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(Circulation. 1996;94:83-87.)
© 1996 American Heart Association, Inc.

Articles

Aspirin-Stimulated Nitric Oxide Production by Neutrophils After Acute Myocardial Ischemia in Rabbits

Antonio Lopez-Farre, PhD; Amparo Riesco, PhD; Enzo Digiuni, MD; Juan R. Mosquera, MD; Carlos Caramelo, MD; Lourdes S. de Miguel, PhD; Inmaculada Millas, MS; Trinidad de Frutos, PhD; Maria R. Cernadas, MD; Mercedes Monton, PhD; Javier Alonso, PhD; Santos Casado, MD

the Laboratorio de Nefrologia-Hipertension, Fundacion Jimenez Diaz, Madrid, Spain.

Correspondence to Antonio Lopez-Farre, PhD, Laboratorio de Nefrologia-Hipertension, Fundacion Jimenez Diaz, Avda Reyes Catolicos, 2, 28040 Madrid, Spain.

	Abstract

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Background In recent studies, it has been hypothesized that the protective anti-ischemic effects of aspirin outweigh the effects of inhibition of platelet thromboxane A₂ synthesis. Recently, we have found that the antiaggregating effects of aspirin significantly affect nitric oxide (NO) generation by neutrophils.

Methods and Results The present study used circulating neutrophils from myocardial ischemic rabbits to assess the effect of aspirin on the circulating neutrophil–derived NO production and, subsequently, on the modulation of platelet activation. Neutrophils were obtained after 60 minutes of coronary artery occlusion followed by 60 minutes of reperfusion. Sham-operated animals were used as controls. The results demonstrated that aspirin stimulated the production of NO by neutrophils obtained from both sham-operated rabbits and rabbits with myocardial ischemia. However, neutrophils isolated from animals with myocardial ischemia showed an enhanced ability to generate NO in the presence of aspirin. As a functional in vitro marker, we observed that neutrophils had a NO-dependent, platelet-antiactivating effect in the presence of aspirin. In the absence of aspirin, ischemic neutrophils did not modify platelet activation, even though they produced increased amounts of NO. An inhibitory role of superoxide anion on the neutrophil-related antiplatelet effect was suggested because superoxide dismutase induced significant platelet inhibition by myocardial ischemic neutrophils in the absence of aspirin.

Conclusions Our results show that myocardial ischemia/reperfusion stimulates production of NO by circulating neutrophils, an effect that was enhanced in the presence of aspirin. These results suggest a novel interpretation of the protective effect of aspirin on myocardial ischemia damage.

Key Words: aspirin • ischemia • thrombosis • leukocytes

	Introduction

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Thrombosis is an integral part of the development of acute myocardial ischemia. In vivo platelet activation and occlusive coronary thrombi have been demonstrated both in humans and in animals with acute myocardial ischemia.^{1 2 3} However, it has been shown^{4 5} that neutrophils, in addition to platelets, are the main constituents of intracoronary thrombus in acute myocardial ischemia.

Inhibition of platelet activation has been the focus of several pharmacological strategies in the prevention and treatment of myocardial ischemia. Aspirin remains a keystone of these preventive and damage-limiting strategies. During the last few years, different reports have demonstrated the protective role of aspirin in the control of the complications of myocardial ischemia^{6 7} and in the prevention of coronary bypass closure.⁸ However, inhibition of cyclooxygenase activity by aspirin does not fully explain the antithrombotic effects of this drug.^{9 10 11} In this regard, we recently obtained new evidence that demonstrates that the antiaggregating effects of aspirin are not dependent solely on the described selective inhibitory effect of platelets on thromboxane A₂ production. We found that aspirin inhibits platelet activation through an increase in NO production by peripheral neutrophils.¹² Therefore, although clinical and experimental models of myocardial ischemia described the detrimental role of neutrophils in the progression of this pathology,^{13 14 15} neutrophils would potentially provide defense properties against thrombosis in the presence of aspirin. The aim of the present study was to evaluate the effect of acute myocardial ischemia on the ability of circulating rabbit neutrophils to produce NO and to achieve platelet antiaggregation and to examine the effect of aspirin in this setting.

	Methods

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Chemicals
Ficoll-Hypaque medium was obtained from Flow Laboratories. Acetylsalicylic acid, L-arginine, and L-NAME were purchased from Sigma Chemical Co. L-[³H]-arginine was purchased from Amersham. Thrombin was purchased from Ortho Diagnostic Systems. All other chemicals were of the highest quality commercially available from Sigma.

Operative Procedures
Healthy male New Zealand White rabbits (weight, 3 to 3.5 kg) were anesthetized with pentobarbital (30 mg/kg IM). Rabbits were intubated with an intratracheal tube and ventilated mechanically with 95% oxygen (rate, 45 breaths per minute; tidal volume, 20 mL) (Harvard rodent ventilator model 683, Harvard Apparatus). A polyethylene catheter was inserted into the right external jugular vein for supplementary addition of anesthesia as needed.

Under sterile conditions, a left thoracotomy was performed, the pericardium was opened, and a 3-0 silk ligature was placed around the left anterior descending coronary artery 8 to 10 mm from its origin. This ligature was left untied until the beginning of the experimental protocol. A polyethylene catheter was inserted into the left femoral artery to obtain peripheral blood samples. ECG was used to follow up ischemic changes.

Sham-operated animals (on which all operative procedures were performed except tightening of the coronary artery ligature) served as controls.

The experimental procedure was approved by the institutional Animal Care and Use Committee.

Experimental Protocol
After all surgical procedures were completed, the rabbits were allowed to stabilize for 30 minutes, at which time a baseline ECG reading was taken. We induced myocardial ischemia by tightening the ligature around the left anterior descending coronary artery to completely occlude the vessel. After 60 minutes of ischemia, the ligature was untied and the ischemic myocardium was reperfused for 60 minutes. Aspirin (20 mg/kg) was given through a nasogastric polyethylene tube to a group of ischemic animals (n=6) and to a group of sham-operated rabbits (n=5). Aspirin was administered in one dose just after occlusion of the coronary artery.

Neutrophil Isolation
Neutrophils were isolated from blood obtained via the femoral artery as previously described.^{12 16} Neutrophils (95% pure, 98% viable by trypan blue exclusion) were resuspended in calcium-PSS containing (in mmol/L) 140 NaCl, 4.6 KCl, 2.0 CaCl₂, 1.0 MgCl₂, 5.0 D-glucose, and 10.0 HEPES, pH 7.4.

Determination of L-[³H]-Citrulline Content
As detailed elsewhere,^{12 16} isolated neutrophils were incubated (45 minutes, 37°C) in calcium-PSS containing 10^-4 mol/L L-arginine and 1 µCi/mL L-[³H]-arginine. Unincorporated L-[³H]-arginine was washed twice with calcium-PSS buffer. Neutrophils were incubated for 5 minutes at 37°C. Neutrophils were lysed with cold ethanol, and the supernatant was evaporated to dryness under N₂ at 37°C. As previously described,¹² the extracts were resuspended in 20 mmol/L HEPES/KOH (pH 5.5) and applied to columns of Dowex AG50WX8 (sodium form), which were subsequently eluted with water (L-citrulline fraction) and 0.5 mol/L NaOH (L-arginine fraction). The L-[³H]-citrulline fraction was quantified by liquid-scintillation counting. The percentage of separation between L-arginine and L-citrulline was 92±3%.

PRP Preparation and Platelet Aggregation
Platelet aggregometry was used as a bioassay of neutrophil functionality. PRP was obtained from normal New Zealand White rabbits as previously described.¹⁷ In brief, whole blood was obtained in (by volume) 10% acid-citrate-dextrose and centrifuged at 800g for 15 minutes. PRP was collected and the platelet number counted by a Coulter counter. Platelet number was adjusted with platelet-poor plasma, obtained from the same normal rabbits, to 2.5x10⁸ cells/mL plasma.

Platelet activation was measured by use of a Lumiaggregometer (Aggrecorder, 4 channels) to quantify changes in light transmission. Previously, a platelet-poor sample was used as control for 100% light transmission. To correct the possible light absorption induced by the presence of the neutrophil suspension, the platelet-poor sample contained the same number of neutrophils as the platelet-rich sample.

PRP (500 µL) was incubated at 37°C for 10 minutes in the aggregometer with continuous stirring (500 rpm) and then stimulated with submaximal concentrations of thrombin (0.025 U/mL). Five minutes before incubation, 100 µL of the neutrophil suspension was added to PRP to reach a final concentration of 1.25x10⁸ platelets and 1x10⁶ neutrophils (125:1), which approximates the relative concentrations of platelets and neutrophils in normal blood. For standardization of measurements, only the values obtained by turbidimetry at 5 minutes were used for the calculations. This time period corresponds to the maximal or near-maximal value of the first wave of platelet aggregation. This primary wave is representative of platelet activation rather than platelet aggregation and is partially reversible. When required, aspirin (600 µg/mL) was added to the platelet-neutrophil suspension. This aspirin concentration was chosen on the basis of a previous study in which we observed that the maximal inhibitory effect of neutrophils on platelet activation occurs with this dose.¹²

In every case, the comparative baseline measurements were performed in the presence of the solvent of aspirin, ethanol, in the same dilution used for the incubations with aspirin (final ethanol concentration in the assay <0.1%). No significant effects were detected in any case with these concentrations of ethanol.

When required, neutrophils were preincubated with the NO-production antagonist L-NAME (10^-5 mol/L). Finally, as an estimation of the circulating levels of aspirin, serum salicylate levels were measured by use of a DuPont ACA analyzer at 510-nm wavelength.

Statistical Methods
Results are expressed as mean±SEM. Unless otherwise stated, each value corresponds to a minimum of seven experiments performed. Comparisons were performed by ANOVA or paired and unpaired Student's t test when appropriate. The Bonferroni correction for multiple comparisons was used to determine the level of significance of the probability value.

	Results

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Generation of NO by Neutrophils From Rabbits With Myocardial Ischemia
We determined NO production by neutrophils by measuring the accumulation of L-[³H]-citrulline in L-[³H]-arginine–loaded neutrophils. In the absence of aspirin, L-[³H]-citrulline generation in neutrophils obtained from the ischemic rabbits was significantly higher than that generated by neutrophils isolated from sham-operated animals (Fig 1A). In the presence of aspirin (600 µg/mL), the accumulation of L-[³H]-citrulline was significantly enhanced in neutrophils obtained from both sham-operated and ischemic rabbits (Fig 1A). However, the aspirin-induced increase in NO formation was higher in neutrophils isolated from ischemic rabbits than from sham-operated rabbits (Fig 1A).

View larger version (11K): [in this window] [in a new window]   Figure 1. Determination of NO production by neutrophils was performed by L-[3H]-citrulline formation from L-[3H]-arginine, as described in "Methods." A, Neutrophils isolated from sham-operated or ischemic rabbits were incubated with (solid bars) or without (open bars) aspirin 600 µg/mL or vehicle (ethanol <0.1%). B, Additional experiments were performed with neutrophils isolated from sham-operated and ischemic rabbits treated with oral aspirin. Results are presented as mean±SEM. P<.05 and 1 P<.01 vs neutrophils obtained from sham-operated animals in absence of aspirin.

To determine whether these in vitro findings were also valid under in vivo conditions, a group of sham-operated and a group of ischemic rabbits were treated with aspirin. The serum salicylate level in the aspirin-treated rabbits was 500±5 µg/mL, which corresponds to 568±5 µg/mL aspirin on a molar basis; this concentration was close to that used in the in vitro experiments.

As shown in Fig 1B, the L-[³H]-citrulline–increased accumulation observed after aspirin treatment was enhanced in neutrophils obtained from the ischemic rabbits compared with those obtained from sham-operated animals.

Effect of Circulating Neutrophils on Platelet Activation
In the absence of neutrophils, the percent light transmission observed by the activation of platelets with thrombin (0.025 U/mL) was 80±1%. In the absence of aspirin, neutrophils obtained from sham-operated rabbits did not modify thrombin-induced platelet activation (Fig 2). In these conditions, a slight but not significant inhibition of thrombin-stimulated platelet activation was observed by the incubation of platelets with ischemic neutrophils (Fig 2).

View larger version (14K): [in this window] [in a new window]   Figure 2. Inhibition of thrombin-stimulated platelet activation by circulating neutrophils obtained from sham-operated or ischemic rabbits. Isolated neutrophils were incubated with (solid bars) or without (open bars) aspirin 600 µg/mL or vehicle (ethanol <0.1%). The percent light transmission obtained by thrombin-stimulated platelet activation was 80±0.9% in the absence of neutrophils. Additional experiments were performed with the superoxide anion scavenger SOD (60 U/mL). Results are presented as mean±SEM. P<.05 and 2 P<.01 vs platelet activation observed with neutrophils isolated from sham-operated rabbits in the absence of aspirin.

In the presence of aspirin (600 µg/mL), the neutrophils obtained from sham-operated rabbits significantly inhibited thrombin-induced platelet activation. The addition of aspirin to neutrophils isolated from ischemic animals also favored the platelet-inactivating effect of neutrophils, with a higher intensity demonstrated than for neutrophils isolated from sham-operated rabbits (Fig 2).

In the absence of neutrophils, aspirin alone did not result in changes of thrombin-induced platelet activation (data not shown). In an effort to address the in vivo importance of the above-mentioned in vitro findings, similar experiments with neutrophils obtained from both sham-operated and ischemic rabbits after oral aspirin intake were performed. After aspirin administration, neutrophils obtained from sham-operated rabbits significantly decreased platelet activation (percent inhibition, 25±4%; P<.05). This effect was more marked when the platelets were incubated with neutrophils isolated from aspirin-treated ischemic rabbits (percent platelet inhibition, 44±6%; P<.01 versus sham-operated animals).

Mechanisms of the Effect of Aspirin: Role of NO and Superoxide Anion
To examine the implication of NO in the above-described effects, neutrophils were preincubated with L-NAME (10^-5 mol/L). When aspirin was added in vitro, incubation of neutrophils with L-NAME blocked the aspirin-sensitive inhibition of platelet activation by neutrophils obtained from both sham-operated and ischemic rabbits (percent inhibition, 86±7% and 89±6%, respectively; P<.01).

As in the in vitro studies, the inhibitory effect of neutrophils obtained from the different aspirin-treated groups of rabbits was also suppressed by L-NAME (Fig 3). Additional data support the role of NO in the effect of neutrophils on platelet activation, ie, L-arginine (10^-4 mol/L) restored the aspirin-sensitive inhibitory effect of neutrophils in the presence of L-NAME (10^-5 mol/L) (Fig 3).

View larger version (14K): [in this window] [in a new window]   Figure 3. Inhibition of thrombin-stimulated platelet activation by circulating neutrophils obtained from aspirin-treated (solid bars) sham-operated or ischemic rabbits. Results of experiments with the L-arginine antagonist L-NAME (10-5mol/L) alone or in combination with L-arginine (L-ARG; 10-4mol/L) are also shown. Results are presented as mean±SEM. 3 P<.01 vs platelet activation observed with neutrophils isolated from sham-operated rabbits in the absence of aspirin.

Since superoxide anions are important to accelerate the catabolism of NO, further experiments were performed to analyze their role in the above-described mechanisms. In the absence of aspirin, a small yet significant effect of the superoxide anion scavenger SOD (60 U/mL) was detected on platelet activation in the presence of neutrophils isolated from sham-operated rabbits (Fig 2). Addition of SOD to neutrophils obtained from ischemic rabbits significantly decreased thrombin-stimulated platelet activation (Fig 2). This effect was almost completely reversed by 10^-5 mol/L L-NAME (percent reversion, 90±0.2%; P<.01) No effect was detected on thrombin-stimulated platelet activation by the incubation of platelets with SOD alone (data not shown).

In the presence of aspirin, the platelet-inactivating effect of neutrophils obtained from sham-operated rabbits was increased by the addition of SOD in a small but significant manner (percent inhibition: with aspirin, 25±0.8%; with aspirin and SOD, 32±0.6%; P<.05). A similar effect of SOD in the presence of aspirin was observed when neutrophils isolated from ischemic rabbits were used (Fig 2).

	Discussion

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The data obtained in the present study demonstrated that after myocardial ischemia, circulating neutrophils produced increased amounts of NO. Addition of aspirin either in vivo or in vitro enhanced NO formation by these neutrophils.

The experiments concerning platelet activation provided functional implications of the changes in NO release described above. In spite of increased NO formation by neutrophils isolated from ischemic rabbits, these neutrophils did not seem to modify thrombin-stimulated platelet activation in the absence of aspirin. However, incubation with the superoxide anion scavenger SOD favored the appearance of an inhibitory effect of these neutrophils on platelet activation. These data suggested the possibility that after myocardial ischemia, the increased NO generation by neutrophils was accompanied by the concomitant release of superoxide radicals that inactivate NO. This hypothesis is supported by reports that observed that neutrophil stimulation resulted in the progressive inactivation of neutrophil-derived NO by superoxide radicals¹⁸ and that myocardial ischemia caused superoxide release by neutrophils.^{19 20 21} In this regard, L-NAME completely reversed the platelet inhibitory activity of ischemic neutrophils in the presence of SOD.

In the presence of aspirin, neutrophils obtained from ischemic rabbits produced enhanced amounts of NO, a phenomenon that correlated with an increased inhibition of platelet activation. The aspirin-mediated, platelet-modulatory response of neutrophils isolated from ischemic rabbits was also potentiated by SOD, although with less intensity than observed in the absence of aspirin. This finding of a less than additive interaction between SOD and aspirin opens the possibility that aspirin might have an action on superoxide effects.

The involvement of NO in the aspirin-sensitive antiplatelet effects of neutrophils obtained from either sham-operated or ischemic rabbits was established by their blockade with the L-arginine competitive antagonist L-NAME. The specificity of this inhibition was demonstrated by the reversal of the L-NAME effect by excess L-arginine.

Experiments with neutrophils obtained from rabbits treated with oral aspirin confirm the in vivo relevance of the in vitro findings and the similarity of the NO-dependent mechanism.

Several reports indicate that aspirin might contribute to increased NO levels or NO effects. In this regard, an antagonistic effect of aspirin has been observed on the vasoconstrictor properties of the L-arginine competitive analogue L-NMMA.²² In addition, aspirin might alter the contractile effects of neutrophils on the pulmonary artery.²³ More recently, we reported a direct demonstration that aspirin increases NO production by neutrophils.¹²

The present data did not allow specific identification of the putative mechanism(s) involved in NO production induced by myocardial ischemia and aspirin. Such identification is beyond the scope of the present research; however, preliminary data from our laboratory suggest a potential role for arachidonic acid and its noncyclooxygenase derivatives.²⁴

Myocardial ischemia/reperfusion results in diminished NO release from endothelial cells.²⁵ Attempts have been made to supplement the reduced NO to attenuate myocardial injury. In this regard, it has been reported that infusion of either organic NO donors, authentic NO, or the substrate of NO synthesis, L-arginine, provides significant cardioprotective effects.^{26 27 28}

On the other hand, it is well established that aspirin decreases the incidence of acute ischemic events.^{29 30 31} To our knowledge, the present study is the first to demonstrate that neutrophil-derived NO generation could be involved in the beneficial effects of aspirin in myocardial ischemia. Moreover, our results raise questions concerning the complete role of neutrophils in this pathology because, in the presence of aspirin, neutrophils actually did possess antiplatelet effects. Our findings, therefore, suggest a novel interpretation of the protective effect of aspirin on damage from myocardial ischemia.

	Acknowledgments

 
This work was supported by grant No. PM 92/041 from Direccion General de Investigaciones Cientificas y Tecnicas, grant No. 94/364 from Fondo de Investigaciones Sanitarias, Fundacion Ramon Areces, Instituto Reina Sofia de Investigaciones Nefrologicas, and Bayer SA. Drs de Miguel, Monton, and Cernadas are fellows of the Fundacion Conchita Rabago. Dr Riesco is a fellow of the Ministerio de Educacion. The authors wish to thank Concepcion San Martin for editorial assistance.

	Selected Abbreviations and Acronyms

 

L-NAME = NW-nitro-L-arginine methyl ester NO = nitric oxide PRP = platelet-rich plasma PSS = physiological saline solution SOD = superoxide dismutase

Received November 7, 1995; revision received January 4, 1996; accepted January 7, 1996.

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