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L-arginine supplementation in hypercholesterolemic rabbits normalizes leukocyte adhesion to non-endothelial matrix
Life Sciences, Vol. 66, No. 16, pp. 1.519-1524.2000 Copyright Q 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0024-3205/OO&see front matter
EISEVIER
PI1 SOO24-3205(00)00469-O
L-ARGININE SUPPLEMENTATION IN HYPERCHOLESTEROLEMIC NORMALIZES LEUKOCYTE ADHESION TO NON-ENDOTHELIAL
RABBITS MATRIX
Ralf P. Brandes a, Stefanie Brandes a, Rainer H. Biiser b, Stefanie M. Bode-Biiger b, and Andreas Miigge a Institut fiir Kar$iovaskulHre Physiologie, Klinikum der J.W. Goethe-Universitat, Frankfurt / Klinische Pharmakologie, Medizinische Hochschule Hannover, Hannover, Main, Germany; Germany; ’ Kardiologie, St. Josef-Hospital, Ruhr-Universitat, Bochum, Germany (Received in final form November 19, 1999)
Summary L-arginine slows the development of atheromatous lesions, improves endotheliumdependent relaxation, and reduces the vascular superoxide anion production in hypercholesterolemic rabbits. These beneficial effects have been attributed to Larginine-dependent formation of nitric oxide within the endothelial layer; a direct effect of L-arginine on other cells, however, has not been investigated. We hypothesised that in hypercholesterolemia L-arginine also specifically acts via a direct inhibitory effect on leukocytes, without affecting endothelial cells. The action of L-arginine was compared to vitamine E and the HMG CoA reductase inhibitor lovastatin which are known to attenuate progression of atherosclerosis. Rabbits were fed cholesterol enriched diet and from week five on lovastatin (lOmg/day), vitamine E (300mg/d) or L-arginine (2% in drinking water) were given. After 16 weeks, blood cholesterol concentration was determined and leukocyte adhesion to cotton wool was measured. In order to exclude any endothelium-mediated effects an adhesion assay to endothelial cells was avoided. Cholesterol-enriched diet increased plasma cholesterol concentration (19&3 vs. 1427&l 17 mg/dl). Cholesterol levels were not affected by L-arginine (1344+163mg/dl) or vitamine E (13 12+243mg/dl). Lovastatin treatment reduced cholesterol concentration by 35% as compared to the cholesterol group (899*51, ~~0.05 vs. cholesterol). Cholesterol diet significantly increased leukocyte adhesion to cotton wool (16+3% vs 27&4%,p<0.05). Lovastatin or vitamine E had no effect on leukocyte adhesion (3 1+4%, 39+5), whereas L-arginine completely normalized adhesion (8.8*3%). Conclusion: Rabbits fed high cholesterol diet have increased leukocyte adhesion, which is not affected by lovastatin or vitamine E treatment, but prevented by L-arginine supplementation. A direct inhibitory effect of Larginine on leukocyte adhesion may contribute to the beneficial effects observed with this substance. Key Words: L-arginine, hypercholesterolemia, leukocyte adhesion
Supplementation with the amino acid L-arginine has been shown to slow the development of atheromatous plaques formation, to improve endothelium-dependent relaxation, and to reduce monocyte adhesion to the vascular endothelium( 1). The mechanisms of these beneficial effects of Corresponding author: Ralf P. Brandes, MD, Institut fiir Kardiovaskulare Physiologie, Klinikum der J.W. Goethe-Universitlt, Theodor Stem Kai 7, D-60596 Frankfurt, Germany, e-mail: [email protected], Phone: ++49-69-6301-6995, Fax: ++49-69-63017668
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L-arginine are not fully understood. Since L-arginine is the substrate of the endothelial NOsynthase, an increased formation of endothelium-derived nitric oxide (NO) induced by excessive L-arginine has been suggested as the underlying mechanism(l). Whether L-arginine does also affect non-endothelial cells is uncertain. Adhesion of monocytes and neutrophils to the endothelium is reduced in the presence of L-arginine(2;3). It has been suggested that this effect is due to an increased endothelial NO formation facilitated by L-arginine rather than a direct effect of L-arginine on the blood cells. In the present study we measured leukocyte adhesion to a nonendothelial matrix in order to determine the direct effect of L-arginine on leukocytes and to exclude any interaction with endothelium. In blood from cholesterol-fed rabbits, the effect of Larginine on leukocyte-adhesion was compared to lovastatin and vitamine E. Both substances are known to slow down the development of atheromatous lesion formation in hypercholesterolemic rabbits. The underlying mechanisms, however, are different to L-arginine, i.e. lovastatin may act anti-proliferative and lowers serum-cholesterol levels(4;5), whereas vitamine E serves as antioxidant preventing LDL-oxidation(6). Methods Animals and study design: Male New Zealand white rabbits of 12 week age were housed in conditions, which conformed with the guide for the care and use of laboratory animals published by the US National Institutes of Health (NIH publication NO 85-23, revised 1985). The study had been approved by the Hannover supervisory committee for studies in animals. Twentytwo rabbits were fed 1% cholesterol enriched diet (Altromin, Lage, Germany), 8 rabbits received normal chow (control group). After 4 weeks, concentration of cholesterol in the diet was reduced to 0.5%, this cholesterol diet was fed for further 12 weeks. From week 5 to 16, cholesterol-fed rabbits were randomly treated with either lovastatin (n=6; lOmg/day lovastatin as oral gavage), Larginine (n=S; given as a 2% supplement to the drinking water 2%) or vitamine E (n=8; 300mgiday vitamine E as oral gavage disolved in olive oil). It has been shown previously that this cholesterol feeding protocol produces aortic intimal hyperplasia and carotid artery plaque formation (7). The concentrations chosen for lovastatin (4), L-arginine (7) and vitamine E (6) have been shown previously to reduce plaque formation in cholesterol-fed rabbits. At the end of the feeding period rabbits were anesthesized with sodium pentobarbital (30mg/kg i.v.) / ketamin (30mg/kg im.). For leukocyte adhesion 5 ml of blood were drawn from the inferior caval vein into commercially available sodium citrate syringes (Sarstedt). Leukocyte adhesion: Citrate blood was diluted with Dulbecco’s minimal essential medium containing 5OmM HEPES (with no bicarbonate and no phenol red) in a ratio of 1:5. 500~1 of sample were aliquoted into test tubes. Two of the tubes contained 5 mg of leukocyte absorbtion wool (Leuko-Pak, Fenwal laboratories, Deerfield, IL, USA) whereas the third tube served as control. The samples were incubated for 30 min at 37°C to allow leukocyte adhesion. Then, the adhesion matrix was removed quickly. 50~1 aliquotes of the samples were mixed with 70~1 modified Turk solution (550 mM acetic acid, 8.5 mM crystal violet). Leukocytes were counted in a Neubauer chamber. Relative adhesion was calculated from the difference in leukocyte content of the control to the cotton containing test tubes. In a sub-group of experiments, L-arginine (300uM) was added to the blood of normal rabbits five minutes prior to the adhesion experiments to determine direct effects of the substance. Cholesterol and L-arginine measurements: Total plasma cholesterol concentrations were determined by a commercially available spectrophotometric assay kit (Boehringer, Mannheim, Germany). Plasma L-arginine concentrations were determined by high-performance liquid chromatography using pre-column derivatization with o-phthalaldehyde after extraction on carboxylic acid solid-phase extraction cartridges (Varian) as described in detail elsewhere(g). Statistics: Values presented are mean + S.E.M. Data were compared by two-way ANOVA followed by Newman-Keuls test. The 0.05 probability level was accepted as significant. The number of experiments refers to the number of animals.
Vol. 66, No. 16,ZOOO
L-arginine Supplementation and Cholesterol
1521
Results Plasma levels of cholesterol and L-arginine: Cholesterol diet induced a pronounced increase in plasma cholesterol concentration after 16 weeks feeding. Supplementation with L-arginine or vitamine E had no effect on plasma cholesterol concentration, whereas lovastatin treatment reduced plasma cholesterol concentration by about 35% (Table 1). The cholesterol diet had no effect on plasma L-arginine levels. L-arginine supplementation to the drinking water approximately doubled plasma levels of L-arginine (Table 1). Plasma levels of vitamine E were not determined. Leukocyte adhesion: As compared to control, cholesterol feeding had no effect on blood leukocyte count. Leukocytes obtained from cholesterol-fed animals showed almost two-fold increase in adhesion to cotton wool as compared to cells from control rabbits (Fig. 1). Supplementation with vitamine E or lovastatin had no effect on the increased leukocyte adhesion. In contrast, L-arginine supplementation completely normalized leukocyte adhesion (Fig. 1). Addition of L-arginine to the blood of normal rabbits however did not change the adhesion to cotton wool (data not shown). Discussion The beneficial effects of L-arginine in hypercholesterolemia have mainly been attributed to an increased generation of NO by the vascular endothelium (1). In the present study we firstly demonstrate a direct beneficial effect of L-arginine on the increased adhesion of hypercholesterolemic leukocytes. An increased adhesion of leukocytes, monocytes and platelets to endothelial cells has been documented in hypercholesterolemic patients and animals (2;3). The underlying mechanism of stimulated blood cell / endothelium interaction in hyperchoelsterolemia is not fully understood, however, an increased breakdown of NO or a decreased endothelial formation of NO has been postulated (9) in addition to changes in adhesion molecule expression (10). In fact, NO is known to prevent polyrnorphonuclear leukocyte and monocyte adhesion to the luminal surface of capillaries and arteries (11). Therefore, decreased bio-availability of NO would be a possible mechanism leading to increased leukocyte adhesion to the endothelium in hypercholesterolemia. In the present study we observed a two-fold increase in leukocyte adhesion to a non-endothelial matrix in hypercholesterolemia. These data suggest that, in addition to the reduced bio-availability of endothelial NO, other factors may be also involved which are related to leukocyte properties, such as an increased expression of adhesion molecules (12) Nevertheless based on the present data no statement about the underlying mechanism can be given. Certainly, in blood vessels adhesion is mediated by a complex interplay of different adhesion molecules on the endothelium as well as on the leukocytes and changes in adhesion molecule expression therefore determine the adhesive properties of neutrophils. Thus, it could be argued that the approach chosen in the present study is inappropriate since the specific interaction between endothelium and neutrophils was not established. It should however be kept in mind that the study was designed to determine a direct effect of L-arginine on leukocytes. In an leukocyte-endothelium adhesion assay the use of TABLE I Plasma Cholesterol Level [mrnol/L] Control 0.5 f 0.08 Cholesterol 36.96 + 3.06* Cholesterol + L-Arginine 34.83 + 4.24* Cholesterol + Lovastatin 23.28? 1.35*$ Cholesterol + Vitamine E 36.34 + 6.72* Plasma levels of cholesterol and L-arginine vs. control, $p
L-Arginine Level [pmol/L] 126.1,ll.O 129.5f6.2 238.5 _+11.5* not performed not performed
and white blood cell count: (*p
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Supplementation
and Cholesterol
Vol. 66, No. 16,200O
Fig. 1 Leukocyte adhesion to cotton wool matrix: Adhesion of leukocytes in diluted blood was determined in samples from rabbits on normal diet and from rabbits on a cholesterol rich diet. In addition to cholesterol Larginine (2%), lovastatin (lOmg/day) or vitamine E (300mg/day) was supplemented in subgroups. (*p
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Supplementation
and Cholesterol
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endothelium. The effect of L-arginine on adhesion of hypercholesterolemic neutrophils was not tested(3). Vitamine E and lovastatin, which both reduced the extent of atheroma development in rabbits(7;15) failed to normalize adhesion of hypercholesterolemic leukocytes in the present study, although lovastatin reduces adhesion of normal leukocytes to hypercholesterolemic endothelium(3). It is therefore likely, that L-arginine exerts specific effects on the adhesion of leukocytes from hypercholerolemic animals. NO-independent effects of L-arginine have been observed at concentration higher than 1mM( 16), therefore NO-dependent effects have to be taken especially since NO is known to inhibit leukocyte adhesion( 11). In into account, hypercholesterolemia an induction of the inducible isoform of the NO-synthase (iNOS) has been observed in lymphocytes and macrophages( 17). iNOS produces excessive amounts of NO as long as the substrate L-arginine is present in sufficient concentrations. Since the beneficial effects of Larginine are observed only under hypercholesterolemic conditions, iNOS-dependent NOformation is likely to be involved in the beneficial effect of L-arginine. In this context, it is interesting to note that although lovastatin which is known to increase endothelial NOS expression( 18; 19), inhibits iNOS expression in macrophages(20). This might also explain the failure of lovastatin to normalize adhesion in hypercholesterolemia. Nevertheless, this issue is still unclear. To prove that an increased formation of NO by iNOS in leukocytes is responsible for the effects of L-arginine, additional experiments especially with NOS inhibition and D-arginine have to be performed. In summary, here we firstly demonstrate that L-arginine normalizes adhesion of leukocytes from hypercholesterolemic rabbits, whereas vitamine E and lovastatin had no effect. This observation adds a new aspect to the beneficial properties of the substance which so far have been thought to be mainly mediated by changes in endothelial properties. References 1. 2. 3. 4. 5.
6. 7. 8. 9. 10. 11. 12.
13.
A.J. MAXWELL and J.P. COOKE, Current Opinion Nephrol Hyperten 7 63-70 (1998). G. THEILMEIER, J.R. CHAN, C. ZALPOUR, B. ANDERSON, B.Y. WANG, A. WOLF, P.S. TSAO and J.P. COOKE, Arter Thromb Vast Biol 17 3557-3564 (1997). A.M. LEFER and X.L. MA, Arter Thromb 13 771-776 (1993). M.P. SENARATNE, A.B. THOMSON and CT. KAPPAGODA, Cardiovasc Res 25 568-587 (1991). E. MUNRO, M. PATEL, P. CHAN, L. BETTERIDGE, G. CLUNN, K. GALLAGHER, A. HUGHES, M. SCHACHTER, J. WOLFE and P. SEVER, Eur J Clin Invest 24 766772 (1994). J.F. KEANEY, Y. GUO, D. CUNNNINGHAM, G.T. SHWAERY, A. XU and J.A. VITA, J Clin Invest 98 386-394 (1996). R.H. BijGER, S.M. BODE-BGGER, R.P. BRANDES, L. PHIVTHONG-NGAM, M. BGHME, R. NAFE, A. MUGGE and J.C. FRGLICH, Circulation 96 1282-1290 (1997). S.M. BODE-BGGER, R.H. BGGER, S. KIENKE, W. JUNKER and J.C. FRGLICH, Biochem Biophys Res Commun 219 598-603 (1996). R.H. BijGER, S.M. BODE-BijGER, A. MUGGE, S. KIENKE, R. BRANDES, A. DWENGER and J.C. FRGLICH, Atherosclerosis 117 273-284 (1995). M.I. CYBULSKY and M.A. GIMBRONE, Science 251788-791 (1991). P. KUBES, M. SUZUKI and D.N. GRANGER, Proc Nat1 Acad Sci USA 88 4651-4655 (1991). H.A. LEHR, F. KROMBACH, S. MUNZING, R. BODLAJ, S.I. GLAUBITT, D. SEIFFGE, C. HUBNER, U.H. VANDRIAN and K. MESSMER, Am J Path01 146 218227 (1995). A. MUGGE, R.P. BRANDES, H. BOGER, A. DWENGER, S. BODE-BGGER, S. KIENKE, J.C. FRGLICH and P.R. LICHTLEN, J Cardiovasc Pharmacol 24 994-998 (1994).
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14. 15.
16. 17. 18. 19.
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X.J. GIRERD, A.T. HIRSCH, J.P. COOKE, V.J. DZAU and M.A. CREAGER, Circ Res 67 1301-1308 (1990). R.H. BOGER, S.M. BODE-BOGER, L. PHIVTHONG-NGAM, R.P. BRANDES, E. SCHWEDHELM, A. MijGGE, M. BijHME, D. TSIKAS and J.C. FRijLICH, Atherosclerosis 141 3 1-43 (1998). G. THOMAS, M. HECKER and P.W. ~MWELL, Biochem Biophys Res Comm 158 177-180 (1989). T. ESAKI, T. HAYASHI, E. MUTO, K. YAMADA, M. KUZUYA and A. IGUCHI, Atherosclerosis 128 39-46 (1997). U. LAUFS, V. LAFATA, J. PLUTZKY and J.K. LIAO, Circulation 97 1129-1135 (1998). 0. HE~ANDEZ-Pact, D. PEREZ-SALA, J. NAVAJO-ANTOL~, R. SANCHEZ-PASCUALA, G. HERNANDEZ, C. DIAZ and S. LAMAS, J Clin Invest 101 271 l-2719 (1998). K. PAHAN, F.G. SHEIKH, A.M. NAMBOODIRI and 1. SINGH, J Clin Invest 100 2671-2679 (1997).
EISEVIER
PI1 SOO24-3205(00)00469-O
L-ARGININE SUPPLEMENTATION IN HYPERCHOLESTEROLEMIC NORMALIZES LEUKOCYTE ADHESION TO NON-ENDOTHELIAL
RABBITS MATRIX
Ralf P. Brandes a, Stefanie Brandes a, Rainer H. Biiser b, Stefanie M. Bode-Biiger b, and Andreas Miigge a Institut fiir Kar$iovaskulHre Physiologie, Klinikum der J.W. Goethe-Universitat, Frankfurt / Klinische Pharmakologie, Medizinische Hochschule Hannover, Hannover, Main, Germany; Germany; ’ Kardiologie, St. Josef-Hospital, Ruhr-Universitat, Bochum, Germany (Received in final form November 19, 1999)
Summary L-arginine slows the development of atheromatous lesions, improves endotheliumdependent relaxation, and reduces the vascular superoxide anion production in hypercholesterolemic rabbits. These beneficial effects have been attributed to Larginine-dependent formation of nitric oxide within the endothelial layer; a direct effect of L-arginine on other cells, however, has not been investigated. We hypothesised that in hypercholesterolemia L-arginine also specifically acts via a direct inhibitory effect on leukocytes, without affecting endothelial cells. The action of L-arginine was compared to vitamine E and the HMG CoA reductase inhibitor lovastatin which are known to attenuate progression of atherosclerosis. Rabbits were fed cholesterol enriched diet and from week five on lovastatin (lOmg/day), vitamine E (300mg/d) or L-arginine (2% in drinking water) were given. After 16 weeks, blood cholesterol concentration was determined and leukocyte adhesion to cotton wool was measured. In order to exclude any endothelium-mediated effects an adhesion assay to endothelial cells was avoided. Cholesterol-enriched diet increased plasma cholesterol concentration (19&3 vs. 1427&l 17 mg/dl). Cholesterol levels were not affected by L-arginine (1344+163mg/dl) or vitamine E (13 12+243mg/dl). Lovastatin treatment reduced cholesterol concentration by 35% as compared to the cholesterol group (899*51, ~~0.05 vs. cholesterol). Cholesterol diet significantly increased leukocyte adhesion to cotton wool (16+3% vs 27&4%,p<0.05). Lovastatin or vitamine E had no effect on leukocyte adhesion (3 1+4%, 39+5), whereas L-arginine completely normalized adhesion (8.8*3%). Conclusion: Rabbits fed high cholesterol diet have increased leukocyte adhesion, which is not affected by lovastatin or vitamine E treatment, but prevented by L-arginine supplementation. A direct inhibitory effect of Larginine on leukocyte adhesion may contribute to the beneficial effects observed with this substance. Key Words: L-arginine, hypercholesterolemia, leukocyte adhesion
Supplementation with the amino acid L-arginine has been shown to slow the development of atheromatous plaques formation, to improve endothelium-dependent relaxation, and to reduce monocyte adhesion to the vascular endothelium( 1). The mechanisms of these beneficial effects of Corresponding author: Ralf P. Brandes, MD, Institut fiir Kardiovaskulare Physiologie, Klinikum der J.W. Goethe-Universitlt, Theodor Stem Kai 7, D-60596 Frankfurt, Germany, e-mail: [email protected], Phone: ++49-69-6301-6995, Fax: ++49-69-63017668
1520
L-arginine Supplementation and Cholesterol
Vol. 66, No. 16,200O
L-arginine are not fully understood. Since L-arginine is the substrate of the endothelial NOsynthase, an increased formation of endothelium-derived nitric oxide (NO) induced by excessive L-arginine has been suggested as the underlying mechanism(l). Whether L-arginine does also affect non-endothelial cells is uncertain. Adhesion of monocytes and neutrophils to the endothelium is reduced in the presence of L-arginine(2;3). It has been suggested that this effect is due to an increased endothelial NO formation facilitated by L-arginine rather than a direct effect of L-arginine on the blood cells. In the present study we measured leukocyte adhesion to a nonendothelial matrix in order to determine the direct effect of L-arginine on leukocytes and to exclude any interaction with endothelium. In blood from cholesterol-fed rabbits, the effect of Larginine on leukocyte-adhesion was compared to lovastatin and vitamine E. Both substances are known to slow down the development of atheromatous lesion formation in hypercholesterolemic rabbits. The underlying mechanisms, however, are different to L-arginine, i.e. lovastatin may act anti-proliferative and lowers serum-cholesterol levels(4;5), whereas vitamine E serves as antioxidant preventing LDL-oxidation(6). Methods Animals and study design: Male New Zealand white rabbits of 12 week age were housed in conditions, which conformed with the guide for the care and use of laboratory animals published by the US National Institutes of Health (NIH publication NO 85-23, revised 1985). The study had been approved by the Hannover supervisory committee for studies in animals. Twentytwo rabbits were fed 1% cholesterol enriched diet (Altromin, Lage, Germany), 8 rabbits received normal chow (control group). After 4 weeks, concentration of cholesterol in the diet was reduced to 0.5%, this cholesterol diet was fed for further 12 weeks. From week 5 to 16, cholesterol-fed rabbits were randomly treated with either lovastatin (n=6; lOmg/day lovastatin as oral gavage), Larginine (n=S; given as a 2% supplement to the drinking water 2%) or vitamine E (n=8; 300mgiday vitamine E as oral gavage disolved in olive oil). It has been shown previously that this cholesterol feeding protocol produces aortic intimal hyperplasia and carotid artery plaque formation (7). The concentrations chosen for lovastatin (4), L-arginine (7) and vitamine E (6) have been shown previously to reduce plaque formation in cholesterol-fed rabbits. At the end of the feeding period rabbits were anesthesized with sodium pentobarbital (30mg/kg i.v.) / ketamin (30mg/kg im.). For leukocyte adhesion 5 ml of blood were drawn from the inferior caval vein into commercially available sodium citrate syringes (Sarstedt). Leukocyte adhesion: Citrate blood was diluted with Dulbecco’s minimal essential medium containing 5OmM HEPES (with no bicarbonate and no phenol red) in a ratio of 1:5. 500~1 of sample were aliquoted into test tubes. Two of the tubes contained 5 mg of leukocyte absorbtion wool (Leuko-Pak, Fenwal laboratories, Deerfield, IL, USA) whereas the third tube served as control. The samples were incubated for 30 min at 37°C to allow leukocyte adhesion. Then, the adhesion matrix was removed quickly. 50~1 aliquotes of the samples were mixed with 70~1 modified Turk solution (550 mM acetic acid, 8.5 mM crystal violet). Leukocytes were counted in a Neubauer chamber. Relative adhesion was calculated from the difference in leukocyte content of the control to the cotton containing test tubes. In a sub-group of experiments, L-arginine (300uM) was added to the blood of normal rabbits five minutes prior to the adhesion experiments to determine direct effects of the substance. Cholesterol and L-arginine measurements: Total plasma cholesterol concentrations were determined by a commercially available spectrophotometric assay kit (Boehringer, Mannheim, Germany). Plasma L-arginine concentrations were determined by high-performance liquid chromatography using pre-column derivatization with o-phthalaldehyde after extraction on carboxylic acid solid-phase extraction cartridges (Varian) as described in detail elsewhere(g). Statistics: Values presented are mean + S.E.M. Data were compared by two-way ANOVA followed by Newman-Keuls test. The 0.05 probability level was accepted as significant. The number of experiments refers to the number of animals.
Vol. 66, No. 16,ZOOO
L-arginine Supplementation and Cholesterol
1521
Results Plasma levels of cholesterol and L-arginine: Cholesterol diet induced a pronounced increase in plasma cholesterol concentration after 16 weeks feeding. Supplementation with L-arginine or vitamine E had no effect on plasma cholesterol concentration, whereas lovastatin treatment reduced plasma cholesterol concentration by about 35% (Table 1). The cholesterol diet had no effect on plasma L-arginine levels. L-arginine supplementation to the drinking water approximately doubled plasma levels of L-arginine (Table 1). Plasma levels of vitamine E were not determined. Leukocyte adhesion: As compared to control, cholesterol feeding had no effect on blood leukocyte count. Leukocytes obtained from cholesterol-fed animals showed almost two-fold increase in adhesion to cotton wool as compared to cells from control rabbits (Fig. 1). Supplementation with vitamine E or lovastatin had no effect on the increased leukocyte adhesion. In contrast, L-arginine supplementation completely normalized leukocyte adhesion (Fig. 1). Addition of L-arginine to the blood of normal rabbits however did not change the adhesion to cotton wool (data not shown). Discussion The beneficial effects of L-arginine in hypercholesterolemia have mainly been attributed to an increased generation of NO by the vascular endothelium (1). In the present study we firstly demonstrate a direct beneficial effect of L-arginine on the increased adhesion of hypercholesterolemic leukocytes. An increased adhesion of leukocytes, monocytes and platelets to endothelial cells has been documented in hypercholesterolemic patients and animals (2;3). The underlying mechanism of stimulated blood cell / endothelium interaction in hyperchoelsterolemia is not fully understood, however, an increased breakdown of NO or a decreased endothelial formation of NO has been postulated (9) in addition to changes in adhesion molecule expression (10). In fact, NO is known to prevent polyrnorphonuclear leukocyte and monocyte adhesion to the luminal surface of capillaries and arteries (11). Therefore, decreased bio-availability of NO would be a possible mechanism leading to increased leukocyte adhesion to the endothelium in hypercholesterolemia. In the present study we observed a two-fold increase in leukocyte adhesion to a non-endothelial matrix in hypercholesterolemia. These data suggest that, in addition to the reduced bio-availability of endothelial NO, other factors may be also involved which are related to leukocyte properties, such as an increased expression of adhesion molecules (12) Nevertheless based on the present data no statement about the underlying mechanism can be given. Certainly, in blood vessels adhesion is mediated by a complex interplay of different adhesion molecules on the endothelium as well as on the leukocytes and changes in adhesion molecule expression therefore determine the adhesive properties of neutrophils. Thus, it could be argued that the approach chosen in the present study is inappropriate since the specific interaction between endothelium and neutrophils was not established. It should however be kept in mind that the study was designed to determine a direct effect of L-arginine on leukocytes. In an leukocyte-endothelium adhesion assay the use of TABLE I Plasma Cholesterol Level [mrnol/L] Control 0.5 f 0.08 Cholesterol 36.96 + 3.06* Cholesterol + L-Arginine 34.83 + 4.24* Cholesterol + Lovastatin 23.28? 1.35*$ Cholesterol + Vitamine E 36.34 + 6.72* Plasma levels of cholesterol and L-arginine vs. control, $p
L-Arginine Level [pmol/L] 126.1,ll.O 129.5f6.2 238.5 _+11.5* not performed not performed
and white blood cell count: (*p
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Fig. 1 Leukocyte adhesion to cotton wool matrix: Adhesion of leukocytes in diluted blood was determined in samples from rabbits on normal diet and from rabbits on a cholesterol rich diet. In addition to cholesterol Larginine (2%), lovastatin (lOmg/day) or vitamine E (300mg/day) was supplemented in subgroups. (*p
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L-arginine
Supplementation
and Cholesterol
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endothelium. The effect of L-arginine on adhesion of hypercholesterolemic neutrophils was not tested(3). Vitamine E and lovastatin, which both reduced the extent of atheroma development in rabbits(7;15) failed to normalize adhesion of hypercholesterolemic leukocytes in the present study, although lovastatin reduces adhesion of normal leukocytes to hypercholesterolemic endothelium(3). It is therefore likely, that L-arginine exerts specific effects on the adhesion of leukocytes from hypercholerolemic animals. NO-independent effects of L-arginine have been observed at concentration higher than 1mM( 16), therefore NO-dependent effects have to be taken especially since NO is known to inhibit leukocyte adhesion( 11). In into account, hypercholesterolemia an induction of the inducible isoform of the NO-synthase (iNOS) has been observed in lymphocytes and macrophages( 17). iNOS produces excessive amounts of NO as long as the substrate L-arginine is present in sufficient concentrations. Since the beneficial effects of Larginine are observed only under hypercholesterolemic conditions, iNOS-dependent NOformation is likely to be involved in the beneficial effect of L-arginine. In this context, it is interesting to note that although lovastatin which is known to increase endothelial NOS expression( 18; 19), inhibits iNOS expression in macrophages(20). This might also explain the failure of lovastatin to normalize adhesion in hypercholesterolemia. Nevertheless, this issue is still unclear. To prove that an increased formation of NO by iNOS in leukocytes is responsible for the effects of L-arginine, additional experiments especially with NOS inhibition and D-arginine have to be performed. In summary, here we firstly demonstrate that L-arginine normalizes adhesion of leukocytes from hypercholesterolemic rabbits, whereas vitamine E and lovastatin had no effect. This observation adds a new aspect to the beneficial properties of the substance which so far have been thought to be mainly mediated by changes in endothelial properties. References 1. 2. 3. 4. 5.
6. 7. 8. 9. 10. 11. 12.
13.
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