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Cardiovascular Research 1998 37(3):756-764; doi:10.1016/S0008-6363(97)00295-2
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Chronic dietary supplementation with L-arginine inhibits platelet aggregation and thromboxane A2 synthesis in hypercholesterolaemic rabbits in vivo

Stefanie M Bode-Böger*, Rainer H Böger, Sven Kienke, Michael Böhme, Laddaval Phivthong-ngam, Dimitrios Tsikas and Jürgen C Frölich

Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

* Corresponding author. Tel.: +49 (511) 5324631; fax: +49 (511) 5325199.

Received 18 June 1997; accepted 7 October 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 References
 
Objectives: L-arginine exerts anti-atherosclerotic effects in hypercholesterolaemic rabbits via modulating endogenous NO production. We investigated whether L-arginine inhibits thromboxane formation in vivo and platelet aggregation ex vivo in this animal model. Methods: The urinary excretion rates of 2,3-dinor-6-keto-PGF1{alpha} (major urinary metabolite of PGI2) and 2,3-dinor-TXB2 (major urinary metabolite of thromboxane A2) were used as indicators of platelet–endothelial cell interactions in vivo. Rabbits were fed 1% cholesterol (Cholesterol group, N=8), 1% cholesterol plus 2,25% L-arginine (Cholesterol+L-arginine, N=8), or normal rabbit chow (Control, N=4) for 12 weeks. Urine samples were collected in weekly intervals. At the end of the study period platelet aggregation ex vivo and endothelium-dependent and -independent vascular function of isolated aortic rings in vitro was assessed. Results: Urinary 2,3-dinor-TXB2 excretion significantly increased in the cholesterol group (p<0.05), and endogenous NO formation (measured as urinary nitrate excretion) decreased (p<0.05). Both parameters were significantly correlated with each other (R=0.48, p<0.01). L-arginine partly restored urinary nitrate excretion and significantly reduced TXA2 production to values even below those in the control group (p<0.001). Urinary 2,3-dinor-6-keto-PGF1{alpha} excretion increased in early hypercholesterolaemia and returned to control values in the second half of the study period. The early increase in urinary 2,3-dinor-6-keto-PGF1{alpha} excretion was attenuated by L-arginine. Platelet aggregation was significantly enhanced in cholesterol-fed rabbits and attenuated by dietary L-arginine. L-arginine also improved the impaired endothelium-dependent relaxations to ADP, and normalized the vasoconstrictor effects of 5-HT in isolated aortic rings. Conclusions: Cholesterol-feeding enhances platelet aggregation and TXA2 formation, and stimulates platelet–endothelial cell interaction in rabbits. These effects are probably due to impaired NO elaboration, as indicated by decreased urinary nitrate excretion. Chronic dietary supplementation with L-arginine elevates systemic NO elaboration and significantly increases the PGI2/TXA2 ratio. It thus beneficially influences the homeostasis between vasodilator and vasoconstrictor prostanoids in vivo.

KEYWORDS Nitric oxide; Prostacyclin; Endothelium; Platelet aggregation; Gas chromatography–mass spectrometry


   1 Introduction
Top
 Abstract
 1 Introduction
2 Material and methods
 3 Results
 4 Discussion
 References
 
Hypercholesterolaemia and atherosclerosis are characterized by impaired endothelium-dependent vascular relaxation [1]due to a reduced ability of the endothelium to elaborate biologically active nitric oxide (NO) [2]and, possibly, prostacyclin [3]. Besides their vasodilator effects, both NO and prostacyclin have been shown to inhibit platelet adhesion and aggregation [4, 5]. This effect may be of importance for the atherosclerotic process, as activation of platelets at the sites of injured endothelium contributes to the progression of atherosclerosis in vivo [6]. Indeed, there is increased platelet aggregability in animal models of hypercholesterolaemia and in human subjects with familial hypercholesterolaemia [7, 8]. Activated platelets also modulate the tone and the structure of the vascular wall by secreting a variety of vasoconstrictor and pro-proliferative mediators (e.g., thromboxane [TX] A2, platelet-derived growth factor [PDGF], and serotonin [5-hydroxytryptamine, 5-HT]) [9, 10]. Moreover, endothelium-dependent vasodilator responses to platelet mediators which induce vasodilatation in healthy blood vessels, like adenosine diphosphate (ADP), are decreased or converted into vasoconstriction in atherosclerotic arteries [11].

Several studies have investigated the production of thromboxane A2 and/or prostacyclin in atherosclerosis [3, 12]. However, the time course of changes in the formation rates of vasoactive prostanoids during the induction of atherosclerosis is unknown. It may be of importance, as prostacyclin is synthesized in part from endoperoxide precursors which are released from platelets and taken up by endothelial cells [5, 13]. By this mechanism, increased platelet activation may also influence prostacyclin formation rates [14, 15]. The balance between prostacyclin and thromboxane has therefore been used as an index of platelet–endothelial cell interactions in vivo [16].

The method of choice to reliably assess whole body formation rates of prostanoids is the quantitation of their main enzymatically formed urinary metabolites, i.e., 2,3-dinor-6-keto-PGF1{alpha} and 2,3-dinor-TXB2, by gas chromatography–tandem mass spectrometry (GC/MS/MS) [17, 18], as these metabolites have been shown to be mainly derived from the endothelium and the platelets, respectively [17, 19], and may be repeatedly and non-invasively analyzed. Similarly, endogenous NO synthesis rates can be assessed by quantifying the urinary excretion rate of nitrate, the oxidative metabolite of NO [20–22].

Chronic dietary administration of L-arginine, the precursor of endogenous NO, has been shown to improve endothelial function and to slow the progression of atherosclerosis in cholesterol-fed rabbits [20, 23], probably by restoring the biological activity of endothelial NO.

The present study was undertaken to investigate whether dietary L-arginine affects thromboxane A2 and/or prostacyclin synthesis in cholesterol-fed rabbits in vivo, as assessed using the GC/MS/MS technique to quantify the major urinary metabolites of TXA2 (2,3-dinor-TXB2) and prostacyclin (2,3-dinor-6-keto-PGF1{alpha}). The relation of these changes to changes in systemic NO formation was assessed by quantifying urinary nitrate excretion. Repetitive urine sampling allowed us for the first time to gain further insight into the time course of these changes during the induction of hypercholesterolaemia. Moreover, we studied whether attenuation of increased platelet aggregability was involved in the effects of L-arginine, and whether the vascular responsiveness to serotonin and ADP, potent platelet-derived vasoactive mediators, was modified by hypercholesterolaemia and by dietary L-arginine.


   2 Material and methods
Top
 Abstract
 1 Introduction
 2 Material and methods
3 Results
 4 Discussion
 References
 
2.1 Study design
20 male New Zealand white rabbits were used for this study, 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) and had been approved by the local supervisory committee for studies in animals (Hannover). Two groups of 8 rabbits were fed rabbit chow enriched with 1% cholesterol (Altromin, Lage, Germany) and had free access to plain tap water (Cholesterol group) or water supplemented with 2.25% L-arginine (Sigma, Munich, Germany; Cholesterol+L-arginine group) for 12 weeks. This cholesterol feeding protocol has been shown previously to produce well-defined lesions throughout the thoracic and abdominal aorta with a marked thickening of the intima [24]. Another group of 4 rabbits was given normal rabbit chow and plain tap water (Control group). Before the beginning of the experimental period and during the feeding period, the rabbits were placed in metabolic cages at weekly intervals to collect 24-h urines. At baseline and the end of the dietary intervention, blood samples were drawn by puncture of the central ear artery into vacutainers containing 1.2 mg EDTA/ml whole blood for the measurement of plasma total cholesterol and L-arginine levels, and into vacutainers containing 3.13% sodium citrate (1:10, v/v) as anticoagulant for platelet aggregation ex vivo. Blood samples were obtained at the end of the urine sampling periods to avoid interference with urinary prostanoid excretion rates. The rabbits were sacrificed, and the aortas were excised for isometric tension recording.

2.2 Platelet aggregation
Platelet rich plasma (PRP) was generated from citrated blood by centrifugation at 200 g for 10 min. Platelet poor plasma (PPP) was prepared from the remaining volume of blood by centrifugation at 1600 g for 10 min. Platelet aggregation was monitored at 37°C using a Born dual-channel aggregometer (Labor, Hamburg, Germany) as described previously [25]. The aggregometer was adjusted before each test so that in each subject the value for light transmission for PRP was 0% and that for PPP was 100%. Aggregation was induced in duplicate using final concentrations of 2–20 µM adenosine diphosphate (ADP), and was monitored for 3 min. As we had previously observed that L-arginine decreased both the maximal extent and the maximal gradient of the aggregation curve, aggregations were evaluated as area under the aggregation curves [25].

2.3 Determination of urinary prostanoid and nitrate excretion rates
Urinary samples were kept at –20°C until analysis of their prostanoid content. Quantification of urinary 2,3-dinor-TXB2 and 2,3-dinor-6-keto-PGF1{alpha} was performed by negative chemical ionization gas chromatography–tandem mass spectrometry (GC–MS/MS) on a triple stage quadrupole mass spectrometer TSQ 45 (Finnigan MAT, San Jose, CA, USA) as described elsewhere [19]. Briefly, endogenous prostanoids and their corresponding tetradeuterated internal standards, which had been externally added to 50 ml aliquots of urine samples, were extracted from acidified urine samples (pH 3.0) by solid-phase extraction on octadecyl silica cartridges (J.T. Baker, Deventer, The Netherlands). After derivatization to their pentafluorobenzyl ester methoxyamine derivatives and separation by reversed-phase HPLC the analytes were converted to their trimethylsilyl ether derivatives. GC–MS/MS was performed by selected reaction monitoring of the characteristic daughter ions generated by collision-activated dissociation of the corresponding parent ions for endogenous prostanoids and their stable-isotope labelled analogues.

Urinary nitrate excretion was determined by GC–MS using the pentafluorobenzyl-(PFB-) derivative of nitrate as described previously [22, 26], using [15N]-NO3– (MSD Isotopes Merck Frosst, Montreal, Canada) as internal standard. 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 nitrate. Intra-assay variability was below 3.8%.

Urinary creatinine was determined spectrophotometrically by the alkaline picric acid reaction with an automatic analyzer (Beckman, Galway, Ireland). The excretion rates of prostanoid metabolites and of nitrate were corrected by urinary creatinine concentration, to exclude changes due to variability in renal excretory function, as described previously [18, 19, 21].

2.4 Determination of plasma arginine and cholesterol
Plasma L-arginine concentrations were determined by HPLC using pre-column derivatization with o-phthalaldehyde (OPA) as described previously [27]. Prior to analysis, lipids were extracted from plasma samples with trifluoroethane (Merck, Darmstadt, Germany). The aqueous phase of the extracted plasma samples, and internal standards were extracted on CBA solid phase extraction cartridges (Varian, Harbor City, CA, USA), and incubated for exactly 30 s with the OPA reagent (5.4 mg/ml OPA in borate buffer, pH 8.5, containing 0.4% 2-mercaptoethanol) before automatic injection into the HPLC. Chromatographic separation was performed on a C6H5 column (Macherey and Nagel, Düren, Germany) with the fluorescence monitor set at {lambda}ex=340 nm and {lambda}em=455 nm. Samples were eluted from the column with 0.96% citric acid/methanol 2:1, pH 6.8, at a flow rate of 1 ml/min. The intra- and inter-assay variability of the method was 5.2% and 5.5%, respectively; the detection limit 0.1 µmol/l.

Plasma total cholesterol was determined by a commercial immunofluorescence assay system (TDX; Abbott Diagnostics, Wiesbaden, Germany).

2.5 Organ bath studies
The aortas were dissected free of adhering fat and connective tissue and placed into organ baths filled with oxygenated (95% O2, 5% CO2) modified Krebs solution (37°C, pH 7.4) of the following composition (in mM): Na+ 145.0, K+ 5.95, Ca2+ 1.7, Mg2+ 1.2, Cl– 128.15, HCO3– 25.0, H2PO4– 1.2, SO42– 1.2, glucose 10.6, EDTA 0.025, within 1 h of death. The vascular preparations were connected to force transducers for isometric tension recording. For 60 min the rings were gradually stretched to a resting tension of 2 g (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 contracted with noradrenaline (1 µM) and relaxed by acetylcholine (1 µM) for testing of endothelial integrity as described previously [20]. Rings from control animals always showed relaxations greater than 70% of the noradrenaline-induced contraction plateau. After wash-out, cumulative concentration response curves were obtained with the endothelium-dependent relaxant ADP and the endothelium-independent relaxant sodium nitroprusside after precontraction with 1 µM noradrenaline, and the vasoconstrictors noradrenaline and serotonin (all drugs 1 nM to 0.1 mM). Relaxations were expressed as per cent of the precontractile tension induced with 1 µM noradrenaline. Noradrenaline, sodium nitroprusside, acetylcholine, and serotonin were purchased from Sigma (Munich, Germany). ADP was purchased from Boehringer (Mannheim, Germany).

2.6 Aortic atherosclerotic plaque formation
Segments of the thoracic aorta immediately distal from the left subclavian artery were fixed in formalin, embedded in paraffin, and stained with haematoxylin/eosin for the morphpometric measurement of intimal and medial cross-sectional areas by planimetry using a semiautomatic system (Zeiss). Four sections of each animal were analyzed, and the values were averaged.

2.7 Calculations and statistics
All values are given as mean±S.E.M. Statistical significance was tested using analysis of variance for repeated measures followed by the Scheffé f-test. For statistical comparison of the time course of urinary prostanoid excretion and of urinary nitrate excretion, the area under the curve (AUC) was calculated for each group, and AUC values were compared using Student's unpaired t-test. EC50 values (concentrations of drugs inducing half-maximal responses) for the organ bath experiments were calculated according to the method of Hafner et al. [28], and compared using ANOVA followed by Fisher's protected least-significant-difference test. Statistical significance was accepted at the 0.05 level of probability.


   3 Results
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
4 Discussion
 References
 
3.1 Urinary prostanoid excretion
Basal urinary 2,3-dinor-TXB2 excretion was 4214.8±275.5 pg/mg creatinine with no significant differences between the three groups. In the control group, 2,3-dinor-TXB2 excretion decreased over time, reaching 1521.6±323.6 pg/mg creatinine in the 12th week. Cholesterol feeding significantly increased urinary 2,3-dinor-TXB2 excretion by 40–60% as compared to the control group during the whole study period (p<0.05 vs. control; Fig. 1a). Supplementation with dietary L-arginine significantly decreased 2,3-dinor-TXB2 excretion to 15–30% below control levels for most of the experimental period (AUC: p<0.05 vs. control and p<0.01 vs. cholesterol).


Figure 1
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  		Fig. 1 Urinary 2,3-dinor-TXB2 excretion (a), 2,3-dinor-6-keto-PGF1{alpha} excretion (b), and ratio of urinary 2,3-dinor-6-keto-PGF1{alpha}/2,3-dinor-TXB2 (c) in 24-h urines from rabbits during 12 weeks of dietary intervention. Values are means±S.E.M. For statistical comparison between the groups, areas under the curves were calculated from the 1st through 12th weeks and compared using ANOVA followed by Fisher's protected least-significant-difference test. *P<0.05 vs. cholesterol.

 
Urinary 2,3-dinor-6-keto-PGF1{alpha} excretion, which was 4830.0±205.9 pg/mg creatinine with no significant differences between the groups at baseline, also decreased in the control group during the study period (to 845.4±178.7 pg/mg creatinine in week 12). In cholesterol-fed rabbits, 2,3-dinor-6-keto-PGF1{alpha} excretion initially increased by 60±15% (p<0.05; Fig. 1b), but starting in week 3, excretion rates returned to levels not significantly different from the control group. Dietary L-arginine inhibited the early increase in 2,3-dinor-6-keto-PGF1{alpha} excretion (p<0.05 vs. cholesterol). 2,3-dinor-6-keto-PGF1{alpha} values in this group were not significantly different from those in the control group.

The ratio of 2,3-dinor-6-keto-PGF1{alpha}/2,3-dinor-TXB2 was 1.2±0.1 at baseline and remained largely unchanged in the control group throughout the experimental period (Fig. 1c). It was not significantly changed in the cholesterol-fed group. In rabbits given cholesterol+L-arginine, the ratio was highly elevated until the 5th week and then returned to control levels (AUC: p<0.05 vs. control and p<0.01 vs. cholesterol).

3.2 Urinary nitrate excretion
Baseline urinary nitrate excretion was 955.5±49.2 µmol/mmol creatinine. After 12 weeks, urinary nitrate excretion was 1053.8±54.0 in the control group (p=n.s. vs. baseline). Urinary nitrate excretion decreased in the cholesterol-fed group during the first 6 weeks of dietary intervention, to arrive at a plateau about 50% below baseline and controls during the second half of the study period (AUC: p<0.05 vs. control; Fig. 2). Dietary L-arginine reduced the extent of this decrease in urinary nitrate excretion by about 50%, but did not completely prevent it (AUC: p<0.05 vs. control, p<0.05 vs. cholesterol). In multiple correlation analyses, the reduction in urinary nitrate excretion rates was correlated with the increase in urinary 2,3-dinor-TXB2 excretion in the cholesterol group (R=0.48, p<0.01). L-arginine supplementation abolished this linear relationship (R=0.08; p=n.s.). There was no significant correlation between nitrate excretion, 2,3-dinor-6-keto-PGF1{alpha} excretion, platelet aggregation and total cholesterol concentrations.


Figure 2
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  		Fig. 2 Urinary nitrate excretion in 24-h urines from rabbits during 12 weeks of dietary intervention. Values are means±S.E.M. For statistical comparison between the groups, areas under the curves were calculated from the 1st through 12th weeks and compared using ANOVA followed by Fisher's protected least-significant-difference test. *P<0.05 vs. cholesterol.

 
3.3 Platelet aggregation
Platelet aggregation in response to ADP was significantly increased in hypercholesterolaemic rabbits as compared to controls over the whole concentration range of ADP (p<0.05; Fig. 3). Dietary L-arginine inhibited both the maximal extent and the maximal slope of the aggregation curves. The area under the aggregation curves was decreased to a level slightly below, but not statistically significantly different from the control group (p<0.05).


Figure 3
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  		Fig. 3 Platelet aggregation induced by ADP (10 µM) in platelet-rich plasma ex vivo from rabbits fed a normal diet (Control), a diet enriched with 1% cholesterol (Cholesterol), or 1% cholesterol+2.25% L-arginine in drinking water (Cholesterol+L-arginine). *P<0.05 vs. control. {dagger}P<0.05 vs. cholesterol.

 
3.4 Vascular reactivity
Endothelium-dependent relaxations in response to ADP were significantly attenuated in the cholesterol-fed rabbits as compared to controls (Fig. 4a). Dietary L-arginine partly, but not completely restored relaxations to ADP (p<0.05 vs. cholesterol). In contrast, no significant differences were observed in endothelium-independent relaxations in response to sodium nitroprusside between all three groups (Fig. 4b).


Figure 4
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  		Fig. 4 Endothelium-dependent relaxations induced by ADP (a), and endothelium-independent relaxations induced by sodium nitroprusside (b) of isolated aortic rings ex vivo from rabbits fed normal rabbit chow (control), or a diet enriched with 1% cholesterol (cholesterol), or a cholesterol-enriched diet plus L-arginine (L-arginine) in drinking water for 12 weeks. Values represent means±S.E.M. of 4 rings from 8 rabbits per group (4 rabbits in the control group).

 
Contractions in response to serotonin were significantly enhanced in the cholesterol-fed group as compared to controls (Fig. 5a). L-arginine resulted in a reduction of the contractile effect of serotonin, which was not significantly different between the L-arginine-treated and the control group. In contrast, noradrenaline contractions were not significantly different between the groups (Fig. 5b). The EC50 values for the endothelium-dependent and -independent vasoconstrictors and vasodilators are given in Table 1.


Figure 5
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  		Fig. 5 Endothelium-dependent contractions induced by serotonin (a), and endothelium-independent contractions induced by noradrenaline (b) of isolated aortic rings ex vivo from rabbits fed normal rabbit chow (control), or a diet enriched with 1% cholesterol (cholesterol), or a cholesterol-enriched diet plus L-arginine (L-arginine) in drinking water for 12 weeks. Values represent means±S.E.M. of 4 rings from 8 rabbits per group (4 rabbits in the control group).

 

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  		Table 1 EC50 values for endothelium-dependent and -independent vasoactive mediators tested in vitro

 
3.5 Aortic plaque formation
In aortic cross-sections from control rabbits, only the endothelial monolayer was visible with no intimal plaques (intima/media ratio, 0). In cholesterol-fed rabbits significant intimal thickening was observed. Intima/media ratio in this group was calculated to be 1.9±0.3. Chronic dietary supplementation with L-arginine significantly reduced intimal atherosclerotic plaque formation (intima/media ratio, 0.7±0.2; p<0.05 vs. cholesterol).

3.6 Plasma cholesterol and L-arginine levels
Plasma total cholesterol was 0.75±0.04 mmol/l at baseline and was not significantly changed in the control group during the study period. It increased to 20.87±0.86 mmol/l in the rabbits fed a cholesterol-enriched diet. Dietary L-arginine had no effect on plasma cholesterol concentrations (Table 2).


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  		Table 2 Plasma L-arginine and cholesterol concentrations

 
Plasma L-arginine concentration was 87.2±8.8 µmol/l at baseline and remained unchanged in the control group. Cholesterol feeding alone did not affect plasma L-arginine concentrations, but dietary L-arginine resulted in an about threefold increase in plasma L-arginine concentrations (Table 2).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
References
 
Our present study suggests that hypercholesterolaemia increases thromboxane A2 synthesis in vivo in cholesterol-fed rabbits. This is paralleled by decreased systemic NO formation, as assessed by measuring the urinary excretion rate of nitrate, the oxidative metabolite of NO, and by platelet hyperactivity. In contrast, prostacyclin synthesis is increased in early hypercholesterolaemia and reduced in later phases of atherogenesis. Dietary L-arginine partly prevents both the decrease in NO synthesis and the elevated thromboxane formation in vivo; it inhibits platelet aggregation ex vivo in response to ADP. Furthermore, endothelium-dependent vascular responses to ADP and serotonin, two potent platelet-derived modulators of vascular tone, which are abnormal in hypercholesterolaemia, are restored by L-arginine.

Platelet activity has previously been shown to be enhanced in hypercholesterolaemic rabbits [29, 30]and in human hypercholesterolaemia and atherosclerosis [7, 8]. Different mechanisms may contribute to this increased platelet activity in hypercholesterolaemia: Changes in lipid content of platelet membranes may facilitate their activation by physiological stimuli [31]. Moreover, superoxide anions released by polymorphonuclear leukocytes have been shown to activate platelets [32]. Superoxide radical production is enhanced in the hypercholesterolaemic vascular wall [20, 24]and may, among other effects, contribute to increased platelet activity. The main underlying cause of platelet activation and endothelial dysfunction in this animal model of atherosclerosis is decreased NO elaboration in the vascular endothelium [20, 23, 30]. Our present results show that the urinary excretion of nitrate is progressively decreased in hypercholesterolaemic rabbits, and NO-dependent relaxations to ADP are impaired, pointing to a progressive deterioration of endothelial NO elaboration during the course of cholesterol feeding. These data are in line with similar observations made by ourselves and others with different endothelium-dependent mediators of vasorelaxation [1, 2, 20]. Furthermore, the finding that dietary supplementation with L-arginine, which restores NO elaboration by the endothelium, inhibits platelet aggregation and thromboxane formation, also indicates that defective activity of NO is the underlying cause for the close interplay between endothelial cell and platelet function in hypercholesterolaemic rabbits. Kaul et al. [11]also demonstrated that atherosclerosis markedly impaired vasodilator responses of perfused rabbit carotid arteries in response to activated human platelets in vitro, probably because vasodilation to ADP released from platelets was impaired due to the NO-deficient, atherosclerotic endothelium. Factors like endothelial platelet adhesion and interactions of platelets with endothelial cells and other cell types may therefore contribute to modulating platelet activity in vivo [33].

Our present data corroborate the finding by Tsao et al. [30]that dietary supplementation with L-arginine reduced aortic intimal plaque formation and platelet activity despite continuously elevated cholesterol levels. Dietary L-arginine was therefore able to profoundly modify factors which are usually accepted as being implicated in atherosclerosis [10]. It may be speculated that differences in L-arginine content in various diets may contribute to the beneficial cardiovascular effects of vegetable/fish diets [34].

Treatment with the antiplatelet agent ticlopidine has also been shown to reduce platelet aggregation in cholesterol-fed rabbits [35], but its antiplatelet effect was attenuated in hypercholesterolaemia as compared to normocholesterolemic control rabbits. Moreover, ticlopidine even increased platelet thromboxane formation in response to ADP. By contrast, L-arginine treatment inhibited both platelet hyperactivity and thromboxane metabolite excretion in vivo in the present study. Therefore, the effects of L-arginine on platelet reactivity and thromboxane formation may be superior to established antiplatelet agents like ticlopidine in hypercholesterolaemia.

The balance between prostacyclin and thromboxane A2 is suggested to be one of the major regulatory mechanisms for platelet activation in vivo [16]. However, radioimmunoassay determinations of the primary metabolites of prostacyclin and TXA2 formed by spontaneous hydrolysis, i.e. 6-keto-PGF1{alpha} and TXB2, have yielded controversial results. Using these methods, it has been shown that slices of aortas from cholesterol-fed rabbits produced significantly less prostacyclin than controls [3]. Biopsy specimens of atherosclerotic human arteries have a reduced capacity to generate prostacyclin in vitro [36]. By contrast, Norman and Miller [12]found no correlation between plasma 6-keto-PGF1{alpha} or TXB2 levels and the progression of coronary atherosclerosis in hypercholesterolaemic swine. Mehta et al. [37]demonstrated that the conversion of [14C]-arachidonic acid to 6-keto-[14C]-PGF1{alpha} and [14C]-TXB2 was increased in aortic segments from hypercholesterolaemic rabbits as compared to non-atherosclerotic segments.

The method of choice to reliably assess prostanoid production rates in vivo is the quantification of their specific, enzymatically formed urinary metabolites, i.e., 2,3-dinor-6-keto-PGF1{alpha} for prostacyclin and 2,3-dinor-TXB2 for TXA2 [17]. This approach avoids sampling artifacts which may occur by vascular injury during the blood sampling procedure when plasma metabolites are measured, and represents a reliable method to assess the synthesis rates of these prostanoids in vivo [38]. Using this non-invasive approach, we have demonstrated for the first time in the present study that the time course of the activation of TXA2 and prostacyclin formation in hypercholesterolemic rabbits differs: TXA2 formation remained elevated throughout the experimental period, whereas prostacyclin production initially increased, but rapidly returned to control levels during the progression of atherosclerosis. Moreover, we showed that dietary supplementation with L-arginine not only inhibited platelet aggregation ex vivo and 2,3-dinor-TXB2 excretion in vivo, by also prevented the early increase in 2,3-dinor-6-keto-PGF1{alpha} excretion.

These phenomena may be explained by the observation that prostacyclin is synthesized in part from platelet-derived endoperoxide precursors, which are shifted from activated platelets to the endothelium [13–15]. Differential activation of this endoperoxide shift in different stages of atherogenesis — and its inhibition by dietary L-arginine — may explain the time-dependent changes in prostanoid metabolite excretion in the present study. In early hypercholesterolaemia, platelet activation at the hypercholesterolaemic endothelium may occur secondarily to impaired NO activity. This results in enhanced release of TXA2 and endoperoxide precursors from platelets, as shown by elevated urinary 2,3-dinor-TXB2 excretion. Endoperoxides may then be taken up by endothelial cells, where they can be utilized as a substrate for prostacyclin synthesis. This results in a transiently increased prostacyclin synthesis, as demonstrated by elevated urinary 2,3-dinor-6-keto-PGF1{alpha} excretion. Later on in the atherosclerotic process, aggravation of endothelial dysfunction may result in decreased prostacyclin biosynthetic capacity, resulting in normalized prostacyclin formation rates in spite of continuously elevated TXA2 formation. Inhibition of platelet activity and TXA2 formation by dietary L-arginine also attenuated PGI2 synthesis in the first half of the study via reduced endoperoxide shift. These differences in the time course of the formation of these two prostanoids may explain in part the differential changes of vascular reactivity observed by others in early hypercholesterolaemia and advanced atherosclerosis [39], and the controversial results found in in vitro studies [3, 12, 36, 37].

Other than platelet sources of TXA2 may contribute to its increased synthesis: It is well known that about 10–20% of 2,3-dinor-TXB2 excreted into the urine is derived from non-platelet sources [17]. Cultured endothelial cells have been shown to release a substance with thromboxane-like immunoreactivity after incubation with LDL cholesterol in vitro [40]. Moreover, other vasoconstrictor mediators released by platelets and other cellular sources may also contribute to enhanced vasoconstrictor tone in hypercholesterolaemia. The effect of hypercholesterolaemia on serotonergic vasoconstriction is of particular interest, since 5-HT together with TXA2 is an important mediator of coronary vasoconstriction in response to platelet aggregation [9, 41], and rabbit platelets contain more 5-HT than human platelets [42]. Hypercholesterolaemia enhances serotonergic vasoconstriction in conductance arteries [43, 44]and resistance vessels [45]. Enhanced vasoconstrictor responses to 5-HT in hypercholesterolaemic porcine coronary arteries have been linked to a reduced basal or 5-HT-induced release of NO from the endothelium [44, 46]. Therefore, the enhanced vasoconstriction in response to 5-HT and its normalization by dietary L-arginine in our present study is indicative of decreased NO formation in the hypercholesterolaemic endothelium and its restoration by chronic dietary L-arginine.

In conclusion, our present study shows that the balance of prostacyclin and TXA2 is disturbed in hypercholesterolaemic rabbits in vivo. In early hypercholesterolaemia, prostacyclin formation is stimulated and may counteract platelet activation, but later on in the atherogenetic process, TXA2 production outweighs prostacyclin formation. This is paralleled by enhanced vasoconstriction in response to serotonin and impaired vasodilation to ADP. Dietary supplementation with L-arginine prevents these changes in prostanoid balance, improves endothelium-mediated vascular responses ex vivo, and enhances urinary nitrate excretion in vivo. Platelet-derived mediators may significantly contribute to the progression of atherosclerosis in hypercholesterolaemic rabbits, and the inhibitory effect of L-arginine on platelet activity may at least partly explain its anti-atherosclerotic effects in this model.

Time for primary review 27 Days.


   Acknowledgements
 
The excellent technical assistance of A. Otten, T. Suchy, and F.-M. Gutzki is gratefully acknowledged. L-Phivthong-ngam is the recipient of a postgraduate exchange grant from the Konrad-Adenauer Foundation.


   References
Top
 Abstract
 1 Introduction
 2 Material and methods
 3 Results
 4 Discussion
 References
 

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