doi:10.1152/ajpendo.00291.2009 297:685-694, 2009. First published Jun 16, 2009;Am J Physiol Endocrinol Metab

Vincenzo Benagiano, Michael J. Quon and Monica Montagnani Edy Tiravanti, Giuseppe Colantuono, Antonio Federici, Loredana Lorusso, Maria A. Potenza, Sara Gagliardi, Leonarda De Benedictis, Addolorata Zigrino,

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Treatment of spontaneously hypertensive rats with rosiglitazone amelioratescardiovascular pathophysiology via antioxidant mechanisms in the vasculature

Maria A. Potenza,1 Sara Gagliardi,1 Leonarda De Benedictis,1 Addolorata Zigrino,1 Edy Tiravanti,2

Giuseppe Colantuono,2 Antonio Federici,1 Loredana Lorusso,3 Vincenzo Benagiano,3 Michael J. Quon,4

and Monica Montagnani11Departments of Pharmacology and Human Physiology, Medical School, 2Emergency and Transplants, and 3Human Anatomyand Histology, University of Bari, Bari, Italy; and 4Diabetes Unit, National Center for Complementary and AlternativeMedicine, National Institutes of Health, Bethesda, Maryland

Submitted 6 May 2009; accepted in final form 14 June 2009

Potenza MA, Gagliardi S, De Benedictis L, Zigrino A, Tira-vanti E, Colantuono G, Federici A, Lorusso L, Benagiano V, QuonMJ, Montagnani M. Treatment of spontaneously hypertensive ratswith rosiglitazone ameliorates cardiovascular pathophysiology viaantioxidant mechanisms in the vasculature. Am J Physiol EndocrinolMetab 297: E685–E694, 2009. First published June 16, 2009;doi:10.1152/ajpendo.00291.2009.—Oxidative stress contributes tocardiovascular complications of diabetes, in part, by reducing thebioavailability of nitric oxide (NO). We investigated the mechanismswhereby the insulin sensitizer rosiglitazone may ameliorate oxidativestress in the vasculature of spontaneously hypertensive rats (SHR).Nine-week-old SHR were treated by gavage for 7 wk with rosiglita-zone (5 mg �kg�1 �day�1) or vehicle control. Treatment of SHR withrosiglitazone lowered systolic blood pressure, reduced fasting plasmainsulin and asymmetrical dimethylarginine, and increased insulinsensitivity (when compared with vehicle treatment). In vessel homog-enates and serum from rosiglitazone-treated SHR, SOD activity wasenhanced, while 8-iso-PGF2� (lipid peroxidation product) was re-duced (when compared with samples from vehicle-treated SHR).Moreover, expression of p22phox (catalytic subunit of NADPHoxidase) as well as nitrotyrosine and superoxide content were allreduced in the aortas of rosiglitazone-treated SHR. In mesentericvascular beds (MVB) isolated ex vivo from rosiglitazone-treatedSHR, NO-dependent vasodilator actions of insulin were improvedwhen compared with MVB from vehicle-treated SHR. Acute pretreat-ment of MVB from vehicle-treated SHR with apocynin (NADPHoxidase inhibitor) enhanced vasodilator actions of insulin (resultscomparable to those in MVB from rosiglitazone-treated SHR). InLangendorff heart preparations from rosiglitazone-treated SHR, isch-emia/reperfusion injury caused infarcts 40% smaller than in heartsfrom vehicle-treated SHR. Acute pretreatment of hearts from vehicle-treated SHR with apocynin produced similar results. Finally, rosigli-tazone treatment of endothelial cells in primary culture reducedsuperoxide induced by insulin-resistant conditions. We conclude thatrosiglitazone therapy in SHR increases SOD activity and decreasesp22phox expression in the vasculature to reduce oxidant stress leadingto an improved cardiovascular phenotype.

oxidative stress

RECIPROCAL RELATIONSHIPS between endothelial dysfunction andinsulin resistance help explain the clustering of cardiovasculardiseases including hypertension, coronary heart disease, andatherosclerosis with diabetes, obesity, and other insulin-resis-tant conditions (34, 50). Endothelial dysfunction, characterized

by reduced bioavailability of nitric oxide (NO), contributes tocardiovascular complications of diabetes (59). NO bioavail-ability is reduced when NO reacts with superoxide anion (O2

�)to generate peroxynitrites (ONOO�), strong oxidants that com-promise cellular function by irreversibly altering nucleic acids,lipids, and proteins (5). Under physiological conditions, O2

�

produced by NADPH oxidases is scavenged by antioxidantenzymes including SOD (23, 43). Imbalance in cell redoxstatus resulting from excessive production of reactive oxygenspecies (ROS) and/or insufficient antioxidant capacity pro-motes both endothelial dysfunction and insulin resistance (4,12, 13, 20, 31). Therefore, therapeutic strategies that diminishoxidative stress by restoring physiological redox balance areattractive treatment approaches for diseases characterized byboth endothelial dysfunction and insulin resistance (see Ref. 36for review).

Thiazolidinediones (TZD) are synthetic ligands for peroxi-some proliferator-activated receptor-� (PPAR�) that act asinsulin sensitizers in metabolic targets including skeletal mus-cle, liver, and adipose tissue (74). In addition, TZDs haveanti-inflammatory actions in macrophages and vascular endo-thelium that may oppose atherosclerosis (35, 48, 76). This isreflected in the ability of TZD treatment to reduce serumC-reactive protein (63) and carotid intima-media thickness(45). Moreover, TZDs have direct actions in vascular endothe-lium to increase expression of endothelial NO synthase (eNOS)and production of NO (8, 22). In spontaneously hypertensiverats (SHR), a genetic model of hypertension with features ofoverweight and insulin resistance resembling human metabolicsyndrome (60), treatment with rosiglitazone restores the bal-ance between phosphatidylinositol 3-kinase and MAPK-de-pendent branches of insulin-signaling pathways in the vascularendothelium resulting in improved endothelial function, de-creased circulating levels of endothelin-1 (ET-1), and reducedsystemic blood pressure (61). Thus the ability of rosiglitazoneto improve insulin action in vascular endothelium may con-tribute to its antihypertensive effects in diabetic patients (65).

Additional mechanisms by which TZDs exert beneficialvascular effects may involve decreased oxidative stress (7). Inprimary endothelial cells, TZDs directly stimulate both in-creased activity and expression of Cu/Zn-SOD (26). In humanumbilical vein endothelial cells, rosiglitazone treatment de-creases expression of NADPH oxidase mRNA as well asNADPH oxidase-dependent O2

� production stimulated by in-cubation with high concentrations of glucose (10, 27). Indiabetic mice, rosiglitazone treatment improves vasodilatoractions of ACh in coronary arterioles by reducing NADPH

Address for reprint requests and other correspondence: M. Montagnani,Dept. of Pharmacology and Human Physiology - Section of Pharmacology,Medical School, Univ. of Bari, Policlinico–Piazza G. Cesare, 11, 70124 Bari,Italy (e-mail: monica@farmacol.uniba.it).

Am J Physiol Endocrinol Metab 297: E685–E694, 2009.First published June 16, 2009; doi:10.1152/ajpendo.00291.2009.

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oxidase-derived O2� (3). Thus rosiglitazone therapy may im-

prove cardiovascular functions through direct actions in thevascular endothelium that decrease oxidative stress and in-crease NO bioavailability. In the present study, we investigatedthe mechanisms whereby rosiglitazone therapy reduces oxida-tive stress in the vasculature to ameliorate endothelial dysfunc-tion and improve cardiovascular function in SHR rats.

METHODS

Animal experiments. All procedures were performed in accordancewith Guidelines and Authorization for the Use of Laboratory Animals(Italian Government, Ministry of Health). Male spontaneously hyper-tensive rats (SHR) and age-matched normotensive Wistar-Kyoto(WKY) control rats were from Charles River (Calco, Italy). Nine-week old SHR were randomized into two groups and treated daily for 7 wkby gavage with vehicle alone or rosiglitazone (5 mg �kg�1 �day�1). Thisdose of rosiglitazone was chosen according to previous studies whereit proved effective in lowering blood pressure, improving endothelialfunction, and decreasing insulin resistance in SHR (61). Nine-week-old WKY controls were given vehicle daily for 7 wk. Daily foodintake was measured by weighing the amount of standard chow leftcompared with the amount provided the previous day. Daily waterintake was measured by calculating the difference between the waterleft in each bottle with respect to the water provided the previous day.Systolic blood pressure was measured using a tail-cuff BP-2000 bloodpressure analysis system (Visitech System; Apex) as described pre-viously (60). Body weight was measured daily during the treatmentperiod.

After 7 wk of treatment, blood samples obtained from rats fastedovernight were collected in test tubes with or without EDTA andcentrifuged (1,000 g, 10 min, 4°C). Serum or plasma samples werefrozen at �70°C until further analysis. Animals were then euthanizedbefore removal of vascular tissue. Plasma insulin and asymmetricaldimethylarginine (ADMA) were measured using ELISA kits (LincoResearch and Alexis Biochemicals, respectively). Blood glucose con-centrations were determined using a diagnostic glucometer (Accu-Chek Active; Roche Diagnostics, Germany). Insulin sensitivity wasestimated using the quantitative insulin-sensitivity check index{QUICKI � 1/[log (insulin) � log (glucose)]} (32).

Reagents. Noradrenaline hydrochloride, ACh, apocynin, SOD, andN�-nitro-L-arginine methyl ester (L-NAME) were from Sigma-Al-drich. Insulin was from Novo Nordisk, and rosiglitazone was fromAlexis Biochemicals. Final dilutions of all drugs were prepared inmodified Krebs-Henseleit solution immediately before use. Stocksolutions of rosiglitazone were in ethanol (1%). Final dilutions werein drinking water (�4� dilution). Vehicle-treated WKY and SHRreceived the same amount of ethanol as drug-treated animals.

Evaluation of vascular function ex vivo. Mesenteric vascular beds(MVB) were isolated and removed from rats after treatment withvehicle or rosiglitazone for 7 wk as described previously (60).

Evaluation of myocardial function in isolated hearts. Hearts fromSHR treated for 7 wk with vehicle or rosiglitazone were isolated andperfused according to the Langendorff technique as described previ-ously (62).

Measurement of SOD activity in serum and tissue homogenates.Total SOD activity in serum and tissue homogenates was measuredusing an immunoenzymatic SOD activity assay kit (Cayman Chemi-cals). Tissues were processed according to instructions provided bythe manufacturer.

Oxidative fluorescence microtopography. Dihydroethidium (DHE;Molecular Probes) was used to evaluate O2

� production in cells and invessels in situ as described previously (6, 52). Bovine aortic endothe-lial cells (BAECs) in primary culture from Lonza (Basel, Switzerland)were maintained in EGM-MV medium with glucose at either normal(4.5 mM) or high concentration (HG; 25 mM) for 24 h. Some groups

of cells under HG conditions were also treated with rosiglitazone (20M) for an additional 24 h before incubation with DHE (3 M, 30min, 37°C). Unfixed, frozen aortas cut into 12-m-thick sections wereprehydrated with PBS (10 min) and then incubated with DHE. Cellsor sections were washed, stained with DAPI (Santa Cruz Biotechnol-ogy, Santa Cruz, CA), and mounted on a coverslip. In each experi-ment, parallel samples were pretreated with SOD (180 U/ml, 30 min).Results visualized using a Zeiss Axiovert TS100 epifluorescent mi-croscope with appropriate filters were captured with a CCD camerausing identical settings for each image acquisition. Densitometricanalysis for merged DHE and DAPI fluorescence was performedusing AxioVision Rel 4.6.3 software. Fluorescence was quantified bycounting the number of pixels in identical fields for each group, anddata were normalized to results from control samples (6).

Measurements of lipid peroxidation in tissue homogenates andserum. Levels of 8-iso-PGF2� were measured in homegenates ofMVB or aorta or in serum using a commercially available ELISA kit(Assay Designs, Ann Arbor, MI) according to instructions providedby the manufacturer.

Immunohistochemistry. Thoracic aortas embedded in paraffin werecut into 5-m sections. Deparaffinized sections heated to unmaskantigens were subsequently blocked (PBS, 1% BSA, 5% FCS, and10% donkey normal serum, 60 min) and then incubated overnight at4°C with primary antibodies against p22phox (Santa Cruz Biotech-nology) or nitrotyrosine (Upstate Biotechnology, Lake Placid, NY).Sections subsequently incubated with donkey anti-rabbit IgG (60 min)and horseradish streptavidin-peroxidase (Vector Labs; 30 min) werecounterstained with Mayer’s hematoxylin (Microstain).

Measurement of p22phox mRNA. Total RNA was prepared fromBAECs by homogenization in the Trizol reagent (Life Technologies)and mRNA quantified using RT-PCR according to standard methods.The following oligonucleotides were used as forward and reverseprimers for bovine p22phox mRNA, respectively: 5-TAT TGG TCTGCC TGC TGG AA-3 and 5-AGG ACC TTC AAA GCT TCAGC-3; 5-GGG AGA AGA TGA CTC AGA TC-3 and 5-AAGCTG TAG CCT CGT TCT GT-3 for �-actin (MWG Synthesis).

Statistical analysis. Results were expressed as means � SE of nexperiments (n � number of rats). Two-way ANOVAs for repeatedmeasures followed by Bonferroni’s correction or Student’s t-tests(paired or unpaired) were used where appropriate. Values of P 0.05were considered to indicate statistical significance.

RESULTS

Treatment of SHR with rosiglitazone improves metabolicand cardiovascular phenotypes. Consistent with our previousfindings (60, 61), 16-wk-old SHR that had undergone treatmentwith vehicle alone for 7 wk were overweight and hypertensivewith substantial fasting hyperinsulinemia (but normoglycemia)when compared with age-matched WKY control rats (Table 1).Insulin sensitivity as assessed by the surrogate index QUICKI(42, 49) was significantly reduced in vehicle-treated SHR.Circulating levels of ADMA (endogenous inhibitor of NOsynthase and circulating marker of oxidative stress; Refs. 70,72) tended to be higher in vehicle-treated SHR when comparedwith WKY rats (although this did not quite reach statisticalsignificance, P � 0.08). When compared with vehicle-treatedSHR, SHR treated with rosiglitazone (5 mg �kg�1 �day�1) for 7wk had comparable body weights and fasting glucose levels.Although a slight tendency to increased food and water intakewas observed in rosiglitazone-treated SHR, no significant dif-ference in these parameters was found between this group andvehicle-treated SHR for the period of our study (Table 1).

In accordance with its known insulin-sensitizing actions,treatment of SHR with rosiglitazone reduced fasting plasma

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insulin to levels that were even below those in WKY rats (P 0.03). This reflected enhanced insulin sensitivity that wasgreater than that of WKY rats (assessed by QUICKI). Treat-ment of SHR with rosiglitazone also caused a significantreduction in systolic blood pressure when compared withvehicle-treated SHR. Moreover, plasma ADMA levels weremarkedly reduced to levels below those observed in WKY rats(Table 1), suggesting that rosiglitazone treatment caused asubstantial decrease in oxidative stress in SHR. In normoten-sive control WKY rats, in vivo treatment with rosiglitazone didnot significantly affect any of the physiological parametersevaluated (data not shown).

We next evaluated vascular function in MVB isolatedex vivo from SHR or WKY rats treated with vehicle orrosiglitazone for 7 wk (Fig. 1). As we reported previously (60),insulin-stimulated vasodilation was significantly impaired inMVB isolated from vehicle-treated SHR (when compared withMVB from WKY rats; Fig. 1). By contrast, in MVB isolatedfrom rosiglitazone-treated SHR, the ability of insulin to stim-ulate vasorelaxation was greater than that observed in MVBisolated from vehicle-treated SHR (Fig. 1). Indeed, at lowerdoses of insulin (1–10 nM), insulin-stimulated vasorelaxationin MVB from rosiglitazone-treated SHR was even greater thanthat observed in MVB from WKY rats. Taken together, thephysiological and biochemical changes observed in SHRtreated with rosiglitazone suggest that therapy with this insulinsensitizer ameliorates both metabolic and hemodynamic de-rangements in a genetic model of hypertension with features ofhuman metabolic syndrome.

Rosiglitazone treatment reduces oxidative products, in-creases SOD activity, and decreases p22phox expression in thecirculation and vasculature of SHR. Oxidative stress may playa key role in cardiovascular pathophysiology associated withinsulin resistance (36). Since rosiglitazone treatment of SHRsubstantially reduced circulating ADMA levels (Table 1), wehypothesized that rosiglitazone treatment of SHR improvescardiovascular function, in part, by reducing oxidative stress in

the circulation and vasculature. When compared with WKYrats, the levels of 8-iso-PGF2� (lipid peroxidation product)were elevated in both serum and MVB homogenates fromvehicle-treated SHR (Table 2). Moreover, activity of SOD (anantioxidant enzyme that scavenges O2

�) was reduced in bothserum and in homogenates of vascular tissues from vehicle-treated SHR when compared with samples from WKY rats.Treatment of SHR with rosiglitazone for 7 wk reduced 8-iso-PGF2� levels in both serum and MVB from SHR when com-

Table 1. Physiological and biochemical parameters obtainedfrom 16-wk-old WKY and SHR rats treated with vehicleor rosiglitazone (5 mg �kg�1 �day�1) for 7 wk

Rat Strain and Treatment

Parameters WKY Vehicle Tx SHR Vehicle TxSHR

Rosiglitazone Tx

Body weight, g 330�10 370�4* 385�3*Daily food intake, g 33�2 35�6 39�8Daily water intake, ml 30�5 29�4 34�9Systolic blood

pressure, mmHg 138�3 220�7* 189�9‡ADMA, M 1.60�0.10 1.95�0.18 0.92�0.07*Fasting blood glucose,

mg/dl 114�3 108�2 104�3Fasting plasma

insulin, ng/ml 1.1�0.2 13.9�2.0† 0.8�0.1§QUICKI 0.283�0.007 0.218�0.003† 0.331�0.004†§

Data shown are means � SE for each group of rats (n � 24 for each group).Statistical comparisons were made using two-tailed unpaired Student’s t-test.Parameters are as described in MATERIALS AND METHODS. ADMA, asymmet-rical dimethylarginine; Tx, treatment. *P 0.001 vs. Wistar-Kyoto(WKY); †P 0.0001 vs. WKY; ‡P 0.001 vs. spontaneously hyperten-sive rats (SHR; vehicle); §P 0.0001 vs. SHR (vehicle).

Fig. 1. Chronic treatment (Tx) of spontaneously hypertensive rats (SHR) withrosiglitazone improved vasodilator actions of insulin. Mesenteric vascular beds(MVB) were isolated from Wistar-Kyoto (WKY) and SHR rats treated dailywith vehicle or rosiglitazone (5 mg �kg�1 �day�1) for 7 wk as described inMATERIALS AND METHODS. Dose-response curves for insulin-induced vasore-laxation obtained in MVB from WKY (F) and SHR (■) treated with vehicle orSHR treated with rosiglitazone (�) are shown. Results are means � SE of atleast 8 independent experiments for each group. Vasodilator actions of insulinwere significantly impaired in MVB from vehicle-treated SHR (vs. WKY, P 0.005). Vasodilator actions of insulin were substantially improved in MVBfrom SHR after treatment of SHR with rosiglitazone for 7 wk (vs. SHR, P 0.001).

Table 2. 8-iso-PGF2� concentration and SOD activityin serum and vasculature of 16-wk-old WKY and SHR ratstreated with vehicle or rosiglitazone (5 mg �kg�1 �day�1)for 7 wk

Rat Strain and Treatment

Parameters WKY Vehicle Tx SHR Vehicle Tx SHR Rosiglitazone Tx

Serum 8-iso-PGF2�,ng/ml 5.2�1.0 40.8�7.6* 22.7�4.0†

Serum SOD activity,U/ml 17.4�1.9 7.3�1.2* 9.6�0.5‡

8-iso-PGF2� in MVB,ng/mg wet tissue 11.5�1.8 27.6�3.7* 21.8�0.3†

SOD activity inMVB, U/g wettissue 23.2�1.5 10.8�3.7* 39.5�11.6†

SOD activity in aorta,U/g wet tissue 89.4�11.1 32.4�5.9* 82.8�7.7‡

Data shown are means � SE for each group (n � 6 for each group).Statistical comparisons were made using two-tailed unpaired Student’s t-test.Parameters are as described in MATERIALS AND METHODS. MVB, mesentericvascular beds. *P 0.001 vs. WKY; †P 0.05 vs. SHR (vehicle); ‡P 0.001 vs. SHR (vehicle).

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pared with samples from vehicle-treated SHR. SOD activity inserum as well as in aorta and MVB homogenates was increasedby treatment of SHR with rosiglitazone (when compared withvehicle-treated SHR). Along these same lines, increased pro-duction of O2

� evident in aortic rings from vehicle-treated SHRwas absent in aortic rings from rosiglitazone-treated SHR (Fig. 2).That is, markedly increased nuclear staining in DHE-treatedsamples (indicating enhanced O2

� production) was observed inaortic rings from vehicle-treated SHR when compared withthose from WKY rats. DHE-treated aortic rings from rosigli-tazone-treated SHR had an appearance similar to that of sam-ples from WKY rats and also to that of aortic rings fromvehicle-treated SHR acutely preincubated in vitro with SOD.Furthermore, as assessed by immunohistochemistry, expres-sion of p22phox (catalytic subunit of NADPH oxidase thatproduces O2

�) and levels of nitrotyrosine (increased by perox-initrite) were both substantially increased in vessels fromvehicle-treated SHR when compared with samples from WKYrats (Fig. 3). By contrast, immunostaining of vessels fromrosiglitazone-treated SHR for p22phox and nitrotyrosineyielded results that were similar to those observed in aorticsections from WKY. Taken together, these findings suggestthat reduced oxidative stress in the vasculature may underlieimprovements in the cardiovascular phenotype of SHR result-ing from rosiglitazone therapy.

Effects of chronic rosiglitazone therapy to improve cardio-vascular function in SHR are mimicked by acute inhibition ofNADPH oxidase. Impaired insulin-mediated vasodilation inMVB from SHR is related to decreased endothelial NO bio-

availability (60). This may be secondary to increased oxidantstress resulting from production of ROS. To help evaluate thispossibility, insulin-stimulated vasorelaxation in MVB fromvehicle-treated SHR was assessed in the absence and presenceof acute pretreatment with apocynin (an inhibitor of NADPHoxidase; Fig. 4A). Importantly, acute pretreatment of MVBfrom vehicle-treated SHR with apocynin significantly aug-mented insulin-dependent vasodilation (Fig. 4A). This effect ofapocynin was completely inhibited in the presence of L-NAME, acompetitive inhibitor for NO synthase (Fig. 4A). These resultssuggest that increased O2

� production from vascular NADPHoxidase may underlie endothelial dysfunction in SHR. Whenthese experiments were repeated using MVB from rosiglita-zone-treated SHR, the maximal insulin-stimulated vasorelax-ation (in the absence of apocynin pretreatment) was compara-ble to that seen in MVB from vehicle-treated SHR acutelypretreated with apocynin (Fig. 4B). Apocynin pretreatment ofMVB from rosiglitazone-treated SHR did not further improveendothelial function (Fig. 4B). However, in the presence ofL-NAME, insulin-stimulated vasorelaxation was substantiallyimpaired in MVB from rosiglitazone-treated SHR (Fig. 4B),resembling the results in MVB from vehicle-treated SHR in theabsence of apocynin pretreatment (Fig. 4A). Thus acute pre-treatment of MVB from vehicle-treated SHR with apocyninmimics the improved NO-dependent vasorelaxation observedin MVB from rosiglitazone-treated SHR.

We next investigated whether a 7-wk therapy with rosigli-tazone may also protect against myocardial ischemia/reperfu-sion injury. Hearts isolated from SHR treated for 7 wk with

Fig. 2. Treatment of SHR with rosiglitazone for 7 wk reduces superoxide production in aorta. In situ detection of O2� in aortic rings was performed by visualizing

red fluorescence of tissue sections loaded with dihydroethidium (DHE) as described in MATERIALS AND METHODS. Representative fluorescent photomicrographsfrom 3 independent sets of experiments are shown. Red fluorescence in sections loaded with DHE is indicative of O2

� production (top); blue fluorescence insections stained with DAPI indicates cell nuclei (middle). Merged images of DHE and DAPI staining are shown at bottom.

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either vehicle or rosiglitazone were perfused according to themethod of Langendorff (Fig. 5). Heart rate and heart weightwere comparable among all groups of animals (data notshown). Isolated hearts from vehicle-treated and rosiglitazone-treated SHR had comparable function during aerobic perfusionas assessed by a variety of hemodynamic parameters (Fig. 5, B,C, and D at baseline time point B). In hearts from vehicle-treated SHR, 30 min of ischemia followed by 120 min reper-fusion resulted in infarction of �50% of left ventricular vol-ume with severely impaired cardiac function (Fig. 5). Bycontrast, myocardial infarct size was �40% smaller in heartsfrom rosiglitazone-treated SHR (Fig. 5A). Consistent withreduction in infarct size, postischemic recovery of ventricularfunction was also improved in hearts from rosiglitazone-treatedSHR, as assessed by coronary flow, left-ventricular end-dia-stolic pressure, and left ventricular developed pressure (Fig. 5,B, C, and D). Acute preincubation of hearts from vehicle-treated SHR with apocynin resulted in reduction in infarct sizeand improvement in left-ventricular end-diastolic pressure andleft ventricular developed pressure that were comparable tothat seen in hearts from rosiglitazone-treated SHR (Fig. 5).Thus as with endothelial function (Fig. 4), the effects ofrosiglitazone therapy to protect against myocardial ischemia/reperfusion injury were mimicked by acute treatment of heartsin vitro with apocynin. This provides additional support for thehypothesis that rosiglitazone therapy in SHR improves cardio-vascular pathophysiology by reducing oxidant stress.

Direct effects of rosiglitazone to reduce ROS in vascularendothelium. To determine whether rosiglitazone has directactions in vascular endothelium to reduce oxidative stress, we

evaluated O2� production in primary endothelial cells incubated

with normal (4.5 mM) or high (25 mM) concentrations ofglucose in the absence or presence of rosiglitazone treatment(Fig. 6). Incubation of BAECs with high glucose concentra-tions increased production of nuclear O2

� levels (as assessed bythe DHE-to-DAPI fluorescence ratio; Ref. 6). Importantly,treatment of BAECs with rosiglitazone (20 M) for 24 hreduced nuclear DHE staining to levels similar to those ob-tained in BAEC cultured with high glucose and treated acutelyin vitro with exogenous SOD (180 U, 30 min). We observedsimilar results in cells where production of O2

� was stimulatedby exposure to high insulin concentrations plus wortmannin(conditions mimicking insulin resistance with compensatoryhyperinsulinemia; Ref. 47; Fig. 6, D and E). In addition tosuperoxide levels, the mRNA expression of p22phox wasincreased in BAEC cells exposed to insulin-resistant condi-tions. Interestingly, in vitro treatment with rosiglitazone sub-stantially reduced mRNA levels of p22phox under these con-ditions (Fig. 6C). Thus rosiglitazone has direct antioxidantactions in the vascular endothelium that may contribute toimproved cardiovascular phenotypes observed in rosiglita-zone-treated SHR.

DISCUSSION

Reciprocal relationships between insulin resistance and en-dothelial dysfunction underlie the clustering of hypertension,overweight, endothelial dysfunction, and insulin resistanceobserved in both SHR rats and humans with the metabolicsyndrome (34, 50). This helps to explain why SHR or humans

Fig. 3. Expression of p22phox and nitrotyrosine containing proteins in aortas from WKY and SHR rats treated with vehicle or rosiglitazone (5 mg �kg�1 �day�1)for 7 wk. Aortas from WKY and SHR rats were prepared as described in MATERIALS AND METHODS. Representatives photomicrographs of aortas immunostainedwith primary antibodies against p22phox or nitrotryosine are shown at �400 magnification (scale bar � 200 m).

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treated with ACE inhibitors, ARBs (antihypertensive agentsthat improve endothelial dysfunction), or TZDs (antidiabeticagents that improve insulin sensitivity) show simultaneousimprovement in both cardiovascular and metabolic phenotypes(37–39, 55, 61). We previously demonstrated that rosiglitazonetherapy in SHR restores the balance between phosphatidylino-sitol 3-kinase-dependent and MAPK-dependent branches ofinsulin-signaling pathways in vascular endothelium to reduceendothelial dysfunction (61). In the present study, we focusedon the ability of rosiglitazone to reduce oxidative stress in thevasculature as an additional mechanism contributing to bene-ficial cardiovascular actions of rosiglitazone therapy.

Rosiglitazone therapy reduces oxidative stress in vascula-ture of SHR. Correlations between increased ROS levels,impaired metabolic control, and higher risk of cardiovascularevents suggest that oxidative stress is a critical mechanismunderlying the pathogenesis of both insulin resistance andendothelial dysfunction (19, 24, 36). Indeed, a number ofstudies suggest that oxidative stress contributes independentlyto both insulin resistance in metabolic and vascular tissues (2,58, 64), as well as to endothelial dysfunction and atheroscle-rosis in the vasculature (14, 33, 75). Consistent with ourprevious report (61), in this study, SHR exhibited systolichypertension, metabolic insulin resistance, and endothelial dys-function that were all reduced by rosiglitazone treatment.Important new findings of the present study include the obser-vations that circulating levels of ADMA and elevated levels of8-iso-PGF2�, O2

�, and nitrotyrosine in serum or vascular tis-sues in SHR were all reduced substantially by rosiglitazonetherapy. High circulating levels of ADMA, increased amountsof lipid peroxides, and nitrosylated proteins in vascular andsmooth muscle tissues are known markers of oxidant stresscharacteristically seen with endothelial dysfunction, athero-sclerosis, and diabetes (11, 53, 69, 72). The effects of rosigli-tazone therapy to reduce markers of oxidative stress in SHRthat we observed were comparable to results obtained by othersusing specific antioxidant treatments in SHR to reduce bloodpressure or prevent progression of hypertension (56, 67). It isunlikely that these changes in markers of oxidative stress weresecondary to the metabolic actions of rosiglitazone, since SHRin our study were not hyperglycemic and fasting glucose levelswere similar among SHR treated with vehicle or with rosigli-tazone. In fact, in primary endothelial cells, we demonstratedthat the effects of rosiglitazone treatment to reduce O2

� pro-duction in cells incubated with insulin plus wortmannin (tomimic conditions of metabolic insulin resistance in SHR) werecomparable to effects seen in cells treated with exogenousSOD. Similar results were obtained when ROS was inducedwith high glucose concentrations. However, this is less relevantto SHR that do not have overt hyperglycemia. Thus the abilityof systemic rosiglitazone therapy to reduce oxidative stress inthe vasculature may be due to direct or indirect antioxidantactions of rosiglitazone in vascular endothelium. In normoten-sive control WKY rats, in vivo treatment with rosiglitazone didnot significantly affect any of the physiological parametersevaluated or other markers of oxidative stress (data not shown).This may be explained by low levels of systemic oxidativestress in WKY. Our results in SHR are in line with previousstudies demonstrating actions of TZDs to oppose oxidativestress in the vasculature of animal models with atherosclerosisor other cardiovascular diseases (46, 51, 76).

Rosiglitazone treatment increases SOD activity and de-creases p22phox expression in the circulation and the vascu-lature of SHR. Increased production of ROS, and of O2

� inparticular, may result from both reduced activity of scavengingenzymes such as SOD and catalase as well as increasedexpression and activation of prooxidant enzymes includingNADPH oxidases (1, 36, 77). Therefore, one potential mech-anism for rosiglitazone therapy to reduce oxidative stress in thevasculature may be to increase expression or activity of scav-enging enzymes and decrease expression of enzymes respon-sible for O2

� production. Of note, in vessels from rosiglitazone-treated SHR, SOD activity was substantially increased, while

Fig. 4. Acute inhibition of NADPH oxidase improved NO-dependent vasodi-lator actions of insulin in SHR to an extent comparable to that achieved withchronic rosiglitazone treatment. MVB were isolated from SHR treated dailywith vehicle (A) or rosiglitazone (B; 5 mg �kg�1 �day�1) for 7 wk as describedin MATERIALS AND METHODS. Dose-response curves for insulin-stimulatedvasorelaxation were obtained in MVB before and after acute pretreatment withthe NADPH oxidase inhibitor apocynin (APO, 100 M, 30 min) in the absenceor presence of the NOS inhibitor N�-nitro-L-arginine methyl ester (L-NAME;100 M, 30 min). Results shown are the means � SE of 8 independentexperiments. Apocynin significantly improved insulin-mediated vasorelax-ation in MVB from vehicle-treated SHR (vs. vehicle-treated SHR, P 0.05).This effect was abrogated by simultaneous pretreatment with L-NAME (vs.APO-treated vehicle-SHR, P 0.05). Insulin-mediated vasodilation wassignificantly reduced by combined pretreatment of MVB from rosiglitazone-treated SHR with apocynin and L-NAME (vs. untreated MVB from rosiglita-zone-treated SHR, P 0.001).

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p22phox expression was markedly decreased (when comparedwith vessels from vehicle-treated SHR). In addition top22phox, several other components of the NOX complex maybe involved in oxidative stress under insulin-resistant condi-tions (36). However, among the numerous elements of theNADPH oxidase enzyme, p22phox is considered the catalyticsubunit (23). Therefore, p22phox levels are more directlyrelated to NADPH oxidase activity. Nevertheless, it remainspossible that other NOX subunits may also be altered byrosiglitazone therapy. Changes in enzyme activity and expres-sion may help to explain the reductions in markers of oxidativestress in the vasculature we observed after rosiglitazone ther-apy. Our findings are consistent with previous reports demon-strating effects of PPAR� ligands to reduce vascular oxidativestress by altering expression and/or activation of key redoxenzymes. For example, increased expression of p22phox ob-served in endothelial cells incubated with phorbol esters isopposed by troglitazone treatment (30). Along these samelines, rosiglitazone treatment of DOCA-salt rats decreasesproduction of O2

� in the vasculature (29), while treatment ofdb/db mice with rosiglitazone reduces NADPH oxidase-de-rived production of O2

� and enhances catalase activity incoronary arterioles (3). TZD treatment also reduces expressionof several subunits of NADPH oxidase (including p47phox andNox-2) in the vasculature of obese or diabetic rodents (16, 27)and hypercholesterolemic rabbits (71).

Effects of chronic rosiglitazone therapy to improve cardio-vascular function in SHR are mimicked by acute inhibition ofNADPH oxidase. In this study, we found that the salutarycardiovascular effects of rosiglitazone therapy included im-proved vasodilator actions of insulin similar to results wereported previously (61). Although MVB is considered as aprototype of resistance vessels (60), distinct vascular districtspossess vessel specificity that may account for important dif-ferences in organ and tissue functions. Thus changes in the

vascular reactivity of MVB do not necessarily imply thatsimilar changes may occur in other vascular districts. Never-theless, the fact that parameters of vascular oxidative stress areameliorated by rosiglitazone in a vascular district not directlyinvolved in glucose uptake (such as skeletal muscle arteries)provides additional evidence that the vascular effects of ros-iglitazone do not completely depend on metabolic ameliora-tion. Endothelial dysfunction and hypertension contribute im-portantly to impaired cardiac function and increase suscepti-bility to myocardial ischemia/reperfusion injury (18). Oxidativestress may also play a role in the pathogenesis of contractiledysfunction and structural damage in the myocardium (28, 44).Since endothelial dysfunction, systolic blood pressure, andmarkers of oxidative stress in the vasculature were all im-proved by rosiglitazone treatment, we reasoned that rosiglita-zone therapy may also afford cardiac protection in SHR.Indeed, we found that rosiglitazone treatment conferred sub-stantial protection against myocardial ischemia/reperfusion in-jury in SHR that has not previously been reported. To helpsupport the idea that the antioxidant actions of rosiglitazonemay be responsible for its ability to improve cardiovascularphenotypes, we performed ex vivo experiments where MVBand hearts from vehicle-treated SHR were preincubated withapocynin. Apocynin is an inhibitor of NADPH oxidase thatprevents the assembly of the NADPH oxidase subunitsp47phox and gp91phox resulting in decreased NADPH-oxi-dase-dependent O2

� formation (73). In addition, apocynin mayhave direct antioxidant properties in the endothelium (25, 66).Interestingly, the results from experiments with MVB andhearts from vehicle-treated SHR that were preincubated withapocynin mimicked the results from similar experiments per-formed with MVB and hearts isolated from rosiglitazone-treated SHR. That is, vessels and hearts from vehicle-treatedSHR subjected to acute antioxidant treatment ex vivo hadimproved function similar to that resulting from chronic ros-

Fig. 5. Chronic treatment of SHR with rosiglitazonereduces infarct size in hearts subjected to myocardialischemia/reperfusion injury. Langendorff-perfusedhearts isolated from SHR treated for 7 wk with eithervehicle or rosiglitazone (5 mg �kg�1 �day�1) weresubjected to ischemia/reperfusion injury as describedin MATERIALS AND METHODS. A: infarct volume afterischemia/reperfusion injury was significantly reducedin hearts isolated from rosiglitazone-treated SHR (vs.vehicle-treated SHR, P 0.001). Acute apocyninpretreatment (100 M, 30 min) of hearts isolatedfrom vehicle-treated SHR also significantly reducedinfarct volume (P 0.001). Coronary flow (B), leftventricular end-diastolic pressure (LVEDP; C), andleft ventricular developed pressure (LVDP; D) wererecorded twice before ischemia (aerobic perfusionbaseline; B), and after reperfusion. Data shown aremeans � SE of 5 independent experiments for eachgroup of animals. Chronic treatment with rosiglita-zone significantly improved all measures of cardiacfunction examined (vs. vehicle-treated SHR, P 0.001). Acute pretreatment of hearts from vehicle-treated SHR with apocynin significantly improvedboth LVEDP and LVDP (vs. untreated hearts fromvehicle-treated SHR, P 0.001).

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iglitazone therapy in vivo. Apocynin pretreatment did notresult in further improvement of endothelial function in MVBfrom rosiglitazone-treated SHR. This may be due to the factthat rosiglitazone therapy had already substantially decreasedexpression of p22phox.

Despite cell and animal studies suggesting that rosiglita-zone may have beneficial cardiovascular actions (74), re-sults from several recent large clinical trials have raised thealarming possibility that TZD therapy in patients with dia-betes may paradoxically increase, rather than decrease,cardiovascular morbidity and mortality (17, 21, 40, 41, 54,57, 68). Given the preclinical data available on the potentialcardiovascular benefits of TZDs, it is not clear why somelarge studies (9, 15) show an increased risk for adversecardiovascular outcomes after TZD therapy. One possibilityis that the cardiovascular risk/benefit profile of TZD therapymay change depending on whether patients are early in thecourse of the disease with reversible functional alterations

or late in the course of disease with irreversible structuraldamage. However, it should also be pointed out that differ-ences in the rosiglitazone-dependent effect on vascularfunction between our investigation and large clinical trialsmay result from the relatively short period of rosiglitazoneadministration in this study. It remains possible that a subsetof patients with reversible cardiovascular disease may de-rive both metabolic and cardiovascular benefits from ros-iglitazone therapy.

In conclusion, rosiglitazone therapy in SHR reducesmarkers of oxidative stress in the circulation and vasculaturethat may be explained by increased SOD activity and de-creased expression of p22phox. Thus in addition to improv-ing insulin signaling in metabolic tissues and the vascula-ture, rosiglitazone may have important antioxidant mecha-nisms of action. These properties may contribute to thebeneficial effects of rosiglitazone therapy on endothelial andcardiac function in SHR.

Fig. 6. Treatment of bovine aortic endothelial cells (BAECs) with rosiglitazone protects against O2� production and p22phox overexpression.

A: representative fluorescent photomicrographs from experiments repeated independently 3 times are shown for BAECs cultured with normal glucose(basal, 4.5 mM glucose) and high glucose (25 mM glucose) concentrations and treated with either rosiglitazone (20 M/24 h) or exogenous SOD (180U/ml, 30 min). Red fluorescence in cells loaded with DHE is indicative of O2

� production (top); blue fluorescence in cells stained with DAPI indicatescell nuclei (middle). Merged images of DHE and DAPI staining are shown at bottom. B: data shown are means � SE of the value of fluorescence relativeto the merging of DHE (red) and DAPI (blue) staining normalized to basal conditions from analysis of 4 fields (at �40 magnification). C: mRNA levelsof p22phox in BAECs cultured under normal conditions, high insulin concentrations (100 nM/24 h) without or with wortmannin (100 M/24 h), andtreated with rosiglitazone (20 M/24 h). D: merged images of DHE and DAPI staining are shown. E: densitometric determination of DHE fluorescenceshown in D.

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GRANTS

This work was supported, in part, by research grant awards from the ItalianMinistry for University and Research and the Juvenile Diabetes ResearchFoundation (CDA 2-2006-32 to M. Montagnani) and by the IntramuralResearch Program, National Center for Complementary and Alternative Med-icine, National Institutes of Health (to M. J. Quon).

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