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Cardiovascular Research 1997 35(2):351-359; doi:10.1016/S0008-6363(97)00122-3
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

The effects of L-arginine on neointimal formation and vascular function following balloon injury in heritable hyperlipidaemic rabbits

Carol Greenleesa, Roger M Wadswortha, Piero A Martoranab and Cherry L Wainwrighta,*

aDepartment of Physiology and Pharmacology, University of Strathclyde, 204 George Street, Glasgow G1 1XW, Scotland, UK
bCardiovascular Agents, Hoechst Marion Roussel, Frankfurt, Germany

* Corresponding author. Tel.: +44 (141) 548 2405; fax: +44 (141) 552 2562; e-mail: c.l.wainwright@strath.ac.uk

Received 21 November 1996; accepted 22 April 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The aims of this study were to determine the morphological and functional consequences of balloon angioplasty of the left subclavian artery of Froxfield heritable hyperlipidaemic (FHHL) rabbits and the influence of oral L-arginine therapy on these changes. Methods: Sixteen-week-old FHHL rabbits were subjected to balloon injury of the left subclavian artery under halothane anaesthesia. Control rabbits (n = 7) were given free access to food and normal tap water. L-Arginine-treated rabbits were given L-arginine (5 g·l–1) in the drinking water for 2 days prior to angioplasty and then for either 2 weeks (n = 7) or 4 weeks (n = 7) after surgery. All rabbits were euthanised 28–30 days after surgery and blood and tissue removed for quantification of neointimal size and determination of endothelial function using isolated vessel tension studies. The ability of the endothelium to prevent platelet aggregation was determined by challenging a vessel ring with carbachol when incorporated into a whole blood sample in which platelet aggregation was induced with collagen. Results: Balloon injury in non-treated rabbits resulted in the development of marked intimal hyperplasia (18.8[3.6]% of the area within the internal elastic lamina) while endothelial function remained intact. Maximum responses to carbachol and calcimycin were, respectively, a 66.6[14.7]% and 46.9[12.9]% relaxation of 5HT-induced tone, compared to 58.0[3.2]% and 39.8[9.4]% in non-injured vessels. Maximum contractile responses to 5HT and KCl were unaffected by injury. L-Arginine therapy alone had no effect on the vasodilator function of the endothelium, but reduced the endothelium-dependent inhibition of platelet aggregation (68.4[7.8] vs 109[10]% of the maximum extent of platelet aggregation in non-treated and 2-week L-arginine-treated non-injured vessels, respectively). L-Arginine significantly reduced the extent of neointimal formation (7.2[3.9]% of the area within the IEL; P<0.05 vs. non-treated group). However, L-arginine significantly attenuated the relaxant responses to both carbachol (26.5[10.4]% and 31.4[9.4]% for 2- and 4-week L-arginine groups) and calcimycin (38.7[15.4]% and 16.4[10.7]%) in the injured artery (P<0.05 compared to non-treated controls). Conclusions: L-Arginine reduces neointimal formation following balloon catheter injury in heritable hypercholesterolaemic rabbits, which is consistent with previous findings in normocholesterolaemic models. However, in the presence of hypercholesterolaemia, L-arginine has a detrimental effect on endothelial function following injury. This may be a consequence of the presence of lipids in the vascular wall on nitric oxide synthase activity.

KEYWORDS Vascular injury; Rabbit, genetically hyperlipidemic; L-Arginine; Neointimal formation; Balloon injury; Hyperplasia; Endothelial function


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Angioplasty is the process of controlled injury aimed at increasing the internal diameter of atherosclerotic coronary arteries in patients suffering from coronary disease. Angioplasty-induced vascular intimal injury is characterised by the prolonged reduction of endothelial cell nitric oxide (NO) release [1]and the concurrent development of an intimal hyperplasia and smooth muscle cell proliferation and mitogenesis which characterises restenosis. As a species the rabbit has been widely used to study restenosis and it has been demonstrated that the development of the intimal hyperplasia maximises 15 days after the initial angioplasty-induced injury [2].

The endothelium plays a crucial role in modulating the responses of vascular smooth muscle cells by the release of modulators such as nitric oxide [3, 4]. In vitro studies have illustrated that, in addition to inhibiting vascular contraction [5]and platelet aggregation and adhesion to collagen fibrils and endothelial cells [6], NO also inhibits smooth muscle cell proliferation and mitogenesis by inhibiting DNA synthesis (G1/S blockage) in smooth muscle cells through elevation of cellular cGMP content [7, 8]. Thus impaired NO production in newly regenerating endothelial cells following balloon angioplasty [1, 9, 10]may contribute to the development of restenosis, whereas stimulation of the endogenous NO pathway may improve endothelial cell function and simultaneously reduce the intimal hyperplastic response to the injury induced.

Administration of L-arginine, the precursor of nitric oxide, has been shown to suppress the formation of vascular neointima and improve endothelial function in a normotensive rat model of balloon injury [11]and normocholesterolaemic rabbit models of restenosis [12–14]. However, since most patients undergoing an angioplasty procedure have some degree of primary lesion in the target vessel, it may not be appropriate to extrapolate results obtained from normocholesterolaemic animals whose vessels have no previous lesion formation.

Cholesterol-fed rabbits are frequently used to emulate clinical atheroma, but these frequently encounter criticism due to the fact that the ‘lesions’ seen in these animals are often rich in foam cells [15], unlike the lesions seen in patients, although the morphology of the lesions will depend very much on the dietary conditions of the experiments (see, for example, [16]). The dysfunction of the endothelium associated with hypercholesterolaemia appears to differ between dietary- and genetically-induced hypercholesterolaemia. With respect to both the vasodilator capacity and antiaggregatory properties of the endothelium, the dysfunction in genetically hypercholesterolaemic rabbits bears a greater similarity to those seen in patients with advanced atherosclerotic lesions [17]. Thus, any drug effects on endothelial function following vascular injury in normocholesterolaemic, or dietary-hypercholesterolaemic, animals may not necessarily reflect what happens in patients. A more appropriate model for the assessment of drugs on neointimal formation and vascular function following balloon injury, therefore, may be animals with genetically-induced hypercholesterolaemia, such as the Watanabe heritable hyperlipidaemic rabbit (WHHL; [18]), which show the typical features of atherosclerotic fibrous plaques with and without calcifications [19, 20]but without marked foam cell infiltration. The Froxfield heritable hyperlipidaemic (FHHL) rabbit is a cross-breed of the WHHL rabbit with a coloured half-lop rabbit which retains the genetic abnormality of a lack of LDL receptors while avoiding some of the problems commonly encountered with the WHHL, namely difficulty in breeding and susceptibility to sudden death. As with the WHHL rabbit, the spontaneous lesions that develop in the FHHL rabbit resemble closely those seen in patients. The WHHL rabbit has been assessed as a potential model for angioplasty [21]and the morphological changes induced in the vasculature following balloon angioplasty of the left subclavian artery are almost identical to those seen in humans following coronary balloon angioplasty. To date, however, the studies with heritable hyperlipidaemic rabbits have considered only the morphology of the restenotic lesion while the functional consequences of balloon injury have not yet been investigated.

The aims of this study, therefore, were to determine, firstly, how the development of restenosis following balloon injury modifies endothelial function in FHHL rabbits and, secondly, to determine whether supplementation of dietary L-arginine can reduce the development of restenosis following angioplasty-induced vascular injury in this model. Since restenosis is also accompanied by both vascular smooth muscle and endothelial cell dysfunction, the study included an assessment of the influence of L-arginine therapy on these parameters using isolated vessel and platelet aggregometry techniques.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Balloon angioplasty
All surgical procedures were performed using aseptic technique. Twenty-one male FHHL rabbits (16 weeks of age) were anaesthetised with Hypnorm® (fentanyl citrate and fluanisone, 0.3 ml·kg–1) and maintained with an oxygen (1%)/nitrous oxide (2%)/halothane (1.5%) mixture in room air. Just prior to anaesthesia an injection of heparin sulphate (1000 U) was given intravenously to prevent early vessel occlusion. The left femoral artery was exposed and ligated distally and an angioplasty catheter with a 3.0 mm balloon and 0.014'' steerable guidewire was introduced and advanced to the upper region of the thoracic aorta using a fluoroscopic monitor (Siemens Memoskop and Siremobil 2). The guidewire was advanced into the left subclavian artery followed by the angioplasty balloon which was manoeuvred to the centre of the vessel. The angioplasty balloon was inflated twice, to 12 atm, for 30 s and then for a third time to 10 atm, during which inflation the angioplasty balloon was withdrawn the length of the subclavian artery to ensure endothelial denudation. Inflation of the angioplasty balloon was confirmed using the contrast medium, Niopam 370 (iopamidol; diluted in a 50:50 ratio with saline). The catheter was then removed from the animal, the femoral artery was ligated, the wound closed and the rabbits allowed to recover from the anaesthetic.

2.2 Experimental protocols
All animals were fed a standard rabbit diet (BS and S, Scotland) and given water ad libitum. Prior to commencement of the study the rabbits were allocated to one of 3 treatment groups (n = 7 per group). Test animals were administered L-arginine orally in the drinking water (5 g·l–1) for 2 days prior to surgery and for either 2 or 4 weeks following surgery. Control animals (n = 7) were given normal drinking water. Throughout the period of L-arginine treatment, water intake was measured daily to estimate the consumption of the L-arginine; the animals consumed an average of 350–450 ml of water per day, reflecting an intake of 1.75–2.25 g L-arginine. All animals were euthanised 28–30 days following the angioplasty procedure by an overdose of sodium pentobarbitone. Blood and tissue were removed for functional analysis and morphological studies. Blood (9.5 ml) was withdrawn from the pulmonary artery, via a thoracotomy, into 10 ml syringes containing 0.5 ml of heparin (200 U). The left (injured) and right (non-injured) subclavian arteries were then dissected free of fat and connective tissue and ring preparations of 3–4 mm in length were cut. All surgical procedures were carried out under a Project Licence in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986.

2.3 Morphological studies
2.3.1 Tissue preparation
All vessel segments were coded to make morphological processing blind. Immediately following dissection, vessel segments were fixed in neutral-buffered formalin (2.0 g NaH2PO4·2H2O, 3.2 g Na2HPO4 in 450 ml distilled water and 50 ml 40% formaldehyde, pH 7.4). The vessels were then processed in an enclosed tissue processor through graded alcohols and chloroform to paraffin wax. Serial sections (5 µm) were cut using a rotary microtome and mounted from water at 50°C onto glass slides and dried overnight at 37°C. Sections were stained using a Masson Goldner Trichrome stain.

2.3.2 Masson Goldner trichrome stain
This stain is a combination of the Masson trichrome stain and the Goldner stain which was manipulated to differentiate elastin fibres from collagen and smooth muscle cells, where nuclei stain blue-black, cytoplasm, muscle and erythrocytes stain red and collagen stains blue/green. Briefly, tissues were stained using Weigert haematoxylin, differentiated in 1% alcohol, stained with Masson Acid Fuchsin, Light green and subsequently Orange G. The segments were dehydrated in 95% and 100% alcohol, cleared in xylene and mounted in DPX. Photomicrographs of the vessel segments which had been stained were taken for quantification of the vessel wall using computerised planimetry (Tracer 1.11). Each photomicrograph was placed on an electronic bit-pad and outlines of the luminal, neo-intimal and medial layers were traced. The media was determined as the area bounded by the internal and external elastic laminae and the neo-intima as the area bounded by the internal elastic lamina and lumen. The areas traced are expressed as a percentage of the total area bounded by the external elastic lamina.

2.4 Studies with isolated subclavian artery rings
Endothelium-containing vessel rings were suspended in an organ chamber on two intraluminal parallel wires, one of which was fixed, and the other attached to an isometric transducer. The rings were placed under an optimum resting force of 3 g [2]. The organ chamber was filled with Krebs-Henseleit solution (37°C) of composition (in mM); NaCl, 118.3; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2; K2HPO4, 1.2 and glucose 11.1 gassed with 95%O2:5%CO2. The vessel rings were equilibrated at their optimum resting force for 30 min and then sensitised by contracting with 40 mM KCl until consistent responses were obtained.

Cumulative concentration–response curves to the endothelium-dependent vasodilators, carbachol (0.01–10 µM) and calcimycin (0.01–0.1 µM) were constructed in vessels pre-contracted with 5HT (0.1 µM). Endothelium-independent relaxations to sin-1 (3-(4-morpholinyl)-sydnonimine-hydrochloride, 0.01–10 µM) were assessed in the vessel rings with intact endothelium and pre-contracted with 5HT (0.1 µM). In addition to studying the responses to vasodilator agonists the ability of the vessels to contract to KCl (10–100 mM) and 5HT (0.01–10 µM) were assessed. These responses were measured as an increase in force (g).

2.5 Platelet aggregometry studies in whole blood
Platelet aggregation in rabbit whole blood was measured by electronic impedance aggregometry (Chrono-log Corporation, Havertown, PA) according to the method by Cardinal and Flower [22]. Aliquots of whole blood (0.5 ml) were transferred to a cuvette containing 0.9% NaCl (0.5 ml) and stirred continuously with a magnetic stir bar. The blood was warmed at 37°C for 10 min and then transferred to the measuring chamber of the aggregometer where platelet aggregation in response to collagen (5 µg/ml) producing ~60–70% of the maximal aggregation response was monitored. The extent of platelet aggregation was measured as the change in electrical impedance ({Omega}) 4 min after the addition of collagen. To eliminate any contribution from prostanoids to the responses obtained, indomethacin (0.1 µM) was present in all studies.

To assess the anti-aggregatory capacity of the vascular endothelium, a single subclavian artery ring (3–4 mm) was suspended by means of an ‘S’-shaped wire in the cuvette containing the heparinised blood. Nitric oxide release from the vessel was triggered by the addition of carbachol (10 µM) 30 s prior to the initiation of aggregation by the addition of collagen. The effects of sin-1 (10 µM) on collagen-induced platelet aggregation were also studied in the presence of a vessel ring since, in this whole blood model of platelet aggregation, sin-1 does not elicit a response in the absence of a vessel ring [15]. This procedure was repeated with vessel rings pre-incubated for 15 min with L-NO Arg (NG-nitro-L-arginine; 100 µM). This was to demonstrate that the anti-aggregatory capacity of the vascular endothelium when stimulated by carbachol is due to the production of nitric oxide. The effect of sin-1 in the presence of an L-NO Arg treated vessel was also assessed. Subclavian artery rings utilised in these aggregometry studies were pre-incubated for 10 min with indomethacin (0.1 µM).

2.6 Sudan red staining
The thoracic aorta was removed from animals selected at random and opened longitudinally and pinned out with the vascular lumen uppermost. The vessels were stained with Sudan red to give an indication of the lesion cover in these heritable hyperlipidaemic rabbits [23]. The aorta was rinsed with 70% ethanol and then immersed in Sudan red solution for 15 min. The vessel was then rinsed and immersed in 80% ethanol for 20 min and left in running tap water for 1 h.

2.7 Cholesterol measurements
Plasma cholesterol levels were measured by the breeder immediately prior to despatch. The rabbits were put on to the appropriate experimental protocol after 1 week of acclimatisation. The average plasma cholesterol levels for the 3 groups were 13.8, 16.8 and 13.0 mmol·l–1 for the control, 2-week and 4-week L-arginine groups, respectively.

2.8 Materials
Heparin (mucous, 1000 units/ml) was purchased from Leo-Laboratories (Buckinghamshire, England, UK). Carbachol, calcimycin, 5HT and collagen were all purchased from Sigma (Poole, Dorset, England, UK). Sin-1 was a gift from Dr Martorana (Hoechst Marion Roussel, Frankfurt, Germany). All drugs were dissolved in distilled water. Calcimycin was dissolved in 10% ethanol solution and stored at –20°C.

2.9 Statistics
Results are shown as the mean (s.e.m.) where n refers to the number of rabbits. The group sizes were based on previous studies which have demonstrated that a group size of 6 is sufficient to detect significant differences between groups. When analysing vasodilator responses in vessels precontracted with 5HT, the responses are given as a percentage of the contractile tone where contraction to 5HT is taken as 100%. The use of a time-control vessel ring from each rabbit allowed each point on the concentration–response curve to be corrected for the loss of 5HT-induced tone that occurs as the experiment progresses using the following equation:


Formula

where TCL= corrected test value (%); T0= peak contraction of test vessel to 5HT (g); T1= uncorrected test value (g); C0 = peak contraction of control vessel to 5HT (g) and C1 = control value corresponding to T1 (g).

For isolated vessel studies the results are shown as either the maximum relaxation or contraction observed. When assessing the effect of balloon angioplasty in the L-arginine treated and non-treated rabbits a Student's unpaired t-test was employed to determine if there were any significant differences between the injured and non-injured arteries. The effects of L-arginine on the responses of either the injured or non-injured group of arteries were assessed using one-way ANOVA followed by the Tukey comparison test.

For platelet aggregometry studies responses were analysed as a percentage of collagen-induced platelet aggregation in the presence of either the injured or non-injured unstimulated vessels. A Student's one-sample t-test was employed with the Bonferroni correction factor (where applicable) to determine significance of drug intervention on platelet aggregation in the presence of the artery ring.

For the morphological studies the data were analysed using one-way ANOVA with a Tukey multiple comparisons test to compare results between the control and various treatment groups. Student's paired t-test was used to compare responses in each left artery with its contralateral control. For all studies, differences were considered significant when P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Morphological studies
Quantification of neointimal size 28–30 days following angioplasty by planimetric analysis demonstrated that vessels from all 3 groups of rabbits had neointimal lesions (Fig. 1). There were no neointimal lesions observed in the contralateral arteries in any of the treatment groups. In the 4-week L-arginine-treated group there was a significant reduction in the size of the neointima (Fig. 2) compared to the control group (no drug therapy). There was also a decrease in the size of the neointimal lesions in the rabbits treated with L-arginine for 2 weeks, although this was not statistically significant compared to the control rabbits. Medial area in the right and left arteries for each group of rabbits was similar; however, the luminal area was significantly reduced in the left artery from the control group of rabbits when compared to the right contralateral control artery. In neither of the drug-treated groups was there any significant difference in the luminal area between the right and left arteries (Fig. 2).


Figure 1
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  		Fig. 1 Examples of micrographs of sections of left subclavian artery obtained from FFHL rabbits subjected to balloon injury 28–30 days previously and given either no drug treatment (top panel) or L-arginine therapy for 2 days prior to angioplasty followed by 2 weeks (middle panel) or 4 weeks (bottom panel) oral L-arginine. N = neointima; A = adventitia; M = media; IEL = internal elastic lamina. Magnification x10.

 

Figure 2
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  		Fig. 2 The effect of L-arginine therapy on neointimal growth following balloon angioplasty of the FHHL rabbit subclavian artery. The areas of lumen (open columns), media (stippled columns) and neointima (solid columns) are expressed as a percentage of the area bounded by the external elastic lamina (EEL). *P<0.05 vs. control group.

 
Sudan red staining of the thoracic aorta from rabbits representing each of the experimental groups showed marked fatty streak development in all animals.

3.2 Isolated vessel studies
For the ex vivo studies, one rabbit from each group failed to yield sufficient tissue for all the experiments. Therefore the n number for the isolated vessel tension and the platelet aggregation studies is 6 per group. In the control group of rabbits, 28–30 days after angioplasty, the response of the injured artery to carbachol was not significantly different from that in the non-injured artery. However, following administration of L-arginine for both 2 and 4 weeks there was a significant reduction in the relaxant response of the injured vessel to carbachol when compared to the non-injured control (Table 1). This was not dependent upon the duration of the L-arginine therapy since the responses of the injured vessels in these groups were not significantly different. In contrast, L-arginine administration for either 2 or 4 weeks did not significantly affect the response of the non-injured arteries when compared with the corresponding vessel from control rabbits (Table 1).


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  		Table 1 The effect of balloon injury and l-arginine on relaxant responses in the FHHL rabbit subclavian artery

 
A similar picture emerged when the effect of angioplasty and L-arginine therapy on the relaxant responses to calcimycin were assessed. In the control group of rabbits angioplasty did not affect the responses of the injured artery to calcimycin when compared to the non-injured artery. With the administration of L-arginine for either 2 or 4 weeks there was a significant reduction in the relaxant response of the injured vessel to calcimycin when compared to the non-injured control vessel (Table 1). L-Arginine administration for 2 weeks, but not 4 weeks, significantly enhanced the response of the non-injured vessel when compared to the control artery from non-treated rabbits.

In the control untreated rabbits the angioplasty procedure sensitised the response of the injured artery to the vasodilator action of sin-1 compared to the non-injured artery. However, with the administration of L-arginine for either 2 or 4 weeks this sensitisation was not apparent (Table 1).

In the control group of rabbits, there was no significant difference in the contractile responses of the injured and non-injured rabbit subclavian artery to 5HT. The administration of L-arginine for either 2 or 4 weeks did not affect the contractile responses to 5HT of either the non-injured or injured arteries (Table 2). A similar picture was observed with the responses to KCl, where neither angioplasty nor L-arginine therapy affected the contractile responses of the non-injured and injured arteries (Table 2).


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  		Table 2 The effects of balloon injury and L-arginine therapy on contractile responses of the FHHL rabbit subclavian artery

 
3.3 Platelet aggregometry studies
Inclusion of an unstimulated, non-injured rabbit subclavian artery ring into the blood samples did not affect collagen-induced platelet aggregation. When carbachol or sin-1 was added to the cuvette, there was a significant reduction in collagen-induced platelet aggregation with both these drugs. However, with vessel rings from rabbits given L-arginine for either 2 or 4 weeks this anti-aggregatory effect of either drug was no longer apparent (Fig. 3).


Figure 3
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  		Fig. 3 The effect of balloon injury and L-arginine therapy on the ability of the endothelium to inhibit platelet aggregation in whole blood in the absence (upper panel) and presence (lower panel) of L-NO Arg. In the presence of the unstimulated vessel (open bars) aggregation was taken as 100%. Aggregation in the presence of a vessel stimulated with carbachol (solid bars) or sin-1 (stippled bars) was calculated as a percentage of that seen with the unstimulated vessel. *P<0.05 vs. unstimulated vessel.

 
Injured, unstimulated, subclavian artery rings did not affect collagen-induced platelet aggregation. When the vessels from untreated rabbits were stimulated, there was a significant reduction in the extent of aggregation in the presence of sin-1 but not carbachol (Fig. 3). Administration of L-arginine for either 2 or 4 weeks resulted in a loss of this anti-aggregatory effect of sin-1. Furthermore, the lack of anti-aggregatory effect of carbachol was not affected by this treatment (Fig. 3).

Following co-incubation of the vessels with L-NO Arg, the anti-aggregatory capacity of the vascular endothelium was lost in both the injured and non-injured subclavian artery rings from all 3 groups when compared to the unstimulated vessel (Fig. 3). L-Arginine administration itself did not affect the extent of collagen-induced aggregation.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The first aim of this study was to assess the development of neointima and alterations in the function of the vascular endothelium following balloon injury in Froxfield heritable hyperlipidaemic (FHHL) rabbits. Subclavian artery angioplasty was found to induce neointimal lesions after 28–30 days which were similar in size (18[1]% of the area within the external elastic lamina) to those which occur in cholesterol-fed NZW rabbits (12[3]% [2]). At this time after angioplasty, in both models, considerable restoration of vascular function occurs, including contraction (to KCl and 5HT) and endothelium-dependent vascular relaxation. However, in contrast to the NZW model of restenosis, angioplasty in the FHHL rabbits increased the vasodilator effect of sin-1. This suggests an alteration in the response of the smooth muscle of the injured FHHL artery to NO.

The second aim of this study was to assess the effects of the NO precursor, L-arginine, on the morphology and function of vessels subjected to injury in FHHL rabbits. Administration of L-arginine for 4 weeks after the angioplasty procedure significantly reduced neointimal size measured after 28–30 days. There was also a tendency towards a reduced neointima after 2 weeks of treatment. These results are in agreement with studies in normocholesterolaemic rabbits [12–14]and in rats subjected to carotid artery balloon injury [11]. The beneficial effect of L-arginine administration is probably due to restoration of NO production by the endothelium, resulting in inhibition of smooth muscle cell proliferation [12, 25].

In contrast to normocholesterolaemic rabbits, in which L-arginine has a beneficial effect on endothelial function following balloon injury [13, 14], the present study in genetically hyperlipidaemic rabbits found a marked attenuation of vasodilator and antiaggregatory activity of the endothelium in angioplastied arteries. A possible explanation is that in the genetic hyperlipidaemic state there may be a combination of NOS induction and NO-induced enzyme inhibition. Induction of NOS has been reported in non-endothelial cells (possibly smooth muscle cells and infiltrating macrophages) following balloon angioplasty [26–29]. If NOS was induced in the present experiments following injury, then L-arginine administration would generate large amounts of NO. NO is known to be able to negatively regulate NOS [30, 31]. Moreover, NO donors can inhibit the endothelium-dependent relaxations of arterial rings, apparently by inhibition of NOS activity [32, 33]. In addition, L-arginine itself may control NOS haem iron activity by slowing ligand interactions and increasing the reduction potential of iron [34]. Thus, in the genetically hyperlipidaemic state, L-arginine would increase NO formation in smooth muscle, but also decrease the release of NO from the endothelium in response to agonists. Consistent with this interpretation, it has been shown that in vessel rings which are refractory to the relaxant capacity of acetylcholine, the tissue levels of L-arginine are 3-fold higher than those observed in vessels that respond normally to these agents [35]. Alternative explanations are that in the injured vessels L-arginine is converted to endogenous inhibitors of NOS [36], or that, in addition to reducing neointimal formation, NO may be proatherogenic by oxidising LDL to oxidised LDL [37], resulting in endothelial dysfunction. One further possibility is that, since the vessel tension studies were not performed in the presence of indomethacin, increased generation of a constrictor prostanoid cannot be ruled out as the cause of the endothelial dysfunction.

In the present studies, L-arginine therapy alone had no marked effect on endothelium-dependent or endothelium-independent relaxation in non-injured vessels. Thus L-arginine per se does not appear to affect the ability of either the endothelium to generate NO or the smooth muscle cells to respond to it. By contrast, in the dietary-induced hypercholesterolaemic rabbit, L-arginine feeding restored cholinergic relaxation of the aorta [38]and a single in vitro dose of L-arginine normalized endothelial function in cerebral vessels [39]. Thus dietary manipulation may reduce endothelial arginine availability or metabolism, a feature not seen in the FHHL. However, there are at present no data available in the literature to either support or refute this possibility.

L-Arginine therapy alone caused a reduction in the capacity of the endothelium to inhibit collagen-induced platelet aggregation. This is unlikely to be caused by decreased NO release, since endothelium-dependent vascular relaxations were not impaired by L-arginine. It is possible that L-arginine attenuated the responsiveness of platelets to NO, since this has been demonstrated to occur in dietary hypercholesterolaemic rabbits [40]. The present experiments did not reveal any direct effect of L-arginine on platelet aggregation in response to collagen, in contrast to the inhibition by L-arginine of platelet aggregation reported in healthy young men [41]. This may be related to an effect of oxidised LDL, which has been shown to decrease L-arginine uptake and NOS expression in human platelets [42].

In conclusion, the present study demonstrates for the first time that L-arginine therapy in Froxfield heritable hyperlipidaemic rabbits reduces the extent of neointimal formation but also impairs vasorelaxant and platelet antiaggregatory functions following balloon injury. This is in contrast to the beneficial effects of L-arginine therapy on endothelial function in normocholesterolaemic rabbits. Given that clinical angioplasty procedures are carried out on severely diseased arteries, and that an improvement in both vessel patency and vascular function are desirable, the results of this study do not support the use of L-arginine as putative therapy for restenosis.

Time for primary review 21 days.


   Acknowledgements
 
C. Greenlees was supported by an A.J. Clark Studentship from the British Pharmacological Society. We are grateful to Professor B. Schölkens (Hoechst AG, Germany) for his support of this project.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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