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(Circulation. 1997;96:1282-1290.)
© 1997 American Heart Association, Inc.

Articles

Dietary L-Arginine Reduces the Progression of Atherosclerosis in Cholesterol-Fed Rabbits

Comparison With Lovastatin

Rainer H. Böger, MD; Stefanie M. Bode-Böger, MD; Ralf P. Brandes, MD; Laddaval Phivthong-ngam, PhD; Michael Böhme; Reinhold Nafe, MD; Andreas Mügge, MD; ; Jürgen C. Frölich, MD

From the Institute of Clinical Pharmacology (R.H.B., S.M.B.-B., L.P., M.B., J.C.F.) and Departments of Cardiology (R.P.B., A.M.) and Pathology (R.N.), Medical School, Hannover, Germany.

	Abstract

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Background We investigated whether L-arginine induces regression of preexisting atheromatous lesions and reversal of endothelial dysfunction in hypercholesterolemic rabbits, whether similar effects can be obtained by cholesterol-lowering therapy with lovastatin, and which mechanism leads to these effects.

Methods and Results Rabbits were fed 1% cholesterol for 4 weeks and 0.5% cholesterol for an additional 12 weeks. Two groups of cholesterol-fed rabbits were treated with L-arginine (2.0% in drinking water) or lovastatin (10 mg/d) during weeks 5 through 16. Systemic nitric oxide (NO) formation was assessed as the urinary excretion rates of nitrate and cGMP in weekly intervals. Cholesterol feeding progressively reduced urinary nitrate excretion to 40% of baseline (P<.05) and increased plasma concentrations of asymmetrical dimethylarginine (ADMA), an endogenous NO synthesis inhibitor. Dietary L-arginine reversed the reduction in plasma L-arginine/ADMA ratio and partly restored urinary excretion of nitrate and cGMP (each P<.05 vs cholesterol) but did not change plasma cholesterol levels. L-Arginine completely blocked the progression of carotid intimal plaques, reduced aortic intimal thickening, and preserved endothelium-dependent vasodilator function. Lovastatin treatment reduced plasma cholesterol by 32% but did not improve urinary nitrate or cGMP excretion or endothelium-dependent vasodilation. Lovastatin had a weaker inhibitory effect on carotid plaque formation and aortic intimal thickening than L-arginine. L-Arginine inhibited but lovastatin potentiated superoxide radical generation in the atherosclerotic vascular wall.

Conclusions Dietary L-arginine improves NO-dependent vasodilator function in cholesterol-fed rabbits and completely blocks the progression of plaques via restoration of NO synthase substrate availability and reduction of vascular oxidative stress. Lovastatin treatment has a weaker inhibitory effect on the progression of atherosclerosis and no effect on vascular NO elaboration, which may be due to its stimulatory effect on vascular superoxide radical generation.

Key Words: endothelium-derived factors • endothelium • free radicals • lipoproteins • plaque

	Introduction

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The precursor of endothelial NO is L-arginine, which plays an important role in maintaining an active vasodilator tone in healthy blood vessels.¹ In hypercholesterolemia and atherosclerosis, the biological activity of NO is severely impaired, leading to attenuated endothelium-dependent vasodilation and intimal thickening.^{2 3} Because this defect in vascular NO elaboration occurs at a very early stage in hypercholesterolemia, it is suggested to critically determine the progression of atherosclerosis.⁴ Consequently, efforts have been made to slow the progression of atherosclerosis by restoring vascular NO formation. Dietary supplementation of hypercholesterolemic rabbits with L-arginine has been shown to restore endothelium-dependent, NO-mediated vascular relaxations,^{2 3 5} to partly restore NO formation rates and reduce the vascular release of superoxide radicals,³ and to reduce the formation of intimal lipid plaques.^{3 5} However, in all of these studies, L-arginine has been administered chronically, starting at the beginning of the cholesterol-feeding period. In addition, a recent study by Candipan et al⁶ showed that L-arginine induces regression of preexisting lesions when treatment is started after the initiation of atheromatous plaques, a situation more similar to the clinical setting. The mechanism of these pharmacological effects of L-arginine in hypercholesterolemia has remained unclear. We have recently demonstrated that the plasma concentrations of ADMA, an endogenous inhibitor of NO synthesis,⁷ are elevated in hypercholesterolemic rabbits.⁸

The impairment of endothelial NO elaboration in hypercholesterolemia is related to the degree of vascular LDL cholesterol accumulation,⁹ and both native and oxidized LDLs interfere with the biological activity of NO in vitro.^{10 11} Therefore, cholesterol-lowering therapy may also improve endothelial vasodilator function. Recently, it has been shown that cholesterol-lowering therapy with HMG-CoA reductase inhibitors like lovastatin reduces vascular LDL cholesterol accumulation and atheromatous plaque formation¹² ; however, it has been debated whether this results in improved endothelial function.^{13 14} Studies in hypercholesterolemic patients have also yielded conflicting results in that some studies^{15 16} showed a beneficial effect of HMG-CoA reductase inhibition on endothelium-dependent coronary vasomotion, whereas others¹⁷ failed to show such an effect.

Therefore, we investigated in the present study whether long-term dietary administration of L-arginine or lovastatin improves endothelial function and induces regression of atherosclerosis in cholesterol-fed rabbits when treatment is started after the beginning of diet-induced hypercholesterolemia. We have further studied whether changes in vascular oxidative stress may be responsible for the effects of L-arginine and lovastatin and whether competition with ADMA may be involved in the effects of dietary L-arginine in this model. To assess NO formation rates in vivo, we measured the urinary excretion rates of its final metabolite, NO₃^-, and of its second messenger, cGMP.^{3 18}

	Methods

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Animals and Study Design
Forty-four male New Zealand White rabbits were used in this study, which conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 85-23, revised 1985) and had been approved by the Hannover supervisory committee for studies in animals. Thirty-two rabbits were fed 1% cholesterol for 4 weeks; the other 12 rabbits received normal rabbit chow. After 4 weeks, 8 cholesterol-fed rabbits (cholesterol-4 group) and 4 control rabbits (control-4 group) were killed. The other animals received 0.5% cholesterol (n=24) or normal rabbit chow (n=8; control-16 group) for an additional 12 weeks. During this second part of the study period, one group of 8 cholesterol-fed rabbits received 2.0% L-arginine in drinking water (L-arginine group), and a second group of 8 rabbits received 10 mg/d of lovastatin by oral gavage (lovastatin group); the other 8 rabbits received no additional treatment (cholesterol-16 group). At the beginning of the experimental period and in weekly intervals thereafter, the rabbits were placed in metabolic cages for 24-hour urine collection. Plasma samples were drawn from the central ear artery every 4 weeks. At the end of the feeding period, the rabbits were killed, and the aorta and carotid arteries were excised and immediately rinsed in freshly prepared, ice-cold Krebs buffer. A segment of the thoracic aorta immediately distal from the left subclavian artery was used for isometric tension recording, and additional segments of the thoracic and abdominal aortas were fixed in buffered formalin for histological examination. The carotid arteries were opened longitudinally for morphometric assessment of intimal plaque area.

Biochemical Analyses
Urinary nitrate excretion was determined by gas chromatography–mass spectrometry using the pentafluorobenzyl derivative of nitrate as described previously.¹⁹ Briefly, aliquots of urine were spiked with [¹⁵N]NO₃^– (MSD isotopes, Merck Frosst) as internal standard and treated with cadmium to reduce NO₃^– to NO₂^–. The suspension was alkalinized, allowed to react with 5 µL of pentafluorobenzyl bromide (75 minutes, 50°C), and extracted with toluene. The toluene phase was taken up, dried, and injected into the gas chromatograph–mass spectrometer. Quantitation was performed by selected ion monitoring at m/z 46 for endogenous NO₂^–/NO₃^– and m/z 47 for the internal standard. The detection limit of the method was 20 fmol of nitrite or nitrate. Intra-assay variability was <3.8%.

Urinary cGMP concentrations were measured by specific radioimmunoassay using [¹²⁵I]cGMP as a tracer and globulin precipitation. The detection limit of the assay was 160 fmol/mL.

Urinary creatinine was determined spectrophotometrically with the alkaline picric acid method in an automatic analyzer (Beckman). The urinary excretion rates of NO₃^– and cGMP were corrected by urinary creatinine concentration to limit variability due to changes in renal excretory function, as described previously.¹⁸

Plasma L-arginine and dimethylarginine 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.⁸ Samples and standards were incubated for exactly 30 seconds with o-phthalaldehyde before automatic injection into the chromatograph and were separated on a C₆H₅ column (Macherey and Nagel) with the fluorescence monitor set at ^ex=340 nm and ^em=455 nm. Samples were eluted from the column with 0.96% citric acid/methanol 2:1 (vol/vol), pH 6.8, at a flow rate of 1 mL/min. The intra-assay and interassay variability of the method was 5.2% and 5.5%, respectively; the detection limit of the assay was 0.1 µmol/L.

Plasma total, LDL, and HDL cholesterol concentrations were determined by use of a commercially available spectrophotometric assay kit (Boehringer-Mannheim).

Endothelial Function
The aortas were dissected free of adhering fat and connective tissue and placed into organ baths filled with oxygenated (95% O_2, 5% CO₂) modified Krebs solution (37°C, pH 7.4) of the following composition (in mmol/L): Na⁺ 145.0, K⁺ 5.95, Ca²⁺ 1.7, Mg²⁺ 1.2, Cl^– 128.15, HCO₃^– 25.0, H₂PO₄^– 1.2, SO₄^2– 1.2, glucose 10.6, and EDTA 0.025. The vascular preparations were connected to force transducers for isometric tension recording. For 60 minutes, the rings were gradually stretched to a resting tension of 2g (which had previously been determined to be the optimum of their length-tension relation) and repeatedly washed with fresh Krebs solution. The rings were then precontracted with norepinephrine (1 µmol/L) and relaxed by acetylcholine (1 µmol/L) for testing of endothelial integrity as described previously.³ Rings from control animals always showed 70% relaxation in response to 1 µmol/L acetylcholine. After washout, cumulative concentration response curves were obtained with the endothelium-dependent vasodilators acetylcholine and calcium ionophore A23187 and the endothelium-independent vasodilator sodium nitroprusside (all drugs 1 nmol/L to 0.1 mmol/L). Relaxations were expressed as a percentage of the precontractile tension induced with 1 µmol/L norepinephrine. All drugs were purchased from Sigma.

Vascular Superoxide Radical Production
The release of superoxide radicals from isolated aortic rings was measured using the lucigenin-enhanced chemiluminescence technique in a Biolumat LB 9505 (Berthold) as described previously.^{3 20} Briefly, the rings were positioned in test vials with the lumen directed toward the detector. After 5 to 10 minutes of equilibration, photon emission was recorded continuously for 5 minutes. Thereafter, photon emission was determined after the addition of PMA, a stimulator of leukocyte respiratory burst, in a final concentration of 2 µmol/L dissolved in dimethyl sulfoxide. The specific chemiluminescence response was expressed as counts per minute minus the average background activity. After the end of the measurements, the rings were blotted and weighed; data were expressed as counts per minute per milligram of dry weight.

Histological and Morphometric Analyses
Segments of the thoracic and abdominal aorta were excised, fixed in formalin, embedded in paraffin, and stained with hematoxylin/eosin for the morphometric measurement of intimal and medial cross-sectional areas by planimetry.²¹ Four sections from each animal were analyzed, and the values were averaged.

The proximal common carotid arteries were dissected free of adventitial tissue and rinsed free of any remaining blood. Thereafter, the arteries were opened longitudinally and placed on an even surface for photography of intimal lesions. Photographs were digitized, and measurements of total intimal area and plaque area were made by planimetry of the digitized images.

The investigators performing the histomorphological measurements (R.H.B. and R.N.) were blinded to the treatment groups.

Calculations and Statistics
All values are given as mean±SE. Statistical significance was tested by use of ANOVA for repeated measures followed by Scheffé's F test. The time course of urinary nitrate and cGMP excretion rates was analyzed by calculating the AUC for each group; AUC values were calculated separately for weeks 0 through 4 (induction phase) and 5 through 16 (treatment phase) and compared by use of Student's unpaired t test. Statistical significance was accepted at the .05 level of probability.

	Results

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Urinary Nitrate and cGMP Excretion Rates
Urinary nitrate excretion significantly decreased, by 60% within the first 4 weeks of the 1% cholesterol-enriched diet (P<.05; Fig 1A), whereas it remained relatively stable in the control groups. After the fifth week, 0.5% cholesterol feeding resulted in stabilization of urinary nitrate excretion on a low level that was 55% lower than in the control group (P<.05). Treatment with L-arginine partly restored urinary nitrate excretion, which reached a level 40% higher than in the cholesterol-16 group (P<.05). In contrast, lovastatin treatment did not influence urinary nitrate excretion (P=NS versus cholesterol-16).

View larger version (23K): [in this window] [in a new window]   Figure 1. Urinary excretion rates of (A) nitrate, the oxidative metabolite of NO, and (B) cGMP, its second messenger, in rabbits fed a cholesterol-enriched diet or normal rabbit chow. The diet contained 1% cholesterol during weeks 1 through 4 and 0.5% cholesterol during weeks 5 through 16. Twenty-four–hour urine samples were collected at weekly intervals. The urinary excretion rates of nitrate and cGMP were related to urinary creatinine concentration to reduce variability due to changes in renal excretory function. Statistical analysis of the excretion data was performed by calculation of the area under the concentration-time curves separately for weeks 0 through 4 and 5 through 16 and comparing AUC data by Student's unpaired t test. *P<.05.

Urinary cGMP excretion was slightly but significantly reduced by cholesterol feeding during the first 4 weeks (Fig 1B). Treatment with L-arginine slightly but significantly increased cGMP excretion after week 5 (P<.05 versus cholesterol-16), whereas lovastatin had no effect on urinary cGMP excretion.

Urinary nitrate and cGMP excretion rates were significantly correlated (r=.471, P<.01).

Plasma L-Arginine, Dimethylarginine, and Cholesterol Concentrations and Creatinine Clearances
Baseline plasma L-arginine concentration was 137.9±3.6 µmol/L, with no significant differences between the groups. Neither cholesterol feeding nor lovastatin therapy had any significant effect on plasma L-arginine levels, whereas dietary L-arginine increased L-arginine plasma concentrations approximately twofold (Table 1). The plasma concentrations of ADMA and symmetrical dimethylarginine were 1.40±0.05 and 1.41±0.08 µmol/L, respectively, at baseline. Mean plasma dimethylarginine concentrations doubled in rabbits fed the cholesterol-enriched diet (Table 1). The addition of dietary L-arginine supplementation resulted in significantly elevated plasma L-arginine/ADMA ratios in weeks 8, 12, and 16 (Fig 2). ADMA plasma concentrations showed a negative linear correlation with urinary nitrate excretion rates (r=.339, P<.01).

View this table: [in this window] [in a new window]   Table 1. Plasma Concentrations of L-Arginine, ADMA, and Symmetrical Dimethylarginine

View larger version (25K): [in this window] [in a new window]   Figure 2. Plasma L-arginine/ADMA ratios in rabbits fed a cholesterol-enriched diet or normal rabbit chow. *P<.05 vs control; P<.05 vs cholesterol.

Plasma total cholesterol (baseline: 44.0±1.4 mg/dL) increased to 1901.5±179.4 mg/dL after 4 weeks of the 1% cholesterol-enriched diet (Table 2). During the 0.5% cholesterol diet, plasma total cholesterol concentrations slightly decreased to a plateau between 1400 and 1600 mg/dL. Lovastatin therapy reduced plasma total cholesterol by 32%, whereas dietary L-arginine had no significant effect on the plasma cholesterol level. Similar changes were also observed for plasma LDL cholesterol, whereas HDL cholesterol levels were not significantly different between the groups at any time point.

View this table: [in this window] [in a new window]   Table 2. Plasma Total Cholesterol Concentrations and Creatinine Clearances

Creatinine clearance was not significantly affected by cholesterol feeding or the pharmacological treatments in any of the groups at any time point compared with controls (Table 2).

Endothelial Function
After 4 weeks of 1% cholesterol feeding, endothelium-dependent relaxations to acetylcholine were slightly but significantly impaired compared with the control-4 group (Fig 3A), whereas endothelium-dependent relaxations induced by calcium ionophore A23187 were unchanged at this time point (Fig 3B). After an additional 12 weeks of 0.5% cholesterol feeding, endothelium-dependent relaxations to both acetylcholine and calcium ionophore A23187 were severely impaired (Fig 3C and 3D). Treatment with L-arginine partly but incompletely restored endothelium-dependent relaxations to acetylcholine and to calcium ionophore A23187, whereas treatment with lovastatin did not affect endothelial vasodilator function. Endothelium-independent relaxations in response to sodium nitroprusside were not significantly different between the groups at any time point. The maximal relaxations to acetylcholine, A23187, and sodium nitroprusside and the respective probability values are given in Table 3.

View larger version (29K): [in this window] [in a new window]   Figure 3. Endothelium-dependent relaxations of isolated aortic rings from rabbits fed a cholesterol-enriched diet or normal rabbit chow. Relaxations were induced by acetylcholine at weeks 4 (A) and 16 (C) and by the calcium ionophore A23187 at weeks 4 (B) and 16 (D). Significance levels for comparisons between the treatment groups are given in Table 3.

View this table: [in this window] [in a new window]   Table 3. Maximum Endothelium-Dependent and -Independent Relaxations of Isolated Aortic Rings

Vascular Superoxide Radical Production
For logistical reasons, we were unable to measure superoxide radical production in the control-4 and cholesterol-4 groups. Baseline chemiluminescence in unstimulated isolated aortic rings was significantly increased, by 67.4±1.5% in the cholesterol-16 group compared with the control-16 group (P<.01). Dietary L-arginine did not significantly affect baseline superoxide radical production, but treatment with lovastatin induced a significant further increase (P<.01 versus cholesterol-16; Fig 4A). Stimulation with PMA induced only a slight, insignificant increase in chemiluminescence response in aortic rings from control animals, whereas in aortic rings from cholesterol-fed animals, the addition of PMA resulted in a significantly increased superoxide release (P<.05; Fig 4B). L-Arginine treatment completely abolished this effect of PMA on superoxide anion release, whereas the PMA response was augmented in aortic rings from lovastatin-treated animals (P<.001 versus cholesterol-16). The differences between the groups in response to PMA were abolished by endothelial denudation (Fig 4B).

View larger version (16K): [in this window] [in a new window]   Figure 4. Chemiluminescence response of isolated rabbit aortic rings under baseline conditions (A) and after stimulation with the protein kinase C activator PMA (2 µmol/L) (B). Photon emission is expressed as counts per minute per milligram of dry weight for baseline values and as change from baseline values for PMA stimulation (, cpm/mg of dry weight). Data are mean±SE. *P<.05 vs control; P<.05 vs cholesterol.

Histological and Morphometric Analyses
In both control groups, no plaques were detectable in the common carotid arteries, with the exception of one rabbit in the control-16 group that showed a small lipid streak in one carotid artery (3.3% of the total intimal area). Intimal plaque area was increased to 15.8±4.6% after 4 weeks of 1% cholesterol feeding (cholesterol group-4), and additionally to 44.0±5.2% after 12 more weeks of 0.5% cholesterol feeding (cholesterol-16 group) (Fig 5A). Dietary L-arginine completely suppressed the aggravation of intimal plaque formation during the second part of the study period, but it did not induce regression of preexisting plaques. Lovastatin treatment significantly reduced the progression of plaque formation but did not completely block it (P<.05 versus cholesterol-16).

View larger version (12K): [in this window] [in a new window]   Figure 5. A, Carotid arterial intimal plaque area assessed by planimetry of digitized photographs of the intimal surfaces of carotid arteries opened longitudinally. B, Aortic intima/media ratio assessed by micromorphometry of semithin sections of the thoracic aortas stained with hematoxylin-eosin. Data are mean±SE of 4 sections from each rabbit. *P<.05 vs cholesterol-16; P<.05 vs cholesterol-4.

In control rabbits, no intimal thickening of thoracic aortic cross sections was observed at either 4 or 16 weeks. Cholesterol feeding significantly and progressively increased intima/media ratios to 0.3±0.2 (4 weeks) and 3.1±0.7 (16 weeks). Both L-arginine and lovastatin reduced the intimal thickening during the second part of this study (each P<.05 versus cholesterol-16); however, the inhibitory effect of L-arginine (intimal/medial ratio, 0.97±0.19) was significantly stronger than that of lovastatin (intimal/medial ratio, 1.40±0.13; P<.05 versus cholesterol plus L-arginine; Fig 5B).

	Discussion

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The salient findings of our study include the following: (1) dietary L-arginine restores endogenous NO production when treatment is started after the induction of atheromatous plaques; (2) this results in significantly improved endothelium-dependent vasodilator function and completely abolishes the progression of plaques; (3) competition between L-arginine and the accumulating endogenous inhibitor of NO synthesis, ADMA, for the NO synthase may cause reduced NO elaboration in hypercholesterolemia and its restoration by exogenous L-arginine; (4) in contrast, cholesterol-lowering therapy with lovastatin slows the progression of atherosclerosis but does not completely block it, and lovastatin treatment has no effect on systemic NO elaboration or endothelium-dependent vasodilator function; and (5) increased vascular superoxide radical production by lovastatin may explain why this drug does not improve endothelial function in this model.

Endothelial dysfunction is due to the decreased ability of the endothelium to release biologically active NO, resulting in elevated vasoconstrictor tone, enhanced platelet aggregation and leukocyte adhesion, and intimal thickening (for a review, see Reference 4⁴ ). Our present study confirms previous findings by Osborne et al¹³ and Galle et al²² that endothelial dysfunction can be detected in rabbits as early as 2 to 4 weeks after starting a high-cholesterol diet. In the present study, this early defect was confined to muscarinic receptor–mediated relaxations, whereas receptor-independent, NO-mediated relaxations induced by the calcium ionophore A23187 were still intact. This is in accordance with data reported by Bossaller et al²³ and Shimokawa et al.²⁴ A similar, early defect of endothelium-mediated vasodilation is found in young, asymptomatic, hypercholesterolemic humans.²⁵

After 16 weeks on the high-cholesterol diet, endothelium-dependent vasodilator function was almost completely abolished (irrespective of the stimulus applied), and intimal thickening significantly increased. Starting dietary supplementation with L-arginine at the same time as the high-cholesterol diet has been shown by us and others to inhibit intimal thickening as well as the deterioration of endothelial function.^{3 5} Our present data show that even in the presence of preexisting lesions, L-arginine significantly preserves endothelium-dependent vasodilator function and completely blocks the progression of plaques; however, we did not find an effect of L-arginine on the regression of preexisting lesions. Candipan et al⁶ recently reported that after 10 weeks of 0.5% cholesterol feeding, treatment with L-arginine for an additional 4, 8, or 13 weeks (during which the high-cholesterol diet was continued) resulted in a reduction of preexisting intimal lesions. After 13 weeks of treatment, this effect was present in half of the rabbits given L-arginine, whereas the other half showed progression. The differences in dietary cholesterol content and the duration of L-arginine treatment may explain the differences in outcome between our present study and the one by Candipan et al.⁶ Other studies^{26 27} have demonstrated that intimal lesions are reversible in cholesterol-fed rabbits after they return to a normal diet. However, pharmacological treatment (including calcium channel blockers,²⁸ ACE inhibitors,²⁹ and cholesterol-lowering drugs¹³ ) has usually resulted in a slowed progression of disease; regression was only reported in those studies in which the rabbits had simultaneously been returned to a normal diet.^{14 30} L-Arginine is the first substance for which a regression of preexisting lesions⁶ or a complete blockade of progression (as in the present study) was demonstrated despite the continued intake of a high-cholesterol diet.

One mechanism by which endothelial NO formation is impaired in hypercholesterolemia may depend on elevated levels of the endogenous NO synthase inhibitor ADMA, which occurs concurrently with decreased NO elaboration in hypercholesterolemic rabbits.⁸ Accumulation of dimethylarginines may be an early event in atherogenesis, since dimethylarginine levels were already elevated within the first 4 weeks on the high-cholesterol diet. In the same time interval, urinary nitrate excretion rates decreased to the low plateau on which they remained until the end of the study period in the cholesterol-fed group; plasma ADMA levels and urinary nitrate excretion rates were negatively correlated. Although the significance of urinary nitrate is limited in that it does not enable us to draw conclusions on the origin of the NO and may be influenced by dietary nitrate, and although urinary cGMP may also be influenced by the activity of the particulate guanylyl cyclase, we have previously shown that both parameters are useful markers for systemic NO production in vivo.^{3 8 18} ADMA may competitively inhibit NO synthase and/or L-arginine uptake into endothelial cells and thus at least partly explain the inhibitory effects of cholesterol on NO-related vascular functions and their reversal by L-arginine.^{31 32} Although there is no conclusive evidence from the present study that ADMA is causally related to the decreased NO formation, preliminary evidence from our laboratory suggests that ADMA inhibits endothelial NO formation in the concentration range occurring under pathophysiological conditions in vivo (R.H. Böger, MD, et al, unpublished observations, 1997). Moreover, two recent studies showed that ADMA at concentrations detected in plasma under pathophysiological conditions is capable of inhibiting NO synthesis in rat mesenteric venules³³ and in rat brain.³⁴ The mechanism leading to this elevation of dimethylarginine, which is normally excreted via the kidneys,⁷ has not been further investigated in the present study; however, impaired renal function does not seem to be involved, given the unchanged creatinine clearances of our rabbits. It is important to note that in contrast to a recent study by Jeremy and coworkers,³⁵ L-arginine plasma levels remained elevated throughout the entire study period in our study, suggesting that L-arginine may also be effective for long-term treatment in humans.

Another important mechanism contributing to the reduced biological activity of NO is enhanced superoxide radical production in the atherosclerotic vascular wall. Superoxide anions inactivate NO once it is released by endothelial cells and thereby determine its biological activity.³⁶ Superoxide radical formation was significantly reduced by L-arginine, which is in accordance with earlier findings by us and others.^{3 6} The dual action of L-arginine on the biological activity of NO (by enhancing its production due to competition with the endogenous NOS inhibitor, ADMA, and by reducing the rate of its oxidative inactivation by superoxide radicals) may explain the strong antiatherosclerotic effects of exogenous L-arginine. These two mechanisms, however, may be related. It has been shown that superoxide radicals are produced by NO synthase at the same time as NO generation is decreased in the presence of competitive NO synthase inhibitors³⁷ and native LDL.³⁸ Evidence for an endothelial origin of superoxide is also supplied by our present finding that endothelial denudation abolished the differences in PMA-stimulated O₂^– release between the groups. This finding is in accordance with previous observations by Ohara et al.^{39 40}

Because dietary correction of hypercholesterolemia has been reported to induce regression of intimal lesions and restore endothelial vasodilator function,^{26 41} we had expected that lowering plasma cholesterol levels with lovastatin might also produce a beneficial effect on the endothelium. However, although lovastatin reduced serum cholesterol levels by approximately one third and reduced the progression of intimal plaque formation, in our study it had no effect at all on endothelium-dependent relaxation of isolated aortic rings ex vivo nor on urinary nitrate excretion in vivo. Again, this finding is in contrast to studies in which this drug had been administered from the beginning of the cholesterol-feeding period.^{12 13} A reason for the weaker effect of lovastatin on lesion formation and its lack of preservation of NO-related function compared with L-arginine may be the significant increase in superoxide radical release from the aortic wall in this group. Superoxide radical generation in aortas from lovastatin-treated rabbits was increased under baseline conditions as well as after stimulation with the protein kinase C activator PMA. In this context, it is interesting that Latruffe et al⁴² found that compactin, a lovastatin analogue, stimulated protein kinase C activity in cultured smooth muscle cells. This finding suggests a possible mechanism by which lovastatin might have increased baseline O₂^– generation and potentiated the stimulatory effect of PMA in the present study of cholesterol-fed rabbits. It remains unclear whether a similar stimulatory effect on superoxide generation may be induced by lovastatin in humans. However, treatment with an HMG-CoA reductase inhibitor in humans with coronary artery disease has been shown to result in an overall improvement of endothelium-dependent coronary vasodilator function.^{15 16} Interestingly, combination therapy with lovastatin and the antioxidant probucol in humans has been shown to induce a greater improvement of endothelium-dependent coronary vasomotion than cholesterol-lowering therapy alone.¹⁷ Taken together, these data support the notion that vascular oxidative stress is a critical factor in the promotion of atherosclerosis and endothelial dysfunction. Clearly, the effects of HMG-CoA reductase inhibitors on these processes deserve further investigation.

In conclusion, our study shows that dietary L-arginine strongly inhibits the progression of atherosclerosis in cholesterol-fed rabbits. Mechanisms involved in this effect may include competitive displacement of ADMA, an endogenous NO synthase inhibitor, from NO synthase, resulting in increased NO formation (as assessed by urinary nitrate excretion), as well as decreased endothelial superoxide radical release, which both result in improved endothelium-dependent vasodilator function. Cholesterol-lowering therapy with lovastatin reduced the progression of vascular lesion formation to a lesser extent than L-arginine, and it had no beneficial effect on endothelial vasomotor function, probably due to its stimulatory effect on endothelial superoxide elaboration. These effects may be of relevance for the use of these substances in the treatment of atherosclerotic vascular disease.

	Selected Abbreviations and Acronyms

 

ADMA = asymmetrical dimethylarginine AUC = area under the curve Cholesterol-4 group = rabbits fed 1% cholesterol for 4 weeks Cholesterol-16 group = rabbits fed 1% cholesterol for 4 weeks, then 0.5% cholesterol for 12 weeks Control-4 group = rabbits fed normal rabbit chow for 4 weeks Control-16 group = rabbits fed normal rabbit chow for 16 weeks HMG-CoA = ß-hydroxy-ß-methylglutaryl coenzyme A L-Arginine group = rabbits fed 1% cholesterol for 4 weeks, then 0.5% cholesterol plus 2.0% L-arginine for 12 weeks Lovastatin group = rabbits fed 1% cholesterol for 4 weeks, then 0.5% cholesterol plus 10 mg/d lovastatin for 12 weeks NO = nitric oxide PMA = phorbol 12-myristate 13-acetate

	Acknowledgments

 
L. Phivthong-ngam is the recipient of a postgraduate grant from the Konrad Adenauer foundation. This study was supported in part by the Deutsche Forschungsgemeinschaft (grant TS 60/1-1). The excellent technical assistance of A. Otten, F.-M. Gutzki, and K.-M. Pütz is gratefully acknowledged.

	Footnotes

 
Reprint requests to Rainer H. Böger, MD, Institute of Clinical Pharmacology, Hannover Medical School, Konstanty-Gutschow-Str 8, 30625 Hannover, Germany.

Presented in part at the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 10-13, 1996, and previously published in abstract form (Circulation. 1996;94[suppl I]:I-522).

Received October 22, 1996; revision received February 13, 1997; accepted March 2, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Moncada S, Higgs EA. The L-arginine–nitric oxide pathway. N Engl J Med. 1993;329:2002-2012.[Free Full Text]

2. Cooke JP, Andon NA, Girerd XJ, Hirsch AT, Creager MA. Arginine restores cholinergic relaxation of hypercholesterolemic rabbit thoracic aorta. Circulation. 1991;83:1057-1062.[Abstract/Free Full Text]

3. Böger RH, Bode-Böger SM, Mügge A, Kienke S, Brandes R, Dwenger A, Frölich JC. Supplementation of hypercholesterolaemic rabbits with L-arginine reduces the vascular release of superoxide anions and restores NO production. Atherosclerosis. 1995;117:273-284.[Medline] [Order article via Infotrieve]

4. Böger RH, Bode-Böger SM, Frölich JC. The L-arginine–nitric oxide pathway: role in atherosclerosis and therapeutic implications. Atherosclerosis. 1996;127:1-11.[Medline] [Order article via Infotrieve]

5. Cooke JP, Singer AH, Tsao P, Zera P, Rowan RA, Billingham ME. Antiatherosclerotic effects of L-arginine in the hypercholesterolemic rabbit. J Clin Invest. 1992;90:1168-1172.

6. Candipan RC, Wang B, Buitrago P, Tsao PS, Cooke JP. Regression or progression: dependency on nitric oxide. Arterioscler Thromb Vasc Biol. 1996;16:44-50.[Abstract/Free Full Text]

7. Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet. 1992;339:572-575.[Medline] [Order article via Infotrieve]

8. Bode-Böger SM, Böger RH, Kienke S, Junker W, Frölich JC. Elevated L-arginine/dimethylarginine ratio contributes to enhanced systemic NO production by dietary L-arginine in hypercholesterolemic rabbits. Biochem Biophys Res Commun. 1996;219:598-603.[Medline] [Order article via Infotrieve]

9. Ragazzi E, Chinellato A, De Biasi M, Pandolfo L, Prosdocimi M, Norido F, Caparotta L, Fassina G. Endothelium-dependent relaxation, cholesterol content and high energy metabolite balance in Watanabe hyperlipidemic rabbit aorta. Atherosclerosis. 1989;80:125-134.[Medline] [Order article via Infotrieve]

10. Jacobs M, Plane F, Bruckdorfer KR. Native and oxidized low-density lipoproteins have different inhibitory effects on endothelium-derived relaxing factor in the rabbit aorta. Br J Pharmacol. 1990;100:21-26.[Medline] [Order article via Infotrieve]

11. Liao JK, Shin WS, Lee WY, Clark SL. Oxidized low-density lipoprotein decreases the expression of endothelial nitric oxide synthase. J Biol Chem. 1995;270:319-324.[Abstract/Free Full Text]

12. Zhu BQ, Sievers RE, Sun YP, Isenberg WM, Parmley WW. Effect of lovastatin on suppression and regression of atherosclerosis in lipid-fed rabbits. J Cardiovasc Pharmacol. 1992;19:246-255.[Medline] [Order article via Infotrieve]

13. Osborne JA, Lento PH, Siegfried MR, Stahl GL, Fusman B, Lefer AM. Cardiovascular effects of acute hypercholesterolemia in rabbits: reversal with lovastatin treatment. J Clin Invest. 1989;83:465-473.

14. Senaratne MPJ, Thomson ABR, Kappagoda CT. Lovastatin prevents the impairment of endothelium dependent relaxation and inhibits accumulation of cholesterol in the aorta in experimental atherosclerosis in rabbits. Cardiovasc Res. 1991;25:568-578.[Abstract/Free Full Text]

15. Treasure CB, Klein JL, Weintraub WS, Talley D, Stillabower ME, Kosinski AS, Zhang J, Boccuzzi SJ, Cedarholm JC, Alexander RW. Beneficial effects of cholesterol-lowering therapy on the coronary endothelium in patients with coronary artery disease. N Engl J Med. 1995;332:481-487.[Abstract/Free Full Text]

16. Egashira K, Hirooka Y, Kai H, Sugimachi M, Suzuki S, Inou T, Takeshita A. Reduction in serum cholesterol with pravastatin improves endothelium-dependent coronary vasomotion in patients with hypercholesterolemia. Circulation. 1994;89:2519-2524.[Abstract/Free Full Text]

17. Anderson TJ, Meredith IT, Yeung AC, Frei B, Selwyn AP, Ganz P. The effect of cholesterol-lowering and antioxidant therapy on endothelium-dependent coronary vasomotion. N Engl J Med. 1995;332:488-493.[Abstract/Free Full Text]

18. Böger RH, Bode-Böger SM, Gerecke U, Gutzki FM, Tsikas D, Frölich JC. Urinary NO₃^– excretion as an indicator of nitric oxide formation in vivo during oral administration of L-arginine or L-NAME in rats. Clin Exp Pharmacol Physiol. 1996;23:11-15.[Medline] [Order article via Infotrieve]

19. Tsikas D, Böger RH, Bode-Böger SM, Gutzki FM, Frölich JC. Quantification of nitrite and nitrate in human urine and plasma as pentafluorobenzyl derivatives by gas chromatography–mass spectrometry using their ¹⁵N-labelled analogs. J Chromatogr B. 1994;661:185-191.[Medline] [Order article via Infotrieve]

20. Brandes RP, Dwenger A, Mügge A. The basal and stimulated release of the endothelium-derived relaxing factor from isolated pig coronary arteries does not interfere with the vascular release of superoxide as measured by a lucigenin-enhanced chemiluminescence technique. Naunyn Schmiedebergs Arch Pharmacol. 1993;349:183-187.

21. Nafe R. Planimetry in pathology: a method in its own right besides stereology and automatic image analysis. Exp Pathol. 1991;43:239-246.[Medline] [Order article via Infotrieve]

22. Galle J, Busse R, Bassenge E. Hypercholesterolemia and atherosclerosis change vascular reactivity in rabbits by different mechanisms. Arterioscler Thromb. 1991;11:1712-1718.[Abstract/Free Full Text]

23. Bossaller C, Habib GB, Yamamoto H, Williams C, Wells S, Henry PD. Impaired muscarinic endothelium-dependent relaxation and cyclic guanosine 5'-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta. J Clin Invest. 1987;79:170-174.

24. Shimokawa H, Flavahan NA, Vanhoutte PM. Loss of endothelial pertussis toxin–sensitive G-protein function in atherosclerotic porcine coronary arteries. Circulation. 1991;83:652-660.[Abstract/Free Full Text]

25. Celermajer DS, Sorensen KE, Bull C, Robinson J, Deanfield JE. Endothelium-dependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors and their interaction. J Am Coll Cardiol. 1994;24:1468-1474.[Abstract]

26. Harrison DG, Armstrong ML, Freiman P, Heistad DD. Restoration of endothelium-dependent relaxation by dietary treatment of atherosclerosis. J Clin Invest. 1987;80:1808-1811.

27. Dowell FJ, Hamilton CA, Reid JL. Effects of manipulation of dietary cholesterol on the function of the thoracic aorta from New Zealand White rabbits. J Cardiovasc Pharmacol. 1996;27:235-239.[Medline] [Order article via Infotrieve]

28. Habib JB, Bossaller C, Wells S, Williams C, Morrisett JD, Henry PD. Preservation of endothelium-dependent vascular relaxation in cholesterol-fed rabbits by treatment with the calcium blocker PN 200110. Circ Res. 1986;58:305-309.[Abstract/Free Full Text]

29. Riezebos J, Vleeming W, Beems BB, van Amsterdam JG, Meijer GW, deWildt DJ, Porsius AJ, Wemer J. Comparison of the anti-atherogenic effect of isradipine and ramipril in cholesterol-fed rabbits, II: effect on regression of atherosclerosis and restoration of endothelial function. J Cardiovasc Pharmacol. 1994;23:424-431.[Medline] [Order article via Infotrieve]

30. Sievers RE, Rashid T, Garret J, Blumlein S, Parmley WW. Verapamil and diet halt progression of atherosclerosis in cholesterol-fed rabbits. Cardiovasc Drugs Ther. 1987;1:65-69.[Medline] [Order article via Infotrieve]

31. Mehta J, Bryant JL Jr, Mehta P. Reduction of nitric oxide synthase activity in human neutrophils by oxidized low-density lipoproteins: reversal of the effect of oxidized low-density lipoproteins by high-density lipoproteins and L-arginine. Biochem Pharmacol. 1995;50:1181-1185.[Medline] [Order article via Infotrieve]

32. Chen LY, Mehta P, Mehta JL. Oxidized LDL decreases L-arginine uptake and nitric oxide synthase protein expression in human platelets: relevance of the effect of oxidized LDL on platelet function. Circulation. 1996;93:1740-1746.[Abstract/Free Full Text]

33. Kurose I, Wolf R, Grisham MB, Granger DN. Effects of an endogenous inhibitor of nitric oxide synthesis on postcapillary venules. Am J Physiol. 1995;268:H2224-H2231.[Abstract/Free Full Text]

34. Faraci FM, Brian JE, Heistad DD. Response of cerebral blood vessels to an endogenous inhibitor of nitric oxide synthase. Am J Physiol. 1995;269:H1522-H1527.[Abstract/Free Full Text]

35. Jeremy RW, McCarron H, Sullivan D. Effects of dietary L-arginine on atherosclerosis and endothelium-dependent vasodilatation in the hypercholesterolemic rabbit: response according to treatment duration, anatomic site, and sex. Circulation. 1996;94:498-506.[Abstract/Free Full Text]

36. Gryglewski RJ, Palmer RMJ, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature. 1986;320:454-456.[Medline] [Order article via Infotrieve]

37. Niu XF, Smith CW, Kubes P. Intracellular oxidative stress induced by nitric oxide synthesis inhibition increases endothelial cell adhesion to neutrophils. Circ Res. 1994;74:1133-1140.[Abstract/Free Full Text]

38. Pritchard KA, Groszek L, Smalley DM, Sessa WC, Wu M, Villalon P, Wolin MS, Stemerman MB. Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. Circ Res. 1995;77:510-518.[Abstract/Free Full Text]

39. Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest. 1993;91:2546-2551.

40. Ohara Y, Peterson TE, Sayegh HS, Subramanian RR, Wilcox JN, Harrison DG. Dietary correction of hypercholesterolemia in the rabbit normalizes endothelial superoxide anion production. Circulation. 1995;92:898-903.[Abstract/Free Full Text]

41. Armstrong ML, Warner ED, Connor WE. Regression of coronary atheromatosis in rhesus monkeys. Circ Res. 1970;27:59-67.[Abstract/Free Full Text]

42. Latruffe N, Boscoboinik D, Azzi A. Stimulation of protein kinase C activity by compactin in vascular smooth muscle cells. Biochem Biophys Res Commun. 1995;217:459-465.[Medline] [Order article via Infotrieve]

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