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(Hypertension. 1995;25:764-768.)
© 1995 American Heart Association, Inc.

Articles

Cyclosporin A Inhibits Nitric Oxide Synthase Induction in Vascular Smooth Muscle Cells

Takeshi Marumo; Toshio Nakaki; Keiichi Hishikawa; Hiromichi Suzuki; Ryuichi Kato; Takao Saruta

From the Departments of Internal Medicine (T.M., K.H., H.S., T.S.) and Pharmacology (T.M., T.N., R.K.), Keio University School of Medicine, Tokyo, Japan.

Correspondence to Toshio Nakaki, MD, PhD, Department of Pharmacology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan.

	Abstract

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Abstract The effect of cyclosporin A on induction of nitric oxide synthase in rat aortic smooth muscle cells was examined. A combination of interleukin-1 (100 U/mL) and tumor necrosis factor- (5000 U/mL) induced accumulation of nitrite/nitrate, the stable end products of nitric oxide, in culture media within 48 hours. Cyclosporin A inhibited this nitrite/nitrate accumulation in a concentration-dependent manner with an IC₅₀ of 4x10^-7 mol/L when applied simultaneously with the cytokines. The expression of inducible nitric oxide synthase messenger RNA (mRNA) induced by the combination of interleukin-1 and tumor necrosis factor– was inhibited by the cyclosporin A cotreatment. Cyclosporin A did not decrease inducible nitric oxide synthase mRNA stability in the presence of transcription inhibitor actinomycin D (5 µg/mL). Induction of nitrite/nitrate production by the combination of tumor necrosis factor– and bacterial lipopolysaccharide or that of interleukin-1 and interferon gamma (100 U/mL) was also inhibited by cyclosporin A cotreatment. Another inhibitor of calcineurin, FK506 (up to 10^-6 mol/L), had no effect on the induction of nitrite/nitrate production, suggesting the possibility that the inhibitory effect of cyclosporin A may be exerted by means of a novel pathway other than inhibition of calcineurin. These results indicate that cyclosporin A inhibits inducible nitric oxide synthase induction at the mRNA level and that inducible nitric oxide synthase in vascular smooth muscle cells can be a target for cyclosporin A, providing a possible mechanism for the interference of the drug with the balance of vasoactive substances.

Key Words: cyclosporine • nitric oxide • interleukin-1 • tumor necrosis factor • interferon type II

	Introduction

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Arterial hypertension and renal toxicity are the main side effects of cyclosporin A (CsA), a widely used immunosuppressive agent. The precise mechanisms by which CsA induces hypertension are not well understood. Activation of the sympathetic nervous system, alteration in the prostaglandin-thromboxane balance, increased endothelin production, and increased cytosolic calcium concentration have all been suggested.¹ Impaired endothelium-dependent relaxation and defective nitric oxide (NO) production from endothelium by CsA have also been shown.¹

NO, synthesized from L-arginine, is a molecule with diverse biological functions in the cardiovascular system, exerting vasodilation, inhibition of adhesion and aggregation of platelets,² and inhibition of vascular smooth muscle cell growth.^{3 4} Three NO synthase (NOS) isoforms encoded by distinct genes have been identified thus far⁵ : inducible NOS (iNOS) and two constitutive NOS isoforms constitutively expressed in the brain and vascular endothelial cells. Immunological and inflammatory stimuli such as bacterial lipopolysaccharide (LPS) and cytokines induce iNOS in the vasculature, including endothelial and smooth muscle cells.^{2 5 6} Recent reports suggest the potential roles of iNOS in vascular remodeling and control of blood pressure under certain conditions. Overproduction of NO by induced iNOS in the vasculature is partly responsible for hypotension in sepsis.^{2 6} NOS activity is induced in the vasculature of cholesterol-fed animals⁷ and after balloon angioplasty.^{8 9} Furthermore, Chen and Sanders¹⁰ reported that defective generation of NO by iNOS produced salt-sensitive hypertension in Dahl/Rapp rats.

Although iNOS is a possible target for CsA, knowledge about the effects of CsA on iNOS induction in vascular smooth muscle cells is still lacking. Therefore, we investigated the effect of CsA on induction of iNOS in rat aortic smooth muscle cells.

	Methods

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Smooth Muscle Cells
Rat aortic smooth muscle cells (RACS-1) were plated on 10-cm dishes for Northern blot analysis or on 9-cm² flasks for other experiments at an initial density of approximately 2.0x10³ cells/cm² and grown in Earle's M199 medium supplemented with 10% fetal calf serum, penicillin (100 U/mL), and streptomycin (100 U/mL) until the cells reached confluence, as previously described.^{11 12} All RACS-1 cultures were used at passages 4 to 5 from stock cells, as described in previous reports.^{11 12} Glassware was used after being heated at 250°C for 2 hours, and LPS-free pipette tips were used throughout the experiments. LPS-free water was used for culture media, as described previously.¹³

Induction of NOS in RACS-1
Cells were washed once with Dulbecco's modified essential medium (DMEM) without phenol red, supplemented with sodium selenite (5 ng/mL), insulin (5 µg/mL), transferrin (5 µg/mL), penicillin (100 U/mL) and streptomycin (100 U/mL) (SIT), and were incubated in DMEM with SIT for 24 or 48 hours, with or without reagents. Because iNOS is effectively induced by the combination of cytokines and LPS in RACS-1,¹⁴ cells were stimulated by the addition of a combination of human interleukin-1 (IL-1) and human tumor necrosis factor–1 (TNF-) or that of IL-1 and rat interferon gamma (IFN-) or that of LPS and TNF- with or without CsA or FK506, an inhibitor of calcineurin. The drugs were dissolved in dimethyl sulfoxide at a concentration of 0.1% (vol/vol). This concentration of dimethyl sulfoxide was added to all culture media in experiments with these drugs. For measurement of iNOS messenger RNA (mRNA) stability, cells were stimulated with a combination of IL-1 (100 U/mL) and TNF- (5000 U/mL) for 24 hours followed by 1 hour of coincubation with CsA (10^-5 mol/L) or vehicle, after which actinomycin D (5 µg/mL) was added to stop transcription. After indicated incubation periods, cells were harvested and Northern blot analysis was carried out. Aliquots of the culture media were taken for LPS determination soon after the reagents had been added and for nitrite/nitrate at the end of each incubation time. The samples for LPS measurement were stored until assay at -80°C.

Assays
LPS was quantified with a commercially available kit (Endospecy ES-6 set and Toxicolor DIA set, Seikagaku Corp). LPS concentrations in the culture media were less than 20 pg/mL in all experiments. Nitrate was reduced to nitrite by passage of the samples through a cadmium column, and the total amounts of nitrite were measured based on the Griess reaction, as described previously.^{15 16 17} Nitrite/nitrate values at 0 hours were subtracted from cumulative values obtained from the same flask. The nitrite/nitrate accumulations observed in flasks without RACS-1, which ran in parallel with those containing RACS-1, were then subtracted from each cumulative value.

Northern blot analysis was performed with a 700-bp fragment of the 5' portion of cloned rat liver iNOS complementary DNA (cDNA)¹⁸ and a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe, as previously described.¹⁴ The iNOS cDNA probe was kindly provided by Dr Hiroyasu Esumi (National Cancer Center Research Institute, Matsudo, Japan). The GAPDH cDNA probe, known to hybridize with rat GAPDH mRNA, was purchased from Clontech Laboratories, Inc. Radioactivities of the membranes were measured by an imaging analyzer (BAS2000, Fuji Photo Film Co Ltd).

Untreated cells that ran parallel in each experiment were washed three times with 0.9% NaCl, and the protein content was determined with a Bio-Rad protein assay kit.

Reagents
Human recombinant IL-1 was a gift from Dainippon Pharmaceutical Co, Ltd. Human recombinant TNF- was a gift from Suntory. Rat IFN- was purchased from Gibco BRL. LPS-free water (<1 pg/mL) was purchased from Otsuka Pharmaceutical Co Ltd. CsA was a gift from Sandoz. FK506 was obtained from Fujisawa Pharmaceuticals. LPS-free pipette tips and LPS-free bovine serum albumin were from Seikagaku Co. E coli LPS (serotype 055:B5) was purchased from Sigma Chemical Co. Dimethyl sulfoxide was purchased from Kanto Chemical Co.

Concentrated IL-1, TNF-, and IFN- were diluted in DMEM containing 0.1% LPS-free bovine serum albumin and aliquoted before storage at less than -20°C.

Statistics
Results are presented as mean±SEM. Statistical significance was evaluated with Student's t test (P<.05).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Because NOS was not induced by IL-1, TNF-, or LPS in RACS-1 when each was applied alone,¹⁴ we used the cytokines in combinations. The combination of IL-1 (100 U/mL) and TNF- (5000 U/mL) induced nitrite/nitrate production in RACS-1 within 48 hours (Fig 1A), which is consistent with our previous report.¹⁴ CsA, when added simultaneously with IL-1 and TNF-, concentration-dependently inhibited the nitrite/nitrate accumulation (Fig 1A). With CsA at a maximum concentration (10^-5 mol/L), a small number of cells detached at the end of a 48-hour incubation. More than 95% of cells, including the detached cells, were negative with trypan blue. To clarify whether the inhibitory effect of CsA on nitrite/nitrate accumulation is specific for the combination of IL-1 and TNF-, we next investigated the effect of CsA on nitrite/nitrate production by other inducers of iNOS. CsA concentration-dependently inhibited nitrite/nitrate accumulation induced by the combination of TNF- (5000 U/mL) and LPS (100 ng/mL) (Fig 1B) or that of IL-1 (100 U/mL) and IFN- (100 U/mL) (Fig 1C). The proportion of trypan blue–positive cells was less than 5% with all these treatments.

View larger version (15K): [in this window] [in a new window]   Figure 1. Concentration-response curves for cyclosporin A (CsA) show inhibition of production of nitrite/nitrate in cytokine-treated rat aortic smooth muscle cells. Cells were treated with a combination of 100 U/mL interleukin-1 and 5000 U/mL tumor necrosis factor– (A), with a combination of 5000 U/mL tumor necrosis factor– and 100 ng/mL lipopolysaccharide (B), or a combination of 100 U/mL interleukin-1 and 100 U/mL interferon gamma (C) for 48 hours with or without various concentrations of CsA. Data are mean±SEM (n=3). For symbols without bars, the SEM is within the symbols. Protein content was 0.1 mg per flask.

Next, the effect of FK506, another calcineurin inhibitor, on nitrite/nitrate production by RACS-1 was examined. No effect of FK506 up to 10^-6 mol/L was detected on nitrite/nitrate production induced by IL-1 (100 U/mL) and TNF- (5000 U/mL) in RACS-1 (Fig 2).

View larger version (15K): [in this window] [in a new window]   Figure 2. Plot shows effect of FK506 on production of nitrite/nitrate in cytokine-treated rat aortic smooth muscle cells. Cells were treated with a combination of 100 U/mL interleukin-1 and 5000 U/mL tumor necrosis factor– for 48 hours with or without various concentrations of FK506. Data are mean±SEM (n=3). For symbols without bars, the SEM is within the symbols. Protein content was 0.2 mg per flask.

Northern blot analysis of iNOS mRNA showed that the combination of IL-1 (100 U/mL) and TNF- (5000 U/mL) induced iNOS mRNA (Fig 3), which is consistent with our previous report.¹⁴ As was the case with nitrite/nitrate accumulation in culture media, CsA (10^-6, 10^-5 mol/L) inhibited the induction of iNOS mRNA at 24 hours (Fig 3). The gel stained by ethidium bromide after electrophoresis revealed that equal amounts of RNA had been loaded. At the end of a 24-hour incubation, the number of detached cells was negligible and more than 95% of cells excluded trypan blue.

View larger version (41K): [in this window] [in a new window]   Figure 3. Northern blot shows inhibitory effect of cyclosporin A on inducible nitric oxide synthase (iNOS) messenger RNA induction in cytokine-treated vascular smooth muscle cells. A, Rat aortic smooth muscle cells were either not treated (lane 1) or treated with a combination of 100 U/mL interleukin-1 and 5000 U/mL tumor necrosis factor– in the absence (lane 2) and presence of 10-6 mol/L (lane 3) or 10-5 mol/L (lane 4) cyclosporin A for 24 hours. B, The gel was stained with ethidium bromide. Cellular protein content was 0.8 mg per dish.

To determine the effect of CsA on iNOS mRNA stability, we measured mRNA levels in the presence of the transcription inhibitor actinomycin D (5 µg/mL) (Fig 4). CsA (10^-5 mol/L) did not decrease the stability of iNOS mRNA induced by the 24-hour stimulation with a combination of IL-1 (100 U/mL) and TNF- (5000 U/mL), but iNOS mRNA decreased slightly more slowly in the CsA-treated cells than in the untreated cells.

View larger version (22K): [in this window] [in a new window]   Figure 4. Northern blots show effect of cyclosporin A on the decay of inducible nitric oxide synthase (iNOS) messenger RNA in rat aortic smooth muscle cells. Cells were stimulated for 24 hours in the presence of a combination of 100 U/mL interleukin-1 and 5000 U/mL tumor necrosis factor–. Vehicle or 10-5 mol/L cyclosporin A was then added to the culture medium and coincubated with the cytokines for 1 hour. After the coincubation period, actinomycin D (5 µg/mL) was added to the culture medium. Total RNA was extracted at the indicated time points after addition of actinomycin D. Northern blot analysis for iNOS messenger RNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA in rat aortic smooth muscle cells treated with vehicle (A) or cyclosporin A (B) was then performed. To correct for differences in loading, the signal density of each RNA sample hybridized to the iNOS probe was divided by that of each RNA sample hybridized to the GAPDH probe. The corrected density for each time point was then divided by that of 0 hour and was plotted as a percentage of the 0 hour value against time (C). indicates vehicle; , cyclosporin A. Cellular protein content was 1.5 mg per dish.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrated for the first time that CsA inhibits iNOS expression at the levels of mRNA and nitrite/nitrate, the stable end products of NO in vascular smooth muscle cells. Both CsA and FK506, after binding to immunophilins (cyclophilin and FK506-binding protein, respectively), inhibit calcineurin, and calmodulin-dependent phosphatase, and their immunosuppressive activity is strictly correlated with inhibition of calcineurin.¹⁹ Our finding that CsA but not FK506 inhibits iNOS induction suggests the possibility that the inhibitory effect of CsA may be exerted by means of a novel pathway other than inhibition of calcineurin in vascular smooth muscle cells. Because the expression of FK506-binding protein 12 has been recently shown to vary in different cell types,¹⁹ we cannot completely exclude the possibility that the discrepancy in inhibition of iNOS induction between CsA and FK506 may be due to a lack of FK506-binding protein in RACS-1. Thus, convincing evidence for the involvement of a novel pathway in the effect of CsA awaits proof that FK506 is, in fact, capable of inhibiting calcineurin in vascular smooth muscle cells under these conditions.

Incidence of hypertension has been reported to be less frequent in FK506-treated patients than in those treated with CsA,^{20 21} although one group recently showed that the difference is not statistically significant.²² A lower incidence of hypertension in patients treated with FK506 suggests involvement of a mechanism other than inhibition of calcineurin or the possibility that FK506 might be less effective in the cardiovascular system.

It is reported that CsA inhibits NO production in cytokine-treated L929 cells²³ and induction of NO production and iNOS mRNA in mesangial cells by IL-1ß.²⁴ However, the effect of CsA on iNOS mRNA stability has not been assessed in these reports. Furthermore, it is impossible to extrapolate from these studies to predict effects in vascular smooth muscle cells, because regulation of iNOS induction appears to vary between different cell types.^{2 25} The cytokine requirement to effectively induce iNOS is different among cell types. Although cyclic AMP–elevating agents have been reported to enhance cytokine-stimulated NO production in vascular endothelial cells,²⁶ smooth muscle cells,²⁷ and Kupffer cells,²⁸ these agents inhibit the effects of cytokines in macrophages²⁹ and astroglial cells.³⁰ Conflicting effects of protein kinase C on iNOS induction have also been reported among different cell types.^{31 32 33} The present demonstration of the inhibition of iNOS induction by CsA in vascular smooth muscle cells, together with reports on L929 cells²³ and mesangial cells,²⁴ suggests that the inhibitory effect of CsA on iNOS induction is a widespread phenomenon.

It is reported that coinduction of tetrahydrobiopterin synthesis with iNOS contributes to increased NO production in vascular smooth muscle cells.^{6 34} Our results indicate that CsA inhibits NO production at least in part at the level of mRNA in these cells. The effects of CsA on iNOS cofactor synthesis, translational regulation, and posttranslational regulation await further study.

CsA did not decrease iNOS mRNA stability in the presence of actinomycin D, suggesting that CsA inhibits iNOS induction by inhibition of transcription. A recent report showed destabilization of IL-3 mRNA by CsA in tumor cell lines, mediated in part by the AUUUA motif in the 3' portion.³⁵ This mechanism also has a potential importance for iNOS and endothelial constitutive NOS, because both contain the mRNA-destabilizing motif AUUUA in the 3' portion.^{36 37} However, FK506 has also been found to downregulate IL-3 mRNA in those cells,³⁵ a result different from that in the present study. Although it is possible that CsA destabilizes iNOS mRNA by a mechanism similar to that of IL-3 mRNA under certain conditions, it is unlikely that the decrease in iNOS mRNA can be explained by the mRNA-destabilizing effect of CsA.

Analysis of the promoter-regulatory region of mouse iNOS shows that numerous binding sites for transcription factors are present, such as NF-B, NF-IL6, the Pu.1/IFN- element, the TNF response element, and AP-1.^{38 39} CsA inhibits transcription of the IL-2 gene by inhibiting translocation of transcription factor NF-AT from the cytoplasm to the nucleus.¹⁹ The activities of other transcription factors such as Oct-1/OAP or NF-B are also sensitive to CsA.¹⁹ It may be that CsA, after binding to its receptor protein, inhibits iNOS induction by inhibiting activation of particular transcription factors such as NF-B.

The combination of IL-1 and TNF- was the most efficient inducer of nitrite production among the combinations tested in RACS-1. In macrophages, the iNOS promoter is reported to be functionally divided into two regions.³⁸ Activation of transcription factors, which bind each region, synergistically induces transcription of the iNOS gene.³⁸ Considering these findings, it is possible that in RACS-1, the combination of IL-1 and TNF- may activate a highly efficient set of transcription factors to induce the iNOS gene compared with the other combinations of cytokines and LPS tested in the present study. The potency of CsA varied depending on which cytokines were used. CsA was less potent in inhibiting nitrite production by the cells treated with the combination of IL-1 and IFN- than was the combination of IL-1 and TNF- or that of TNF- and LPS. It is possible that CsA inhibits transcription factors activated by the combination of IL-1 and TNF- or that of TNF- and LPS more effectively than it inhibits transcription factors activated by the combination of IL-1 and IFN-. Full elucidation of the underlying mechanisms requires analysis of transcription factors in each cell type. In some pathological situations in vivo, where different combinations of cytokines may occur, the efficiency of CsA-mediated inhibition of iNOS induction is likely to vary among tissue types.

Recommended trough whole blood concentrations of CsA are 8.30x10^-8 mol/L to 2.50x10^-7 mol/L (100 to 300 ng/mL), although target concentrations of CsA depend on the clinical indication for treatment and time after initiation of therapy.⁴⁰ Peak concentration of CsA after oral administration reaches more than 8.30x10^-7 mol/L (1000 ng/mL).⁴¹ Because the observed IC₅₀ for iNOS induction was 4.00x10^-7 mol/L (480 ng/mL) in the present study, it is possible that CsA inhibits iNOS induction in some in vivo settings.

There is precise evidence that iNOS is induced in the vasculature in particular conditions.^{6 7 8 9} It has also been shown that iNOS plays an important role in the prevention of salt-sensitive hypertension.¹⁰ Although some steps require further elucidation, the inhibition of iNOS induction in vascular smooth muscle cells shown in the present study may prove to be one of the mechanisms underlying CsA-induced hypertension.

	Acknowledgments

 
This work was supported in part by the Mitsukoshi Prize for Medicine 1992 and by a Grant-in-Aid for General Scientific Research from the Ministry of Education, Science and Culture of Japan.

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