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J Am Coll Cardiol, 2002; 39:818-825
© 2002 by the American College of Cardiology Foundation
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CLINICAL STUDY: ACUTE CORONARY SYNDROMES

Nitric oxide production by neutrophils obtained from patients during acute coronary syndromes: expression of the nitric oxide synthase isoforms

Lourdes Sánchez de Miguel, PhD, M. a Mar Arriero, PhD*, Jerónimo Farré, MD, PhD{dagger}, Petra Jiménez, PhD, Antonio García-Méndez, PhD, Trinidad de Frutos, PhD, Ana Jiménez, PhD, Rosa García, PhD, Fernando Cabestrero, MD{dagger}, Juan Gómez, MD{ddagger}, Raimundo de Andrés, MD{ddagger}, Mercedes Montón, PhD*, Edita Martín, RN{dagger}, Luz M. De la Calle-Lombana, RN{ddagger}, Luis Rico, MD§, José Romero, MD{dagger} and Antonio López-Farré, PhD*,*

* Cardiovascular Research and Hypertension Laboratory, Fundación Jiménez Díaz, Madrid, Spain
{dagger} Cardiology Department, Fundación Jiménez Díaz, Madrid, Spain
{ddagger} Cardiovascular Research Hypertension Laboratory, Emergency Department, Fundación Jiménez Díaz, Madrid, Spain
§ Cardiovascular Research Hypertension Laboratory, Internal Medicine Department, Fundación Jiménez Díaz, Madrid, Spain

Manuscript received May 18, 2001; revised manuscript received November 20, 2001, accepted December 6, 2001.

* Reprint requests and correspondence: Dr. Antonio López-Farré, Fundación Jiménez Díaz, Cardiovascular Research and Hypertension Laboratory, Avda. Reyes Católicos, 2, 28040 Madrid, Spain.
alopeza{at}fjd.es


   Abstract
Top
 Abstract
Materials and methods
 Results
 Discussion
 References
 
OBJECTIVES: To analyze the differences in the nitric oxide (NO) forming system between neutrophils obtained from patients during unstable angina (UA) and during acute myocardial infarction (AMI).

BACKGROUND: Neutrophils are involved in the regulation of thrombus formation through the release of active substances such as NO. Acute myocardial infarction is the result of an occlusive thrombus; unstable angina is attributed to intermittent thrombus formation.

METHODS: We studied 49 patients admitted to hospital within 24 h after the onset of chest pain: 31 experienced AMI and 18 experienced UA. Acute myocardial infarction was defined as CK greater than two-fold the upper limit of normal value of biochemical laboratory, with CK-MB >10% total CK. Unstable angina was defined as transient ST segment changes without significant increases in CK and CK-MB.

RESULTS: The amount of NO generated by neutrophils from AMI patients was significantly higher than that generated by neutrophils from UA patients. Neutrophils from UA and AMI patients showed low levels of endothelial-like NO synthase protein expression and a marked expression of the inducible NO synthase (iNOS) isoform. Although neutrophils from patients during acute coronary syndromes generated high amounts of NO, they did not demonstrate an increased ability to stimulate cyclic guanosine monophosphate (cGMP) synthesis in platelets. This lack of activity to release NO by neutrophils from patients during AMI was unrelated to a defect in the platelet cGMP-forming system; sodium nitroprusside, an exogenous NO donor, similarly increased cGMP levels in platelets from AMI patients and healthy donors.

CONCLUSIONS: Neutrophils from patients during AMI and UA showed an increased production of NO and a marked expression of the iNOS isoform. However, NO released from these neutrophils showed a deficient functionality. These findings could have clinical implications because they show differences in thrombus growth in patients with UA versus patients with AMI.

Abbreviations and Acronyms
  cGMP
  AMI
  acute myocardial infarction
  cGMP
  cyclic guanosine monophosphate
  eNOS
  endothelial nitric oxide synthase
  iNOS
  inducible nitric oxide synthase
  NO
  nitric oxide
  PRP
  platelet-rich plasma
  SOD
  superoxide dismutase
  UA
  unstable angina

Acute coronary syndromes are the result of thrombus formation within a coronary artery (1,2). In acute myocardial infarction (AMI) there is a complete, long-lasting, thrombotic occlusion of a coronary artery. Unstable angina (UA), on the other hand, is attributed to an incomplete or short-lived thrombotic coronary occlusion. This variability could be related to different degrees of platelet activation.

The thrombotic arterial event is a multicellular phenomenon in which, in addition to platelets, neutrophils and endothelium are implicated (3,4). Clinical and experimental models of coronary ischemia have shown the detrimental role of neutrophils in the progression of myocardial damage through their ability to generate oxygen-derived free radicals, such as superoxide anion (5,6). Recently, it has been demonstrated that neutrophils also produce nitric oxide (NO) (7). Nitric oxide released by neutrophils has been demonstrated to prevent neutrophil adhesion to vascular endothelium and to control aggregation and adhesion of neighboring platelets (8,9).

Nitric oxide is produced by the metabolic conversion of L-arginine into L-citrulline due to the activity of NO-synthesizing enzymes (NO synthase, NOS) (10,11). Functional evidence has demonstrated the presence of a constitutive NOS isoform in neutrophils that we have recently identified as an endothelial-like NOS isoform (eNOS) (9,12,13). Under inflammatory conditions, several types of cells, including neutrophils, express a second NOS isoform, an inducible NOS isoform (iNOS) (14,15). Whereas eNOS activity generates small amounts of NO for short periods of time and is coupled to the endothelium-dependent relaxation, iNOS stimulation results in a delayed and prolonged release of large amounts of NO (10,11).

As mentioned, NO released by neutrophils has been shown to inhibit the activation of neighboring platelets through the stimulation of cyclic guanosine monophosphate (cGMP) formation in platelets (8,16,17). The ability of neutrophils to produce NO during acute coronary syndromes, however, has not been previously studied. In the present study we have analyzed the differences between the NO-forming system in neutrophils obtained from patients during UA versus in those obtained from patients during AMI.


   Materials and methods
Top
 Abstract
 Materials and methods
Results
 Discussion
 References
 
Patients.   Forty-nine consecutive patients admitted to the Coronary Care Unit and Emergency Unit of Fundación Jiménez Díaz were investigated. Of these, 31 had transmural AMI diagnosed on the basis of the classical criteria of prolonged chest pain accompanied by serial changes on the standard 12-lead electrocardiogram (ECG) and raised serum CK (greater than two-fold upper limit of normal value of biochemical laboratory) and CK-MB (>10% total CK). Eighteen patients had unstable angina diagnosed on the basis of chest pain at rest and transient ST segment changes, without significant increases in CK (1.5-fold more than the upper normal range) and CK-MB. The control group included 20 healthy volunteers from the hospital staff. The results obtained from healthy volunteers were only used as reference for normal values because the aim of the study was to analyze the differences between the neutrophil NO-forming system in AMI versus in UA.

The criteria for enrollment were admission within 24 h after the onset of chest pain. The criteria for exclusion were a previous episode of acute coronary syndrome or the recent exposure (during the preceding two weeks) to antithrombotic, steroidal and nonsteroidal anti-inflammatory drugs or heparin. Moreover, the present study did not include patients or control subjects with a history of neoplastic, infectious or autoimmune diseases or any surgical procedure in the preceding six months. The study was performed between January 1998 and May 2000. All subjects gave fully informed consent and the study was approved by the Ethics Committee of Fundación Jiménez Díaz.

Blood sampling.   Blood samples were drawn from a peripheral vein on admission. A 20 ml blood sample was obtained for neutrophil isolation by peripheral venipuncture with a syringe containing 0.2 ml of EDTA. A further 10 ml blood sample was also obtained in acid-citrate-dextrose for platelet-rich plasma (PRP) isolation.

Neutrophil isolation and PRP preparation.   Neutrophils were isolated as previously described (7,8). Isolation and manipulation of neutrophils were always performed under sterile conditions. Neutrophils were isolated by Ficoll/Hypaque centrifugation. Neutrophils (95% pure, 98% viable by trypan blue exclusion) were resuspended in RPMI 1640 medium supplemented with 0.25% bovine serum albumin, 5 mmol/l glutamine, 2 x 10–5 µg/l penicillin and 2 x 10–5 µg/l streptomycin.

Platelet-rich plasma was obtained from the same donor for each experiment as described (8,17). In brief, blood samples were collected in acid-citrate-dextrose (10% vol/vol) and centrifuged at 800 g for 15 min. Platelet-rich plasma was obtained and platelet number counted by a coulter counter. The platelet number was adjusted with platelet-poor plasma obtained from the same individual to 2.5 x 108 cells/ml plasma.

Determination of [3H]-L-citrulline content.   As detailed elsewhere (8,17), isolated neutrophils were counted and incubated during 45 min at 37°C in the above-described RPMI 1640 medium containing 1 µCi/ml [3H]-L-arginine. Unincorporated [3H]-L-arginine was washed out twice with the same medium free of [3H]-L-arginine; 3 x 106 neutrophils/sample were incubated during 10 min at 37°C.

After incubation, the neutrophils were lysed with cold ethanol and the supernatants collected and evaporated until dryness under N2 atmosphere. As previously described (7), the extracts were resuspended in 20 mmol/l Hepes/KOH, pH 5.5 and applied to columns of Dowex AG50WX8 (Na+ form) which were subsequently eluted with water (L-citrulline fraction) and 0.5 mol/l NaOH (L-arginine fraction). The [3H]-L-citrulline fraction was quantified by liquid-scintillation counting. This column method separates L-arginine from L-citrulline by up to 92 ± 3%.

Determination of eNOS and iNOS protein expression.   Endothelial NO synthase and inducible NO synthase protein expression were analyzed in human neutrophils by Western blotting, as previously described (18,19). Neutrophils were lysed in Laemmli buffer containing 2-mercaptoethanol (20). Proteins were separated on denaturing sodium dodecyl sulfate-10% polyacrylamide gels. Equal amounts of proteins (10 µg/lane) estimated by bicinchoninic acid reagent (Pierce, Rockford, Illinois) were loaded in the gel. A parallel gel with identical samples was run and stained with Commassie to compare the intensities of the protein bands. The separated proteins were then blotted into nitrocellulose (Immobilon-P, Millipore Corp., Bedford, Massachusetts). Blots were blocked overnight at 4°C with 5% nonfat dry milk in TBS-T (20 mmol/l Tris [hydroxymethyl] aminomethane [Tris-HCl], 137 mmol/l NaCl, 0.1% Tween 20). Western blot analysis was performed with monoclonal antibodies against eNOS or iNOS proteins (Transduction Laboratories, Lexington, United Kingdom). We have previously reported that these monoclonal antibodies specifically recognized the eNOS and iNOS isoforms (18,19,21).

Blots were incubated with the first antibody (1:2,500) for 1 h at room temperature and, after extensive washing, with the second antibody (horseradish peroxidase-conjugated antimouse immunoglobulin antibody) at a dilution of 1:1,500 for a further 1 h. Specific eNOS protein and iNOS protein were detected by enhanced chemiluminescence (ECL, Amersham Iberica, Madrid, Spain) and evaluated by densitometry (Molecular Dynamics, Sunnyvale, California). Prestained protein markers were used for molecular mass determinations.

Measurement of cGMP.   The measurement of cGMP was used as a bioassay of NO functionality. Nitric oxide produced by neutrophils increases cGMP production in platelets via activation of soluble guanylate cyclase; therefore, the amount of cGMP in the neutrophil/platelet suspension could be considered an index of the NO bioactivity. Cyclic GMP concentrations in the platelet/neutrophil suspension were determined as previously described (8,19). In brief, 100 µl of the neutrophil suspension were added to PRP to reach a final amount of 1.25 x 108 platelets and 1 x 106 neutrophils (125:1), which approximates the relative concentrations in normal blood. The suspension of neutrophils and platelets was incubated for 15 min at 37°C with continuous stirring (1,000 rpm). Parallel samples of PRP in the absence of neutrophils were also tested. Both platelets and neutrophils were always obtained from the same donor. To stop the incubation, cells were pelleted by centrifugation (2,500 rpm, 4°C, 5 min). The supernatant was aspirated and cells were extracted at 4°C with a 49:1 (v/v) mixture of 0.1 mol/l ethanol/HCl. The extracts were evaporated using a speed vac evaporator (Model VR-1/120/240, Heto Lab-Equipment A/S, Denmark). Cyclic GMP concentrations were measured in acetylated samples by means of a radioimmunoassay kit (Amersham International, Buckinghamshire, United Kingdom). The sensitivity of the assay was 0.5 fmol. The intra-assay and interassay variations were <8.9% and <16%, respectively.

Neutrophil superoxide radical generation.   The amount of superoxide anion generated by neutrophils was determined by measuring the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome C. In brief, neutrophils at a concentration of 3 x 106 cells/ml were placed in a water bath at 37°C in the above-described RPMI-1640 medium containing 0.1 mmol/l ferricytochrome C. The generation of superoxide anion was calculated as the difference in absorbance between aliquots of cells incubated with and without SOD 100 µg/ml. The difference was then divided by the molar extinction coefficient change between ferricytochrome C and ferrocytochrome C to determine nmoles of superoxide radicals produced by 5 x 106 neutrophils over 20 min. All observations were made in triplicate and the data averaged. The absorbance was measured in a spectrophotometer at 550 nm.

Nitrotyrosine protein formation in neutrophils.   Under circumstances where both NO and superoxide are cogenerated, they react with each other and yield the harmful peroxynitrite which has a very short half-life (~1.9 s at pH 7.4) (22). However, peroxynitrite reacts with L-tyrosine to produce nitrotyrosine, which is a stable substance and can be used as an in vivo marker of peroxynitrite formation. Thus, we measured the content of nitrotyrosine proteins in neutrophils derived from UA and AMI patients by Western blotting, as mentioned, for eNOS and iNOS protein. Western blot was developed using a monoclonal antibody against nitrotyrosine groups (1:200, Calbiochem, San Diego, California) (23).

Statistical methods.   Results are expressed as means ± SEM. Unless otherwise stated, each value corresponds to a duplicate assay of each sample. To determine the statistical significance, we have performed ANOVA with Bonferroni’s correction for multiple comparisons or a Student’s t test, paired or unpaired. A p value <0.05 was considered statistically significant.


   Results
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 Abstract
 Materials and methods
 Results
Discussion
 References
 
NOS expression and NO generation in neutrophils.   Table 1 shows the clinical features and previous medication of the UA and AMI patients. The healthy volunteers from the hospital staff, whose mean age was 42 ± 5 years (range 28 to 52 years), had not ingested any drug for at least two weeks before the study.


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  		Table 1 Characteristics of the Study Group

 
We determined the ability of neutrophils to produce NO by measuring the accumulation of [3H]-L-citrulline in [3H]-L-arginine-loaded neutrophils. A significant increase in the accumulation of [3H]-L-citrulline was found in neutrophils obtained from patients during AMI compared with that in patients during UA ([3H]-L-citrulline: AMI: 64 ± 5, UA: 45 ± 6 nmol/min, p < 0.05). The amount of [3H]-L-citrulline generated by neutrophils from the healthy volunteers was always lower than that generated by neutrophils from patients during the acute coronary syndromes ([3H]-L-citrulline in control neutrophils: 7 ± 2 nmol/min; p < 0.05 with respect to AMI and UA). In the presence of L-NAME (10–4 mol/l), the conversion of [3H]-L-arginine to [3H]-L-citrulline was inhibited by 85 ± 4% in the neutrophils isolated from any experimental group, indicating the specificity of the reaction.

Neutrophils from UA and AMI patients showed low levels of eNOS protein expression (Figs. 1A and 1B), but they demonstrated a marked iNOS protein expression (Figs. 2A and 2B). Inducible NO synthase protein expression was almost absent in the neutrophils from control volunteers (Figs. 2A and 2B), which demonstrated a marked eNOS protein expression (Figs. 1A and 1B).



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  		Figure 1 (Top) Representative Western blot demonstrating endothelial nitric oxide synthase (eNOS) expression in human neutrophils obtained from healthy volunteers (control) and patients during acute myocardial infarction (AMI) and unstable angina (UA). (Bottom) Bar graph showing the densitometric analysis of the Western blot. Results are presented as mean ± SEM. *p < 0.05 with respect to neutrophils obtained from healthy donors.

 


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  		Figure 2 (Top) Representative Western blot demonstrating inducible nitric oxide synthase (iNOS) expression in human neutrophils obtained from healthy volunteers (control) and patientes during acute myocardial infarction (AMI) and unstable angina (UA). (Bottom) Bar graph showing the densitometric analysis of the Western blot. Results are presented as mean ± SEM. *p < 0.05 with respect to neutrophils obtained from healthy donors. **p < 0.05 with respect to neutrophils from UA patients.

 
CGMP formation in the suspension of platelets and neutrophils.   The antiplatelet effect of cGMP has been shown to be the main mediator of the cellular effects of NO (8,16). Therefore, we determined the cGMP levels in coincubations of platelets and neutrophils as an index of NO functionality.

Cyclic GMP levels in platelets isolated from acute coronary syndromes were higher than in platelets isolated from control volunteers (Fig. 3). In this regard, platelets obtained from AMI patients showed a significant increase in the content of cGMP compared with platelets obtained from UA patients (Fig. 3).



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  		Figure 3 Cyclic guanosine monophosphate (cGMP) levels in platelets and platelets plus neutrophils obtained from healthy volunteers and patients during acute coronary syndromes. Determination of cGMP levels were performed in platelets alone (PLT) or coincubated with neutrophils (PMN). Platelets and neutrophils were obtained from peripheral blood of healthy volunteers (control) and patients during unstable angina (UA) and during acute myocardial infaction (AMI). Results are represented as mean ± SEM. *p < 0.05 with respect to the corresponding platelets alone.

 
When cGMP was measured in the coincubation of platelets and neutrophils, it was found increased in UA patients and healthy volunteers with respect to the level observed in their respective platelets alone (Fig. 3). The increase in the cGMP levels observed in the presence of neutrophils from UA patients was very similar to that obtained in the presence of neutrophils from healthy donors (cGMP increase: healthy 10 ± 4, UA: 9 ± 2 fmol; p = NS). The increase in cGMP content in the coincubation of platelets and neutrophils was inhibited by an L-arginine competitor, 10–4 mol/l L-NAME (% inhibition: 91 ± 3%), supporting the involvement of NO.

As shown in Figure 3, the presence of neutrophils from AMI patients failed to modify the cGMP content with respect to that measured in platelets alone. Because the cGMP content in platelets from AMI patients was not increased by the addition of neutrophils, we analyzed whether the cGMP generating system in platelets from these patients could be saturated. We thus determined the ability of platelets from patients during AMI to generate cGMP in response to the exogenous NO-donor sodium nitroprusside (10–5 mol/l). Sodium nitroprusside increased the cGMP content in platelets from patients during AMI to a level similar to that seen in platelets from the healthy volunteers (cGMP increase: healthy 24 ± 5, AMI 20 ± 3 fmol; p = NS).

Superoxide anion released from neutrophils.   As shown in Figure 4, neutrophils from patients during AMI demonstrated a significantly higher ability to release superoxide anion than did neutrophils obtained from either control volunteers or patients during UA. Although neutrophils from UA patients tended to release higher amounts of superoxide anion than those of healthy volunteers, the difference did not reach statistical significance.



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  		Figure 4 Bar graph showing superoxide anion production by neutrophils. Neutrophils were obtained from control subjects (control), acute myocardial infartion (AMI) and unstable angina (UA) patients. Results are presented as mean ± SEM. *p < 0.05 with respect to neutrophils obtained from healthy donors.

 
Nitrotyrosine protein formation in neutrophils.   Neutrophils from patients during an acute coronary syndrome showed higher levels of nitrotyrosilated proteins than those from control volunteers (Fig. 5). Three bands with approximate molecular mass of 16, 24, 84 KDa, respectively, were observed with the antinitrotyrosine antibody. Significant increased levels of these tyrosine-nitrated proteins were detected in neutrophils from patients during AMI compared with neutrophils from patients during UA.



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  		Figure 5 (Top) Representative Western blot demonstrating the presence of nitrotyrosilated proteins in neutrophils from healthy volunteers (control) and patients during acute myocardial infarction (AMI) and unstable angina (UA). (Bottom) Bar graph showing the densitometric analysis of the nitrotyrosilated proteins with apparent molecular weights of 16, 24, 84 KDa that was observed in the Western blot. Results are mean ± SEM. Asterisk = p < 0.05 with respect to neutrophils from healthy donor; star = p < 0.05 with respect to neutrophils from UA patients. White bars = 16 KDa; grey bars = 24 KDa; black bars = 84 KDa.

 

   Discussion
Top
 Abstract
 Materials and methods
 Results
 Discussion
References
 
In the present study, we evaluated the NO-synthesizing system in a group of patients during an acute coronary syndrome, that is, unstable angina or acute myocardial infarction. Neutrophils from patients during AMI produced greater amounts of NO than neutrophils from patients during UA, but all neutrophils showed an increased ability to form NO compared with neutrophils from healthy donors.

eNOS and iNOS expression in neutrophils.   Functional evidence has demonstrated the ability of neutrophils to produce NO through a constitutively expressed NOS isoform (7,9,12). In a previous study, we have identified the presence of an eNOS-like isoform in human neutrophils (13). In the present study, we observed that neutrophils from patients during an acute coronary syndrome showed a diminished eNOS protein expression compared with neutrophils from healthy volunteers.

The mechanisms underlying the downregulation of eNOS expression in neutrophils from patients during acute coronary syndromes were not established in the present study. Although the eNOS isoform had been initially described as constitutive, in recent years it has been demonstrated that cytokines downregulate eNOS expression in the endothelium, mesothelium and neutrophils by destabilizing eNOS mRNA (13,24–26). Therefore, the potential causes for the downregulation of eNOS protein could include cytokines, which have been reported elevated during acute coronary syndromes (27–29).

The Western blot experiments also demonstrated that neutrophils from patients during AMI contained higher amounts of iNOS protein than neutrophils from patients during UA. The iNOS isoform has been shown to be stimulated by the same cytokines that downregulate eNOS expression (11). The differences observed in the level of expression of iNOS protein between neutrophils from patients during UA and during AMI could be related to a different type and/or different level of cytokines generated during each specific coronary syndrome. Further studies are warranted to clarify this issue.

NO activity and cGMP formation.   The experiments related to the level of cGMP in platelets provided a parameter to determine the biological activity of NO generated by neutrophils. Whereas neutrophils from UA patients increased cGMP levels in the platelet/neutrophil system, neutrophils from AMI patients failed to modify it. Platelets from patients during acute coronary syndromes demonstrated higher basal cGMP levels, which could be attributable not only to the exposure to high levels of NO but also to the nitrate treatment. The results obtained with the exogenous NO-donor, sodium nitroprusside, suggested that there is no defect in the cGMP-forming system in platelets from AMI. In this regard, the increment in the cGMP levels observed with sodium nitroprusside stimulation was very similar in platelets from healthy donors and AMI patients. Taken together, these results could suggest that the increased generation of NO by neutrophils during acute coronary syndromes could be accompanied by the concomitant inactivation of this molecule.

Nitric oxide is catabolized by free radicals and more particulary by superoxide anion (10). We determined ex vivo the ability of neutrophils to release superoxide anion. Neutrophils from AMI patients demonstrated an increased ability to release superoxide anion, which was associated with their incapacity to increase cGMP in platelets. Neutrophils from UA patients also tended to release higher amounts of superoxide anion than those from healthy volunteers; although the difference did not reach statistical significance, it might be enough to decrease NO activity. In this regard, it is noteworthy that, although the level of NO generated by neutrophils from patients during UA was significantly higher than that generated by neutrophils from healthy donors, the increase in the cGMP accumulated in the platelet/neutrophil coincubation was very similar between them. The measurement of superoxide anion was based on an in vitro assay and therefore we should interpret these results with caution. However, these data suggested the possibility that during acute coronary syndromes the greater generation of NO by neutrophils could be accompanied by an increased catabolism of NO due to the concomitant release of superoxide radicals. It was further supported by the fact that the level of nitrotyrosilated proteins, a marker of peroxynitrite formation, was greater in neutrophils from patients during acute coronary syndromes. Interestingly, the different levels of nitrotyrosilated proteins observed between neutrophils from patients during AMI and during UA were associated with the different ability of their neutrophils to increase cGMP formation in their platelets.

Previous studies in animal models of coronary occlusion have postulated the involvement of NO in the pathogenesis of ischemic heart disease. In this regard, Pearson et al. have reported the impairment of NO-dependent relaxation in ischemic canine coronary arteries (30). Bauerssachs et al. (31) have demonstrated that the addition of superoxide dismutase to isolated coronary arteries from rats with heart failure partially restored the endothelium-dependent relaxation. In the same line of evidences, a substantial reduction of endothelial SOD and endothelial-mediated dilation in patients with coronary artery disease has been reported (32). In this regard, it is also noteworthy that in addition to serving as a biomarker of peroxynitrite, nitration of tyrosine residues is known to be an inhibitor of several biochemical pathways, including SOD (33). However, we have not performed experiments with superoxide anion scavengers, such as the mentioned SOD, and therefore the present study did not allow us to identify whether the production of superoxide anion is the mechanism responsible for the deficient activity of the NO released by neutrophils from patients during acute myocardial infarction.

Nitric oxide produced by neutrophils appears to have a relevant role in inhibiting platelet activation through cGMP mediation (8,15). Langford et al. (34) have previously observed a different degree of platelet activation in patients with AMI and UA. Moreover, administration of exogenous NO-donors reduced platelet P-selectin expression in patients with acute coronary syndromes (34). Interestingly, it has been demonstrated that the antioxidant enzyme glutathione peroxidase potentiates the inhibition of platelet function by NO via the inhibition of reactive oxygen species (35). There is also evidence that loss of endothelium-derived NO contributes to the pathogenesis of acute coronary syndromes (36,37). To our knowledge, however the NO-forming system in neutrophils has not been previously studied in patients during AMI and UA.

In summary, neutrophils from patients during AMI and UA showed an increased production of NO and have a marked expression of the iNOS isoform. However, NO released from these neutrophils had a deficient functionality to stimulate cGMP formation by platelets. This fact was more marked in neutrophils from patients during AMI than in those from patients during UA. The different functionality of the NO released by neutrophils from patients during UA and AMI could contribute to the different growth of the thrombus within the coronary vessels and may be a suitable target for therapy.


   Acknowledgments
 
The authors thank Begoña Larrea for secretarial assistance.


   Footnotes
 
Supported by grants from Ministerio de Ciencia y Tecnología (SAF 2000-024), Fundación Ramón Areces and Fundación Mapfre Medicina. Ma Mar Arriero and Ana Jiménez are fellows from Fundación Conchita Rabago de Jiménez Díaz. Mercedes Monton is a postdoctoral fellow of Comunidad Autonoma de Madrid.


   References
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 Abstract
 Materials and methods
 Results
 Discussion
 References
 

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