Arginine or citrulline associated with a statin stimulates nitric oxide production in bovine aortic endothelial cells

European Journal of Pharmacology 670 (2011) 566–570

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Cardiovascular Pharmacology

Arginine or citrulline associated with a statin stimulates nitric oxide production in bovine aortic endothelial cells Marie-Clotilde Berthe a, b,⁎, Mélisande Bernard a, Carole Rasmusen a, Sylviane Darquy a, Luc Cynober a, c, Rémy Couderc a, b a b c

Laboratoire de Biologie de la Nutrition, EA2498, Université Paris Descartes, France Service de Biochimie, Hôpital Armand Trousseau, AP-HP, Paris, France Service de Biochimie, Hôpitaux Hôtel-Dieu et Cochin, AP-HP, Paris, France

a r t i c l e

i n f o

Article history: Received 10 June 2011 Received in revised form 29 July 2011 Accepted 17 August 2011 Available online 2 September 2011 Keywords: Arginine Citrulline eNOS Endothelial cell Nitric oxide

a b s t r a c t Nitric oxide (NO) is an antiatherogenic vasodilator synthesized from arginine and, indirectly, from citrulline through argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL). Hypercholesterolemia-induced atherosclerosis is usually treated by statins, which decrease cholesterolemia and increase endothelial NO synthase (eNOS) activity. Therefore, a treatment associating a statin with arginine or citrulline could be more efficient than statin alone. The aim of this study was to optimize NO production in bovine aortic endothelial cells (BAEC) by a combination of simvastatin with arginine or citrulline and to identify the molecular mechanisms involved. NO production was measured after stimulation of BAEC in different conditions (simvastatin 0 to 10 μM associated with arginine or citrulline 0 to 5 mM) after 24-hour incubation. Intracellular levels of specific proteins were evaluated by Western-Blot analysis, and mRNA levels of eNOS, iNOS, caveolin-1, ASS and ASL were assessed by RT-PCR. Simvastatin co-administrated with arginine or citrulline increased NO production, but at simvastatin 10 μM, 1 mM arginine-induced NO production was significantly (P b 0.01) higher than 1 mM citrulline-induced NO production. Simvastatin induced an increase in eNOS mRNA expression and protein levels in the presence of arginine or citrulline. ASS and ASL mRNA levels were increased by simvastatin, whereas a high substrate concentration (1 mM) strongly decreased ASL mRNA levels. Combining statin with arginine or citrulline increased NO production in endothelial cells by increasing eNOS protein levels. These results form a strong rationale to evaluate the potential utilization of these in atherosclerosis prevention and treatment. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Nitric oxide (NO) has known antiatherogenic properties. In endothelial cells, NO is produced from arginine with simultaneous production of citrulline by endothelial NO synthase (eNOS). Endothelial NO decreases in atherosclerosis (Liu and Huang, 2008), leading to the endothelial dysfunction implicated in cardiovascular disease (Liu and Huang, 2008). Therefore, maintaining NO production in blood vessels represents a therapeutic target in atherosclerosis prevention (Gewaltig and Kojda, 2002). Several studies performed in animals and humans (Preli et al., 2002) have shown that L-arginine supplementation increases NO production and inhibited atherosclerosis progression and leukocyte adhesion in hypercholesterolemia. The efficiency of arginine administered to produce NO stems from the fact that the CAT-1 transporter responsible for 60 to 80% of arginine transport into endothelial cells (McDonald et al., ⁎ Corresponding author at: Hôpital Armand Trousseau, 26 avenue du Dr Arnold Netter, 75571 Paris Cedex 12, France. Tel.: +33 1 44 73 63 56; fax: +33 1 44 73 66 87. E-mail address: [email protected] (M.-C. Berthe). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.08.018

1997) interacts directly with eNOS. Thus, this eNOS/CAT-1 interaction decreases the inhibitory interaction of caveolin-1 with eNOS which is activated (Li et al., 2005). Thus, NO production from arginine is tightly compartmentalized, which explains the “arginine paradox” (Husson et al., 2003; Shen et al., 2005). In addition, in endothelial cells, arginine recycling from citrulline via successive actions of argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) is the main substrate source for NO production via eNOS (Shen et al., 2005) indicating a close relationship between eNOS activity and arginine recycling. Moreover, Goodwin et al. (2007) demonstrated that a decrease in ASS activity induced an 80% decrease in NO production. Indeed, Flam et al. (2001) showed that eNOS, ASS and ASL were colocalized in caveolae. Furthermore, from a therapeutic perspective, citrulline offers a number of advantages compared to arginine: better gastrointestinal tolerance (Grimble, 2007) and better whole-body bioavailability (Bode-Boger et al., 1998; Moinard et al., 2008; Tangphao et al., 1999). Statins are currently used to treat atherosclerosis in primary and secondary prevention (Vaughan et al., 2000). Cholesterol lowering is the predominant mechanism underlying the beneficial effects of statins in cardiovascular diseases (Igel et al., 2002) but several studies

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have shown that statins also have pleiotropic effects, including effects on eNOS (Ii and Losordo, 2007; Rasmusen et al., 2007). By inhibiting the biosynthesis of L-mevalonate and isoprenoid intermediates, statins increase eNOS mRNA stability and therefore eNOS activity (Rasmusen et al., 2007). Moreover, some statins as pitavastatin induce eNOS phosphorylation by inducing Akt pathway (Wang et al., 2008). Moreover, cholesterol synthesis is strongly related to caveolin-1 expression and a decrease in cholesterol caveolae reduces cell surface caveolin-1 expression. Consequently, interactions between eNOS and caveolin-1 decrease, and thus eNOS activity increases (Goligorsky et al., 2002). A study performed on Watanabe hypercholesterolemic rabbits in our laboratory (Rasmusen et al., 2007) demonstrated that the association of arginine with atorvastatin significantly limited atherosclerosis development compared with statin or arginine alone. Our working hypothesis was that the association of statin with an eNOS substrate could potentiate NO production. This study was therefore designed to evaluate the combined effects of the L-arginine or L-citrulline with simvastatin on NO production in bovine aortic endothelial cells and to identify the molecular mechanisms involved. 2. Materials and methods 2.1. Products All chemicals and antibodies were purchased from Sigma-Aldrich (St Quentin Fallavier, France). Since simvastatin is a prodrug, its use in vitro requires an activation step in order to open its lactone cycle to active dihydroxy acid form. This was performed by alkaline hydrolysis, as described previously (Dichtl et al., 2003). 2.2. Cell culture Bovine aortic endothelial cells (BAECs) obtained courtesy of Pr Fruchart (Institut Pasteur, Lille, France) were grown in confluence in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (complete DMEM) and incubated at 37 °C in a humidified 5% CO2–95% O2 atmosphere. BAECs were used from 6th to 10th passages. At confluence, cells were incubated with serum-free media for 24 h and then incubated under experimental conditions as described below. During experiments, cells were incubated in an arginine, citrulline, and glutamine free-DMEM named minimum DMEM (Invitrogen, Cergy-Pontoise, France). Arginine and citrulline concentrations were selected to cover both physiological and pharmacological ranges (Barr et al., 2007; Bode-Boger et al., 2007; Smith et al., 2006). Simvastatin was used at a concentration commonly found in the literature (Dichtl et al., 2003). The homogeneity of cultured endothelial cells was checked by their typical cobblestone morphology under an optical microscope (CK2, Olympus, Japan). Cellular toxicity of statin was tested by trypan blue exclusion. No significant changes were observed with simvastatin at the concentration range used in this work (data not shown). 2.3. NO measurement Cells were grown in complete DMEM. After reaching confluence, they were seeded onto 96-well culture plates (200 000 cells/well) and incubated in serum-free medium during 24 h. Then, cells were rinsed with PBS (1×) and again incubated for 24 h in minimum DMEM, supplemented with different arginine-plus-statin or citrulline-plus-statin concentrations. Calcium ionophore A23187

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was then added to the wells (10 μmol/l) for 30 min. The supernatants were then removed and stored at − 80 °C until analysis. Extracellular NO assessed by measurement of nitrite was determined using 2,3-diaminonaphtalene (DAN), which reacts rapidly with nitrites in acid to form the fluorescent compound 2,3-naphtotriazole (NAT) (Misko et al., 1993). Briefly, medium culture supernatants were collected and centrifuged; then 10 μl of DAN (0.05 mg DAN/ml in HCl 0.62 M) was added to 90 μl of supernatant in 96-well flat black plates. After a 15-minute incubation at room temperature, the reaction was stopped by adding 5 μl NaOH 2.8 M, and fluorescence was measured on a microplate reader (Saphire®, Tecan, Maennedorf, Suisse) at 375 nm excitation and 415 nm emission (Misko et al., 1993). Results were given as percentage in comparison with fluorescence from minimum DMEM after DAN action. 2.4. Western Blot assays Proteins extracted from cultured BAECs with Leammli buffer (1,2×) containing 2-mercaptoethanol. Equal amounts of protein (10 μg/lane) were separated on denaturing SDS-10% polyacrylamide gel and blotted onto a nitrocellulose membrane (Amersham Biosciences, Orsay, France) as described by de Frutos et al. (1999). Then, nitrocellulose membrane was incubated with anti-eNOS (1/1000), anti-iNOS (1/2000), anti-caveolin-1 (1/4000) and anti-βactin (1/400, used as standard) primary rabbit antibodies. The complex was revealed by a secondary rabbit anti-IgG antibody (1/4000) coupled with peroxidase. Staining was then performed on the membrane using an ECL kit (Amersham Biosciences, Orsay, France) and relative intensities of proteins bands were analyzed by Genetool software (Syngene, ST Quentin, France) and expressed as the ratio to βactin protein band. 2.5. RT-PCR assays Total RNA (1 μg) extracted from cultured BAECs using the RNAeasy kit (Ambion, Huntington, Cambridgeshire, UK) was reverse-transcripted with oligo dT and SuperScript® First-Strand reverse transcriptase (SuperScript® First-Strand, Invitrogen, Cergy-Pontoise, France). Reverse-transcripts were amplified with Hi-Taq DNA polymerase using eNOS, caveolin-1, ASS, ASL and L19 (as reference mRNA) specific primers (Invitrogen). Internal control was L19 mRNA encoding a ribosomal protein. – eNOS: sense: 5′-CCAGCTCAAGACTGGAGACC-3′, antisense: 5′ TCAATGTCATGCAGCCTCTC-3′ – iNOS: sense: 5′-GGATCTTCACCTGCTTGCTC-3′, antisense: 5′CTGGGCCTGACTTTTCACAT-3′ – Caveolin-1: sense: 5′-AGCCCAACAACAAGGCTATG-3′, antisense: 5′-GATGCCATCGAAACTGTGTG-3′ – ASS: sense: 5′-CTGATGGAGTATGCGAAGCA-3′, antisense: 5′GGGTCCTGGGTCTTTGTGTA-3′ – ASL: sense: 5′-GTTTGTGGGCACAGTGGA-3′, antisense: 5′-CAGCTGTAGGCTTTGCTG-3′ – L19: sense: 5′-AAGATCGATCGCCACATGTATCA-3′, antisense: 5′TGCGTGCTTCCTTGGTCTTAGA-3′ PCR-conditions were the following: after an initial step denaturing, 40 cycles were performed as follow: 15 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C and 10 min at 72 °C (Smart Cycler®, Cepheid Europe, Maurens-Scopont, France). Amplification gene quantity was evaluated by using SYBER Green (Smart® Kit fot SYBER® Green I, Eurogentec, Angers, France). The PCR-amplified samples were visualized on 1.5% agarose gels using ethidium bromide. Results were expressed as the ratio between the studied gene and the reference gene (L19) amplification levels.

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Concentration (mM) Fig. 1. Combined effects of arginine (Arg) or citrulline (Cit) plus simvastatin (SIM) on NO production in bovine aortic endothelial cells. Results are expressed as means (n = 4 per series) ± S.E.M. Combined effects were tested by 2-way ANOVA (simvastatin and arginine/citrulline) ✳ versus SIM 0 μM for a given arginine or citrulline concentration; + versus arginine 1 mM for a given SIM concentration.

2.6. Statistical analysis Statistical data analysis was performed using StatView Software (version 5.0). All values were expressed as means ± S.E.M. Betweengroup comparisons were performed using a Mann–Whitney test. Combined effects were tested by 2 way-ANOVA. The level of statistically significant difference was set at P b 0.05. 3. Results 3.1. NO production A significant doubling of NO production after BAEC's incubation during 30 min with calcium ionophore compared with non stimulated

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cells was observed (data not shown). Therefore, all experiments were realized on stimulated cells. NO production increased with the substrate concentration, and arginine induced a higher NO production increase than citrulline. On the other hand, no simvastatin effect in the absence of substrate was observed. A dose-dependent relationship of simvastatin on NO production was observed in the presence of arginine or citrulline, whereas there was no simvastatin effect in the absence of substrate. At the same simvastatin concentration, NO production increased higher with arginine, plateauing at 0.5 mM, than with citrulline. In the presence of 1 mM arginine or citrulline, there was a significant (P b 0.01 for arginine and citrulline) increase in NO production with simvastatin 10 μM compared to absence of simvastatin. With arginine, this difference was also significant (Pb 0.05) for simvastatin 1 μM (Fig. 1).

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Fig. 2. Combined effects of simvastatin plus arginine or citrulline on eNOS. a) eNOS protein level b) eNOS mRNA level c) eNOS Western-Blot gel. Results are expressed as means (n = 3 per series) ± S.E.M. ✳ versus SIM 0 μM; + versus control; × versus arginine 0.5 mM.

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There was a significant increase in eNOS protein quantity for all substrate concentrations in simvastatin-treated cells compared to non-treated cells. Moreover, this effect of simvastatin was dosedependent by the presence of substrate (Fig. 2a). Mirroring protein levels, there was a significant increase in eNOS mRNA levels with simvastatin-treated cells at all substrate concentrations. Moreover, eNOS mRNA level was significantly increased by citrulline 1 mM combined with simvastatin compared to simvastatin alone (Fig. 2b). 3.3. Inducible NO synthase iNOS mRNA and protein were not detectable whatever the cell treatment (with or without simvastatin) or substrate concentration (data not shown). 3.4. Caveolin-1 There was a non-significant difference in caveolin protein quantity between simvastatin-treated and untreated cells. There was no arginine or citrulline effect on caveolin-1. In contrast, simvastatin led to a significant increase in caveolin-1 mRNA levels, but only in combination with arginine 0.5 mM. This result was not reproduced with citrulline or arginine alone, nor with simvastatin alone (data not shown). 3.5. Arginine recycling enzymes Arginine and citrulline 1 mM significantly decreased ASL mRNA in 10 μM simvastatin-treated cells. In contrary, substrate has no effect on ASS mRNA in 10 μM simvastatin-treated cells (Fig. 3a). Moreover, there was a simvastatin dose-dependent relationship on ASS and ASL mRNA levels in the presence of arginine 0.5 mM. Furthermore, 0.5 mM arginine-treated cells showed significantly increased ASS and ASL mRNA levels with simvastatin 10 μM compared to non-treated simvastatin cells (Fig. 3b and c).

Arginine or citrulline without statin did not significantly alter eNOS levels. However, NO production increased gradually with substrate concentration, plateauing at 0.5 mM arginine and 1 mM citrulline. NO production was higher with arginine than with citrulline, and a plateau appeared at higher concentrations with citrulline. This indicates that in our experimental conditions, the recycling of citrulline to arginine directly available for NO production by eNOS was not total, that is in accordance with Flam et al. (2007) who have estimated at 80% the rate of citrulline recycled into arginine, indicating a loss of intermediates during recycling. Simvastatin and eNOS substrate have a synergistic action on eNOS protein levels (significant combined effect), but this effect was not found for mRNA level. It may be hypothesized that the statin induces an increase in eNOS mRNA levels via mRNA stabilization, and that the substrate induces eNOS protein stabilization. There was no significant effect of simvastatin or substrate on caveolin-1 protein and an increase in mRNA levels with arginine 0.5 mM on simvastatin-treated cells. This runs contrary to literature reports (Feron et al., 2001; Pelat et al., 2003). However, in our study, before the experiments, the cells were incubated for 24-h in a serum-free medium to deplete their cholesterol content, probably minimizing the role of the regulation pathway via extracellular cholesterol (SREBP-1 pathway). Féron et al. (Brouet et al., 2001) have shown that the action of atorvastatin on caveolin-1 was dependent on type of endothelial cell and may be ineffective in endothelial

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To the best of our knowledge, this is the first study to compare citrulline and arginine as potentiators of statin effects on NO production focusing on recycling enzymes. This study on BAECs has shown that arginine or citrulline associated with simvastatin induced a stronger increase in NO production compared to arginine, citrulline or statin alone. This is in line with the increase in eNOS protein and eNOS mRNA levels. This observation suggested that statin and substrate have a synergic role. Moreover, we have demonstrated an increase in arginine recycling enzyme expression in the presence of simvastatin, inducing a better arginine bioavailability for eNOS and consequently an increase of NO production. In agreement with results from previous studies (Kalinowski et al., 2002; Laufs et al., 1998; Takemoto and Liao, 2001; Wolfrum et al., 2003), simvastatin had a dose-dependent relationship on NO production, whatever the substrate. This effect was correlated with an increase in eNOS mRNA and protein expression. Laufs et al. (1998) also reported an increase in eNOS mRNA and protein levels in simvastatin (1 μM for 48 h) and 0.5 mM arginine-treated human endothelial cells (HUVEC). They hypothesized that simvastatin could induce an increase in eNOS mRNA half-life. Indeed, Kosmidou et al. demonstrated that simvastatin leads to increase eNOS mRNA stability by polyadenylation (Kosmidou et al., 2007). Moreover, these effects were reversible by addition of mevalonate, showing that it could be due to hydroxymethylglutaryl coenzyme A reductase inhibition by statin and a decrease in isoprenylic derivatives. This point was also confirmed by Kalinowski et al. (2002) using cerivastatin in HUVEC cells.

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Arginine concentration (mM) Fig. 3. Combined effects of simvastatin plus arginine or citrulline on enzyme regeneration. a) Substrate and simvastatin 10 μM b) ASS in arginine 0.5 mM and simvastatintreated cells c) ASL in arginine 0.5 mM and simvastatin-treated cells. Results were expressed as means (n = 3 per series) ± S.E.M. ✳ versus control; + versus SIM 0 μM.

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cells presenting high caveolin levels. Nevertheless, the BAECs used in our study may have a small caveolin pool, and should be more sensitive to statin than some other endothelial cell types. This is the first study to show a significant increase in ASS and ASL mRNA with simvastatin, which could constitute a novel pleiotropic effect of this drug family. Moreover, Schwartz et al. (2006) have demonstrated an increase of protein CAT-1 level by arginine or atorvastatin in a model of uremic rat. These results show that statins contribute to increase NO production by different mechanisms. In combination with arginine, by increasing eNOS activity, and in combination with citrulline, by increasing eNOS activity and arginine recycling enzyme activity. 5. Conclusion This study confirmed at cellular level that statin associated with an NO precursor could enhance the NO pathway. We now need to determine the more efficient substrate: citrulline or arginine. Although arginine seemed more efficient than citrulline at the same concentration in our in vitro study, citrulline could be a best candidate in vivo since it does not undergo hepatic catabolism and, unlike arginine, does not present side effects. References Barr, F.E., Tirona, R.G., Taylor, M.B., Rice, G., Arnold, J., Cunningham, G., Smith, H.A., Campbell, A., Canter, J.A., Christian, K.G., Drinkwater, D.C., Scholl, F., KavanaughMcHugh, A., Summar, M.L., 2007. Pharmacokinetics and safety of intravenously administered citrulline in children undergoing congenital heart surgery: potential therapy for postoperative pulmonary hypertension. J. Thorac. Cardiovasc. Surg. 134, 319–326. Bode-Boger, S.M., Boger, R.H., Galland, A., Tsikas, D., Frolich, J.C., 1998. L-arginine-induced vasodilation in healthy humans: pharmacokinetic–pharmacodynamic relationship. Br. J. Clin. Pharmacol. 46, 489–497. Bode-Boger, S.M., Scalera, F., Ignarro, L.J., 2007. The L-arginine paradox: importance of the L-arginine/asymmetrical dimethylarginine ratio. Pharmacol. Ther. 114, 295–306. Brouet, A., Sonveaux, P., Dessy, C., Moniotte, S., Balligand, J.L., Feron, O., 2001. Hsp90 and caveolin are key targets for the proangiogenic nitric oxide-mediated effects of statins. Circ. Res. 89, 866–873. de Frutos, T., de Miguel, L.S., Garcia-Duran, M., Gonzalez-Fernandez, F., Rodriguez-Feo, J.A., Monton, M., Guerra, J., Farre, J., Casado, S., Lopez-Farre, A., 1999. NO from smooth muscle cells decreases NOS expression in endothelial cells: role of TNF-alpha. Am. J. Physiol. 277, H1317–H1325. Dichtl, W., Dulak, J., Frick, M., Alber, H.F., Schwarzacher, S.P., Ares, M.P., Nilsson, J., Pachinger, O., Weidinger, F., 2003. HMG-CoA reductase inhibitors regulate inflammatory transcription factors in human endothelial and vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 23, 58–63. Feron, O., Dessy, C., Desager, J.P., Balligand, J.L., 2001. Hydroxy-methylglutaryl-coenzyme A reductase inhibition promotes endothelial nitric oxide synthase activation through a decrease in caveolin abundance. Circulation 103, 113–118. Flam, B.R., Hartmann, P.J., Harrell-Booth, M., Solomonson, L.P., Eichler, D.C., 2001. Caveolar localization of arginine regeneration enzymes, argininosuccinate synthase, and lyase, with endothelial nitric oxide synthase. Nitric Oxide 5, 187–197. Flam, B.R., Eichler, D.C., Solomonson, L.P., 2007. Endothelial nitric oxide production is tightly coupled to the citrulline-NO cycle. Nitric Oxide 17, 115–121. Gewaltig, M.T., Kojda, G., 2002. Vasoprotection by nitric oxide: mechanisms and therapeutic potential. Cardiovasc. Res. 55, 250–260. Goligorsky, M.S., Li, H., Brodsky, S., Chen, J., 2002. Relationships between caveolae and eNOS: everything in proximity and the proximity of everything. Am. J. Physiol. Renal Physiol. 283, F1–F10.

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