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Pimobendan inhibits the activation of transcription factor NF-κB
Life Sciences 67 (2000) 2513–2519
Pharmacology letters Accelerated communication
Pimobendan inhibits the activation of transcription factor NF-kB A mechanism which explains its inhibition of cytokine production and inducible nitric oxide synthase Akira Matsumoria,*, Youichi Nunokawab, Shigetake Sasayamaa a
Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho Shogoin, Sakyo-ku, Kyoto 606-8397, Japan b Suntory Institute for Biomedical Research, Osaka 618-8503, Japan (Submitted February 10, 2000; accepted May 2, 2000; received in final form July 25, 2000)
Abstract Pimobendan, an inhibitor of phosphodiesterase III with calcium sensitizing properties, inhibits the production of cytokines and nitric oxide. In the present study, the effects of pimobendan and other inotropic agents on the activation of transcription factor NF-kB were examined. Pimobendan significantly decreased the expression of luciferase protein in A549 cells transfected with the NF-kB reporter plasmid, stimulated with interleukin (IL)-1b, tumor necrosis factor-a, or phorbol 12-myristate 13 acetate. However, high concentrations of amrinone, vesnarinone, or NKH 477 decreased promoter activity only slightly. Electrophoretic mobility shift assay also showed inhibition of NF-kB activation by pimobendan. Pimobendan possesses the unique property of inhibiting NF-kB, which may be independent of phosphodiesterase inhibition. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Heart failure; Inotropic drugs; Pimobendan; Interleukins; Nitric oxide; NF-kB
Introduction NF-kB was first identified as a regulator of the expression of the kappa light-chain gene in murine B lymphocyte [1], and has subsequently been found in many different cell types. It regulates the expression of several genes involved in immune and inflammatory responses. NF-kB is activated by many of the factors that increase the inflammatory response, including * Corresponding author. Tel.: (81) 75-751-3186; fax: (81) 75-751-6477. E-mail address: [email protected] (A. Matsumori) 0024-3205/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 8 3 4 -1
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viral infection, oxidants, and antigens. This activation, in turn, leads to the coordinated expression of several genes that encode proteins (such as cytokines, chemokines, adhesion molecules, and enzymes) involved in mediator synthesis, and to further amplification and perpetuation of the inflammatory response. NF-kB is therefore an obvious target for new types of anti-inflammatory treatments. Recently, activation of NF-kB has been demonstrated in the myocardium of the failing human heart [2], and sites of NF-kB activation were associated with those of cyclooxgenase expression. Induction of NF-kB and cyclooxygenase appears to be associated with the presence of inflammation and scar formation. Pimobendan, an inhibitor of phosphodiesterase III (PDE III) with calcium-sensitizing properties, has been found effective in the treatment of heart failure [3–5]. We have shown that it inhibits the production of tumor necrosis factor (TNF)-a in human peripheral mononuclear cells in vitro [6], and of nitric oxide in cultured macrophages [7]. More recently, we have also found that pimobendan inhibits the production of interleukin (IL)-1b, IL-6, TNF-a and nitric oxide in a murine model of heart failure induced by viral myocarditis [8]. Since, in inflammation, NF-kB is critical for the inducible expression of multiple genes, including IL-1, IL-6, TNF-a, adhesion molecules, and nitric oxide synthase [9], we examined whether pimobendan has an inhibitory effect on the activation of NF-kB.
Material and methods Cell culture, transfection, and luciferase assay A549 cells, a human epithelium-like lung carcinoma cell line (American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Rockville, MD, USA) containing 10% FCS, transfected in 35-mm culture dishes with 2 mg of the NF-kB reporter plasmid (pNF-kB-Luc, Stratagene, La Jolla, CA, USA), which contains luciferase gene driven by promoters containing a TATA element and 5 copies of the kB cis-acting element (GGGGACTTTCC), and co-transfected with 0.2 mg of pSV2neo vector (Clontech, Palo Alto, CA, USA) using LipofectAMINE (Life Technologies). Selection with 1 mg/mL of G418 (Life Technologies) was carried out over 3 weeks, by which time visible colonies had formed. Stably transfected cells were seeded at 104 cells/well in 96-well culture plates and used for experiments 24 h later. The cells were cultured in 100 mL of DMEM containing 10% FCS, and stimulated with human IL-1b (1 ng/mL, Genzyme, Cambridge, MA, USA), TNF-a (50 ng/mL, Suntory Institute, Osaka, Japan) or phorbol 12-myristate 13-acetate (PMA, 100 ng/mL, Wako Pure Chemicals, Osaka, Japan) with or without test compounds in DMEM containing 10% FCS. Amrinone (contributed by Yamanouchi Pharmaceutical Co., Tokyo, Japan), pimobendan (contributed by Boehringer Ingelheim, Kawanishi, Japan), vesnarinone (contributed by Otsuka Pharmaceutival Co., Tokushima, Japan), and a forskolin derivative, NKH477 (contributed by Nippon Kayaku Co., Ltd, Tokyo, Japan) were prepared as 10 mmol/L in DMSO and added to the cells 1 h before the stimulation. The final concentrations of DMSO in medium were less than 0.3%. The luciferase activity was determined 3 h after stimulation, using the Luciferase assay system (Promega, Southampton, UK). Briefly, the cells were washed twice with phosphatebuffered saline and incubated for 5 min in 0.5 mL of lysis buffer containing 100 mmol/L
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K2HPO4 and 1 mmol/L dithiothreiol. The supernatant (100 mL) was assayed for luciferase activity with D-luciferin as a substrate in an ARGUS-59 luminometer (Hamamatsu Photonics, Hamamatsu, Japan). Preparation of nuclear extracts and electrophoretic mobility shift assay RAW264.7 mouse macrophage cell lines were seeded in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum. The cells were cultured in the presence or absence of pimobendan, 10 or 30 mmol/L, for 60 min, followed by stimulation with 10 mg/ mL of lipopolysaccharide (LPS) for 60 min. The nuclear extracts were then isolated according to a slightly modified method described by Schreiber et al. [9]. Briefly, cells were collected with a cell scraper, washed with phosphate buffered saline, and pelleted by centrifuge. The cell pellet was resuspended in cold buffer A (10 mmol/L HEPES pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonylfluoride). The cells were allowed to swell on ice for 10 min, mixed with 10% Nonidet-40, and vortexed for 10 s. The cell homogenate was centrifuged for 10 min in a microfuge. After removal of the supernatant, the nuclear pellets were resuspended in buffer B (20 mmol/L HEPES pH 7.9, 0.4 M NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonylfluoride). The nuclear extracts were centrifuged, and the supernatants were used. The NF-kB probe used for gel shift assay consisted of the NF-kB consensus sequence (59-AGTTGAGGGGACTTTCCCAGGC-39). The oligonucleotides 59-labeled with rhodamine were purchased from Amersham Pharmacia Biotech (Tokyo, Japan). The nuclear extracts (2 mg/mL) were incubated with rhodamine-labeled NF-kB probe in the binding buffer (20 mmol/L HEPES, pH7.6, 1 mmol/L EDTA, 10 mmol/L (NH4)2SO4, 1 mmol/L dithiothreitol, 30 mmol/L KCl, 0.2%(w/v) Tween 20) at room temperature for 15 min. The nuclear protein and oligonucleotides complexes were separated from free probes on native 5% polyacrylamide gel (BioRad, Richmond, CA) in 0.25 3 TBE (Tris-borateEDTA) buffer. The gel was scanned using a fluorescent image analyzer (FMBIO II, Hitachi, Yokohama, Japan). All values are presented as mean 6 SD. Statistical analyses were performed by one-way ANOVA, with multiple comparisons by Fisher’s protected least significance difference test using Stat View (Abacus Concept Inc., Berkeley, CA). A p value , 0.05 was considered significant. Results Fig. 1 shows the effect of pimobendan, amrinone, vesnarinone and NKH477 on transcriptional responses mediated by NF-kB. Pimobendan significantly decreased the expression of luciferase protein stimulated with IL-1 compared with controls: 6964% at 1 mmol/L, 5862% at 3 mmol/L, 3161% at 10 mmol/L and 2061% at 30 mmol/L (p,0.0001). The inhibitory effect of pimobendan on promoter activity was concentration-dependent with a maximal effect obtained at 30 mmol/L. However, amrinone, NKH477 or vesnarinone decreased luciferase activity only slightly, even in high concentrations (8262%, 8862% or 7462% at 30 mmol/L respectively, Fig. 1A). A comparable inhibitory effect of pimobendan was observed when cells were stimulated with TNF-a (7462% at 1 mmol/L, 5861% at 3 mmol/L, 2761% at 10 mmol/L and 2161% at 30 mmol/L, Fig. 1B) or PMA (6862% at 1 mmol/L,
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Fig. 1. Effects of pimobendan, vesnarinone, amrinone and NKH477 on transcriptional responses of NF-kB stimulated with IL-1 (A), PMA (B) and TNF-a (C). See text for further explanations.
5960% at 3 mmol/L, 3260% at 10 mmol/L and 2961% at 30 mmol/L, Fig. 1C). NF-kB/ DNA-binding activity was weakly detectable in controls, but increased markedly after treatment with LPS (Fig. 2). Treatment with pimobendan, 10 or 30 mmol/L, reduced NF-kB/DNA binding significantly. Discussion Despite the demonstration of favorable short-term hemodynamic effects, multicenter trials of several PDE III inhibitors have yielded disappointing results. In contrast, a multicenter study of patients with severe congestive heart failure found a long-term increase in exercise duration and peak oxygen uptake, and an improvement in quality of life by pimobendan [4]. This drug possesses the unique additional property of increasing the affinity of cardiac contractile proteins for calcium, which adds to its inotropic activity [11]. Thus far, the advantage of pimobendan over the other PDE III inhibitors in the treatment of chronic heart failure has been attributed to its calcium-sensitizing property. Recent studies have clarified the roles played by various cytokines in diverse cardiovascular disorders [12], and have found increased levels of circulating TNF-a, IL-1b, IL-6 and other proinflammatory cytokines in patients with myocarditis and acute myocardial infarction [13], and increased circulating TNF-a in patients with chronic congestive heart failure, including transplant rejection and dilated cardiomyopathy [14]. In a murine model of encephalomyocarditis virus-induced myocarditis, the majority of mice die of congestive heart failure in the acute stage [15]. The severity of the course of the disease appears to correlate with the degree of intracardiac production of proinflammatory cytokines [16]. Several proinflammatory cytokines are known to cause myocardial contractile dysfunction, with negative inotropic effects thought to be mediated by nitric oxide [17,18]. We have
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Fig. 2. Effect of pimobendan on LPS-stimulated NF-kB activation in macrophage cell line. RAW264.7 cells were incubated for 60 min with pimobendan at indicated concentrations followed by treatments with LPS for 60 min and then tested for NF-kB activation by gel shift analysis. Lane a: without LPS; Lane b: LPS (10 mg/mL); Lane c: LPS (10 mg/mL) 1 pimobendan (30 mmol/mL); Lane d: LPS (10 mg/mL) 1 pimobendan (10 mmol/mL). Arrows, oligonucleotides specific binding. Results are representative of 3 separate experiments.
recently studied the effects of the PDE III inhibitors amrinone, pimobendan and vesnarinone on the production of such cytokines, and have observed differential modulations of their production [6]. We have also studied the effects of amrinone, pimobendan, vesnarinone, and the cell permeable cyclic nucleotide analogue, 8-bromo adenosine 3959-cyclic monophosphate (8 Br-cAMP) on the induction of nitric oxide synthase by LPS in J774A.1 macrophages in vitro. Although the 3 inotropic agents inhibited nitrite accumulation, the degree of inhibition was variable, pimobendan being the most potent inhibitor and amrinone the least. 8 Br-cAMP increased nitrite production in high concentrations, suggesting that the inhibitory effects of the inotropic agents were not due to an increase in cAMP [7]. Thus, the variable inhibition of inducible nitric oxide synthase by inotropic agents suggested different effects of these drugs in patients with heart failure. In our murine model of heart failure due to viral myocarditis, pimobendan improved survival, attenuated inflammatory lesions, and decreased production of intracardiac IL-1b, IL-6 and TNF-a and nitric oxide [8]. However, the inhibitory mechanism of pimobendan on these mediators was not clarified. In the present study, pimobendan, but not the other PDE III inhibitors, inhibited activation of NF-kB. Like forskolin, NKH477 directly activates the catalytic unit of adenylate cyclase and increases cAMP. Since NKH did not suppress the activation of NF-kB either, inhibition of NF-kB activation by pimobendan was apparently not attributable to an increase in cAMP. The mechanism by which pimobendan inhibits NF-kB remains unclear. Calcium channel blockers such as amlodipine, diltiazem, and verapamil have been shown to activate NF-kB in
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human vascular smooth muscle cells [19]. Although the action on signal transduction pathway by these calcium channel blockers remains to be clarified, modulatory effects on cAMPdependent protein kinase, calmodulin-dependent protein kinase, and protein kinase C have been reported [20,21]. It is, thus, plausible that these kinases contribute to the activation of NF-kB. More recently, the importance of intracellular free calcium in the regulation of cytokine expression has been established in human monocytes, and changes in calcium levels have modulated the phosphorylation of I-kBa, which regulates the activation of NF-kB [22]. Since pimobendan possesses calcium-sentitizing properties, it may inhibit the activation of NF-kB by modulating these signal transduction pathways. Activation of NF-kB is critical for the expression of proinflammatory cytokines such as IL-1b, Il-6 and TNF-a, and inducible nitric oxide synthase, and plays an important role in the pathogenesis of inflammation and immunological diseases [23,24]. Thus, the inhibitory effect of pimobendan on the production of proinflammatory cytokines and nitric oxide is explained by its inhibitory effect on the activation of NF-kB. The effect of pimobendan in heart failure may also be partly explained by this effect. The activation of NF-kB in rheumatoid arthritis, and the nearly perfect match between the list of inducers and targets of NF-kB and the profile of mediators of inflammation in rheumatoid arthritis [25] suggests an important role of NF-kB in the control of synovial inflammation. Furthermore, we have observed a remarkable abatement of symptoms and signs of rheumatoid arthritis in 3 patients with heart failure associated with rheumatoid arthritis treated with pimobendan (unpublished observations). After 2 months of treatment, arthralgias, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) had decreased significantly in all 3 patients (ESR from 60618 to 40614 mm/ hr, CRP from 4.660.5 to 1.666.4 mg/dl, mean6SEM, p,0.05 for both), effects which persisted long after the start of the treatment. These observations, which suggest that pimobendan is effective in the treatment of rheumatoid arthritis by inhibiting activation of NF-kB, should be pursued in further studies. Acknowledgments This work was supported in part by a Research Grant from the Japanese Ministry of Health and Welfare and a Grant for Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture. We would like to thank Ms. S. Sakai and Y. Okazaki for preparing the manuscript. References 1. Sen R, Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 1986;46(5):705–716. 2. Wong SC, Fukuchi M, Melnyk P, Rodger I, Giaid A. Induction of cyclooxygenase-2 and activation of nuclear factor-kappaB in myocardium of patients with congestive heart failure. Circulation 1998;98(2):100–103 3. Walter M, Liebens I, Goethals H, Renard M, Dresse A. Bernard R. Pimobendane (UD-CG 115 BS) in the treatment of severe congestive heart failure. An acute haemodynamic cross-over and double-blind study with two different doses. Br J Clin Pharmacol 1988;25(3):323–329. 4. Kubo SH, Gollub S, Bourge R, Rahko P, Cobb F, Jessup M, Brozena S, Brodsky M, Kirlin P, Shanes J, Konstam M, Gradman A, Morledge J, Cinquegrani M, Singh S, Lejemtel T, Nicklas J, Troha J, Cohn J, for the
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Pimobendan Multicenter Research Group. Beneficial effects of pimobendan on exercise tolerance and quality of life in patients with heart failure: Results of a multicenter trial. Circulation 1992;85(3):942–949. Sasayama S, Asanoi H, Kihara Y, Yokawa S, Terada Y, Yoshida S, Ejiri M, Horikoshi I. Clinical effects of long-term administration of pimobendan in patients with moderate congestive heart failure. Heart Vessels 1994;9(3):113–20. Matsumori A, Ono K, Sato Y, Shioi T, Nose Y, Sasayama S. Differentialmodulation of cytokine production by drugs: Implications for therapy in heart failure. J Mol Cell Cardiol 1996;28(12):2491–2499. Matsumori A, Okada I, Shioi T, Furukawa Y, Nakamura T, Ono K, Iwasaki A, Sasayama S. Inotropic agents differentially inhibit the induction of nitric oxide synthase by endotoxin in cultured macrophages. Life Sci 1996;59(10):PL121–125. Iwasaki A, Matsumori A, Yamada T, Shioi T, Wang W, Ono K, Nishio R, Okada M, Sasayama S. Pimobendan inhibits the production of proinflammatory cytokines and gene expression of inducible nitric oxide synthase in a murine model of viral myocarditis. J Am Coll Cardiol 1999;33(5):1400–1407. Kopp E, Ghosh S. Inhibition of NF-kappa B by sodium salicylate and aspirin. Science 1994;265(5174):956–959. Schreiber E, Matthias P, Muller MM, Schaffner W. Rapid detection of octamer binding proteins with ‘miniextracts’, prepared from a small number of cells. Nucleic Acids Res 1989;17(15):6419. Fujino K, Sperelakis N, Solaro RJ. Sensitization of dog and guinea pig heart myofilaments to Ca21 activation and the inotropic effect of pimobendan: comparison with milrinone. Circ Res 1988;63(5):911–922. Matsumori A. Molecular and immune mechanisms in the pathogenesis of cardiomyopathy role of viruses, cytokines, and nitric oxide. Jpn Circ J 1997;61(4):275–291. Matsumori A, Yamada T, Suzuki H, Matoba Y, and Sasayama S. Increased circulating cytokines in patients with myocarditis and cardiomyopathy. Br Heart J 1994;72(6):561–566. Levine B, Kalman J, Mayer L, Fillit HM, and Packer M. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med 1990;323(4):236–241. Matsumori A, Kawai C. An animal model of congestive (dilated) cardiomyopathy: dilatation and hypertrophy of the heart in the chronic stage in DBA/2 mice with myocarditis caused by encephalomyocarditis virus. Circulation 1982;66(2):355–360. Shioi T, Matsumori A, Sasayama S. Persistent expression of cytokine in the chronic stage of viral myocarditis in mice. Circulation 1996;94(11):2930–2937. Finkel MS, Oddis CV, Jacob TD, Watkins SC, Hattler BG, Simmons RL. Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science 1992;257(5068):387–389. Ungureanu-Longrois D, Balligand JL, Kelly RA, Smith TW. Myocardial contractile dysfunction in the systemic inflammatory response syndrome: role of a cytokine-inducible nitric oxide synthase in cardiac myocytes. J Mol Cell Cardiol 1995;27(1):155–167. Eickelberg O, Roth M, Mussmann R, Rüdiger JJ, Tamm M, Perruchoud AP, Block LH. Calcium channel blockers activate the interleukin-6 gene via the transcription factors NF-IL6 and NF-kB in primary human vascular smooth muscle cells. Circulation1999;99(17):2276–2282. Sculptoreanu A, Rotman E, Takahashi M, Scheuer T, Catterall WA. Voltage-dependent potentiation of the activity of cardiac L-type calcium channel a1 subunits due to phosphorylation by cAMP-dependent protein kinase. Proc Natl Acad Sc. USA 1993;90(21):10135–10139. Imagawa T, Leung AT, Campbell KP. Phosphorylation of the 1,4-dihydropyridine receptor of the voltagedependent Ca21 channel by an intrinsic protein kinase in isolated triads from rabbit skeletal muscle J. Biol. Chem. 1987;262(17):8333–8339. Asea A, Kraeft SK, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, Koo GC, Calderwood SK. HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nat Med 2000;(6):435–442. Baldwin AS Jr. The NF-kB and I-kB proteins: New discoveries and insights. Annu Rev Immunol 1996;14(1):649–681. Baeuerle PA, Baichwal AV. NF-kB as a frequent target for immunosuppressive and anti-inflammatory molecules. Adv immunol 1997;65:111–137. Barnes PJ, Karin M. Nuclear factor-kB: A pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 1997;336(15):1066–1071.
Pharmacology letters Accelerated communication
Pimobendan inhibits the activation of transcription factor NF-kB A mechanism which explains its inhibition of cytokine production and inducible nitric oxide synthase Akira Matsumoria,*, Youichi Nunokawab, Shigetake Sasayamaa a
Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho Shogoin, Sakyo-ku, Kyoto 606-8397, Japan b Suntory Institute for Biomedical Research, Osaka 618-8503, Japan (Submitted February 10, 2000; accepted May 2, 2000; received in final form July 25, 2000)
Abstract Pimobendan, an inhibitor of phosphodiesterase III with calcium sensitizing properties, inhibits the production of cytokines and nitric oxide. In the present study, the effects of pimobendan and other inotropic agents on the activation of transcription factor NF-kB were examined. Pimobendan significantly decreased the expression of luciferase protein in A549 cells transfected with the NF-kB reporter plasmid, stimulated with interleukin (IL)-1b, tumor necrosis factor-a, or phorbol 12-myristate 13 acetate. However, high concentrations of amrinone, vesnarinone, or NKH 477 decreased promoter activity only slightly. Electrophoretic mobility shift assay also showed inhibition of NF-kB activation by pimobendan. Pimobendan possesses the unique property of inhibiting NF-kB, which may be independent of phosphodiesterase inhibition. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Heart failure; Inotropic drugs; Pimobendan; Interleukins; Nitric oxide; NF-kB
Introduction NF-kB was first identified as a regulator of the expression of the kappa light-chain gene in murine B lymphocyte [1], and has subsequently been found in many different cell types. It regulates the expression of several genes involved in immune and inflammatory responses. NF-kB is activated by many of the factors that increase the inflammatory response, including * Corresponding author. Tel.: (81) 75-751-3186; fax: (81) 75-751-6477. E-mail address: [email protected] (A. Matsumori) 0024-3205/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 8 3 4 -1
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viral infection, oxidants, and antigens. This activation, in turn, leads to the coordinated expression of several genes that encode proteins (such as cytokines, chemokines, adhesion molecules, and enzymes) involved in mediator synthesis, and to further amplification and perpetuation of the inflammatory response. NF-kB is therefore an obvious target for new types of anti-inflammatory treatments. Recently, activation of NF-kB has been demonstrated in the myocardium of the failing human heart [2], and sites of NF-kB activation were associated with those of cyclooxgenase expression. Induction of NF-kB and cyclooxygenase appears to be associated with the presence of inflammation and scar formation. Pimobendan, an inhibitor of phosphodiesterase III (PDE III) with calcium-sensitizing properties, has been found effective in the treatment of heart failure [3–5]. We have shown that it inhibits the production of tumor necrosis factor (TNF)-a in human peripheral mononuclear cells in vitro [6], and of nitric oxide in cultured macrophages [7]. More recently, we have also found that pimobendan inhibits the production of interleukin (IL)-1b, IL-6, TNF-a and nitric oxide in a murine model of heart failure induced by viral myocarditis [8]. Since, in inflammation, NF-kB is critical for the inducible expression of multiple genes, including IL-1, IL-6, TNF-a, adhesion molecules, and nitric oxide synthase [9], we examined whether pimobendan has an inhibitory effect on the activation of NF-kB.
Material and methods Cell culture, transfection, and luciferase assay A549 cells, a human epithelium-like lung carcinoma cell line (American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Rockville, MD, USA) containing 10% FCS, transfected in 35-mm culture dishes with 2 mg of the NF-kB reporter plasmid (pNF-kB-Luc, Stratagene, La Jolla, CA, USA), which contains luciferase gene driven by promoters containing a TATA element and 5 copies of the kB cis-acting element (GGGGACTTTCC), and co-transfected with 0.2 mg of pSV2neo vector (Clontech, Palo Alto, CA, USA) using LipofectAMINE (Life Technologies). Selection with 1 mg/mL of G418 (Life Technologies) was carried out over 3 weeks, by which time visible colonies had formed. Stably transfected cells were seeded at 104 cells/well in 96-well culture plates and used for experiments 24 h later. The cells were cultured in 100 mL of DMEM containing 10% FCS, and stimulated with human IL-1b (1 ng/mL, Genzyme, Cambridge, MA, USA), TNF-a (50 ng/mL, Suntory Institute, Osaka, Japan) or phorbol 12-myristate 13-acetate (PMA, 100 ng/mL, Wako Pure Chemicals, Osaka, Japan) with or without test compounds in DMEM containing 10% FCS. Amrinone (contributed by Yamanouchi Pharmaceutical Co., Tokyo, Japan), pimobendan (contributed by Boehringer Ingelheim, Kawanishi, Japan), vesnarinone (contributed by Otsuka Pharmaceutival Co., Tokushima, Japan), and a forskolin derivative, NKH477 (contributed by Nippon Kayaku Co., Ltd, Tokyo, Japan) were prepared as 10 mmol/L in DMSO and added to the cells 1 h before the stimulation. The final concentrations of DMSO in medium were less than 0.3%. The luciferase activity was determined 3 h after stimulation, using the Luciferase assay system (Promega, Southampton, UK). Briefly, the cells were washed twice with phosphatebuffered saline and incubated for 5 min in 0.5 mL of lysis buffer containing 100 mmol/L
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K2HPO4 and 1 mmol/L dithiothreiol. The supernatant (100 mL) was assayed for luciferase activity with D-luciferin as a substrate in an ARGUS-59 luminometer (Hamamatsu Photonics, Hamamatsu, Japan). Preparation of nuclear extracts and electrophoretic mobility shift assay RAW264.7 mouse macrophage cell lines were seeded in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum. The cells were cultured in the presence or absence of pimobendan, 10 or 30 mmol/L, for 60 min, followed by stimulation with 10 mg/ mL of lipopolysaccharide (LPS) for 60 min. The nuclear extracts were then isolated according to a slightly modified method described by Schreiber et al. [9]. Briefly, cells were collected with a cell scraper, washed with phosphate buffered saline, and pelleted by centrifuge. The cell pellet was resuspended in cold buffer A (10 mmol/L HEPES pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonylfluoride). The cells were allowed to swell on ice for 10 min, mixed with 10% Nonidet-40, and vortexed for 10 s. The cell homogenate was centrifuged for 10 min in a microfuge. After removal of the supernatant, the nuclear pellets were resuspended in buffer B (20 mmol/L HEPES pH 7.9, 0.4 M NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonylfluoride). The nuclear extracts were centrifuged, and the supernatants were used. The NF-kB probe used for gel shift assay consisted of the NF-kB consensus sequence (59-AGTTGAGGGGACTTTCCCAGGC-39). The oligonucleotides 59-labeled with rhodamine were purchased from Amersham Pharmacia Biotech (Tokyo, Japan). The nuclear extracts (2 mg/mL) were incubated with rhodamine-labeled NF-kB probe in the binding buffer (20 mmol/L HEPES, pH7.6, 1 mmol/L EDTA, 10 mmol/L (NH4)2SO4, 1 mmol/L dithiothreitol, 30 mmol/L KCl, 0.2%(w/v) Tween 20) at room temperature for 15 min. The nuclear protein and oligonucleotides complexes were separated from free probes on native 5% polyacrylamide gel (BioRad, Richmond, CA) in 0.25 3 TBE (Tris-borateEDTA) buffer. The gel was scanned using a fluorescent image analyzer (FMBIO II, Hitachi, Yokohama, Japan). All values are presented as mean 6 SD. Statistical analyses were performed by one-way ANOVA, with multiple comparisons by Fisher’s protected least significance difference test using Stat View (Abacus Concept Inc., Berkeley, CA). A p value , 0.05 was considered significant. Results Fig. 1 shows the effect of pimobendan, amrinone, vesnarinone and NKH477 on transcriptional responses mediated by NF-kB. Pimobendan significantly decreased the expression of luciferase protein stimulated with IL-1 compared with controls: 6964% at 1 mmol/L, 5862% at 3 mmol/L, 3161% at 10 mmol/L and 2061% at 30 mmol/L (p,0.0001). The inhibitory effect of pimobendan on promoter activity was concentration-dependent with a maximal effect obtained at 30 mmol/L. However, amrinone, NKH477 or vesnarinone decreased luciferase activity only slightly, even in high concentrations (8262%, 8862% or 7462% at 30 mmol/L respectively, Fig. 1A). A comparable inhibitory effect of pimobendan was observed when cells were stimulated with TNF-a (7462% at 1 mmol/L, 5861% at 3 mmol/L, 2761% at 10 mmol/L and 2161% at 30 mmol/L, Fig. 1B) or PMA (6862% at 1 mmol/L,
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Fig. 1. Effects of pimobendan, vesnarinone, amrinone and NKH477 on transcriptional responses of NF-kB stimulated with IL-1 (A), PMA (B) and TNF-a (C). See text for further explanations.
5960% at 3 mmol/L, 3260% at 10 mmol/L and 2961% at 30 mmol/L, Fig. 1C). NF-kB/ DNA-binding activity was weakly detectable in controls, but increased markedly after treatment with LPS (Fig. 2). Treatment with pimobendan, 10 or 30 mmol/L, reduced NF-kB/DNA binding significantly. Discussion Despite the demonstration of favorable short-term hemodynamic effects, multicenter trials of several PDE III inhibitors have yielded disappointing results. In contrast, a multicenter study of patients with severe congestive heart failure found a long-term increase in exercise duration and peak oxygen uptake, and an improvement in quality of life by pimobendan [4]. This drug possesses the unique additional property of increasing the affinity of cardiac contractile proteins for calcium, which adds to its inotropic activity [11]. Thus far, the advantage of pimobendan over the other PDE III inhibitors in the treatment of chronic heart failure has been attributed to its calcium-sensitizing property. Recent studies have clarified the roles played by various cytokines in diverse cardiovascular disorders [12], and have found increased levels of circulating TNF-a, IL-1b, IL-6 and other proinflammatory cytokines in patients with myocarditis and acute myocardial infarction [13], and increased circulating TNF-a in patients with chronic congestive heart failure, including transplant rejection and dilated cardiomyopathy [14]. In a murine model of encephalomyocarditis virus-induced myocarditis, the majority of mice die of congestive heart failure in the acute stage [15]. The severity of the course of the disease appears to correlate with the degree of intracardiac production of proinflammatory cytokines [16]. Several proinflammatory cytokines are known to cause myocardial contractile dysfunction, with negative inotropic effects thought to be mediated by nitric oxide [17,18]. We have
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Fig. 2. Effect of pimobendan on LPS-stimulated NF-kB activation in macrophage cell line. RAW264.7 cells were incubated for 60 min with pimobendan at indicated concentrations followed by treatments with LPS for 60 min and then tested for NF-kB activation by gel shift analysis. Lane a: without LPS; Lane b: LPS (10 mg/mL); Lane c: LPS (10 mg/mL) 1 pimobendan (30 mmol/mL); Lane d: LPS (10 mg/mL) 1 pimobendan (10 mmol/mL). Arrows, oligonucleotides specific binding. Results are representative of 3 separate experiments.
recently studied the effects of the PDE III inhibitors amrinone, pimobendan and vesnarinone on the production of such cytokines, and have observed differential modulations of their production [6]. We have also studied the effects of amrinone, pimobendan, vesnarinone, and the cell permeable cyclic nucleotide analogue, 8-bromo adenosine 3959-cyclic monophosphate (8 Br-cAMP) on the induction of nitric oxide synthase by LPS in J774A.1 macrophages in vitro. Although the 3 inotropic agents inhibited nitrite accumulation, the degree of inhibition was variable, pimobendan being the most potent inhibitor and amrinone the least. 8 Br-cAMP increased nitrite production in high concentrations, suggesting that the inhibitory effects of the inotropic agents were not due to an increase in cAMP [7]. Thus, the variable inhibition of inducible nitric oxide synthase by inotropic agents suggested different effects of these drugs in patients with heart failure. In our murine model of heart failure due to viral myocarditis, pimobendan improved survival, attenuated inflammatory lesions, and decreased production of intracardiac IL-1b, IL-6 and TNF-a and nitric oxide [8]. However, the inhibitory mechanism of pimobendan on these mediators was not clarified. In the present study, pimobendan, but not the other PDE III inhibitors, inhibited activation of NF-kB. Like forskolin, NKH477 directly activates the catalytic unit of adenylate cyclase and increases cAMP. Since NKH did not suppress the activation of NF-kB either, inhibition of NF-kB activation by pimobendan was apparently not attributable to an increase in cAMP. The mechanism by which pimobendan inhibits NF-kB remains unclear. Calcium channel blockers such as amlodipine, diltiazem, and verapamil have been shown to activate NF-kB in
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human vascular smooth muscle cells [19]. Although the action on signal transduction pathway by these calcium channel blockers remains to be clarified, modulatory effects on cAMPdependent protein kinase, calmodulin-dependent protein kinase, and protein kinase C have been reported [20,21]. It is, thus, plausible that these kinases contribute to the activation of NF-kB. More recently, the importance of intracellular free calcium in the regulation of cytokine expression has been established in human monocytes, and changes in calcium levels have modulated the phosphorylation of I-kBa, which regulates the activation of NF-kB [22]. Since pimobendan possesses calcium-sentitizing properties, it may inhibit the activation of NF-kB by modulating these signal transduction pathways. Activation of NF-kB is critical for the expression of proinflammatory cytokines such as IL-1b, Il-6 and TNF-a, and inducible nitric oxide synthase, and plays an important role in the pathogenesis of inflammation and immunological diseases [23,24]. Thus, the inhibitory effect of pimobendan on the production of proinflammatory cytokines and nitric oxide is explained by its inhibitory effect on the activation of NF-kB. The effect of pimobendan in heart failure may also be partly explained by this effect. The activation of NF-kB in rheumatoid arthritis, and the nearly perfect match between the list of inducers and targets of NF-kB and the profile of mediators of inflammation in rheumatoid arthritis [25] suggests an important role of NF-kB in the control of synovial inflammation. Furthermore, we have observed a remarkable abatement of symptoms and signs of rheumatoid arthritis in 3 patients with heart failure associated with rheumatoid arthritis treated with pimobendan (unpublished observations). After 2 months of treatment, arthralgias, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) had decreased significantly in all 3 patients (ESR from 60618 to 40614 mm/ hr, CRP from 4.660.5 to 1.666.4 mg/dl, mean6SEM, p,0.05 for both), effects which persisted long after the start of the treatment. These observations, which suggest that pimobendan is effective in the treatment of rheumatoid arthritis by inhibiting activation of NF-kB, should be pursued in further studies. Acknowledgments This work was supported in part by a Research Grant from the Japanese Ministry of Health and Welfare and a Grant for Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture. We would like to thank Ms. S. Sakai and Y. Okazaki for preparing the manuscript. References 1. Sen R, Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 1986;46(5):705–716. 2. Wong SC, Fukuchi M, Melnyk P, Rodger I, Giaid A. Induction of cyclooxygenase-2 and activation of nuclear factor-kappaB in myocardium of patients with congestive heart failure. Circulation 1998;98(2):100–103 3. Walter M, Liebens I, Goethals H, Renard M, Dresse A. Bernard R. Pimobendane (UD-CG 115 BS) in the treatment of severe congestive heart failure. An acute haemodynamic cross-over and double-blind study with two different doses. Br J Clin Pharmacol 1988;25(3):323–329. 4. Kubo SH, Gollub S, Bourge R, Rahko P, Cobb F, Jessup M, Brozena S, Brodsky M, Kirlin P, Shanes J, Konstam M, Gradman A, Morledge J, Cinquegrani M, Singh S, Lejemtel T, Nicklas J, Troha J, Cohn J, for the
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