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(Hypertension. 1999;33:943-948.)
© 1999 American Heart Association, Inc.

Scientific Contributions

Troglitazone Upregulates Nitric Oxide Synthesis in Vascular Smooth Muscle Cells

Yoshiyuki Hattori; Sachiko Hattori; Kikuo Kasai

From the Department of Endocrinology, Dokkyo University School of Medicine, Mibu, Tochigi, Japan.

Correspondence to Yoshiyuki Hattori, MD, Department of Endocrinology, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan. E-mail yhattori{at}dokkyomed.ac.jp

	Abstract

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Abstract—We investigated the effects of troglitazone on cytokine-stimulated nitric oxide (NO) production in cultured rat vascular smooth muscle cells (VSMC). The increase in NO formation caused by interleukin-1 (IL-1) was enhanced by troglitazone in a concentration-dependent manner. Bacterial lipopolysaccharide–stimulated NO synthesis was also increased by troglitazone. The combinations of IL-1, tumor necrosis factor-, or lipopolysaccharide with interferon- (IFN) were strong stimuli for induction of NO synthesis in VSMC, which were further potentiated by the presence of troglitazone. When troglitazone was added at increasing intervals after the stimulation of VSMC with IL-1, the enhancement in NO production decreased as the interval lengthened, suggesting that troglitazone alters NO synthase (NOS) expression by VSMC rather than having a direct affect on VSMC NOS activity. Troglitazone had no effect on IL-1–elicited or IL-1/IFN–elicited nuclear factor-B activity in VSMC. Troglitazone inhibited the degradation of cytokine-induced NOS mRNA. Thus troglitazone appears to enhance IL-1–induced NOS mRNA levels by prolonging its half-life rather than activating its transcription, which is nuclear factor -B–dependent. No expression of peroxisome proliferator–activated receptor- (PPAR) was detected in VSMC, and 15-deoxy-D^12,14 prostaglandin J₂, the natural ligand for the PPAR, did not resemble the effect of troglitazone on IL-1–induced NO synthesis. These results indicate that troglitazone upregulates cytokine-stimulated NO synthesis in VSMC through PPAR-independent mechanisms. Considering its inhibitory effects on the action of numerous growth factors on VSMC, the direct vascular effects of troglitazone shown in this study may have important implications for prevention of restenosis and possibly atherosclerosis.

Key Words: troglitazone • nitric oxide • peroxisome proliferator–activated receptor- • cytokines • muscle, smooth, vascular

	Introduction

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The thiazolidinediones are novel insulin-sensitizing agents that have been shown to significantly reduce hyperinsulinemia in insulin-resistant animals and humans and to reduce hyperglycemia in diabetic models, including humans.^{1 2 3}

Migration and proliferation of vascular smooth muscle cells (VSMC) are critical events in the development of restenosis and in the progression of atherosclerosis.⁴ A previous study demonstrated that the thiazolidinedione analogue pioglitazone inhibited insulin, epidermal growth factor, and serum-induced growth of cultured arterial VSMC.⁵ The effect of troglitazone has been investigated on intimal hyperplasia after balloon injury of the vessel wall, a situation in which platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are induced and regulate these VSMC activities. Troglitazone inhibited both bFGF-induced VSMC growth and PDGF-induced VSMC migration as well as VSMC intimal hyperplasia after endothelial injury in rats.⁶

In animal models of restenosis after balloon angioplasty, endogenous nitric oxide (NO) also appears to modulate myointimal hyperplasia.⁷ Although the endothelial source of NO synthesis is removed by angioplasty, the inducible isoform of NO synthase (iNOS) is expressed by VSMC in the vicinity of the injury. This may explain the observation that systemic administration of L-arginine (an NO precursor) can reduce the degree of myointimal hyperplasia after balloon angioplasty in animal models and that the effect of arginine is blocked by coadministration of an NO synthase inhibitor.^{8 9}

Proinflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor- (TNF) induce iNOS in VSMC.^{10 11} The purpose of this study was to determine the effect of the thiazolidinedione troglitazone on the cytokine-induced NO synthesis in VSMC. The thiazolidinediones are ligands for the peroxisome proliferator–activated receptor- (PPAR).¹² PPAR has been found to inhibit macrophage activation.¹³ PPAR activators seem to exert their anti-inflammatory action, in part, by antagonizing the activities of transcription factors such as nuclear factor-B (NF-B),¹³ which has been shown to mediate the induction of iNOS.^{14 15} We therefore investigated the effect of troglitazone, the synthetic PPAR agonist, on cytokine-stimulated iNOS gene expression as well as NF-B activation of VSMC to better understand troglitazone's mechanism of action on NO synthesis by VSMC.

	Methods

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Cell Culture and RNA Extraction
VSMC were isolated by elastase and collagenase digestion of thoracic aortas from male Wistar rats, as previously described.¹⁶ Cells in passages 10 to 15 were used for experiments. Total RNA was extracted from confluent VSMC by use of a modified guanidinium isothiocyanate method.¹⁷

Nitrite Assay
Nitrite accumulation, an indicator of NO synthesis, was measured in the cell culture medium of confluent VSMC.¹⁸ Nitrite was quantified colorimetrically after adding 100 µL of Griess reagent (1% sulfanilamide and 0.1% naphthylenediamine in 5% phosphoric acid) to 100-µL samples. Absorbance at 550 nm was determined with a microplate reader (Molecular Devices). Nitrite concentrations were calculated by comparison with the absorbance of standard solutions of sodium nitrite prepared in cell culture medium.

Northern Blot Analysis of iNOS mRNA
An iNOS cDNA (kindly provided by Dr Nunokawa)¹⁹ labeled with [-³²P]dCTP by random priming was used as a probe. Total RNA (10 µg per lane) was subjected to electrophoresis on a 1.2% agarose gel containing formaldehyde and transferred to nitrocellulose filters. The filters were prehybridized at 68°C for 15 minutes and then hybridized with the ³²P-labeled iNOS cDNA probe in a rapid hybridization solution (QUIKHYB; Stratagene) at 68°C for 1 hour. The hybridized filters were washed twice for 15 minutes at room temperature with 2x SSC/0.1% SDS and then twice for 30 minutes at 60°C with 0.1x SSC/0.1% SDS. The filters were exposed to an imaging plate (Fuji Photo Film Co) at room temperature for 6 hours and analyzed with the use of a FUJIX bioimaging analyzer (BAS2000II, Fuji Photo Film Co).

NF-B Activation
To study NF-B activation, the cells were stably transfected with a cis-reporter plasmid containing the luciferase reporter gene linked to 5 repeats of NF-B binding sites (pNFB-Luc, Stratagene). For this, the pNFB-Luc plasmid was transfected together with a pSV2neo helper plasmid (Clontech, Palo Alto, Calif) into rat VSMC with FuGEN 6 transfection reagent (Boehringer Mannheim, Mannheim, Germany). The cells were cultured in the presence of G418 (Clontech) at a concentration of 500 µg/mL with medium replacement at 2- to 3-day intervals. Approximately 3 weeks later, G418-resistant clones were isolated with the use of a cloning cylinder and analyzed individually for expression of luciferase activity. Thus several clones were selected for analysis of NF-B activation. Luciferase activity was measured with a luciferase assay kit (Stratagene).

Reverse Transcription-Polymerase Chain Reaction Assay of PPAR mRNA
Reverse transcription polymerase chain reaction (RT-PCR) was performed by standard methods.¹⁶ The sequences of the forward (21-mer, 5'-AGCCCTTTACCACAGTTGATT-3') and reverse (21-mer, 5'-AGACATCCCCACAGCAAG-3') primers were based on the published mouse PPAR cDNA sequence.²⁰ The predicted amplification product was 425 bp in length, comprising nucleotides 588 to 1012 of mouse PPAR.²⁰ In addition, RT-PCR to distinguish PPAR1 mRNA from PPAR2 mRNA was performed for 3T3-L1 cells with the use of primers for amplification of each entire coding region. PCR products were electrophoresed through a 1.5% agarose gel containing ethidium bromide and visualized by UV-induced fluorescence. To ensure that equal amounts of reverse-transcribed RNA were added to the PCR reaction, we amplified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA in parallel, as a reference, using primers described by Terada et al.²¹ The PCR primers used for the full-length coding regions of PPAR1 and PPAR2 are: PPAR1 forward, AAGACTACCCTTTACTGAAATTACC; PPAR2 forward, AGCAAATCTCTGTTTTATGCTGTT; and reverse, TTCCTGCTAATACAAGTCCTTGTA.

Statistical Analysis
Data are presented as mean±SEM. Multiple comparisons were evaluated by ANOVA followed by Fisher's protected least-significant difference test. Student's unpaired t test was used for comparisons between 2 experiments. A value of P<0.05 was considered statistically significant.

	Results

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We examined the effects of troglitazone on NO synthesis by rat VSMC stimulated with IL-1, TNF, and lipopolysaccharide (LPS) alone or in combination with interferon- (IFN). As shown in Figure 1, the increase in NO formation caused by IL-1 was enhanced by troglitazone in a concentration-dependent manner. LPS-stimulated NO synthesis was also increased by troglitazone. NO synthesis was not induced in cells treated with TNF alone or IFN alone in the presence and absence of troglitazone. The combination of IL-1, TNF, or LPS with IFN were strong stimuli for induction of NO synthesis in VSMC, confirming prior reports that immuno-stimulants induce iNOS activity in rat VSMC by a mechanism that is synergistic with IFN.¹⁸ The increased induction of NO synthesis caused by IL-1, LPS, or TNF in combination with IFN was also enhanced in the presence of troglitazone.

View larger version (21K): [in this window] [in a new window]   Figure 1. Effects of troglitazone on cytokine-induced nitrite production in VSMC. VSMC were stimulated with IL-1 (10 ng/mL), LPS (30 µg/mL), or TNF (10 ng/mL) alone or in combination with IFN (100 U/mL) for 24 hours, after which nitrite accumulation in culture medium was measured. Data are mean±SEM of triplicate observations. *P<0.05 and **P<0.01 compared with control (no troglitazone).

Time-course studies, with the use of a final concentration of 25 µmol/L troglitazone, were conducted to determine whether troglitazone has a direct affect on VSMC NOS activity or alters iNOS expression by VSMC (Figure 2). When troglitazone was added at increasing intervals after the stimulation of VSMC with IL-1, the enhancement in nitrite production decreased as the interval lengthened. When troglitazone was added 8 hours after stimulation with IL-1, the amounts of nitrite in the supernatants were the same as in supernatants from the activated controls (no troglitazone).

View larger version (52K): [in this window] [in a new window]   Figure 2. Effect of adding troglitazone after stimulation of VSMC with IL-1. Troglitazone (25 µmol/L) was added at the indicated time after stimulation of VSMC with 10 ng/mL IL-1. Nitrite accumulation was measured 24 hours after administration of IL-1. Data are mean±SEM of triplicate observations. **P<0.01 compared with control (time=0 hours).

Next we investigated the effects of troglitazone on iNOS mRNA induction and its stability. We stimulated VSMC with IL-1 in the presence or absence of troglitazone. After 6 hours, iNOS mRNA levels were assessed by Northern blot analysis for IL-1–stimulated or (IL-1+troglitazone)–stimulated cells. Thereafter, the rate of disappearance of iNOS mRNA was evaluated after blocking further transcription with actinomycin D (5 µg/mL). As shown in Figure 3, the rate of decay of iNOS mRNA was blunted markedly by treatment with troglitazone. In the cells treated with troglitazone, iNOS mRNA decayed much more slowly, and the half-life increased >4-fold after the addition of troglitazone from 2 hours to 8 hours compared with control cells.

View larger version (27K): [in this window] [in a new window]   Figure 3. Effect of troglitazone on iNOS mRNA stability in rat aortic smooth muscle cells. Cells were incubated for 6 hours with IL-1 in the presence or absence of troglitazone (50 µmol/L) to increase the levels of iNOS mRNA and then exposed to 5 µg/mL of actinomycin D (ACTD). After the indicated times, total RNA was isolated and analyzed by Northern blot analysis with an iNOS-specific probe. Quantitation was performed with a bioimaging analyzer, and data represent mean of duplicate determinations from each of 2 RNA preparations (IL-1 only, ; IL-1+troglitazone, ).

In addition, we investigated the effects of troglitazone on regulation of NF-B activation by using VSMC stably transfected with an NF-B response element-luciferase reporter plasmid. Since troglitazone is reported to have antioxidant properties,^{22 23} its effect on NF-B activity was compared with 2 antioxidants, N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), which have been shown to prevent NF-B activation.^{24 25} IL-1 either alone or in combination with IFN markedly induced NF-B activity, though IFN by itself did not show any effects. Troglitazone had no effects on IL-1–induced or IL-1/IFN–induced NF-B activity at concentrations examined (Figure 4). In contrast, NAC (1 mmol/L) or PDTC (50 µmol/L) markedly attenuated IL-1–induced NF-B activity, by 68% and by 88%, respectively.

View larger version (14K): [in this window] [in a new window]   Figure 4. Effect of troglitazone on NF-B–dependent transcriptional activity. VSMC were transfected with pNFB-Luc (a cis-reporter plasmid containing the luciferase reporter gene linked to 5 repeats of an NF-B binding site). The cells were left untreated () or were treated with IL-1 () or IL-1/IFN () in the presence of different concentrations of troglitazone. After 4 hours, cells were lysed and luciferase activities were measured. Data are mean±SEM of triplicate observations.

We next determined whether the effects of 15-deoxy-D^{12 13} prostaglandin J₂ (15 days-PGJ₂; Cayman Chemical, Ann Arbor, Mich), the natural ligand for PPAR,^{26 27} resemble those of troglitazone on IL-1–induced NO synthesis in VSMC. The 15 days-PGJ2 did not increase IL-1–induced NO production but rather decreased it at the higher concentrations (Figure 5).

View larger version (12K): [in this window] [in a new window]   Figure 5. Effects of 15 days-PGJ2 on cytokine-induced nitrite production in VSMC. VSMC were stimulated with IL-1 (10 ng/mL) for 24 hours, after which nitrite accumulation in the culture medium was measured. Data are mean±SEM of triplicate observations. *P<0.05 and **P<0.01 compared with control (no troglitazone).

Using RT-PCR, we examined whether PPAR mRNA is present in VSMC. Although PPAR mRNA is clearly expressed in 3T3-L1 fibroblasts and its expression is substantially increased by 3-day treatment, which induces differentiation into adipocytes (3T3-L1/D),²⁸ it was barely detectable in VSMC. We did not find any increase in PPAR mRNA abundance in VSMC even after the cells were stimulated with IL-1 or IL-1/IFN (data not shown). To confirm that adipocytes really express PPAR mRNA, RT-PCR for amplification of the entire coding regions of both PPAR1 and PPAR2 was performed separately with 3T3-L1/D cells, which express both mRNAs (Figure 6).

View larger version (42K): [in this window] [in a new window]   Figure 6. RT-PCR analysis of PPAR and GAPDH expression in rat VSMC and mouse 3T3-L1 cells. RNA was isolated from 3T3-L1 cells untreated (3T3-L1) or treated for 3 days with dexamethasone/insulin/3-isobutyl-1-methylxanthine (3T3-L1/D) as well as VSMC (in triplicate) and amplified with the use of PPAR-specific primers (top). Separate experiment shows RT-PCR amplification of the full-length coding regions for PPAR1 and PPAR2 with RNA from 3T3-L1/D cells (bottom).

	Discussion

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The present investigation demonstrates that troglitazone increases cytokine-stimulated NO synthesis in VSMC. To learn whether these effects of troglitazone on cytokine-induced NO synthesis by VSMC are mediated by PPAR, we examined whether the effects of the natural PPAR ligand, 15 days-PGJ2,^{26 27} resemble those of troglitazone and whether PPAR mRNA is expressed in VSMC. We found that 15 days-PGJ2 does not enhance cytokine-stimulated NO synthesis by VSMC and that with the use of RT-PCR, the expression of PPAR mRNA is very low in VSMC, suggesting that the stimulatory effect of troglitazone on cytokine-induced NO generation is unlikely to be mediated by PPAR. The potential importance of the NF-B system as a key player in control of transcription of genes for mediators of a variety of inflammatory responses, from those mediated by cytokine pathways to atherogenesis and thrombogenesis, is receiving increasing recognition. Interestingly, it has been shown that macrophage expression of PPAR is markedly upregulated on cell activation and that PPAR-specific ligands inhibit expression of certain genes, including iNOS, by antagonizing the transcription factors such as NF-B in activated macrophage.¹³ By contrast, VSMC appear to express very low levels of PPAR both under basal and activated conditions, and troglitazone had no effects on IL-1–induced NF-B activities in VSMC. Alternatively, we found that troglitazone inhibits the degradation of cytokine-induced iNOS mRNA. Thus troglitazone appears to enhance IL-1–induced iNOS mRNA levels by prolonging mRNA half-life rather than activating iNOS transcription, which is NF-B dependent.^{14 15} Unlike NAC and PDTC,^{24 25} troglitazone does not suppress cytokine-induced NO production by inhibiting the activation of NF-B as an antioxidant but enhances NO generation by potentiating iNOS induction.

In clinical studies, within the therapeutic range of 200 to 600 mg once daily, troglitazone improves glycemic control and insulin sensitivity. The postdose plasma drug concentrations in humans observed 2.5 to 3.5 hours after 200-mg troglitazone ranged from 0.5 to 0.7 µg/mL (:11.3 to 15.9 µmol/L).²⁹ In the present study, troglitazone potentiated cytokine-induced formation of NO in VSMC with a 2.6-fold increase at 50 µmol/L and initial detectable effects at 10 µmol/L. These concentrations that enhance NO formation in vitro are likely to be attained in plasma or vascular tissue under therapeutic conditions in humans.

It is unknown whether agents that inhibit VSMC growth prevent the development of advanced atherosclerotic lesions; thiazolidinediones may be useful agents to address this question. This issue is particularly relevant to the diabetic who has a 2- to 4-fold increased incidence of coronary artery disease compared with the nondiabetic.³⁰ Coronary balloon angioplasty is associated with a high rate of restenosis that detracts from its clinical value in the treatment of coronary artery disease. A recent clinical study shows that NO donors, which inhibit VSMC proliferation and decrease platelet aggregation in addition to their vasodilator effect, appear to have beneficial effects on restenosis after coronary balloon angioplasty.³¹ More recently, it has been demonstrated that troglitazone has a potent inhibitory effect on carotid arterial wall thickness in type 2 diabetes.³² Taken together, the demonstration that troglitazone increases the NO generation in VSMC raises strong interest in its potential utility to inhibit human restenosis.

Troglitazone is an agent that not only improves the insulin-resistant state in diabetic patients³ but also inhibits the action of numerous growth factors on VSMC.⁶ The finding that troglitazone potentiates the induction of NO synthesis may have important implications for inhibition of restenosis and atherosclerosis, pathological states in which NO plays a preventative role. Thiazolidinediones may be useful for their metabolic effects as well as for their direct vascular effects in insulin-resistant subjects, who have an increased incidence of coronary artery diseases.

	Acknowledgments

 
This work was supported in part by a grant from the Japan Private School Promotion Foundation. Troglitazone was a generous gift from Sankyo Pharmaceutical Co (Tokyo, Japan).

Received October 27, 1998; first decision November 23, 1998; accepted December 1, 1998.

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