This Article Abstract Full Text (PDF) 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 Ikeda, U. Articles by Shimada, K. Search for Related Content PubMed PubMed Citation Articles by Ikeda, U. Articles by Shimada, K. Related Collections Pathophysiology Cell biology/structural biology Gene expression Growth factors/cytokines Smooth muscle proliferation and differentiation Endothelium/vascular type/nitric oxide Mechanism of atherosclerosis/growth factors

(Hypertension. 2000;35:1232.)
© 2000 American Heart Association, Inc.

Scientific Contributions

Peroxisome Proliferator–Activated Receptor- Ligands Inhibit Nitric Oxide Synthesis in Vascular Smooth Muscle Cells

Uichi Ikeda; Masahisa Shimpo; Yoshiaki Murakami; Kazuyuki Shimada

From the Department of Cardiology, Jichi Medical School, Tochigi, Japan.

Correspondence to Uichi Ikeda, MD, PhD, Department of Cardiology, Jichi Medical School, Minamikawachi-Machi, Tochigi 329-0498, Japan. E-mail uikeda{at}jichi.ac.jp

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—Peroxisome proliferator–activated receptor- (PPAR) is a key player in glucose metabolism. If PPAR ligands modulate nitric oxide (NO) synthesis in the vascular tissue, they may affect the process of plaque formation and postangioplasty restenosis. We investigated the effects of PPAR ligands on NO synthesis in vascular smooth muscle cells. Incubation of cultures with interleukin-1ß (10 ng/mL) for 24 hours caused a significant increase in the production of nitrite, a stable metabolite of NO, in cultured rat vascular smooth muscle cells. The PPAR agonists troglitazone and 15-deoxy-^12,14-prostaglandin J₂ (15d-PG J₂) dose-dependently inhibited nitrite production by interleukin-1ß–stimulated vascular smooth muscle cells. Decreased interleukin-1ß–induced nitrite production by the PPAR agonists was accompanied by decreased inducible NO synthase mRNA and protein accumulation. Interleukin-1ß induced nuclear factor-B activation in vascular smooth muscle cells, and both troglitazone and 15d-PG J₂ markedly suppressed this nuclear factor-B activation. PPAR ligands inhibit NO synthesis in cytokine-stimulated vascular smooth muscle cells, suggesting that these agonists may act directly on the vascular smooth muscle and influence the process of atherosclerosis and restenosis.

Key Words: interleukins • nitric oxide • muscle, smooth • atherosclerosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Nitric oxide (NO), the extensively characterized endothelium-derived relaxing factor, is a short-lived free radical. NO is synthesized from L-arginine by 3 isoenzymes expressed either constitutively (neuronal, type I cNOS; endothelial, type III cNOS) or after stimulation by cytokines (inducible, type II iNOS).^{1 2} iNOS has been identified in endotoxin- and cytokine-treated neutrophils, hepatocytes, endothelial cells, and myocardium.^{1 3} iNOS activity is also induced in aortic rings and cultured vascular smooth muscles by cytokines and endotoxins.^{4 5} It has also been demonstrated that iNOS is expressed after in vivo balloon injury of rat carotid arteries⁶ and in human atherosclerotic lesions.⁷ NOS induction in vascular smooth muscle cells may play a role in local vascular injury associated with atherosclerosis by inhibiting smooth muscle cell proliferation as well as by limiting thrombus formation by preventing platelet adhesion and aggregation.⁸

Peroxisome proliferator–activated receptors (PPARs) are key players in lipid and glucose metabolism and have been implicated in metabolic disorders, resulting in a predisposition to atherosclerosis such as dyslipidemia and diabetes.^{9 10} Three types of PPARs have been described in rodents, humans, and amphibians: PPAR, Nuc1 (also called PPARß or PPAR), and PPAR. Recently, the existence of PPAR has been reported in human and rat vascular smooth muscle cells.^{11 12 13} In these cells, PPAR agonists inhibit gene expression and migration in vascular smooth muscle cells in vivo and in vitro.^{11 14 15} If PPAR ligands affect NO synthesis in the vascular tissue, they may influence the process of plaque formation and postangioplasty restenosis. We investigated the effects of PPAR agonists on NO production by cultured rat vascular smooth muscle cells.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
Human recombinant interleukin-1ß (specific activity 2x10⁷ units/mg) was a gift from Otsuka Pharmacy (Tokushima, Japan). Troglitazone was a gift from Sankyo Co (Tokyo, Japan). A monoclonal anti-mouse iNOS antibody, which cross-reacts with rat iNOS, was obtained from Transduction Laboratory (Lexington, Ky). 15-deoxy-^12,14-prostaglandin J₂ (15d-PG J₂) and Wy14643 were purchased from Caymen Chemical. Fenofibrate was purchased from Sigma Co. All other chemicals used were of the highest grade commercially available.

Culture of Cells
Primary cultures of vascular smooth muscle cells were obtained from the media of thoracic aortas of Sprague-Dawley rats (200 to 250 g), as described previously.¹⁶ The cells were grown in Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cultures were harvested twice per week by treatment with 0.125% trypsin and passaged at a ratio of 1:3 in 100-mm culture dishes. A typical experiment was performed with cultured cells at passages 5 to 10. Cells (3x10⁴/mL) were plated in 24-well or 100-mm culture dishes in DMEM, supplemented as described above, and allowed to grow to subconfluence for 24 to 48 hours, after which they were preincubated in DMEM containing 0.5% fetal bovine serum and supplemented with insulin (5 µg/mL) and transferrin (5 µg/mL) for 24 hours, and used for the experiments described below.

This investigation was performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

Measurements of Nitrite
NO production by the cultured cells was determined by measuring the nitrite contents of the culture media.¹⁷ Vascular smooth muscle cells plated in 24-well dishes were incubated in DMEM containing 0.5% fetal bovine serum at 37°C. The nitrite contents of culture media were determined by mixing 500 µL of medium with an equal volume of Griess reagent (1 part 0.1% naphthylethylene-diamine dihydrochloride to 1 part 1% sulfanilamide in 5% phosphoric acid).¹⁸ The absorbance at 550 nm was measured, and the nitrite concentration was determined by interpolation of a calibration curve of standard sodium nitrite concentrations against absorbance. After washing, cells were dissolved in 0.2 mL of 1% SDS and used for protein assay (Bio-Rad assay kit) with bovine serum albumin as a standard. Nitrite levels were corrected by protein measurement, and data are shown as nanomoles per milligram of protein.

Assay for iNOS mRNA
Total RNA was extracted from vascular smooth muscle cells plated in 100-mm culture dishes by the acid guanidinium isothiocyanate-phenol-chloroform method, and 20-µg aliquots were subjected to electrophoresis on 1% agarose gels. After electrophoretic separation, RNA was transferred onto nylon filters, which were then hybridized with a random-primed [³²P]-labeled mouse macrophage iNOS cDNA probe for 24 hours, followed by washing twice with an aqueous solution of 150 mmol/L NaCl, 15 mmol/L sodium citrate, and 0.1% SDS at 65°C.¹⁷ The filters were exposed to Kodak XAR-5 film for 1 to 2 days at -70°C with the use of an intensifying screen.

Assay for iNOS Protein
The expression of iNOS protein was analyzed by immunoblotting with an anti-iNOS antibody as described previously.¹⁹ Briefly, cells were lysed in a buffer containing 50 mmol/L Tris/Cl, pH 7.5, 1 mmol/L EDTA, 1 µmol/L leupeptin, 1 µmol/L pepstatin A, 0.1 mmol/L phenylmethylsulfonyl fluoride, and 1 mol/L dithiothreitol; the buffer was then sonicated. The homogenates were then centrifuged at 100 000g for 20 minutes, and the supernatants (60 µg protein) were subjected to 10% SDS-polyacrylamide gel electrophoresis. The separated proteins were electrophoretically transferred onto nitrocellulose membranes, and the resultant blots were incubated with the anti-iNOS antibody for 2 hours followed by peroxidase-labeled donkey anti-rabbit IgG for 1 hour. Peroxidase-labeled proteins were detected by means of the enhanced chemiluminescence detection system (Amersham Int) on x-ray film, and the results were quantified by densitometry.

Gel Retardation Assays
The levels of nuclear factor (NF)-B proteins in nuclear extracts from vascular smooth muscle cells were analyzed by gel retardation assays. Nuclear extract preparation and gel retardation experiments were performed as described previously.²⁰ A chemically synthesized oligonucleotide that contained a recognition site for NF-B (5'-TCAACAGAGGGGACTTTCCGAGGCCA-3') was annealed with its complementary sequence oligonucleotide, which was 5'-labeled with [-³²P]ATP with polynucleotide kinase and used for probe. For 15-µL DNA-binding reaction mixture, a 5- to 8-fmol probe was incubated with nuclear extract (6 µg protein) and 1 µg poly[d(I-C) · d(I-C)] at room temperature for 30 minutes in 60 mmol/L KCl, 20 mmol/L HEPES (pH 8.4), and 4% Ficoll. The DNA-bound protein complexes in the reaction mixtures were resolved by electrophoresis on a 4% polyacrylamide gel in 0.25xTris/Borate/EDTA buffer. NF-B consensus oligonucleotides (5'-AGTTGAGGCGAC-TTTCCCAGGC-3', Santa Cruz Biotechnology Inc) were used as competitors. An anti–NF-B p65 antibody (goat polyclonal IgG, 2 mg/mL) was used for supershift assay.

Statistical Analysis
Data are expressed as mean±SEM of 4 samples, which represented 3 separate experiments. Differences were analyzed by 1-way ANOVA combined with Scheffé’s test, and a probability value of <0.05 was considered to be statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects of PPAR Agonists on Nitrite Production
Incubation of cultured rat vascular smooth muscle cells with interleukin-1ß for 24 hours caused a significant increase in the production of nitrite, a stable metabolite of NO, as reported previously.⁴ Figure 1A shows the dose-response relation of the effect of the PPAR agonist troglitazone on the basal and interleukin-1ß–induced nitrite production by vascular smooth muscle cells. Although troglitazone alone did not affect the basal level of nitrite, it significantly decreased interleukin-1ß–induced nitrite accumulation in a dose-dependent manner. Another PPAR agonist, 15d-PG J₂, also significantly decreased nitrite production by interleukin-1ß–stimulated vascular smooth muscle cells (Figure 1B).

View larger version (33K): [in this window] [in a new window]   Figure 1. Dose-dependent effects of PPAR agonists on nitrite accumulation. Vascular smooth muscle cells were incubated for 24 hours with (diagonal bars) or without (stippled bars) 10 ng/mL interleukin-1ß in presence of various concentrations of troglitazone (A) or 15d-PG J2 (B), as indicated. Nitrite accumulation in culture medium was measured; values were normalized to protein content per well. Data represent mean±SEM (n=4). *P<0.05 compared with control cells without agonists.

On the other hand, PPAR agonists fenofibrate and Wy14643 showed no effect on the basal and interleukin-1ß–induced nitrite production (data not shown).

Effects of PPAR Agonists on iNOS mRNA and Protein Levels
We then examined whether PPAR agonists decreased iNOS mRNA accumulation in vascular smooth muscle cells. As shown in Figure 2, unstimulated cells did not express iNOS mRNA. Incubation with interleukin-1ß for 24 hours resulted in an induction of iNOS mRNA expression, and its expression was significantly suppressed in the presence of troglitazone or 15d-PG J₂ at 10^-4 mol/L.

View larger version (92K): [in this window] [in a new window]   Figure 2. Expression of iNOS mRNA in vascular smooth muscle cells. Cells were incubated with 10 ng/mL interleukin-1ß for 24 hours in presence of troglitazone or 15d-PG J2 (10-4 mol/L). iNOS mRNA and 28S and 18S ribosomal RNAs visualized by ethidium bromide staining under UV transillumination are shown. Lane 1, Vehicle; lane 2, interleukin-1ß; lane 3, interleukin-1ß plus troglitazone; and lane 4, interleukin-1ß plus 15d-PG J2. Two independent experiments yielded identical results.

The expression of iNOS protein in vascular smooth muscle cells was also analyzed by immunoblotting with the anti-iNOS antibody (Figure 3). No immunoreactive band of iNOS was detected in unstimulated vascular smooth muscle cells. The iNOS protein band with a molecular mass of 125 kDa appeared clearly after exposure to interleukin-1ß for 24 hours. Interleukin-1ß–induced iNOS protein accumulation was significantly decreased by troglitazone and 15d-PG J₂.

View larger version (37K): [in this window] [in a new window]   Figure 3. Expression of iNOS protein in vascular smooth muscle cells. A, Western blot depicting iNOS protein accumulation. Cells were incubated with 10 ng/mL interleukin-1ß for 24 hours with 10-4 mol/L troglitazone or 15d-PG J2. iNOS protein was detected as band with molecular mass of 125 kDa. Lane 1, Vehicle; lane 2, interleukin-1ß; lane 3, interleukin-1ß plus troglitazone; lane 4, interleukin-1ß plus 15d-PG J2; and lane 5, positive control (mouse macrophage iNOS). B, Densitometry values (in arbitrary densitometry units) of iNOS protein on Western blots. Data represent mean±SEM (n=3). *P<0.05 compared with interleukin-1ß–stimulated cells (lane 2).

Involvement of Activation of NF-B
It has been reported that transcription of NF-B is critical for the transcriptional regulation of iNOS.^{21 22} We thus examined whether PPAR agonists modulate NF-B activity in vascular smooth muscle cells. Figure 4 shows the results of the gel retardation assay. Addition of interleukin-1ß induced specific retardation complexes (lane 2, compared with lane 1). Supershift experiments with anti–NF-B p65 antibody confirmed that these complexes contained NF-B (lane 5). These complexes competed with nonradiolabeled NF-B consensus oligonucleotides (lane 7) but not with mutated NF-B oligonucleotides (lane 6). Addition of PPAR agonists troglitazone (lane 3) and 15d-PG J₂ (lane 4) markedly suppressed this interleukin-1ß–induced activation of NF-B.

View larger version (103K): [in this window] [in a new window]   Figure 4. Analysis of NF-B activation by gel retardation assays. Vascular smooth muscle cells were incubated for 1 hour with 10 ng/mL interleukin-1ß in presence or absence of 10-4 mol/L troglitazone or 15d-PG J2. 32P-labeled NF-B consensus oligonucleotides were incubated with nuclear extracts from nonstimulated (lane 1), interleukin-1ß–stimulated (lanes 2, 5 to 7), interleukin-1ß plus troglitazone (lane 3), and interleukin-1ß plus 15d-PG J2 (lane 4) costimulated vascular smooth muscle cells. Anti–NF-B antibody was used for supershift (lane 5). NF-B consensus oligonucleotides (lane 7) and mutated NF-B oligonucleotides (lane 6) were used as competitors. Gel retardation complexes of NF-B are indicated by closed arrows. Supershift complex is indicated by open arrow.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study, we demonstrated that PPAR agonists troglitazone and 15d-PG J₂ decreased NO synthesis in cytokine-stimulated rat vascular smooth muscle cells. On the other hand, PPAR agonists fenofibrate and Wy14643 did not affect the NO production. The decreased NO production by the PPAR agonists was accompanied by decreased iNOS mRNA and protein accumulation. The concentration required to mediate the observed effects was within the therapeutic plasma concentration (10^-5 mol/L) reported for troglitazone.²³

NF-B is critical for the transcriptional regulation of iNOS. It has been shown that iNOS induction depends on the unique NF-B sequence containing nucleotides -85 to -76 of the murine iNOS promoter and binding to this region of a cycloheximide-sensitive complex containing both p50/c-rel and p50/RelA heterodimers of NF-B, in partnership with additional unidentified nuclear protein(s). Additionally, 2 NF-B consensus sequences have been demonstrated in the murine iNOS promoter.^{21 22} The cytokines interleukin-1 and tumor necrosis factor have signal transduction pathways that culminate in the activation of NF-B.²⁴ Previously, Su et al²⁵ reported that PPAR ligands inhibited the activation of NF-B in colonic epithelial cells. We investigated the effects of troglitazone and 15d-PG J₂ on NF-B in interleukin-1ß–stimulated vascular smooth muscle cells and found that both agonists markedly suppressed NF-B activity.

In contrast to our observations, Hattori et al²⁶ recently reported that 15d-PG J₂ decreased whereas troglitazone upregulated NO synthesis in cytokine-stimulated rat vascular smooth muscle cells, with no effect on NF-B activity. The discrepancy between the results of Hattori et al and ours may lie in the phenotypic heterogeneity of rat vascular smooth muscle cell preparations. We thus addressed this issue of phenotypic heterogeneity by examining 3 independently derived rat vascular smooth muscle preparations. Both troglitazone and 15d-PG J₂ decreased NO production in all vascular smooth muscle cell preparations. Differences in cell isolation and preparation, culture conditions, and other factors may all have contributed to the discrepancy in the results between this study and that of Hattori et al.²⁶ Recently, Takano et al²⁷ reported that both troglitazone and 15d-PG J₂ inhibited the activation of NF-B in rat cardiac myocytes, which is compatible with our observation in rat vascular smooth muscle cells.

iNOS activity is induced in blood vessel walls and cultured vascular smooth muscle cells by endotoxins and cytokines.⁵ Joly et al⁶ demonstrated that in vivo balloon injury induced NOS activity in rat carotid arteries, even in the absence of endothelium. Hansson et al²⁸ reported that arterial smooth muscle cells in the neointima formed after deendothelializing balloon injury of the rat carotid artery expressed the cytokine-inducible isoform of NOS. Recently, Buttery et al⁷ reported that iNOS mRNA and protein were present within human arteriosclerotic lesions. Excess NO may prevent atherosclerosis by inhibiting smooth muscle cell proliferation and leukocyte and platelet adhesion. However, NO also has toxic and cytolytic effects, and increased expression of iNOS may promote the process of atherogenesis by increasing cell death and necrosis. Some investigators consider that iNOS activity in the plaque is deleterious because of the formation of peroxynitrite,^{29 30} the product of NO and superoxide, which enhances platelet adhesion and aggregation^{31 32} and induces vascular hyperreactivity.^{33 34} NO also stimulates matrix metalloproteinase activity,³⁵ which might contribute to the weakening of plaque caps by degrading the extracellular matrix and lead to plaque rupture.^{36 37 38} Depre et al³⁹ reported that iNOS expression was induced in the coronary atherosclerotic plaque from patients with unstable angina, and its expression was associated with increased presence of thrombus in the plaque and a higher prevalence of chest pain at rest.

Recently, Colveille-Nash et al⁴⁰ and Ricote et al⁴¹ reported that PPAR agonists reduced NO synthesis in murine macrophages. Ricote et al⁴² and Marx et al⁴³ demonstrated that PPAR was expressed in macrophages in human atherosclerotic lesions. Although PPAR expression in smooth muscle cells in atherosclerotic lesions has not been determined, the present observations suggest that PPAR ligands may influence the progression of atherosclerosis through inhibition of NO production by vascular smooth muscle cells. Indeed, evidence from human patients treated with the PPAR ligand troglitazone and studies of a balloon injury model of atherosclerosis in rats suggested its protective effects on lesion development.^{44 45}

The potential pathological role of excess NO derived from iNOS in the vascular tissue has not been fully characterized and is still controversial.⁸ However, the present study revealed that PPAR agonists such as antidiabetic troglitazone inhibit NO synthesis in cytokine-stimulated vascular smooth muscle cells, suggesting that these agonists may directly act on the vascular smooth muscle and influence the process of atherosclerosis and postangioplasty restenosis.

	Acknowledgments

 
This study was supported by Research Grants for Cardiovascular Diseases (10C-1, 11C-1) from the Ministry of Health and Welfare, a Research Grant from the Ministry of Education, Science, Sports, and Culture, and a grant from Takeda Medical Foundation. We thank Toshiko Kambe for technical assistance.

Received July 26, 1999; first decision September 16, 1999; accepted January 24, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev. 1991;43:109–140.[Medline] [Order article via Infotrieve]

2. Nathan C, Xie Q. Regulation of biosynthesis of nitric oxide. J Biol Chem. 1994;269:13725–13728.[Free Full Text]

3. Shindo T, Ikeda U, Ohkawa F, Takahashi M, Funayama H, Nishinaga M, Kawahara Y, Yokoyama M, Shimada K. Nitric oxide synthesis in rat cardiac myocytes and fibroblasts. Life Sci. 1994;55:1101–1108.[Medline] [Order article via Infotrieve]

4. Ikeda U, Yamamoto K, Maeda Y, Shimpo M, Kanbe T, Shimada K. Endothelin-1 inhibits nitric oxide synthesis in vascular smooth muscle cells. Hypertension. 1997;24:65–69.

5. Dinerman JL, Lowenstein CJ, Snyder SH. Molecular mechanisms of nitric oxide regulation. Circ Res. 1993;73:217–222.[Free Full Text]

6. Joly GA, Schini VB, Vanhoutte PM. Balloon injury and interleukin-1ß induce nitric oxide synthase activity in rat carotid arteries. Circ Res. 1992;71:331–338.[Abstract/Free Full Text]

7. Buttery LD, Springall DR, Chester AH, Evans TJ, Standfield EN, Parums DV, Yacoub MH, Polak JM. Inducible nitric oxide synthase is present within human atherosclerotic lesions and promotes the formation and activity of peroxynitrite. Lab Invest. 1996;75:77–85.[Medline] [Order article via Infotrieve]

8. Ikeda U, Maeda Y, Shima K. Inducible nitric oxide synthase and atherosclerosis. Clin Cardiol. 1998;21:473–476.[Medline] [Order article via Infotrieve]

9. Schoonjans K, Martin G, Steals B, Auwerx J. Peroxisome proliferator-activated receptors, orphans with ligands and functions. Curr Opin Lipidol. 1997;8:159–166.[Medline] [Order article via Infotrieve]

10. Staels B, Koenig W, Habib A, Merval R, Lebret M, Torra IP, Delerive P, Fadel A, Chinetti G, Fruchart JC, Najib J, Maclouf J, Tedgui A. Activation of human aortic smooth-muscle cells is inhibited by PPAR but not by PPAR activators. Nature. 1998;393:790–793.[Medline] [Order article via Infotrieve]

11. Marx N, Schonbeck U, Lazar MA, Libby P, Plutzky J. Peroxisome proliferator-activated receptor activators inhibit gene expression and migration in human vascular smooth muscle cells. Circ Res. 1998;83:1097–1103.[Abstract/Free Full Text]

12. Iijima K, Yoshizumi M, Ako J, Eto M, Kim S, Hashimoto M, Sugimoto N, Liang Y, Sudoh N, Toaba K, Ouchi Y. Expression of peroxisome proliferator-activated receptor (PPAR) in rat aortic smooth muscle cells. Biochem Biophys Res Commun. 1998;247:353–356.[Medline] [Order article via Infotrieve]

13. Couturier C, Brouillet A, Couriad C, Koumanov K, Gereziat G, Andreani M. Interleukin 1ß induces type II-secreted phospholipase A2 gene in vascular smooth muscle cells by a nuclear B and peroxisome proliferator-activated receptor-mediated process. J Biol Chem. 1999;274:23085–23093.[Abstract/Free Full Text]

14. Goetze S, Xi XP, Kawano H, Gotlibowski T, Fleck E, Hsueh WA, Law RE. PPAR gamma-ligands inhibit migration mediated by multiple chemoattractants in vascular smooth muscle cells. J Cardiovasc Pharmacol. 1999;33:798–806.[Medline] [Order article via Infotrieve]

15. Law RE, Meehan WP, Xi XP, Graf K, Wuthrich DA, Coats W, Faxon D, Hsueh WA. Troglitazone inhibits vascular smooth muscle cell growth and intimal hyperplasia. J Clin Invest. 1996;98:1897–1905.[Medline] [Order article via Infotrieve]

16. Ikeda U, Ikeda M, Oohara T, Oguchi A, Kamitani T, Tsuruya Y, Kano S. Interleukin 6 stimulates growth of vascular smooth muscle cells in a PDGF-dependent manner. Am J Physiol. 1991;260:H1713–H1717.[Abstract/Free Full Text]

17. Ikeda U, Kanbe T, Kawahara Y, Yokoyama M, Shimada K. Adrenomedullin augments inducible nitric oxide synthase expression in cytokine-stimulated cardiac myocytes. Circulation. 1996;94:2560–2565.[Abstract/Free Full Text]

18. Green LC, Wagner DA, Glogowski J, Skiper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite and [¹⁵N]nitrate in biological fluids. Anal Biochem. 1982;126:131–138.[Medline] [Order article via Infotrieve]

19. Ikeda U, Kurosaki K, Shimpo M, Okada K, Saito T, Shimada K. Adenosine stimulates nitric oxide synthesis in rat cardiac myocytes. Am J Physiol. 1997;273:H59–H56.[Abstract/Free Full Text]

20. Murakami Y, Ikeda U, Shimada K, Kawakami K. Promoter of the Na, K-ATPase 3 subunit gene is composed of cis elements to which NF-Y and Sp1/Sp3 bind in rat cardiocytes. Biochim Biophys Acta. 1997;1352:311–324.[Medline] [Order article via Infotrieve]

21. Xie QW, Whisnant R, Nathan C. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide. J Exp Med. 1993;177:1779–1784.[Abstract/Free Full Text]

22. Lowenstein CJ, Alley EW, Raval P, Snowman AM, Snyder SH, Russell SW, Murphy WJ. Macrophage nitric oxide synthase gene: 2 upstream regions mediate induction by interferon gamma and lipopolysaccharide. Proc Natl Acad Sci U S A. 1993;90:9730–9734.[Abstract/Free Full Text]

23. Izumi T, Enomoto S, Hosiyama K, Sasahara K, Shibukawa A, Nakagawa T, Sugiyama Y. Prediction of the human pharmacokinetics of troglitazone, a new and extensively metabolized antidiabetic agent, after oral administration, with an animal scale-up approach. J Pharmacol Exp Ther. 1996;277:1630–1641.[Abstract/Free Full Text]

24. Siebenlist U, Franzoso G, Brown K. Structure, regulation and function of NF-B. Annu Rev Cell Biol. 1994;10:405–455.

25. Su CG, Wen X, Bailey ST, Jiang W, Rangwala SM, Keilbaugh SA, Flanigan A, Murthy S, Lazar MA, Wu GD. A novel therapy for colitis utilizing PPAR ligands to inhibit the epithelial inflammatory response. J Clin Invest. 1999;104:383–389.[Medline] [Order article via Infotrieve]

26. Hattori Y, Hattori S, Kasai K. Troglitazone upregulates nitric oxide synthesis in vascular smooth muscle cells. Hypertension. 1999;33:943–948.[Abstract/Free Full Text]

27. Takano H, Asakawa M, Toyozaki T, Nagai T, Saito T, Masuda Y. PPAR ligands inhibit LPS-induced expression of TNF in cardiac myocytes. Circulation. 1999;100(suppl I):I-429. Abstract.

28. Hansson GK, Geng Y, Holm J, Hårdhammar P, Wennmalm Å, Jennische E. Arterial smooth muscle cells express nitric oxide synthase in response to endothelial injury. J Exp Med. 1994;180:733–738.[Abstract/Free Full Text]

29. Sobey CG, Brooks RM, Heistad DD. Evidence that expression of inducible nitric oxide synthase in response to endotoxin is augmented in atherosclerotic rabbits. Circ Res. 1995;77:536–543.[Abstract/Free Full Text]

30. Keaney JF, Vita JA. Atherosclerosis, oxidative stress, and anti-oxidant protection in endothelium-derived relaxing factor action. Prog Cardiovasc Dis. 1995;38:129–154.[Medline] [Order article via Infotrieve]

31. Salvemini D, De Nucci G, Sneddon JM, Vane JR. Superoxide anions enhance platelet adhesion and aggregation. Br J Pharmacol. 1989;97:1145–1150.[Medline] [Order article via Infotrieve]

32. Shatos MA, Doherty JM, Hoack JC. Alteration in human endothelial cell function by oxygen free radicals: platelet adherence and prostacyclin release. Arteroscler Thromb. 1991;11:594–601.[Abstract/Free Full Text]

33. Verbeuren TJ, Jordaens FH, Zonnekeyn LL, Van-Hove CE, Coene MC, Herman AG. Effect of hypercholesterolemia on vascular reactivity in the rabbit. Circ Res. 1986;58:552–564.[Abstract/Free Full Text]

34. Ludmer PL, Selwyn AP, Shook TL, Wayne RR, Mudge GH, Alexander RW, Ganz P. Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic coronary arteries. N Engl J Med. 1986;315:1046–1051.[Abstract]

35. Trachtman H, Futterweit S, Garg P, Reddy K, Singhal PC. Nitric oxide stimulates the activity of a 72-kDa neutral matrix metalloproteinase in cultured rat mesangial cells. Biochem Biophys Res Commun. 1996;218:704–708.[Medline] [Order article via Infotrieve]

36. Shu YE, Humphries S, Henney A. Matrix metalloproteinases: implication in vascular matrix remodeling during atherogenesis. Clin Sci. 1998;94:103–110.[Medline] [Order article via Infotrieve]

37. Stary HC, Chandler AB, Glagov S, Guyton JR, Insull W Jr, Rosenfeld ME, Schaffer SA, Schwartz CJ, Wagner WD, Wissler RW. A definition of initial, fatty streak, and intermediate lesions of atherosclerosis: a report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation. 1994;89:2462–2478.[Abstract/Free Full Text]

38. Libby P. Molecular bases of the acute coronary syndromes. Circulation. 1995;91:2844–2850.[Free Full Text]

39. Depre C, Havaux X, Renkin J, Vanoverschelde JLJ, Wijns W. Expression of inducible nitric oxide synthase in human coronary atherosclerotic plaque. Cardiovasc Res. 1999;41:465–472.[Abstract/Free Full Text]

40. Colveille-Nash PR, Qureshi SS, Willis D, Willoughby DA. Inhibition of inducible nitric oxide synthase by peroxisome proliferator-activated receptor agonists: correlation with induction of heme oxygenase 1. J Immunol. 1998;161:978–984.[Abstract/Free Full Text]

41. Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK. The peroxisome proliferator-activated receptor- is a negative regulator of macrophage activation. Nature. 1998;391:79–82.[Medline] [Order article via Infotrieve]

42. Ricote M, Huang J, Fajas L, Li A, Welch J, Najib J, Witztum JL, Auwerx J, Palinski W, Glass CK. Expression of the peroxisome proliferator-activated receptor (PPAR) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein. Proc Natl Acad Sci, U S A. 1998;95:7614–7619.[Abstract/Free Full Text]

43. Marx N, Sukhova G, Murphy C, Libby P, Plutzky J. Macrophages in human atheroma contain PPAR: differentiation-dependent peroxisome proliferator-activated receptor g (PPAR) expression and reduction of MMP-9 activity through PPAR activation in mononuclear phagocytes in vitro. Am J Pathol. 1998;153:17–23.[Abstract/Free Full Text]

44. Minamikawa J, Yamauchi M, Inoue D, Koshiyama H. Another potential use of troglitazone in non–insulin-dependent diabetes mellitus. J Clin Endocrinol Metab. 1998;83:1041–1042.[Free Full Text]

45. Law RE, Meehan WP, Xi XP, Graf K, Wuthrich DA, Coats W, Faxon D, Hsueh WA. Troglitazone inhibits vascular smooth muscle cell growth and intimal hyperplasia. J Clin Invest. 1996;98:1897–1905.

This article has been cited by other articles:

				H. Tomita, T. Osanai, T. Toki, S. Sasaki, N. Maeda, R. Murakami, K. Magota, M. Yasujima, and K. Okumura Troglitazone and 15-deoxy-{Delta}12,14-prostaglandin J2 inhibit shear-induced coupling factor 6 release in endothelial cells Cardiovasc Res, July 1, 2005; 67(1): 134 - 141. [Abstract] [Full Text] [PDF]

				S. Verma, M. A. Kuliszewski, S.-H. Li, P. E. Szmitko, L. Zucco, C.-H. Wang, M. V. Badiwala, D. A.G. Mickle, R. D. Weisel, P. W.M. Fedak, et al. C-Reactive Protein Attenuates Endothelial Progenitor Cell Survival, Differentiation, and Function: Further Evidence of a Mechanistic Link Between C-Reactive Protein and Cardiovascular Disease Circulation, May 4, 2004; 109(17): 2058 - 2067. [Abstract] [Full Text] [PDF]

				D.-H. Cho, Y. J. Choi, S. A. Jo, and I. Jo Nitric Oxide Production and Regulation of Endothelial Nitric-oxide Synthase Phosphorylation by Prolonged Treatment with Troglitazone: EVIDENCE FOR INVOLVEMENT OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR) {gamma}-DEPENDENT AND PPAR{gamma}-INDEPENDENT SIGNALING PATHWAYS J. Biol. Chem., January 23, 2004; 279(4): 2499 - 2506. [Abstract] [Full Text] [PDF]

				M. Mendez and M. C. LaPointe PPAR{gamma} Inhibition of Cyclooxygenase-2, PGE2 Synthase, and Inducible Nitric Oxide Synthase in Cardiac Myocytes Hypertension, October 1, 2003; 42(4): 844 - 850. [Abstract] [Full Text] [PDF]

				D. S. Calnek, L. Mazzella, S. Roser, J. Roman, and C. M. Hart Peroxisome Proliferator-Activated Receptor {gamma} Ligands Increase Release of Nitric Oxide From Endothelial Cells Arterioscler. Thromb. Vasc. Biol., January 1, 2003; 23(1): 52 - 57. [Abstract] [Full Text] [PDF]

				J. J. Schlezinger, B. A. Jensen, K. K. Mann, H.-Y. Ryu, and D. H. Sherr Peroxisome Proliferator-Activated Receptor {gamma}-Mediated NF-{kappa}B Activation and Apoptosis in Pre-B Cells J. Immunol., December 15, 2002; 169(12): 6831 - 6841. [Abstract] [Full Text] [PDF]

				C. M. Komar and T. E. Curry Jr Localization and Expression of Messenger RNAs for the Peroxisome Proliferator-Activated Receptors in Ovarian Tissue from Naturally Cycling and Pseudopregnant Rats Biol Reprod, May 1, 2002; 66(5): 1531 - 1539. [Abstract] [Full Text]

				C. M. Komar, O. Braissant, W. Wahli, and T. E. Curry Jr. Expression and Localization of PPARs in the Rat Ovary During Follicular Development and the Periovulatory Period Endocrinology, November 1, 2001; 142(11): 4831 - 4838. [Abstract] [Full Text] [PDF]

				K. Berge, K. J. Tronstad, E. N. Flindt, T. H. Rasmussen, L. Madsen, K. Kristiansen, and R. K. Berge Tetradecylthioacetic acid inhibits growth of rat glioma cells ex vivo and in vivo via PPAR-dependent and PPAR-independent pathways Carcinogenesis, November 1, 2001; 22(11): 1747 - 1755. [Abstract] [Full Text] [PDF]

				J. G. Hunter, M. F. van Delft, R. A. Rachubinski, and J. P. Capone Peroxisome Proliferator-activated Receptor gamma Ligands Differentially Modulate Muscle Cell Differentiation and MyoD Gene Expression via Peroxisome Proliferator-activated Receptor gamma -dependent and -independent Pathways J. Biol. Chem., October 5, 2001; 276(41): 38297 - 38306. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) 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 Ikeda, U. Articles by Shimada, K. Search for Related Content PubMed PubMed Citation Articles by Ikeda, U. Articles by Shimada, K. Related Collections Pathophysiology Cell biology/structural biology Gene expression Growth factors/cytokines Smooth muscle proliferation and differentiation Endothelium/vascular type/nitric oxide Mechanism of atherosclerosis/growth factors