

MethodsA detailed Methods section can be found in the online Data Supplement(please see http://hyper.ahajournals.org).

Animal Models and Experimental DesignAnimal use protocols were approved by the New York MedicalCollege Institutional Animal Care and Use Committee. To assesspreventive effects of resveratrol on MCT-induced pulmonary hyper-tension, adult male Sprague-Dawley rats (300 g) were randomlyassigned to the following groups (n�6 animals in each group) toreceive either vehicle or resveratrol in the drinking water: (1) controlanimals for 14 and 21 days; (2) MCT (60 mg/kg SC)-treated animalsfor 14 and 21 days; or (3) MCT-treated animals receiving resveratrol(25 mg/kg per day, PO, in the drinking water) from day 1 to day 14or day 21 after MCT injection.

Assessment of Right Ventricular HemodynamicsTwo or 3 weeks after MCT administration, rats were anesthetizedwith ketamine (60 mg/kg IM) and xylazine (3 mg/kg IM). A microtipMillar pressure catheter was introduced into the right ventriclethrough the jugular vein to measure right ventricular (RV) pressure.After hemodynamic measurements, the thorax was opened, andpieces of the left lung and isolated segments of small intrapulmonaryarteries were processed for histological evaluation or frozen in liquidnitrogen for subsequent biochemical measurements.

Measurement of RV HypertrophyThe heart was dissected, and the ratio of the RV free wall weightdivided by the length of the tibia was calculated as an index of RVhypertrophy (which is unaffected by changes in body weight or leftventricular mass).

Cell Proliferation and Apoptosis AssaysImmunofluorescent labeling for �-smooth muscle actin was per-formed to visualize the medial layer of the vessels. Index of smoothmuscle hypertrophy of small pulmonary arteries (diameter: 50 to150 �m) was calculated. Cell proliferation was also assessed in thewalls of distal pulmonary vessels by use of a monoclonal antibodyagainst proliferating cell nuclear antigen (PCNA). The percentage ofPCNA-positive cells was calculated in 10 randomly chosen fields.To quantify the rate of endothelial cell apoptosis, TUNEL assay wasperformed, as described.7

To assess the antiproliferative effects of resveratrol in vitro,primary human PASMCs were stimulated in vitro with platelet-derived growth factor (PDGF; 10 ng/mL) in the presence or absenceof resveratrol. Cultured cells were stained with the membrane-permeable nucleic acid stain 4�,6�-diamidino-2-phenylindole, andcell number was determined using a hemocytometer. The inhibitoryeffect of resveratrol on cell proliferation was also tested using acolony formation assay. In other experiments, PASMCs grown in a

96-well plate were stimulated in vitro with transforming growthfactor (TGF)-� (10 ng/mL), interleukin (IL) 1� (10 ng/mL), or IL-6(10 ng/mL) in the presence or absence of resveratrol. Cell prolifer-ation was assessed by measuring the fluorescence of the DNA-binding CyQUANT dye (Invitrogen) in cell lysates with a fluores-cence microplate reader. The effects of resveratrol on apoptotic celldeath (annexin V and TUNEL staining) in PASMCs and pulmonaryarterial endothelial cells were quantified by flow cytometry (GuavaEasycyte). Resveratrol-induced inhibition of the cell cycle inPASMCs was assessed by the Guava Cell Cycle Assay by flowcytometry.

Functional StudiesAcetylcholine-induced relaxation was assessed in segments of smallintrapulmonary arteries precontracted by phenylephrine. In separateexperiments, the vasorelaxant effect of in vitro administration ofresveratrol (10�7 to 3�10�4 mol/L) was also assessed.

Measurement of Tissue H2O2 andSuperoxide ProductionProduction of superoxide in isolated intrapulmonary arteries wasassessed using the dihydroethidine staining method, as describedpreviously.10 The cell-permeant oxidative fluorescent indicator dye5-(and-6)-carboxy-2�, 7�-dichlorodihydrofluorescein diacetate (C-H2DCFDA) was used to assess peroxide levels in isolated intrapul-monary arteries, as we reported previously.5 Pressure overload–induced oxidative stress in the right ventricle was assessed bydihydroethidine staining and lucigenin chemiluminescence assay, asreported previously.11

Western BlottingWestern blotting was performed to assess protein expression ofNox-1, gp91phox, and endothelial NO synthase (eNOS) in smallintrapulmonary arteries.

Quantitative Real-Time PCRReal time RT-PCR technique was used to analyze mRNA expres-sion, as reported previously.11

Assessment of Leukocyte InfiltrationImmunolabeling on lung sections was performed for the rat mono-cyte/macrophage marker ED-1. The number of ED-1–positive cellswas determined in 10 randomly chosen fields.

Data AnalysisData were normalized to the respective control mean values and areexpressed as mean�SD or SEM. Statistical analyses of data wereperformed by Student’s t test or by 2-way ANOVA followed by aTukey’s posthoc test, as appropriate. P�0.05 was considered statis-tically significant.

ResultsResveratrol Prevents the Development ofPulmonary HypertensionRats challenged with MCT consistently developed significantpulmonary hypertension within 14 days, which progressivelyincreased until day 21. Consequently, RV systolic pressurewas increased significantly as compared with the saline-challenged group (Figure 1A). Resveratrol treatment fromday 1 normalized RV systolic pressure in MCT-injected ratsat both the 2- and 3-week periods (Figure 1A). Mean systemicarterial pressure was comparable in each group (data notshown). In the MCT groups, a significant RV hypertrophy(Figure 1B) associated with RV oxidative stress (Figure S1)developed as a consequence of increased pulmonary pres-

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sures. Resveratrol treatment prevented MCT-induced RVhypertrophy (Figure 1B) and oxidative stress.

Resveratrol Inhibits Proliferation of PASMCsSmooth muscle cell mass in the small pulmonary arteries wasmarkedly increased in the MCT groups, both at day 14 andday 21 (Figure 1C and 1D), as compared with controlanimals. Resveratrol treatment normalized medial wall thick-ness, preventing the increase in PASMC mass in vessels ofMCT-treated rats (Figure 1C and 1D). Medial hypertrophy ofpulmonary resistance vessels was associated with an increasednumber of PCNA-positive proliferating vascular cells in MCT-induced pulmonary hypertension (Figure 1E and Figure S2). Inparallel to normalization of vessel morphology, the number ofPCNA-positive cells was considerably reduced in animalstreated with resveratrol (Figure 1E and Figure S2). Neither MCTpulmonary hypertension nor resveratrol treatment elicited apoptoticcell death in small pulmonary arteries (Figure 1F and Figure S2).

PDGF (10 ng/mL for 48 hours) stimulated proliferation incultured PASMCs, which was prevented by resveratrol (Fig-ure 2A). The inhibitory effect of resveratrol on PASMCproliferation was also tested using a colony formation assay(Figure 2B). We confirmed that resveratrol potently inhibitedPASMC colony formation and proliferation in a physiologicallyrelevant concentration range (Figure 2B and 2C). Resveratrolalso inhibited proliferation of PASMCs stimulated by IL-1�,TGF-�, or IL-6 (Figure 2D). In the concentration range, inwhich the antiproliferative effects were evident, resveratrol didnot significantly increase apoptotic cell death either in PASMCsor pulmonary arterial endothelial cells (Figure 2E and 2F). Usingflow cytometry, we demonstrated that the inhibitory effect ofresveratrol on cell proliferation could be attributed to the cellcycle arrest in S phase (Figure 2G through 2I). Resveratrolsignificantly inhibited cytokine-induced nuclear factor �B(NF-�B) activation in PASMCs (Figure S3).

Development of pulmonary hypertension was associatedwith dysregulation of the expression of components of thebone morphogenetic protein (BMP)-4 signaling pathway.Resveratrol treatment normalized alterations in BMP receptors(ACVR1 and ACVRL1), BMP antagonists (chordin), andSMAD signaling molecules (SMAD1/4) in the lung (Figure S4).

Resveratrol Attenuates Inflammatory GeneExpression and Monocyte InfiltrationDevelopment of pulmonary hypertension was associated withupregulation of mRNA expression of IL-6, IL-1, tumornecrosis factor-�, PDGF-�, PDGF-�, TGF-�, MCP-1, induc-ible NO synthase, and intercellular adhesion molecule 1 in thelung of MCT-treated rats (Figure 3). Resveratrol treatment

Figure 1. A, Pulmonary hypertension developed progressively inMCT-injected rats, as shown by increases in the RV systolic pres-sure at 14 and 21 days post-MCT injection. Resveratrol treatment(25 mg/kg per day from day 1) prevented development of pulmo-nary hypertension. B, Adaptive RV hypertrophy was demonstratedby RV weight:tibia length ratio in MCT-treated rats. Resveratroltreatment prevented RV hypertrophy. Data are mean�SEM.*P�0.05 vs control, #P�0.05 vs no resveratrol. C, Immunofluores-cent labeling (red) for �-smooth muscle actin for identifying

Figure 1 (Continued). vascular smooth muscle cells (blue fluo-rescence: nuclei). Medial wall thickness of small pulmonaryarteries was significantly increased in MCT-treated rats. Res-veratrol treatment prevented increases in vascular smooth mus-cle mass. Scale bar: 20 �m. D, Bar graphs are summary datafor medial hypertrophy index in control, MCT-, and MCT plusresveratrol–treated rats. E and F, Relative ratio of PCNA-positivenuclei (E) or TUNEL-positive nuclei (F) in the wall of small pul-monary arteries of MCT- and MCT plus resveratrol–treatedrats (n�6 animals for each group). Data are expressed asmean�SEM. *P�0.05 vs control, #P�0.05 vs no resveratrol.

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significantly attenuated expression of each inflammatorymarker (Figure 3).

In the lungs of MCT-treated rats, the number of ED-1–positive leukocytes was significantly increased (Figure 4).Resveratrol treatment prevented infiltration of ED-1–positivecells (Figure 4.). Thus, signaling for leukocyte invasion intolung tissue is markedly attenuated by resveratrol treatment.

Resveratrol Upregulates eNOS, AttenuatesOxidative Stress, and Improves EndothelialFunction in Small Pulmonary ArteriesAcetylcholine-induced relaxation was impaired in smallpulmonary arteries of MCT-treated rats, and relaxationwas restored after resveratrol treatment (Figure 5A). Vas-cular relaxations to the NO donor S-Nitroso-N-Acetyl-D,L-Penicillamine (SNAP) were unaffected in MCT rats(data not shown). In small pulmonary arteries of MCT-treated rats, reactive oxygen species generation was ele-

vated, whereas resveratrol treatment significantly attenuatedoxidative stress (detected with dihydroethidine [Figure 5B] andC-H2 DCFDA fluorescence [data not shown] methods). Res-veratrol in vitro did not elicit significant vasorelaxation in thephysiological relevant concentration range (�10 �mol/L; Figure5C). In small pulmonary arteries of MCT-treated rats, expressionof NAD(P)H oxidase subunits was upregulated (Figures 5D, 5E,and S4). Attenuation of vascular oxidative stress by resveratrol(Figure 5B) was associated with downregulation of NOX-1 andgp91phox, as well as improved eNOS expression (Figures 5Dthrough 5F and S5).

DiscussionResveratrol belongs to a new class of drugs12 that exhibitscytoprotective, antioxidative, and anti-inflammatory vasopro-tective properties in the systemic circulation.4 Our studiesshow that resveratrol in the pulmonary arteries of MCT-treated rats improved endothelial function, attenuated oxida-

Figure 2. A, PDGF (10 ng/mL for 48hours) stimulates human PASMC prolif-eration, which is prevented by resvera-trol (10 �mol/L). Data are mean�SEM.*P�0.05 vs no resveratrol, #P�0.05 vsPDGF only. B, Original photomicro-graphs showing colonies of PDGF-stimulated PASMCs (on day 14). Res-veratrol in a concentration-dependentmanner inhibited PASMC proliferation.Bar graphs (C) are summary data. Dataare mean�SEM. *P�0.05. D, Prolifera-tion of human PASMCs, stimulated byIL-1� (10 ng/mL), TGF-� (10 ng/mL), orIL-6 (10 ng/mL) was inhibited by resvera-trol (10 �mol/L). The DNA-binding fluo-rescent dye CyQUANT was used toassess cell mass. E and F, Resveratroldoes not stimulate apoptotic cell deathin PASMCs and pulmonary arterial endo-thelial cell (PAECs). Apoptosis wasquantified by flow-cytometry (E, annexinV; F, TUNEL staining). G through I, Res-veratrol arrests PASMC proliferation pre-dominantly in S phase (flow cytometrydata). Data are expressed asmean�SEM. *P�0.05 vs no resveratrol(n�4 to 8 for each group).

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tive stress, and inhibited inflammation. Resveratrol treatmentalso inhibited vascular remodeling, thus preventing develop-ment of pulmonary hypertension. In addition, treatment ofcultured PASMCs with resveratrol inhibited cell prolifera-tion, recapitulating in vitro the effects observed in vivo onMCT-treated animals.

Previous studies focused on the effects of resveratrol on thesystemic circulation but provided little information on itspulmonary effects. In our study, we found a prominentprevention of progression of pulmonary hypertension inresponse to resveratrol therapy (Figure 1A). Accordingly,reducing right heart load also prevented adaptive hypertrophy

Figure 3. Analysis of mRNA expression ofIL-6, IL-1�, tumor necrosis factor (TNF)-�,PDGF-�, PDGF-�, TGF-�, MCP-1, induc-ible NO synthase (iNOS), and intercellularadhesion molecule (ICAM) 1 in the lungsof control rats and MCT-injected rats (21days post-MCT). Analysis of mRNAexpression was performed by real-timequantitative RT-PCR. �-Actin was usedfor normalization. Data are shown asmean�SEM. *P�0.05 vs control,#P�0.05 vs no resveratrol (n�6 for eachgroup).

Figure 4. A through C, Representative fluo-rescent images compare the presence ofED-1–positive leukocytes (green fluores-cence, arrows) in control rats (A) and lungsof MCT (B)-treated rats. C, Resveratrol treat-ment substantially reduced the number ofED-1–positive cells in the lungs of MCT-treated rats. Immunofluorescent labeling (red)for �-smooth muscle actin was used foridentifying vascular smooth muscle cells(blue fluorescence: nuclei). Scale bar:100 �m. D, Bar graphs are summary data(mean�SEM). *P�0.05 vs control, #P�0.05vs no resveratrol (n�6 for each group).

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(Figure 1B) and strain-induced oxidative stress (Figure S1) inthe right ventricle of MCT-treated rats. Additional studies areneeded to determine whether resveratrol treatment can re-verse/delay the progression of the disease once pulmonaryhypertension is established, which is the ultimate clinicalrelevance of a new therapeutic paradigm.

Structural changes observed in MCT-induced pulmonaryhypertension resemble characteristics of human pulmonaryhypertension in terms of marked medial wall thickening (Figure1C and 1D), resulting in a dramatic increase in pulmonaryarterial resistance.13 The current study is in agreement withprevious reports2 in that smooth muscle cell proliferation mark-edly increases in pulmonary resistance vessels of MCT-challenged animals (Figure 1C through 1E). Resveratroltreatment resulted in near normal vessel morphology andreduced pulmonary arterial smooth muscle proliferation (Fig-ure 1C through 1E) without increasing the rate of apoptosis(Figure 1F). The lack of increased apoptotic cell death mayexplain the lack of adverse effects and good tolerability ofresveratrol treatment in clinical studies.

It is likely that inhibition of pulmonary arterial smoothmuscle proliferation and vascular remodeling is predomi-nantly a direct effect of resveratrol, because resveratrol alsoeffectively inhibited proliferation of cultured PASMCs, ar-resting the cell cycle in the S phase (Figure 2). These findingsare in agreement with our previous studies that demonstratethat the antiproliferative properties of resveratrol are attrib-uted to induction of p53 and heat shock protein HSP27 inhuman aortic smooth muscle cells.8,14

Current literature supports contributory effects of inflam-mation on the development and progression of pulmonaryhypertension.15 In animal models of pulmonary hypertension,an increased presence and activity of inflammatory cells(including macrophages, polymorphonuclear neutrophils,

lymphocytes, and mast cells) are routinely observed.15 In-deed, our study documents a substantial increase in ED-1–positive cells in the lungs of MCT-treated rats (Figure 4),which is accompanied by a significant upregulation of in-flammatory cytokines16,17 and growth factors (tumor necrosisfactor-�, IL-1�, IL-6, PDGF-�, PDGF-�, and TGF-�), aswell as adhesion molecules (Figure 3). There is increasingappreciation of inflammation in various forms of clinicalpulmonary hypertension on the basis of evidence rangingfrom increased plasma levels of inflammatory cytokines topulmonary infiltration of inflammatory cells.15 In humans, avariety of diverse inflammatory conditions (eg, viral infec-tions and autoimmune diseases) culminate in severe pulmo-nary hypertension. It has been generally accepted that inflam-matory cytokines and growth factors can cause pulmonaryvasoconstriction and promote proliferation of vascular cells.Recent studies suggest that disruption of PDGF signalingpathways prevents development and progression of MCT-induced pulmonary hypertension in rats.2 Importantly, wefound that resveratrol treatment in MCT-treated rats normal-ized expression of inflammatory cytokines and growth factors(including PDGF-�/�; Figure 3) and significantly decreasedthe number of infiltrating ED-1–positive cells in the lung(Figure 4). The mechanisms by which resveratrol interfereswith inflammatory processes in the lung are not well under-stood. Recently we showed that resveratrol effectively blocksNF-�B activation in endothelial cells, thus limiting expres-sion of inflammatory cytokines and adhesion molecules andattenuating monocyte adhesiveness.6 Resveratrol also effec-tively inhibits cytokine-induced NF-�B activation inPASMCs (Figure S3). NF-�B activation is known to regulatePASMC proliferation in vitro.18,19 Because development ofpulmonary hypertension in MCT-treated rats is associatedwith NF-�B activation,20 and administration of an inhibitor of

Figure 5. A, Relaxation of small intrapulmo-nary arteries from control rats and MCT-injected rats (21 days post-MCT) in responseto acetylcholine. The effect of chronic res-veratrol treatment (25 mg/kg per day; fromday 1 to day 21) on vascular relaxations isalso shown. Data are mean�SEM. *P�0.05vs control, #P�0.05 vs no resveratrol (n�6for each group). B, Production of superoxide(assessed by the dihydroethidine stainingmethod; build-up of nuclear ethidium bromide[EB] fluorescence over time was quantified) issignificantly increased in small intrapulmonaryarteries from MCT-treated rats vs controls(*P�0.05). Resveratrol significantly attenuatedvascular reactive oxygen species generation(#P�0.05). Data are mean�SEM (n�6 foreach group). C, Resveratrol-induced relax-ation in small intrapulmonary arteries.*P�0.05 vs control. D through F, Proteinexpression of NOX-1, gp91phox, and eNOS(assessed by Western blotting, data are pre-sented in densitometric units) in smallintrapulmonary arteries from control, MCT-treated, and MCT plus resveratrol–treatedrats. �-Actin was used for normalization.

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NF-�B prevents pulmonary hypertension in MCT-treatedrodents,21 we speculate that inhibition of NF-�B by resvera-trol may contribute to its therapeutic potential in pulmonaryhypertension.

Mutations in BMP receptors have been identified in pa-tients with primary pulmonary arterial hypertension, impli-cating BMP signaling in pulmonary hypertension. Impor-tantly, recent studies have demonstrated that the expressionand function of the BMP/Smad signaling axis are perturbed inMCT-treated animals as well.22 Because BMP signals antag-onize PASMC proliferation induced by inflammatory cyto-kines, altered BMP/Smad signaling may play a role in thepathogenesis of pulmonary hypertension. Resveratrol normal-ized the expression of many components of the BMP/Smadsignaling pathway (Figure S4). Additional studies are neededto elucidate whether these effects contribute to the therapeuticbenefits of resveratrol treatment.

There is solid evidence in MCT-treated rats that oxidativeinjury to the pulmonary vascular endothelium precedesPASMC proliferation and medial hypertrophy in the distalpulmonary vascular bed and the rise in pulmonary arterypressure. We found that pulmonary hypertension in smallpulmonary arteries is associated with endothelial dysfunction(Figure 5A), oxidative stress (Figure 5B), and upregulation ofNAD(P)H oxidases (Figure 5D and 5E). By contrast, resvera-trol treatment significantly improved pulmonary arterial en-dothelial function (Figure 5A) and attenuated oxidative stressand NADPH oxidase expression (Figures 5D, 5E, and FigureS5). These findings are significant because recent studies onp91phox knockout mice suggest that NAD(P)H oxidases con-tribute to the development of pulmonary hypertension.23 Theactivity and expression of vascular NAD(P)H oxidases areknown to be regulated by inflammatory cytokines (eg, tumornecrosis factor-� and TGF�24), the expression of which isupregulated in lungs of rats with pulmonary hypertension(Figure 2). Thus, we suggest that the observed resveratrol-induced downregulation of NAD(P)H oxidase (Figure 5) isattributed to the resveratrol-induced attenuation of inflamma-tory cytokine production (Figure 3).

We confirmed that eNOS is downregulated (Figure 5E) inrats with pulmonary hypertension.16,25 Importantly, this atten-uation was corrected by resveratrol (Figure 5E). Previousstudies showed that resveratrol directly regulates eNOSexpression at the level of transcription in cultured endothelialcells.5 Because some studies suggest that treatment with NOdonors26 or overexpression of eNOS itself may attenuatepulmonary hypertension,27 it is possible that upregulation ofeNOS contributes to the therapeutic action of resveratrol. Wecannot exclude the possibility that resveratrol, in addition toupregulating eNOS, may also affect BH4 levels and/orcaveolin25 and, thus, directly influence eNOS activity. Previ-ously, resveratrol was shown to upregulate the expression ofNrf2-regulated antioxidant gene battery, including heme ox-ygenase 1 in systemic arteries.5 In the present study, resvera-trol treatment also induced heme oxygenase 1 in the pulmo-nary arteries of MCT-treated rats (Figure S6), which is knownto confer antiproliferative and vasoprotective effects.28

PerspectivesWe have demonstrated that treatment with resveratrol pre-vents the development of pulmonary hypertension in arecognized animal model of the disease. The demonstratedantiproliferative action of resveratrol on PASMCs may becontributory to its beneficial effects. Furthermore, we haveprovided evidence for anti-inflammatory, antioxidative, andendothelial protective effects in the beneficial actions ofresveratrol in pulmonary arteries. Because phase I clinicaltrials are currently underway for anticancer efficacy of oralresveratrol treatment in humans,12 the use of resveratrol as anew therapy for pulmonary hypertension might, therefore, beplausible. Future studies should determine the following: (1)whether resveratrol treatment can reverse or delay the pro-gression of established pulmonary hypertension; (2) whetherresveratrol may potentiate the effects of current therapies; and(3) whether resveratrol can exert direct cardioprotectiveeffects preventing RV failure in patients with pulmonaryhypertension.

Sources of FundingThis work was supported by grants from the American Federation forAging Research (to A.C.), the American Diabetes Association (to Z.U.),the National Institutes of Health (HL077256, HL43023, and HL31069),and the Hungarian Scientific Research Fund (OTKA-K68758).

DisclosuresNone.

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7. Pearson KJ, Baur JA, Lewis KN, Peshkin L, Price NL, Labinskyy N,Swindell WR, Kamara D, Minor RK, Perez E, Jamieson HA, Zhang Y,Dunn SR, Sharma K, Pleshko N, Woollett LA, Csiszar A, Ikeno Y, LeCouteur D, Elliott PJ, Becker KG, Navas P, Ingram DK, Wolf NS,Ungvari Z, Sinclair DA, de Cabo R. Resveratrol delays age-relateddeterioration and mimics transcriptional aspects of dietary restrictionwithout extending life span. Cell Metab. 2008;8:157–168.

8. Wang Z, Chen Y, Labinskyy N, Hsieh TC, Ungvari Z, Wu JM. Regu-lation of proliferation and gene expression in cultured human aorticsmooth muscle cells by resveratrol and standardized grape extracts.Biochem Biophys Res Commun. 2006;346:367–376.

9. Rosenberg HC, Rabinovitch M. Endothelial injury and vascular reactivityin monocrotaline pulmonary hypertension. Am J Physiol. 1988;255:H1484–H1491.

Csiszar et al Resveratrol Prevents Pulmonary Hypertension 7

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10. Csiszar A, Ahmad M, Smith KE, Labinskyy N, Gao Q, Kaley G, EdwardsJG, Wolin MS, Ungvari Z. Bone morphogenetic protein-2 induces proin-flammatory endothelial phenotype. Am J Pathol. 2006;168:629–638.

11. Csiszar A, Labinskyy N, Podlutsky A, Kaminski PM, Wolin MS, ZhangC, Mukhopadhyay P, Pacher P, Hu F, de Cabo R, Ballabh P, Ungvari Z.Vasoprotective effects of resveratrol and SIRT1: attenuation of cigarettesmoke-induced oxidative stress and proinflammatory phenotypic alter-ations. Am J Physiol Heart Circ Physiol. 2008;294:H2721–H2735.

12. Baur JA, Sinclair DA. Therapeutic potential of resveratrol: the in vivoevidence. Nat Rev Drug Discov. 2006;5:493–506.

13. Jeffery TK, Wanstall JC. Pulmonary vascular remodeling: a target fortherapeutic intervention in pulmonary hypertension. Pharmacol Ther.2001;92:1–20.

14. Zou J, Huang Y, Cao K, Yang G, Yin H, Len J, Hsieh TC, Wu JM. Effectof resveratrol on intimal hyperplasia after endothelial denudation in anexperimental rabbit model. Life Sci. 2000;68:153–163.

15. Voelkel NF, Cool C, Lee SD, Wright L, Geraci MW, Tuder RM. Primarypulmonary hypertension between inflammation and cancer. Chest. 1998;114:225S–230S.

16. Nishimura T, Vaszar LT, Faul JL, Zhao G, Berry GJ, Shi L, Qiu D,Benson G, Pearl RG, Kao PN. Simvastatin rescues rats from fatal pul-monary hypertension by inducing apoptosis of neointimal smooth musclecells. Circulation. 2003;108:1640–1645.

17. Bhargava A, Kumar A, Yuan N, Gewitz MH, Mathew R. Monocrotalineinduces interleukin-6 mRNA expression in rat lungs. Heart Dis. 1999;1:126–132.

18. Diebold I, Djordjevic T, Hess J, Gorlach A. Rac-1 promotes pulmonaryartery smooth muscle cell proliferation by upregulation of plasminogenactivator inhibitor-1: role of NFkappaB-dependent hypoxia-induciblefactor-1alpha transcription. Thromb Haemost. 2008;100:1021–1028.

19. Ibe BO, Abdallah MF, Portugal AM, Raj JU. Platelet-activating factorstimulates ovine foetal pulmonary vascular smooth muscle cell prolif-eration: role of nuclear factor-kappa B and cyclin-dependent kinases. CellProlif. 2008;41:208–229.

20. Mathew R, Huang J, Gewitz MH. Inhibition of NF-kB not only attenuatesMCT-induced pulmonary hypertension but also prevents the distruptionof endothelial cell membrane proteins. FASEB J. 2006;20:A401.

21. Sawada H, Mitani Y, Maruyama J, Jiang BH, Ikeyama Y, Dida FA,Yamamoto H, Imanaka-Yoshida K, Shimpo H, Mizoguchi A, MaruyamaK, Komada Y. A nuclear factor-kappaB inhibitor pyrrolidine dithiocar-bamate ameliorates pulmonary hypertension in rats. Chest. 2007;132:1265–1274.

22. Morty RE, Nejman B, Kwapiszewska G, Hecker M, Zakrzewicz A, KouriFM, Peters DM, Dumitrascu R, Seeger W, Knaus P, Schermuly RT,Eickelberg O. Dysregulated bone morphogenetic protein signaling inmonocrotaline-induced pulmonary arterial hypertension. ArteriosclerThromb Vasc Biol. 2007;27:1072–1078.

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24. Sturrock A, Cahill B, Norman K, Huecksteadt TP, Hill K, Sanders K,Karwande SV, Stringham JC, Bull DA, Gleich M, Kennedy TP, HoidalJR. Transforming growth factor-beta1 induces Nox4 NAD(P)H oxidaseand reactive oxygen species-dependent proliferation in human pulmonaryartery smooth muscle cells. Am J Physiol. 2006;290:L661–L673.

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26. Mathew R, Gloster ES, Sundararajan T, Thompson CI, Zeballos GA,Gewitz MH. Role of inhibition of nitric oxide production inmonocrotaline-induced pulmonary hypertension. J Appl Physiol. 1997;82:1493–1498.

27. Liu L, Liu H, Visner G, Fletcher BS. Sleeping Beauty-mediated eNOSgene therapy attenuates monocrotaline-induced pulmonary hypertensionin rats. FASEB J. 2006;20:2594–2596.

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8 Hypertension September 2009

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ONLINE SUPPLEMENT

Resveratrol Prevents Monocrotaline-induced Pulmonary Hypertension in Rats Anna Csiszar1,2, Nazar Labinskyy1, Susan Olson3, John T. Pinto3, Sachin Gupte4, Joseph M. Wu3, Furong Hu5, Praveen Ballabh5, Andrej Podlutsky6, Gyorgy Losonczy7, Rafael de Cabo8, Rajamma Mathew5, Michael S. Wolin1, Zoltan Ungvari1,2,7 1) Department of Physiology, New York Medical College, Valhalla, NY 10595 2) Reynolds Oklahoma Center on Aging, Department of Geriatric Medicine, University of Oklahoma Health Science Center, Oklahoma City OK 73104 3) Department of Biochemistry, New York Medical College-Westchester Medical Center, Valhalla, New York 10595, USA 4) Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL 36688, USA 5) Departments of Anatomy and Cell Biology and Pediatrics, New York Medical College, Valhalla, NY 10595 6) The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center, San Antonio, Texas 78245 7) Department of Pulmonology, Semmelweis University; H-1125 Budapest, Hungary 8) Laboratory of Experimental Gerontology, National Institute on Aging, 5600 Nathan Shock Drive, Baltimore, MD

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Extended Materials and Methods Animal Models and Experimental Design Animal use protocols were approved by the Institutional Animal Care and Use Committee of the New York Medical College, Valhalla, NY. To assess the preventive effects of resveratrol on MCT-induced pulmonary hypertension, adult male Sprague-Dawley rats (300 g, purchased from Charles River Co, Austin, TX) were randomized to the following groups (n=6 animals in each group) to receive either vehicle of resveratrol in the drinking water: control animals for 14 and 21 days; MCT (60 mg/kg s.c.) -treated animals for 14 and 21 days; MCT-treated animals receiving resveratrol (25 mg/kg/day, p.o., in the drinking water) from day 1 to day 14 or day 21 after MCT injection. This dose of resveratrol was shown to be vasoprotective in the systemic circulation1, 2. Assessment of right ventricular hemodynamics Three or 5 weeks after MCT administration, rats were anesthetized with ketamine (60 mg/kg i.m.) and xylazine (3 mg/kg i.m.). A microtip Millar pressure catheter was introduced into the right ventricle through the jugular vein to measure right ventricular pressure. After hemodynamic measurements the thorax was opened and pieces of the left lung and isolated segments of small intrapulmonary arteries were processed for histological evaluation or frozen in liquid nitrogen for subsequent biochemical measurements. Measurement of right ventricular hypertrophy The heart was dissected, and the ratio of the right ventricular free wall weight divided by the length of the tibia was calculated as an index of right ventricular hypertrophy (which is unaffected by changes in body weight). Cell proliferation and apoptosis assays Immunofluorescent labeling for α-smooth muscle actin was performed to visualize the medial layer of the vessels. Index of smooth muscle hypertrophy of small pulmonary arteries (d: 50-150 μm) was calculated as follows: (a+b+c+d)x(A+B)-1, where a,b,c,d and A,B are measurements of medial wall thickness (at 3, 6, 9 and 12 o’clock direction, respectively) and external diameter (vertically and diagonally, respectively). Cell proliferation was also assessed in the walls of distal pulmonary vessels by use of a monoclonal antibody against proliferating cell nuclear antigen (PCNA). The percentage of PCNA–positive cells was calculated in 10 randomly chosen fields. To assess the rate of endothelial cell apoptosis, TUNEL assay was performed as described1. To assess the anti-proliferative effects of resveratrol in vitro, primary human pulmonary arterial smooth muscle cells (PASMC; cultured in 0.2% FBS-DMEM as described3) were stimulated in vitro with PDGF (10 ng/mL) in the presence or absence of resveratrol. Cultured cells were stained with the membrane-permeable nucleic acid stain 4',6'-diamidino-2-phenylindole (5 µM; Molecular Probes) and cell number was determined using a hemocytometer. The inhibitory effect of resveratrol on cell proliferation was also tested using a colony formation assay as described by Smith et al.4 with few modifications. In brief, cells were grown in 60-mm dishes for 14 days in the presence or absence of various concentrations of resveratrol. Three dishes were seeded for each treatment, at a concentration of 200 cells per dish. Cells were then fixed and stained and total colony number and cell numbers per colony scored. In other experiments PASMC grown in a 96 well plate were stimulated in vitro with TGFβ (10 ng/mL), IL-1β (10 ng/mL) or IL-6 (10 ng/mL) in the presence or absence of resveratrol (1 to 30 μmol/L). Cell proliferation was assessed by measuring the fluorescence of the DNA-binding CyQUANT dye (Invitrogen) in cell lysates with a fluorescence microplate reader. The effect of resveratrol on apoptotic cell death (annexin V and TUNEL staining) in PASMC and pulmonary arterial endothelial cells (PAEC) were quantified by flow cytometry (Guava Easycyte). Resveratrol-induced inhibition of the cell cycle in PASMC was assessed by the Guava Cell Cycle Assay by flow cytometry.

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Transient transfection and luciferase assays

Effect of PDGF (10 ng/mL, for 24 h) and resveratrol (10 µmol/L, 24 h pre-treatment) on NF-κΒ activity in PASMCs was tested by a reporter gene assay as described5, 6. We used a NF-κΒ reporter comprised of an NF-κB response element upstream of firefly luciferase (NF-κΒ-Luc, Stratagene) and a renilla luciferase plasmid under the control of the CMV promoter (as an internal control). Transfections in PASMCs were performed using the Amaxa Nucleofector technology (Amaxa, Gaithersburg, MD), as we have previously reported5, 6. Firefly and renilla luciferase activities were assessed after 24 h using the Dual Luciferase Reporter Assay Kit (Promega) and a luminometer. Functional studies Acetylcholine-induced relaxation was assessed in segments of small intrapulmonary arteries pre-contracted by phenylephrine (1 μmol/L), as described7. Relaxation of arterial segments to the NO donor S-nitrosopenicillamine (SNAP, from 10-9 to 3x10-5 mol/L mol/L) was also evaluated. In separate experiments the vasorelaxant effect of in vitro administration of resveratrol (10-7 to 3x10-4 mol/L) was also assessed. Measurement of tissue H2O2 and O2

.- production Production of O2

.- in isolated intrapulmonary arteries, RV, and coronary arteries isolated from the RV was assessed using the dihydroethidine staining method, as described8. Untreated tissues were used for determining levels of autofluorescence. The cell-permeant oxidative fluorescent indicator dye C-H2DCFDA (5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester, Invitrogen, Carlsbad CA) was used to assess peroxide levels in isolated intrapulmonary arteries, as we reported9. In addition, pressure overload-induced O2

.- generation in pieces of the right and left ventricles was measured by the lucigenin (10 μmol/L) chemiluminescence (CL) method, as reported5. Quantitative real-time PCR

Total RNA was isolated with Mini RNA Isolation Kit (Zymo Research, Orange, CA) and was reverse transcribed using Superscript III RT (Invitrogen) as described previously5-7, 10.. Real time RT-PCR technique was used to analyze mRNA expression using the Strategen MX3000, as reported2, 10, 11. Efficiency of the PCR reaction was determined using dilution series of a standard vascular sample. Quantification was performed using the efficiency-corrected ΔΔCT method. The housekeeping genes GAPDH and β-actin were used for internal normalization. Fidelity of the PCR reaction was determined by melting temperature analysis and visualization of product on a 2% agarose gel.

Western blotting Western blotting was performed as described8, 10 to assess protein expression of Nox-1, gp91phox and eNOS in small intrapulmonary arteries. Assessment of leukocyte infiltration Immunolabeling on lung sections was performed for the rat monocyte/macrophage marker ED1. The number of ED1-positive cells was determined in 10 randomly chosen fields. Histological analysis was performed in a blinded fashion by two observers. Data analysis

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Data were normalized to the respective control mean values and are expressed as means ± S.D. or S.E.M. Statistical analyses of data were performed by Student’s t-test or by two-way ANOVA followed by a Tukey's post hoc test, as appropriate. p<0.05 was considered statistically significant.

References for Online Supplement 1. Pearson KJ, Baur JA, Lewis KN, Peshkin L, Price NL, Labinskyy N, Swindell WR, Kamara

D, Minor RK, Perez E, Jamieson HA, Zhang Y, Dunn SR, Sharma K, Pleshko N, Woollett LA, Csiszar A, Ikeno Y, Le Couteur D, Elliott PJ, Becker KG, Navas P, Ingram DK, Wolf NS, Ungvari Z, Sinclair DA, de Cabo R. Resveratrol Delays Age-Related Deterioration and Mimics Transcriptional Aspects of Dietary Restriction without Extending Life Span. Cell Metab. 2008;8:157-168.

2. Ungvari ZI, Orosz Z, Labinskyy N, Rivera A, Xiangmin Z, Smith KE, Csiszar A. Increased mitochondrial H2O2 production promotes endothelial NF-kB activation in aged rat arteries. Am J Physiol Heart Circ Physiol. 2007;293:H37-47.

3. Wang Z, Chen Y, Labinskyy N, Hsieh TC, Ungvari Z, Wu JM. Regulation of proliferation and gene expression in cultured human aortic smooth muscle cells by resveratrol and standardized grape extracts. Biochemical and biophysical research communications. 2006;346:367-376.

4. Smith JR, Pereira-Smith OM, Schneider EL. Colony size distributions as a measure of in vivo and in vitro aging. Proc Natl Acad Sci U S A. 1978;75:1353-1356.

5. Csiszar A, Labinskyy N, Podlutsky A, Kaminski PM, Wolin MS, Zhang C, Mukhopadhyay P, Pacher P, Hu F, de Cabo R, Ballabh P, Ungvari Z. Vasoprotective effects of resveratrol and SIRT1: attenuation of cigarette smoke-induced oxidative stress and proinflammatory phenotypic alterations. Am J Physiol Heart Circ Physiol. 2008;294:H2721-2735.

6. Csiszar A, Smith K, Labinskyy N, Orosz Z, Rivera A, Ungvari Z. Resveratrol attenuates TNF-{alpha}-induced activation of coronary arterial endothelial cells: role of NF-{kappa}B inhibition. Am J Physiol. 2006;291:H1694-1699.

7. Csiszar A, Labinskyy N, Jo H, Ballabh P, Ungvari Z. Differential proinflammatory and prooxidant effects of bone morphogenetic protein-4 in coronary and pulmonary arterial endothelial cells. Am J Physiol Heart Circ Physiol. 2008;295:H569-577.

8. Csiszar A, Ahmad M, Smith KE, Labinskyy N, Gao Q, Kaley G, Edwards JG, Wolin MS, Ungvari Z. Bone morphogenetic protein-2 induces proinflammatory endothelial phenotype. Am J Pathol. 2006;168:629-638.

9. Ungvari Z, Orosz Z, Rivera A, Labinskyy N, Xiangmin Z, Olson S, Podlutsky A, Csiszar A. Resveratrol increases vascular oxidative stress resistance. American journal of physiology. 2007;292:H2417-2424.

10. Csiszar A, Smith KE, Koller A, Kaley G, Edwards JG, Ungvari Z. Regulation of bone morphogenetic protein-2 expression in endothelial cells: role of nuclear factor-kappaB activation by tumor necrosis factor-alpha, H2O2, and high intravascular pressure. Circulation. 2005;111:2364-2372.

11. Csiszar A, Labinskyy N, Smith K, Rivera A, Orosz Z, Ungvari Z. Vasculoprotective effects of anti-TNFalfa treatment in aging. The American journal of pathology. 2007;170:388-698.

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Figure S1, A: Representative fluorescent images showing increased nuclear dihydroethidine staining in the right ventricle (RV) of MCT treated rats (middle; 21 days post-MCT), as compared to controls (left). Resveratrol treatment normalized O2

.- production in the RV. B: RV hypertrophy in MCT-treated rats is associated with increased O2

.- production (assessed by the lucigenin chemiluminescence (CL) method) in the RV, but not in the left ventricle (LV). Resveratrol treatment prevented oxidative stress in the RV. Data are mean ± S.E.M. *P<0.05 vs. control, #P<0.05 vs. no resveratrol.; (n=6 for each group). C: Relaxation of intramural coronary arteries from the RV of control rats and MCT-injected rats in response to acetylcholine. The effect of chronic resveratrol treatment on vascular relaxations is also shown. Data are mean ± S.E.M. *P<0.05 vs. control, #P<0.05 vs. no resveratrol; (n=6 for each group). D: Representative fluorescent images showing increased nuclear dihydroethidine staining in the coronary arteries isolated from the RV of MCT treated rats (middle), as compared to controls (left), which was prevented by resveratrol treatment (right). E: endothelium, M: media, Ad: adventitia.

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Figure S2, panel A: Medial hypertrophy (red fluorescence: α-smooth muscle actin) of small pulmonary arteries (d: 50-150 µm) was associated with an increased number of proliferating vascular cells in MCT-induced pulmonary hypertension. Immunolabeling for PCNA (green nuclei are PCNA-positive cells; arrows) revealed increased proliferation in MCT-challenged animals (as compared with controls). Prevention of medial hypertrophy induced by resveratrol was attributed to reduced SMC proliferation.. B: TUNEL assay results showing that neither MCT-PAH nor resveratrol increases apoptotic cell death in small pulmonary arteries (left: arrows point to non-vascular TUNEL positive cell nuclei (green); middle: overlay with nuclear staining (blue); right: overlay with α-smooth muscle actin (red)). Scale bars: 25 μm

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Figure S3: To determine the effect of resveratrol on PDGF-induced NF-κB activation, we transiently transfected PASMCs with a NF-κB-driven reporter gene construct and then pre-treated the cells with resveratrol (10 µmol/L, for 24 h) followed by stimulation with PDGF (10 ng/mL, for 24 h). A significant increase in luciferase activity over the vector control was noted upon stimulation

with PDGF in the absence of resveratrol. Pre-treatment of PASMCs with resveratrol prevented

PDGF-induced NF-κB activation. Data are mean ± S.E.M. *P<0.05 vs. control, #P<0.05 vs. no resveratrol.

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Figure S4. mRNA expression of BMP-4, BMP receptors (BMPR1a, BMPR1b, BMPR2, ACVR2a, ACVR2b, ACVR1, ACVRL1), BMP antagonists (chordin, noggin) and SMAD signaling molecules (SMAD1/4/5/6/7) in the lung of control, MCT-treated and MCT plus resveratrol treated rats. Analysis of mRNA expression was performed by real-time QRT-PCR. Data are mean ± S.E.M. *P<0.05 vs. control, #P<0.05 vs. no resveratrol.

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Figure S5. mRNA expression of Nox-1 (A), gp91phox (B) and Nox-4 (C) in small intrapulmonary arteries from control, MCT-treated and MCT plus resveratrol treated rats. Analysis of mRNA expression was performed by real-time QRT-PCR. Data are presented as mean ± S.E.M. *P<0.05 vs. control, #P<0.05 vs. no resveratrol.

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Figure S6. Expression of HO-1 mRNA in small intrapulmonary arteries from control, MCT-treated and MCT plus resveratrol treated rats. β-actin was used for normalization.

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