This Article Abstract Full Text (PDF) All Versions of this Article: 108/22/2805    most recent 01.CIR.0000097003.49585.5Ev1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tao, L. Articles by Yue, T.-L. Search for Related Content PubMed PubMed Citation Articles by Tao, L. Articles by Yue, T.-L. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information Antioxidants Cholesterol Hazardous Substances DB L-TYROSINE NITRIC OXIDE Related Collections Cardiovascular Pharmacology Oxidant stress Endothelium/vascular type/nitric oxide

(Circulation. 2003;108:2805.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Antioxidative, Antinitrative, and Vasculoprotective Effects of a Peroxisome Proliferator–Activated Receptor- Agonist in Hypercholesterolemia

Ling Tao, MD; Hui-Rong Liu, MD, PhD; Erhe Gao, MD, PhD; Zhi-Ping Teng, MD, PhD; Bernard L. Lopez, MD; Theodore A. Christopher, MD; Xin-Liang Ma, MD, PhD; Ines Batinic-Haberle, PhD; Robert N. Willette, PhD; Eliot H. Ohlstein, PhD; Tian-Li Yue, PhD

From the Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, Pa; the Department of Radiation Oncology, Duke University Medical Center, Durham, NC (I.B.-H.); and GlaxoSmithKline Pharmaceuticals, King of Prussia, Pa (R.N.W., E.H.O., T.-L.Y.).

Correspondence to Xin L. Ma, MD, PhD, Department of Emergency Medicine, 1020 Sansom St, Thompson Building, Room 241, Philadelphia, PA 19107. E-mail xin.ma{at}jefferson.edu

Received April 23, 2003; de novo received June 24, 2003; revision received July 31, 2003; accepted August 1, 2003.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— Peroxisome proliferator–activated receptor (PPAR) signaling pathways have been reported to exert anti-inflammatory effects and attenuate atherosclerosis formation. However, the mechanisms responsible for their anti-inflammatory and antiatherosclerotic effects remain largely unknown. The present study tested the hypothesis that a PPAR agonist may exert significant endothelial protection by antioxidative and antinitrative effects.

Methods and Results— Male New Zealand White rabbits were randomized to receive a normal (control) or a high-cholesterol diet and treated with vehicle or rosiglitazone (a PPAR agonist) 3 mg · kg^-1 · d^-1 for 5 weeks beginning 3 weeks after the high-cholesterol diet. At the end of 8 weeks of a high-cholesterol diet, the rabbits were killed, and the carotid arteries were isolated. Bioactive nitric oxide was determined functionally (endothelium-dependent vasodilatation) and biochemically (the phosphorylation of vasodilator-stimulated phosphoprotein, or P-VASP). Vascular superoxide production, PPAR, gp91^phox, and inducible nitric oxide synthase (iNOS) expression, and vascular ONOO^- formation were determined. Hypercholesterolemia caused severe endothelial dysfunction and reduced P-VASP, despite a marked increase in iNOS expression and total NO_x production. Treatment with rosiglitazone enhanced PPAR expression, improved endothelium-dependent vasodilatation, preserved P-VASP, suppressed gp91^phox and iNOS expression, reduced superoxide and total NO_x production, and inhibited nitrotyrosine formation.

Conclusions— The PPAR agonist rosiglitazone exerted a significant vascular protective effect in hypercholesterolemic rabbits, most likely by attenuation of oxidative and nitrative stresses. The endothelial protective effects of PPAR agonists may reduce leukocyte accumulation in vascular walls and contribute to their antiatherosclerotic effect.

Key Words: hypercholesterolemia • endothelium • inflammation • atherosclerosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Hypercholesterolemia and the subsequent formation of atherosclerosis is one of the most important risk factors for ischemic heart disease.¹ Considerable evidence exists that hypercholesterolemia causes endothelial dysfunction, a prerequisite of atherosclerosis, in conduit vessels and small arteries. Numerous mechanisms have been proposed to explain this pathological alteration, including deficiencies of arginine supply, alteration of signaling mechanisms, alterations of nitric oxide synthase (NOS) expression or one of the cofactors involved in NOS activation, and increased destruction of nitric oxide (NO).¹ Among these proposed mechanisms, superoxide inactivation of NO has been thought to be the most important mechanism of endothelial dysfunction in hypercholesterolemia. However, the enzymatic sources responsible for this increased superoxide production remain largely unknown.

Vasodilator-stimulated phosphoprotein (VASP), a family member of proline-rich proteins, was first characterized as a major phosphorylated protein in platelets and endothelial cells after stimulation with vasodilators such as prostaglandins and NO donors. Recent studies have demonstrated that VASP is the downstream target of both cAMP- and cGMP-dependent protein kinases (cAK and cGK), and reduced VASP phosphorylation (P-VASP) is a sensitive monitor of defective NO/cGMP signaling.² However, the diagnostic and pathological significances of this newly identified phosphoprotein have not been determined in real pathological settings, such as hypercholesterolemia.

Peroxisome proliferator–activated receptors (PPARs) are transcription factors belonging to the nuclear receptor superfamily. Emerging evidence indicates that the PPAR signaling pathways play critical roles in the regulation of a variety of biological processes within the cardiovascular system.³ Considerable evidence indicates that treatment with PPAR agonists improves endothelial function in diabetic animal models and diabetic patients.⁴ However, whether this endothelial protective effect is secondary to improved glucose metabolism by the drug or PPAR agonists exert direct endothelial protection remains unknown.

Several recent studies have demonstrated that PPAR agonists improve atherosclerosis by ameliorating systemic metabolic risk factors for atherogenesis and inflammatory events that occur within the artery wall.^5,6 However, although substantial evidence exists that endothelial dysfunction is one of the earliest events in hypercholesterolemia and plays a critical role in the development of atherosclerosis,¹ whether or not PPAR agonists may preserve vascular endothelial function and thus retard atherosclerosis has not been investigated previously.

Therefore, the purposes of the present study were to (1) provide direct evidence that the NO/cGMP/cGK signaling pathway is impaired in hypercholesterolemia, (2) identify possible enzymatic sources responsible for increased superoxide production in hypercholesterolemic vessels, (3) determine whether the PPAR agonist rosiglitazone (RSG) may attenuate endothelial dysfunction in hypercholesterolemia, and (4) investigate the potential mechanisms by which PPAR agonists may exert their vasoprotective effects.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Animals
Adult male New Zealand White rabbits (Covance, Denver, Pa) were randomly assigned to one of the following groups: control (C, normal diet, n=8), hypercholesterolemia (HC, 1% cholesterol diet for 8 weeks, n=10), and HC treated with RSG, a PPAR agonist (oral gavage, 3 mg · kg^-1 · d^-1 for 5 weeks beginning 3 weeks after HC, n=10). At the end of 8 weeks of a high-cholesterol diet, rabbits were anesthetized, and tissue samples were collected immediately. The experiments were performed in adherence to National Institutes of Health Guidelines on the Use of Laboratory Animals and were approved by the Thomas Jefferson University Committee on Animal Care.

Determination of Endothelium-Dependent, NO-Mediated Vasorelaxation
Freshly harvested carotid arteries were placed into ice-cold Krebs-Henseleit buffer and cut into rings 3 to 4 mm long. Endothelial function was examined as described in our previous studies by comparing the vasorelaxation responses to acetylcholine (ACh), an endothelium-dependent vasodilator, with those to acidified NaNO₂, an endothelium-independent vasodilator.⁷ Endothelial dysfunction was described as reduced vasorelaxation to ACh with a normal response to acidified NaNO₂.

Vascular Superoxide Production
Superoxide production was measured by lucigenin-enhanced chemiluminescence as described previously.⁸ Superoxide production was expressed as relative light units (RLU) per second per mg vessel dry weight (RLU · mg^-1 · s^-1).

Immunoblotting
Proteins from tissue homogenate were separated on SDS-PAGE gels, transferred to nitrocellulose membranes, and Western blotted with monoclonal antibodies against gp91^phox, PPAR, endothelial NOS (eNOS) (Santa Cruz), VASP, and P-VASP (A.G. Scientific). The blot was developed with Super Signal Femto Reagent (Pierce) and visualized with a Kodak Image Station 400. The blot densities were analyzed with Kodak 1D software.

Determination of Total NO_x Content in Carotid Artery Tissue
Vascular NO and its in vivo metabolic products (NO₂ and NO₃), collectively known as NO_x, were determined by use of a chemiluminescent NO detector (Siever 280i) as described in our previous study.⁹

Quantification of Tissue Nitrotyrosine Content
Nitrotyrosine content, a footprint of in vivo ONOO^- formation, was determined by an ELISA method described in our recent publication.¹⁰ The results were presented as pmol nitrotyrosine/mg protein.

Immunohistological Detection of PPAR, Inducible NOS, gp91^phox, and Nitrotyrosine
Carotid arteries were removed and stored in 10% formalin for <48 hours. Fixed artery segments were dehydrated and embedded in paraffin, and sections were cut at 5 µm and mounted onto glass slides. Immunohistochemical detections of PPAR, gp91^phox, inducible NOS (iNOS), and nitrotyrosine were performed.

Statistical Analysis
All values in the text, table, and figures are presented as mean±SEM of n independent experiments. All data (except Western blot density) were subjected to ANOVA followed by Bonferroni correction for post hoc t test. Western blot densities were analyzed with the Kruskal-Wallis test followed by Dunn’s post hoc test. Probabilities of P0.05 were considered to be statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
RSG Had No Effects on Plasma Lipid Profiles After a High-Cholesterol Diet
There were no significant differences in plasma lipid profiles before the onset of the study (time 0), after 3 weeks of high-cholesterol diet, or at the end of 5 weeks of RSG treatment (at 8 weeks, cholesterol, P=0.4953; LDL, P=0.3541 between vehicle and RSG-treated group) (Table). These results indicate that RSG failed to improve plasma lipid profiles in this diet-induced hypercholesterolemic model; therefore, any vascular protective effects observed cannot be attributed to this mechanism.

View this table: [in this window] [in a new window]   Lipid Profile in Vehicle-Treated and RSG-Treated Groups

RSG Attenuated Hypercholesterolemia-Induced Endothelial Dysfunction
In carotid artery rings isolated from control rabbits, ACh induced a concentration-dependent vasorelaxation. In contrast, carotid artery rings isolated from hypercholesterolemic rabbits treated with vehicle showed a severe right-shifting of their dose-response curve to ACh. Treatment with RSG markedly preserved ACh-induced vasorelaxation, indicating that the PPAR agonist preserved endothelial function in hypercholesterolemic animals (Figure 1A). Addition of acidified NaNO₂, an exogenous NO donor, resulted in comparable and full relaxations in rings from all 3 groups, indicating that smooth muscle response to exogenous NO was normal in these vascular segments (Figure 1B).

View larger version (18K): [in this window] [in a new window]   Figure 1. Concentration-relaxation responses of rings from 3 experimental groups to ACh (A) and acidified NaNO2 (B). Rings were constricted with U-46619, and maximal relaxation responses to each concentration of ACh (an endothelium-dependent vasodilator) or acidified NaNO2 (an endothelium-independent vasodilator) were determined. *P<0.05, **P<0.01 vs control; ##P<0.01 vs HC (high-cholesterol diet). n=8 to 10 rabbits per group (4 rings for each rabbit).

Previous studies have demonstrated that many antioxidants preserve endothelial function in animals subjected to hypercholesterolemia. To explore the possibility that RSG may act as an antioxidant and thus improve endothelial function, RSG was added directly into the tissue bath containing carotid artery rings from vehicle-treated hypercholesterolemic rabbits. Preincubation of these rings with RSG (5 µmol/L for 30 minutes) failed to improve ACh-induced vasorelaxation (maximal relaxation, 60±5.9%). However, preincubation with MnTE-2-PyP⁵⁺ (50 µmol/L), a cell-permeable superoxide dismutase mimetic, markedly improved ACh-induced vasorelaxation (79±6.9%, P<0.01). Taken together, these results demonstrated that RSG attenuated hypercholesterolemia-induced endothelial dysfunction when administered in vivo via mechanisms other than direct superoxide scavenging.

RSG Preserved Hypercholesterolemia-Induced Impairment of the NO/cGMP/cGK Signaling Pathway
To determine the signaling pathway involved in hypercholesterolemia-induced endothelial dysfunction and to further document the vasoprotective effect of RSG, we examined P-VASP, a novel marker for NO/cGMP/cGK signaling, in vascular tissues. As illustrated in Figure 2A and summarized in Figure 2B, P-VASP was significantly diminished in vascular tissues from vehicle-treated rabbits, suggesting that hypercholesterolemia impaired NO/cGMP/cGK signaling. Treatment with RSG significantly enhanced P-VASP compared with vehicle-treated animals, indicating that RSG treatment maintained the integrity of a critical signaling system by which NO exerts most of its physiological effects.

View larger version (27K): [in this window] [in a new window]   Figure 2. Effect of RSG treatment on total VASP and phosphorylated VASP levels. A, Representative images; B, densitometry analysis. **P<0.01 vs control; ##P<0.01 vs HC.

Antioxidant Effect of RSG as a Mechanism Responsible for Its Vascular Protection
Considerable evidence suggests that increased superoxide generation and subsequent NO degradation is the major cause of endothelial dysfunction associated with hypercholesterolemia. To further identify the enzymatic source(s) responsible for superoxide generation under this particular pathological condition and, more importantly, to determine whether a PPAR agonist may attenuate superoxide generation in hypercholesterolemic vessels, thus improving endothelial function, superoxide generation was assayed directly. Consistent with previously reported results, a 2.2-fold increase in superoxide generation was observed in vessels from HC rabbits receiving vehicle (98.5±7.2 versus 44.3±10.1 RLU · mg^-1 · s^-1 in control, P<0.01). In vivo treatment with RSG markedly reduced superoxide production (56.5±7.1 RLU · mg^-1 · s^-1, P<0.01 versus vehicle). Moreover, in vitro preincubation (30 minutes, 37°C) of the vascular segments from HC rabbits with MnTE-2-PyP⁵⁺ (50 µmol/L, 28.2±3.6 RLU · mg^-1 · s^-1, P<0.01 versus vehicle) or DPI (an NADPH oxidase inhibitor, 100 µmol/L, 37.9±5.8, P<0.01 versus vehicle) but not with oxypurinol (100 µmol/L, 86.6±4.4 RLU · mg^-1 · s^-1, P>0.05 versus vehicle) or N^G-monomethyl-L-arginine (94.6±7.1 RLU · mg^-1 · s^-1, P>0.05 versus vehicle) markedly inhibited superoxide generation from HC rabbits, suggesting that NADPH oxidase is the primary source for increased O₂^- in hypercholesterolemic vessels. Short-term incubation of carotid artery segments from hypercholesterolemic animals with RSG (5 µmol/L, 30 minutes) did not inhibit O₂^- production, further indicating that RSG has no intrinsic antioxidant properties.

To further ensure that NADPH oxidase plays a major role in hypercholesterolemia-induced oxidative stress and that a PPAR agonist may exert its antioxidant effect by suppressing NADPH oxidase expression, 2 additional experiments were performed. As illustrated in Figure 3A, the degree of gp91^phox staining was markedly increased in the vascular endothelium and in the adventitia of hypercholesterolemic rabbits receiving vehicle. This staining was significantly attenuated by RSG treatment. Western blotting demonstrated that hypercholesterolemia resulted in a 3.6-fold increase in gp91^phox expression and treatment with RSG significantly reduced its expression (Figure 3, B and C). Taken together, these results provide direct evidence that NADPH oxidase expression is upregulated by hypercholesterolemia and that PPAR agonists may inhibit NADPH oxide expression in hypercholesterolemic vessels, thus reducing superoxide production, preventing NO degradation, and improving endothelial function.

View larger version (31K): [in this window] [in a new window]   Figure 3. Effect of RSG treatment on vascular gp91phox expression in hypercholesterolemic rabbits. A, Immunohistochemistry; B, Western blot; C, densitometry analysis. *P<0.05, **P<0.01 vs control; ##P<0.01 vs HC. Con. indicates control.

Antinitrative Effect of RSG as a Mechanism Responsible for Its Vasculoprotection
Having demonstrated that RSG exerted significant antioxidant effects and preserved endothelial function in hypercholesterolemia, we further investigated the effect of RSG on tissue nitrative stress associated with hypercholesterolemia. Despite a marked reduction in endothelium-dependent vasorelaxation and an impaired NO/cGMP/cGK signaling pathway, the total NO production, as determined by NO_x content, in vascular tissue was markedly increased (Figure 4). Treatment with RSG reduced total NO_x production compared with HC rabbits treated with vehicle. Hypercholesterolemia did not alter eNOS levels in the carotid artery, and treatment with RSG had no effect on its expression (representative Western blot presented in Figure 4; samples from 5 animals per group were studied). However, endothelial and smooth muscle expression of iNOS was significantly increased in vascular samples from hypercholesterolemic rabbits, and this was markedly attenuated by RSG treatment (Figure 4). To provide direct evidence that increased NO production (probably from iNOS) was inactivated by increased superoxide (probably from NADPH oxidase) before it could reach its normal physiological target (guanylate cyclase) and that RSG could block this reaction, we further determined the tissue nitrotyrosine content by ELISA and its distribution by immunohistology. As illustrated in Figure 5A, strong nitrotyrosine staining was detected in endothelial cells and smooth muscle cells from hypercholesterolemic rabbits, and treatment with RSG reduced nitrotyrosine staining. Quantitative ELISA results indicated that a 6.8-fold increase in nitrotyrosine content was observed in hypercholesterolemic rabbit tissue, and this increased nitrotyrosine content was markedly reduced by in vivo administration of RSG (Figure 5B).

View larger version (45K): [in this window] [in a new window]   Figure 4. Effect of RSG treatment on eNOS expression (Western blot), iNOS expression (immunohistochemistry), and total vascular NOx content. *P<0.05, **P<0.01 vs control; ##P<0.01 vs HC.

View larger version (46K): [in this window] [in a new window]   Figure 5. Effect of RSG treatment on nitrotyrosine staining (A, immunohistochemistry) and vascular nitrotyrosine content (B) in hypercholesterolemic rabbits. **P<0.01 vs control; ##P<0.01 vs HC. Con. indicates control.

RSG Upregulated PPAR Expression in the Carotid Artery
Recent studies have demonstrated that PPAR expression is reduced under certain pathological conditions, such as pulmonary hypertension,¹¹ and that in vitro treatment with PPAR agonists upregulates PPAR expression.^12,13 To determine whether hypercholesterolemia may alter PPAR expression and in vivo treatment with RSG may upregulate PPAR expression, 2 additional experiments were performed. As illustrated in Figure 6, in carotid arteries isolated from control rabbits, PPAR is expressed in both endothelial cells and adventitial tissue. Hypercholesterolemia had no significant effect on PPAR expression in adventitial tissue. However, PPAR expression in endothelial cells was virtually abolished, and the total PPAR protein content as measured by Western blot was significantly reduced (57% of control, n=5). Most interestingly, in vivo treatment with RSG in hypercholesterolemic rabbits upregulated PPAR expression to a level even higher than that seen in control rabbits (1.7-fold increase over control, n=5).

View larger version (106K): [in this window] [in a new window]   Figure 6. Effect of RSG on hypercholesterolemia-induced downregulation of PPAR expression. Con. indicates control.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study confirmed previous reports that hypercholesterolemia induces endothelial dysfunction by increased O₂^- generation rather than decreased NO production. Moreover, using a novel molecular marker of the NO/cGMP/cGK signaling pathway, we have demonstrated that P-VASP at Ser²³⁹ is markedly reduced in diet-induced hypercholesterolemic vessels. In addition, our results provide the first direct evidence that NADPH oxidase but not xanthine oxidase or dysfunctional NOS is the primary source for O₂^- in vascular tissues from diet-induced hypercholesterolemic animals. Most importantly, we have made a novel observation and demonstrated for the first time that a PPAR agonist inhibited gp91^phox expression (Figure 3) and superoxide generation, blocked iNOS expression and excess NO production (Figure 4), attenuated toxic ONOO^- formation (Figure 5), improved endothelial function (Figure 1), and preserved the integrity of NO/cGMP/cGK signaling (Figure 2) in hypercholesterolemic animals. These novel results provide another likely mechanism for its previously reported anti-inflammatory and antiatherosclerotic effects. In addition, we have provided first direct evidence in an in vivo animal model that hypercholesterolemia downregulates and a PPAR agonist upregulates PPAR expression in vascular tissue.

Impaired NO/cGMP/cGK in Hypercholesterolemic Vessels
The VASP was originally discovered as a substrate for both cAMP- and cGMP-dependent protein kinases in human platelets, but subsequent studies have demonstrated that VASP is expressed ubiquitously in many types of cells.¹⁴ Although its downstream signaling molecules and physiological roles remain unknown, P-VASP represents a novel biochemical marker for monitoring the NO-stimulated soluble guanylate cyclase (sGC)/cGK pathway and endothelial integrity in vascular tissue.² Decreased P-VASP has been reported in vascular tissues from nitroglycerin-induced nitrate tolerance,¹⁵ chronic angiotensin II exposure,¹⁶ and spontaneous hyperlipidemic Watanabe rabbits.^2,17 Our present study demonstrated for the first time that diet-induced hypercholesterolemia had no effect on VASP expression (total VASP); however, P-VASP was markedly reduced.

NADPH Oxidase as a Major O₂^- Source in Hypercholesterolemic Vessels
Increasing evidence indicates that NADPH oxidase is the predominant O₂^- source in both endothelial and smooth muscle cells, and increased NADPH oxidase activity has been found in human atherosclerotic vessels¹⁸ and diabetic vessels.¹⁹ In a recent study, Warnholtz et al²⁰ reported that NADPH-oxidase activity in aortic homogenates from hyperlipidemic Watanabe rabbits and cholesterol-fed rabbits was increased significantly. In accordance with this study, we have demonstrated that O₂^- production is markedly inhibited by in vitro treatment with DPI but not N^G-monomethyl-L-arginine in carotid arteries from diet-induced hypercholesterolemic rabbits. In addition, we have provided, for the first time, direct evidence that expression of gp91^phox, a membrane-bound subunit of NADPH oxidase, is markedly increased in endothelial and adventitial tissue from hypercholesterolemic rabbits. These results provide firm evidence that NADPH oxidase is a significant source of O₂^- in hypercholesterolemic vessels.

Vascular Protective Effects of a PPAR Agonist and Its Potential Mechanisms
The most important finding of the present study is that treatment with RSG markedly improved NO bioavailability in a nondiabetic animal model. There are several potential mechanisms by which a PPAR agonist may exert its vascular protection in hypercholesterolemia. PPAR activators have been found to decrease triglyceride concentrations and increase HDL cholesterol.²¹ The improvement in lipid profile may thus attenuate hypercholesterolemia-induced endothelial dysfunction. However, this mechanism is unlikely to contribute to the vascular protection exerted by RSG (a PPAR agonist), because this treatment failed to improve the lipid profile in our experiment. PPAR agonists improve insulin sensitivity and glucose metabolism in the diabetic patient.²² However, it is unlikely that insulin resistance plays a significant role in endothelial dysfunction associated with diet-induced hypercholesterolemia.

The present experiment demonstrated that RSG most likely achieves its endothelial protection via inhibition of O₂^- production and subsequent NO degradation. A variety of antioxidants, such as ascorbate, -tocopherol, and carvedilol, have been reported to improve endothelial dysfunction induced by hypercholesterolemia.^23,24 However, unlike these antioxidants, RSG failed to reduce superoxide production or improve endothelium-dependent vasodilatation after in vitro incubation with hypercholesterolemic vessels. Therefore, our results suggest that PPAR agonists may inhibit superoxide-producing enzyme(s) expression and/or increase antioxidant enzyme(s) expression. In a recent study, Inoue et al²⁵ demonstrated that in vitro treatment of endothelial cells with pioglitazone increased CuZn–superoxide dismutase gene expression and inhibited p22^phox and p47^phox expression. In the present study, we have provided direct evidence that in vivo RSG treatment markedly attenuated the expression of gp91^phox, a membrane-bound subunit of NADPH oxidase, in hypercholesterolemic vessels.

Emerging evidence suggests that in addition to oxidative stress, nitrative stress also plays a significant role in tissue injury.²⁶ Recent in vitro studies have demonstrated that PPAR agonists exert significant anti-inflammatory effects, as evidenced by reduced adhesion molecule expression²⁷ and decreased iNOS expression.^28,29 In addition, Linscheid et al³⁰ recently reported that in addition to its inhibitory effect on iNOS gene expression, RSG may inhibit NO production by reducing tetrahydrobiopterin production in adipocytes. Our present study demonstrated that treatment with a PPAR agonist in vivo exerted significant antinitrative effects, as evidenced by reduced iNOS expression, total NO production, and nitrotyrosine formation. This unique effect has not been reported with other treatments such as angiotensin II type I receptor antagonists (Bay 10-6734) and ascorbate, although these treatments also reduce oxidative stress.^20,23 Taken together, our present study demonstrated for the first time that the PPAR agonist RSG exerted significant endothelial protective effects by reducing both oxidative stress (reduced superoxide generation) and nitrative stress (prevented excessive NO production and ONOO^- formation).

The endothelial protective effects of PPAR agonists are of clinical significance. The endothelium plays a crucial role in the process of atherosclerotic disease. Endothelial dysfunction causes upregulation of adhesion molecules, enhances migration of monocytes into the vessel wall, and increases proliferation of smooth muscle cells.^1,31 Therefore, therapeutic interventions that are capable of preserving the NO/cGMP signaling pathway and improving endothelial function, such as PPAR agonists, may have tremendous application in the treatment of cardiovascular diseases.

	Acknowledgments

 
This research was supported in part by National Institutes of Health grant HL-63828 (Dr Ma).

	Footnotes

 
Dr Batinic-Haberle serves as a consultant for Aeolus/Incara Pharmaceuticals, RTP, NC.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res. 2000; 87: 840–844.[Abstract/Free Full Text]
   
2. Oelze M, Mollnau H, Hoffmann N, et al. Vasodilator-stimulated phosphoprotein serine 239 phosphorylation as a sensitive monitor of defective nitric oxide/cGMP signaling and endothelial dysfunction. Circ Res. 2000; 87: 999–1005.[Abstract/Free Full Text]
   
3. Bishop-Bailey D. Peroxisome proliferator-activated receptors in the cardiovascular system. Br J Pharmacol. 2000; 129: 823–834.[CrossRef][Medline] [Order article via Infotrieve]
   
4. Caballero AE, Saouaf R, Lim SC, et al. The effects of troglitazone, an insulin-sensitizing agent, on the endothelial function in early and late type 2 diabetes: a placebo-controlled randomized clinical trial. Metabolism. 2003; 52: 173–180.[CrossRef][Medline] [Order article via Infotrieve]
   
5. Hsueh WA, Law RE. PPAR and atherosclerosis: effects on cell growth and movement. Arterioscler Thromb Vasc Biol. 2001; 21: 1891–1895.[Abstract/Free Full Text]
   
6. Plutzky J. Peroxisome proliferator-activated receptors in vascular biology and atherosclerosis: emerging insights for evolving paradigms. Curr Atheroscler Rep. 2000; 2: 327–335.[Medline] [Order article via Infotrieve]
   
7. Ma XL, Gao F, Chen J, et al. Endothelial protective and antishock effects of a selective estrogen receptor modulator in rats. Am J Physiol. 2001; 280: H876–H884.
   
8. Lund DD, Faraci FM, Miller FJ Jr, et al. Gene transfer of endothelial nitric oxide synthase improves relaxation of carotid arteries from diabetic rabbits. Circulation. 2000; 101: 1027–1033.[Medline] [Order article via Infotrieve]
   
9. Gao F, Gao E, Yue TL, et al. Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation. Circulation. 2002; 105: 1497–1502.[Abstract/Free Full Text]
   
10. Gao F, Yao CL, Gao EH, et al. Enhancement of glutathione cardioprotection by ascorbic acid in myocardial reperfusion injury. J Pharmacol Exp Ther. 2002; 301: 1–8.[Abstract/Free Full Text]
    
11. Ameshima S, Golpon H, Cool CD, et al. Peroxisome proliferator–activated receptor gamma (PPAR) expression is decreased in pulmonary hypertension and affects endothelial cell growth. Circ Res. 2003; 92: 1162–1169.[Abstract/Free Full Text]
    
12. Park KS, Ciaraldi TP, Lindgren K, et al. Troglitazone effects on gene expression in human skeletal muscle of type II diabetes involve up-regulation of peroxisome proliferator-activated receptor-gamma. J Clin Endocrinol Metab. 1998; 83: 2830–2835.[Abstract/Free Full Text]
    
13. Su JL, Winegar DA, Wisely GB, et al. Use of a PPAR gamma-specific monoclonal antibody to demonstrate thiazolidinediones induce PPAR gamma receptor expression in vitro. Hybridoma. 1999; 18: 273–280.[CrossRef][Medline] [Order article via Infotrieve]
    
14. Gambaryan S, Hauser W, Kobsar A, et al. Distribution, cellular localization, and postnatal development of VASP and Mena expression in mouse tissues. Histochem Cell Biol. 2001; 116: 535–543.[CrossRef][Medline] [Order article via Infotrieve]
    
15. Mulsch A, Oelze M, Kloss S, et al. Effects of in vivo nitroglycerin treatment on activity and expression of the guanylyl cyclase and cGMP-dependent protein kinase and their downstream target vasodilator-stimulated phosphoprotein in aorta. Circulation. 2001; 103: 2188–2194.[Medline] [Order article via Infotrieve]
    
16. Mollnau H, Wendt M, Szocs K, et al. Effects of angiotensin II infusion on the expression and function of NAD(P)H oxidase and components of nitric oxide/cGMP signaling. Circ Res. 2002; 90: E58–E65.
    
17. Warnholtz A, Mollnau H, Heitzer T, et al. Adverse effects of nitroglycerin treatment on endothelial function, vascular nitrotyrosine levels and cGMP-dependent protein kinase activity in hyperlipidemic Watanabe rabbits. J Am Coll Cardiol. 2002; 40: 1356–1363.[Abstract/Free Full Text]
    
18. Sorescu D, Weiss D, Lassegue B, et al. Superoxide production and expression of NOx family proteins in human atherosclerosis. Circulation. 2002; 105: 1429–1435.[Abstract/Free Full Text]
    
19. Guzik TJ, Mussa S, Gastaldi D, et al. Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase. Circulation. 2002; 105: 1656–1662.[Abstract/Free Full Text]
    
20. Warnholtz A, Nickenig G, Schulz E, et al. Increased NADH-oxidase mediated superoxide production in the early stages of atherosclerosis: evidence for involvement of the renin-angiotensin system. Circulation. 1999; 99: 2027–2033.[Medline] [Order article via Infotrieve]
    
21. Fruchart JC, Staels B, Duriez P. PPARS, metabolic disease and atherosclerosis. Pharmacol Res. 2001; 44: 345–352.[CrossRef][Medline] [Order article via Infotrieve]
    
22. Kelly DP. PPARs of the heart: three is a crowd. Circ Res. 2003; 92: 482–484.[Free Full Text]
    
23. Carr AC, Zhu BZ, Frei B. Potential antiatherogenic mechanisms of ascorbate (vitamin C) and -tocopherol (vitamin E). Circ Res. 2000; 87: 349–354.[Abstract/Free Full Text]
    
24. Ma XL, Yue TL, Lopez BL, et al. Carvedilol, a new beta-adrenoreceptor blocker and free radical scavenger, attenuates myocardial ischemia-reperfusion injury in hypercholesterolemic rabbits. J Pharmacol Exp Ther. 1996; 277: 128–136.[Abstract/Free Full Text]
    
25. Inoue I, Goto Si, Matsunaga T, et al. The ligands/activators for peroxisome proliferator-activated receptor (PPAR) and PPAR increase Cu²⁺, Zn²⁺-superoxide dismutase and decrease p22phox message expressions in primary endothelial cells. Metabolism. 2001; 50: 3–11.[CrossRef][Medline] [Order article via Infotrieve]
    
26. Ramachandran A, Levonen AL, Brookes PS, et al. Mitochondria, nitric oxide, and cardiovascular dysfunction. Free Radic Biol Med. 2002; 33: 1465–1474.[CrossRef][Medline] [Order article via Infotrieve]
    
27. Pasceri V, Wu HD, Willerson JT, et al. Modulation of vascular inflammation in vitro and in vivo by peroxisome proliferator-activated receptor-gamma activators. Circulation. 2000; 101: 235–238.[Medline] [Order article via Infotrieve]
    
28. Li M, Pascual G, Glass CK. Peroxisome proliferator-activated receptor gamma-dependent repression of the inducible nitric oxide synthase gene. Mol Cell Biol. 2000; 20: 4699–4707.[Abstract/Free Full Text]
    
29. Petrova TV, Akama KT, Van Eldik LJ. Cyclopentenone prostaglandins suppress activation of microglia: down-regulation of inducible nitric-oxide synthase by 15-deoxy-delta12,14-prostaglandin J2. Proc Natl Acad Sci U S A. 1999; 96: 4668–4673.[Abstract/Free Full Text]
    
30. Linscheid P, Keller U, Blau N, et al. Diminished production of nitric oxide synthase cofactor tetrahydrobiopterin by rosiglitazone in adipocytes. Biochem Pharmacol. 2003; 65: 593–598.[CrossRef][Medline] [Order article via Infotrieve]
    
31. Vinten-Johansen J, Zhao ZQ, Nakamura M, et al. Nitric oxide and the vascular endothelium in myocardial ischemia-reperfusion injury. Ann N Y Acad Sci. 1999; 874: 354–370.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				D. Demozay, J.-C. Mas, S. Rocchi, and E. Van Obberghen FALDH Reverses the Deleterious Action of Oxidative Stress Induced by Lipid Peroxidation Product 4-Hydroxynonenal on Insulin Signaling in 3T3-L1 Adipocytes Diabetes, May 1, 2008; 57(5): 1216 - 1226. [Abstract] [Full Text] [PDF]

				A. H. Remels, H. R. Gosker, P. Schrauwen, R. C. Langen, and A. M. Schols Peroxisome proliferator-activated receptors: a therapeutic target in COPD? Eur. Respir. J., March 1, 2008; 31(3): 502 - 508. [Abstract] [Full Text] [PDF]

				R. Li, W.-Q. Wang, H. Zhang, X. Yang, Q. Fan, T. A. Christopher, B. L. Lopez, L. Tao, B. J. Goldstein, F. Gao, et al. Adiponectin improves endothelial function in hyperlipidemic rats by reducing oxidative/nitrative stress and differential regulation of eNOS/iNOS activity Am J Physiol Endocrinol Metab, December 1, 2007; 293(6): E1703 - E1708. [Abstract] [Full Text] [PDF]

				S. A. Sorrentino, F. H. Bahlmann, C. Besler, M. Muller, S. Schulz, N. Kirchhoff, C. Doerries, T. Horvath, A. Limbourg, F. Limbourg, et al. Oxidant Stress Impairs In Vivo Reendothelialization Capacity of Endothelial Progenitor Cells From Patients With Type 2 Diabetes Mellitus: Restoration by the Peroxisome Proliferator-Activated Receptor-{gamma} Agonist Rosiglitazone Circulation, July 10, 2007; 116(2): 163 - 173. [Abstract] [Full Text] [PDF]

				F. Mittermayer, G. Schaller, J. Pleiner, K. Krzyzanowska, S. Kapiotis, M. Roden, and M. Wolzt Rosiglitazone Prevents Free Fatty Acid-Induced Vascular Endothelial Dysfunction J. Clin. Endocrinol. Metab., July 1, 2007; 92(7): 2574 - 2580. [Abstract] [Full Text] [PDF]

				L. Tao, E. Gao, X. Jiao, Y. Yuan, S. Li, T. A. Christopher, B. L. Lopez, W. Koch, L. Chan, B. J. Goldstein, et al. Adiponectin Cardioprotection After Myocardial Ischemia/Reperfusion Involves the Reduction of Oxidative/Nitrative Stress Circulation, March 20, 2007; 115(11): 1408 - 1416. [Abstract] [Full Text] [PDF]

				I. Imayama, T. Ichiki, K. Inanaga, H. Ohtsubo, K. Fukuyama, H. Ono, Y. Hashiguchi, and K. Sunagawa Telmisartan downregulates angiotensin II type 1 receptor through activation of peroxisome proliferator-activated receptor {gamma} Cardiovasc Res, October 1, 2006; 72(1): 184 - 190. [Abstract] [Full Text] [PDF]

				H. Sourij, R. Zweiker, and T. C. Wascher Effects of Pioglitazone on Endothelial Function, Insulin Sensitivity, and Glucose Control in Subjects With Coronary Artery Disease and New-Onset Type 2 Diabetes Diabetes Care, May 1, 2006; 29(5): 1039 - 1045. [Abstract] [Full Text] [PDF]

				R. Agarwal Anti-inflammatory effects of short-term pioglitazone therapy in men with advanced diabetic nephropathy Am J Physiol Renal Physiol, March 1, 2006; 290(3): F600 - F605. [Abstract] [Full Text] [PDF]

				M. Pesant, S. Sueur, P. Dutartre, M. Tallandier, P. A. Grimaldi, L. Rochette, and J.-L. Connat Peroxisome proliferator-activated receptor {delta} (PPAR{delta}) activation protects H9c2 cardiomyoblasts from oxidative stress-induced apoptosis Cardiovasc Res, February 1, 2006; 69(2): 440 - 449. [Abstract] [Full Text] [PDF]

				N. Nighoghossian, L. Derex, and P. Douek The Vulnerable Carotid Artery Plaque: Current Imaging Methods and New Perspectives Stroke, December 1, 2005; 36(12): 2764 - 2772. [Abstract] [Full Text] [PDF]

				A. C. Calkin, J. M. Forbes, C. M. Smith, M. Lassila, M. E. Cooper, K. A. Jandeleit-Dahm, and T. J. Allen Rosiglitazone Attenuates Atherosclerosis in a Model of Insulin Insufficiency Independent of Its Metabolic Effects Arterioscler. Thromb. Vasc. Biol., September 1, 2005; 25(9): 1903 - 1909. [Abstract] [Full Text] [PDF]

				L. Tao, H.-R. Liu, F. Gao, Y. Qu, T. A. Christopher, B. L. Lopez, and X. L. Ma Mechanical traumatic injury without circulatory shock causes cardiomyocyte apoptosis: role of reactive nitrogen and reactive oxygen species Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2811 - H2818. [Abstract] [Full Text] [PDF]

				M. Abdelrahman, A. Sivarajah, and C. Thiemermann Beneficial effects of PPAR-{gamma} ligands in ischemia-reperfusion injury, inflammation and shock Cardiovasc Res, March 1, 2005; 65(4): 772 - 781. [Abstract] [Full Text] [PDF]

				E. L. Schiffrin Peroxisome proliferator-activated receptors and cardiovascular remodeling Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1037 - H1043. [Abstract] [Full Text] [PDF]

				M.-A. Devynck, A. Simon, M.-G. Pernollet, G. Chironi, J. Gariepy, F. Rendu, and J. Levenson Plasma cGMP and Large Artery Remodeling in Asymptomatic Men Hypertension, December 1, 2004; 44(6): 919 - 923. [Abstract] [Full Text] [PDF]

				B. Sung, S. Park, B. P. Yu, and H. Y. Chung Modulation of PPAR in Aging, Inflammation, and Calorie Restriction J. Gerontol. A Biol. Sci. Med. Sci., October 1, 2004; 59(10): B997 - B1006. [Abstract] [Full Text] [PDF]

H.-R. Liu, L. Ta