Author + information
- Received May 11, 2000
- Revision received September 29, 2000
- Accepted November 10, 2000
- Published online March 1, 2001.
- Trinidad de Frutos, PhDa,
- Lourdes Sánchez de Miguel, PhDa,
- Jerónimo Farré, MD, PhDa,
- Juan Gómez, MD, PhDa,
- José Romero, MDa,
- Pedro Marcos-Alberca, MDa,
- Antonio Nuñez, MDa,
- Luis Rico, MD, PhDa and
- Antonio López-Farré, PhDa,* ()
- ↵*Reprint requests and correspondence: Dr. Antonio López-Farré, Cardiovascular Research and Hypertension Laboratory, Fundación Jiménez Dı́az, Avda. Reyes Católicos 2, Madrid, 28040 Spain
The purpose of this study was to determine whether human neutrophils express an endothelial-type nitric oxide synthase (eNOS), and to study the effect of tumor necrosis factor-alpha (TNF-alpha) on its expression.
Several studies have demonstrated the presence of a constitutively expressed nitric oxide synthase (NOS) in neutrophils. Cardiovascular disease is characterized by increased levels of plasma TNF-alpha, a cytokine that has demonstrated eNOS messenger ribonucleic acid (mRNA) destabilization in cultured endothelial cells.
Neutrophils were obtained from healthy volunteers and from patients with acute myocardial infarction (AMI).
Human neutrophils express eNOS mRNA and eNOS protein. Stimulation of neutrophils with TNF-alpha decreased eNOS protein expression by reducing eNOS mRNA stabilization. In the present study, we also show that the cytosol of human neutrophils contains proteins that bind to a specific region within the 3′-untranslated region (3′-UTR) of eNOS mRNA. Tumor necrosis factor-alpha increased the binding of the cytosolic proteins to the 3′-UTR of eNOS mRNA. Simvastatin reduced the TNF-alpha–related binding activity of neutrophil cytosolic proteins to eNOS mRNA, which was associated with its protective effect on eNOS protein expression. The in vivo reproduction of the in vitro findings was performed in neutrophils obtained from patients with AMI and showed a diminished expression of eNOS protein, which was associated with increased binding of the cytosolic proteins.
These observations demonstrate that human neutrophils express eNOS, which is downregulated by TNF-alpha and during AMI. This effect is associated with increased binding of neutrophil cytosolic proteins to the 3′-UTR of eNOS mRNA.
Nitric oxide (NO) is released from the endothelium through the activity of an initially defined constitutive enzyme called “endothelial-type nitric oxide synthase” (eNOS) (1–3). It has recently been shown that several pathophysiologic stimuli, including cytokines, can reduce eNOS expression at a post-transcriptional level (4,5). The loss of endothelium-derived NO results in vascular abnormalities, including vasoconstriction, smooth muscle cell proliferation and adhesion of blood elements to the vessel wall (1,3,6). Therefore, a decreased expression of eNOS may contribute to the development of cardiovascular disease, including acute myocardial infarction (AMI), in which the presence of cytokines (i.e., tumor necrosis factor-alpha [TNF-alpha]) and a defective production of endothelium-derived NO with a reduction of eNOS expression have been reported (7–9). Interestingly, there is growing evidence that the hydroxymethyl glutaryl coenzyme A reductase inhibitor simvastatin reverses the downregulation of eNOS expression induced by oxidized low density lipoprotein in endothelial cells under cholesterol-clamped conditions (10,11). This occurs by eNOS messenger ribonucleic acid (mRNA) destabilization through a still unknown mechanism (10).
Several studies have shown that protein molecules control mRNA stability by binding to specific sequences contained in each mRNA (12,13). Some of these sequences have been identified within the 3′-untranslated region (3′-UTR) of the mRNAs (12). We recently demonstrated that bovine endothelial cells contain cytosolic proteins that form complexes with the in vitro–transcribed 3′-UTR of eNOS mRNA, which is associated with reduced expression of eNOS protein (14).
Human neutrophils also produce NO through the activity of a constitutive NOS (15,16). Several studies support the importance of NO released by neutrophils to inhibit their adhesion to the endothelium and to inhibit the aggregation of neighboring platelets (17,18). Although the factors regulating expression of eNOS in the endothelium have been widely investigated, little is known about the regulation of the one expressed by neutrophils.
In the present study, we have analyzed whether the constitutive NOS isoform present in human neutrophils is an endothelial-like NOS and whether these cells contain cytosolic proteins that interact with the 3′-UTR of eNOS mRNA, as described in bovine endothelial cells. In addition, we have studied the role of TNF-alpha in the expression of the aforementioned constitutive NOS isoform and the binding of cytosolic proteins to 3′UTR of eNOS mRNA. Because simvastatin has been shown to stabilize eNOS mRNA in the endothelium (10,11), we used this drug to analyze the correlation between the binding of neutrophil cytosolic proteins to eNOS mRNA and the level of eNOS protein expression.
In a second set of experiments, we studied the level of eNOS protein in neutrophils obtained from patients with AMI, a pathologic condition where the level of TNF-alpha is considered to be increased (7,19).
Human neutrophil isolation
Human neutrophils were isolated from the peripheral blood of 15 healthy donors (25 to 40 years old) who had not ingested any drug for at least two weeks before the study. Isolation and manipulation of neutrophils were always performed under sterile and endotoxin-free conditions. The neutrophils were isolated by Ficoll/Hypaque centrifugation, as previously described (6,18). The neutrophils were 95% pure and 98% viable, as determined by exclusion of trypan blue stain. Neutrophils were resuspended in RPMI 1640 medium, supplemented with 0.25% bovine serum albumin, 5 mmol/liter glutamine, 2 × 10−5μg/liter penicillin and 2 × 10−5μg/liter streptomycin (5 × 106neutrophils/ml). The neutrophils were then stimulated with TNF-alpha (500 U/ml).
Determination of eNOS protein expression
Endothelial-type NOS protein expression in human neutrophils was analyzed by Western blotting, as previously described (20,21). Human neutrophils were incubated in the presence and absence of TNF-alpha (500 U/ml). The neutrophils were then lysed in Laemmli buffer containing 2-mercaptoethanol (22). Proteins (10 μg/lane) were separated on denaturing sodium dodecyl sulfate/10% polyacrylamide gels, as previously described (14,21). A parallel gel with identical samples was run and stained with Commassie blue to compare the intensities of the protein bands. Western blot analysis was performed with a specific monoclonal antibody against eNOS (Transduction Laboratories, Lexington, United Kingdom), as previously reported (14,21). Prestained protein markers were used for molecular mass determinations.
Northern blot analysis
Isolated neutrophils were incubated in the presence and absence of TNF-alpha (500 U/ml). The experiments were performed in the presence of the transcriptional inhibitor 5,6-dichloro-1-2-D-ribofuranosylbenzimidazole (DRB; Calbiochem, La Jolla, California). Total RNA was isolated according to the method of Chomczynski and Sacchi (23). Northern blot analysis of 20 μg/sample total RNA was performed as previously described, using as a complementary deoxyribonucleic acid (cDNA) probe the radiolabeled UTR-large (L) RNA (see later discussion) (500,000 cpm/ml) (14). We also used a probe encoding glyceraldehyde-3-phosphate dehydrogenase as a control for loading.
Plasmids and in vitro transcription
Oligonucleotides complementary to bovine eNOS cDNA (GenBank accession no. BTNIOXSY) were purchased from Bio-synthesis, Inc. (Lewisville, Texas). The pNOS-UTR of plasmids was made as previously reported (14). Oligonucleotide 1 (5′-GGATCTAGAACGCTATCACGAGGACATT-3′) and oligonucleotide 2 (5′-AGGAAGCTTAGTAGGTCTCCTAACTTCTG-3′) were used to produce the pNOS–UTR-L, a fragment covering 166 bases of the coding region and 393 bases of the 3′-UTR of eNOS cDNA (Fig. 1)by reverse transcriptase polymerase chain reaction (RT-PCR). The amplification products were purified after agarose gel electrophoresis, subjected to restriction endonuclease digestion with XbaI and HindIII and ligated to PGEM4Z to create pNOS–UTR-L.
Oligonucleotide 3 (5′-GTTGGATCCCCTGTACTATCTCACCCT-3′) and oligonucleotide 2 were used to create pNOS–UTR-small (S), as previously described (14). To make plasmid pNOS–UTR-UC (rich in uridine and cytidine nucleotides), pNOS–UTR-S was cut with AcsI. To make pNOS–UTR-AU (rich in adenine and uridine nucleotides), AcsI–linearized pNOS–UTR-S was purified by agarose gel electrophoresis and religated. Figure 1represents the different probes containing the different segments of the 3′-UTR of eNOS mRNA.
To produce single-stranded RNA, the plasmids were linearized with the corresponding restriction enzyme and transcribed with SP6 or T7 RNA polymerase. Radiolabeled RNA was produced according to the manufacturer’s recommendations (Promega Biotech, Madison, Wisconsin) with 32P-CTP (CTP that is attached to radiolabeled phosphate) (109cpm/μg; Amersham Iberica, Madrid, Spain).
The neutrophils were incubated with TNF-alpha (500 U/ml) for different periods. Then the neutrophils were immediately centrifuged, washed twice in ice-cold phosphate-buffered saline, resuspended in hypotonic buffer (25 mmol/liter Tris-hydrochloride [pH 7.9], 0.5 mmol/liter EDTA and 1 mmol/liter phenilmethylsulfonyl fluoride [PMSF] and lysed by four cycles of freezing and thawing, followed by centrifugation at 12,000 gat 4°C for 15 min. The supernatant was removed, supplemented with glycerol (10% final concentration) and frozen at −80°C until use. As previously reported (14), cytoplasmic lysates of human neutrophils (10 μg) were incubated with different radiolabeled RNA probes (5 to 10 × 104cpm, ∼1 ng RNA) in 15 mmol/liter HEPES (pH 7.9), 10 mmol/liter KCl, 5 mmol/liter Cl2Mg, 1 mmol/liter dithidthretrol (DTT), 1 μg/μl yeast tRNA (transfer RNA), 40 U RNAsin (Promega Biotech, Madison, Wisconsin) and 10% glycerol in a total volume of 15 μl for 10 min at 25°C. Afterwards, 20 U of RNase T1 per reaction (Gibco-BRL, Eggenstein, Germany) was added, and the reaction mixtures were incubated for 30 min at 37°C. The samples were electrophoresed on 4% native polyacrylamide gel, dried and autoradiographed with Kodak X-OMAT-S film.
Eight consecutive patients (7 men and 1 woman) with AMI admitted in the Coronary Care Unit and Emergency Unit of Fundación Jiménez Dı́az were investigated. Acute MI was diagnosed on the basis of the classic criteria of prolonged chest pain accompanied by serial changes on the standard 12-lead electrocardiogram, as well as raised serum creatine kinase (CK) (more than twice the upper limit of the normal value measured in the biochemistry laboratory) and CK-MB (>10% total CK). The mean age of the patients was 57 ± 4 years (range 39 to 80). None of the patients was taking any anti-inflammatory agent, except for aspirin (200 mg/day). The control group included eight volunteers from the hospital staff (mean age 50 ± 6 years [range 33 to 70]).
The criteria for enrollment was hospital admission within 24 h after the onset of chest pain. The criteria for exclusion were a previous episode of acute coronary syndrome and previous treatment with statins. The present study did not include patients or control subjects with a history of neoplastic, infectious or autoimmune disease or any surgical procedure within the preceding six months. All subjects gave fully informed consent, and the study was approved by the Ethics Committee of Fundación Jiménez Dı́az.
Blood was obtained on hospital admission, and the neutrophils were isolated and immediately lysed and frozen at −80°C for determination of the level of eNOS expression and the presence of cytosolic proteins that bind to the 3′-UTR of eNOS mRNA.
Determinations of TNF-alpha
Tumor necrosis factor-alpha was measured in plasma obtained from volunteers and patients with AMI by using an enzyme-linked immunosorbent assay (ELISA) kit (Quantikine HS human TNF-alpha, R&D Systems, Oxon, United Kingdom). The intra- and interassay variabilities of the ELISA kit were 14.4% and 18.7%, respectively. The sensitivity of this kit is <0.18 pg/ml.
Unless otherwise stated, each study was performed in a minimum of six different experiments. Densitometric results of both Northern and Western blots were expressed as the mean value ± SEM. Comparisons were performed by analysis of variance. The Bonferroni correction for multiple comparisons was used to determine the level of significance. A p value <0.05 was considered statistically significant.
Expression of eNOS in human neutrophils: effect of TNF-alpha
Western blot analysis demonstrated the presence of eNOS protein in human neutrophils (Fig. 2). Tumor necrosis factor-alpha reduced eNOS protein expression in the isolated neutrophils in a time-dependent manner (Fig. 2). The maximal decrease in eNOS protein expression was observed 8 h after TNF-alpha incubation (Fig. 2). At this time, the percent neutrophil viability was 86 ± 3%.
Northern blot analysis demonstrated the presence of eNOS mRNA in human neutrophils (Fig. 3A). The post-transcriptional regulation of eNOS mRNA was determined in the presence of the transcriptional inhibitor DRB. Tumor necrosis factor-alpha decreased the levels of eNOS mRNA in a time-dependent manner in the presence of the transcriptional inhibitor DRB (60 μmol/liter), indicating that the effect of TNF-alpha on eNOS mRNA expression was dependent on mRNA destabilization (Fig. 3, A and B).
Binding of neutrophil cytosolic proteins to the 3′-UTR of eNOS mRNA
We first examined the ability of human neutrophil cytoplasmic extracts to bind to a probe corresponding to the entire 3′-UTR by using an RNA gel mobility shift assay. Addition of human neutrophil cytosolic extracts to the labeled UTR-L probe resulted in a gel-shift band (Fig. 4A, lane 1). An excess of unlabeled UTR-L (1,000 ng) prevented the interaction between labeled UTR-L and neutrophil cytosolic extracts (Fig. 4A, lane 2).
Analysis of the nucleotide sequence of the 3′-UTR of eNOS mRNA showed two differentiated regions in its 3′ terminal: an uridine plus cytidine (U + C)-rich region and an adenine plus uridine (A + U)-rich region with two AUUUA pentamers.
The complex between labeled UTR-L and the neutrophil cytosolic extracts was removed by an excess of unlabeled UTR-UC probes, but not by unlabeled UTR-AU RNA (Fig. 4A, lanes 3 and 4). The level of prevention observed with unlabeled UTR-UC was very similar to that obtained when the unlabeled UTR-L RNA was the competitor, suggesting that no additional binding site outside the UTR-UC was present in the 3′-UTR of eNOS mRNA (Fig. 4A, lanes 2 and 3).
Treatment of neutrophil cytosolic extracts with proteinase K (87 μg/ml) before their incubation with labeled UTR-L abolished the complex formation, thus indicating the involvement of cytosolic proteins in it (Fig. 4B).
Tumor necrosis factor-alpha–induced changes in the binding activity of human neutrophil cytosolic proteins to the 3′-UTR of eNOS mRNA
Cytosolic extracts obtained from TNF-alpha-stimulated human neutrophils showed an increased time-dependent binding to the labeled UTR-UC probe (Fig. 5A, lanes 2 to 5). A marked increase in complex formation was observed after 4 h, and maximal activity after 8 h of TNF-alpha incubation (Fig 5A, lanes 2 to 5). Tumor necrosis factor-alpha incubation of human neutrophils for >8 h did not demonstrate any further increase in complex formation.
Complex formation between UTR-UC and the cytosolic proteins obtained from TNF-alpha–incubated neutrophils was prevented by increased concentrations of unlabeled UTR-UC (Fig. 5B). Incubation of the cytosolic extracts obtained from TNF-alpha–stimulated neutrophils with proteinase K also abolished complex formation with the UTR-UC probe (data not shown).
Effect of simvastatin on the binding activity of cytosolic proteins to eNOS mRNA
Human neutrophils were stimulated with TNF-alpha (500 U/ml) for 8 h in the presence and absence of increasing doses of simvastatin. As shown in Figure 6, simvastatin reduced the TNF-alpha–stimulated binding activity of neutrophil cytosolic proteins to labeled UTR-UC. The effect of simvastatin occurred in a dose-dependent manner (Fig. 6). A slight, although significant, decrease of cytosolic protein binding activity was observed at 10−7mol/liter of simvastatin. Doses of simvastatin <10−7mol/liter failed to affect the TNF-alpha–stimulated binding activity of the neutrophil cytosolic proteins to eNOS mRNA (data not shown). The maximal inhibition of simvastatin on cytosolic protein binding activity was reached at 10−5mol/liter (Fig. 6). It is noteworthy that treatment with 10−5mol/liter of simvastatin not only reversed the TNF-alpha–stimulated binding activity of the cytosolic proteins to eNOS mRNA, but also caused a decrease in binding activity with respect to that found at baseline (not stimulated with TNF-alpha) (Fig. 6).
As occurred with the binding of the cytosolic proteins, the presence of simvastatin (10−5mol/liter) resulted not only in a reversal of eNOS downregulation by TNF-alpha, but also in a significant increase in eNOS protein levels above baseline (Fig. 7).
Endothelial-type NOS expression in neutrophils from patients with AMI
Table 1shows the clinical features of the patients with AMI and control volunteers. Neutrophils from patients with AMI showed a decreased expression of eNOS protein, as compared with neutrophils from control volunteers (Fig. 8A). Furthermore, in six patients with AMI, the expression of eNOS protein was almost undetectable in their neutrophils. The reduction in the expression of eNOS protein was associated with a greater increase (about threefold) in the complexes formed between the neutrophil cytosolic extracts and the labeled UTR-UC probe in the patients with AMI as compared with the control volunteers (Fig. 8B). The interaction between UTR-UC and the cytosolic extracts of neutrophils from patients with AMI was prevented by an excess of unlabeled UTR-UC (1,000 ng) and by previous incubation with proteinase K (Fig. 8B). It is noteworthy that, although some of the control subjects had risk factors such as cigarette smoking, hypertension and hypercholesterolemia, in all cases, their neutrophils showed a higher expression of eNOS protein and a reduced presence of neutrophil cytosolic proteins, as compared with the neutrophils from patients with AMI. However, the plasma levels of TNF-alpha in the patients with AMI were not significantly different from those found in the control volunteers (4.0 ± 1.4 vs. 4.5 ± 1.8 pmol/ml; p = NS).
Our study has shown that human neutrophils express the eNOS isoform, and that it is reduced by TNF-alpha. We have also shown that the cytosol of human neutrophils contains proteins that interact with a specific sequence within the 3′-UTR of eNOS mRNA. The binding activity of these cytosolic proteins was markedly enhanced by the stimulation of neutrophils with TNF-alpha.
Endothelial-type NOS expression in human neutrophils: regulation by TNF-alpha
Functional studies have demonstrated the ability of neutrophils to produce NO through constitutively expressed NOS (15,16,24,25). We have demonstrated, at the molecular level, the presence of an endothelial-like NOS in human neutrophils.
Expression of eNOS has been identified not only in the endothelium, but also in other blood cells, including monocytes and macrophages (26,27). The purity of our neutrophil preparations excludes the possibility of the presence of eNOS from contaminating macrophages.
In contrast with our results, Amin et al. (28)recently reported that human neutrophils constitutively expressed the iNOS isoform. Furthermore, Yan et al. (29)could not find detectable amounts of eNOS in neutrophils. The presence of iNOS expression and the lack of eNOS expression reported in the aforementioned studies could be due to the presence of endotoxins contained in the incubation medium, because these investigators did not report the use of endotoxin-free conditions in their experiments. In this regard, we recently reported a downregulation of eNOS expression in conditions in which iNOS protein is upregulated (30). In our study, all of the experimental procedures were performed under sterile and endotoxin-free conditions.
Expression of eNOS mRNA was found to be decreased after TNF-alpha stimulation in the presence of DRB, a transcriptional inhibitor (31), strongly suggesting that a change in stability of the eNOS message is the predominant mechanism for the downregulation of eNOS expression by TNF-alpha.
Several proteins that bind to specific sequences in the 3′-UTR of many mRNAs have been implicated in the regulation of their half-lives (12,13). In a previous study, we have demonstrated that bovine endothelial cytosolic proteins interact with the 3′-UTR of eNOS mRNA (14). In the present study, complexes were formed between the cytosolic lysates of human neutrophils and the complete in vitro–transcribed 3′-UTR of eNOS mRNA. The proteinase K preincubation assay demonstrated that the neutrophil cytosolic components that bind to the 3′-UTR of eNOS mRNA were proteins. In addition, the gel-shifted analysis demonstrated that the cytosolic proteins bind to an U + C–rich region in this 3′-UTR of eNOS mRNA. In this regard, although the AUUUA pentamer is a common sequence that binds proteins within the 3′-UTR, different studies have demonstrated proteins that interact with the 3′-UTR of mRNAs independently of these AUUUA sequences (32).
Our next aim was to analyze whether the binding of neutrophil cytosolic extracts to the 3′-UTR of eNOS mRNA could be modified by TNF-alpha. Cytosolic extracts obtained from TNF-alpha–stimulated human neutrophils showed increased binding to the U + C–rich region of the 3′-UTR of eNOS mRNA, an effect that was associated with a decreased level of eNOS protein and eNOS mRNA.
Simvastatin and eNOS protein expression in neutrophils
In an attempt to study the correlation between the binding of neutrophil cytosolic proteins to eNOS mRNA and the level of expression of the eNOS protein, we performed experiments in the presence of simvastatin, a drug that has been previously reported to stabilize eNOS mRNA in the endothelium (10,11). Simvastatin prevented the binding of neutrophil cytosolic proteins to eNOS mRNA stimulated by TNF-alpha. This was accompanied by an increased expression of eNOS protein, suggesting an association between the binding of cytosolic proteins to the 3′-UTR of eNOS mRNA and the decrease in eNOS protein expression.
The levels of simvastatin that prevented eNOS protein expression in TNF-alpha–stimulated neutrophils are within the range of the expected tissue levels obtained with recommended doses (33). It is noteworthy that the binding region of the neutrophil cytosolic proteins flanks a potential stem-loop structure in the 3′-UTR of eNOS mRNA. Therefore, the binding of these cytosolic proteins to the 3′-UTR may modify the tri-dimensional conformation of this region, showing a RNase active site. A similar mechanism has been described for iron-regulated transferrin receptor mRNA stability (34).
Reduction of eNOS protein expression in patients with AMI
To analyze the in vivo reproduction of the in vitro findings, we obtained neutrophils from patients with AMI. Neutrophils obtained from patients with AMI showed a significant reduction of eNOS protein, accompanied by increased binding of the cytosolic extracts to the 3′-UTR of eNOS mRNA, suggesting a relationship between these two phenomena.
Interestingly, the expression of eNOS protein in neutrophils from patients with AMI was markedly lower than that observed with in vitro TNF-alpha incubation. In this regard, we did not find modifications in the TNF-alpha plasma levels between patients with AMI and control volunteers, as previously reported (19,35). However, although we recognize that the number of the study subjects could be not enough to find such differences, Guillén et al. (36)have also reported that other cytokines (i.e., interleukin-1-beta and interleukin-6), but not TNF-alpha, are increased during the early phase of myocardial infarction. However, we could not rule out the possibility that other cytokines generated during AMI (35,37), in addition to TNF-alpha, could contribute to the reduction in eNOS expression in neutrophils.
Impairment in the production of endothelium-derived NO has been extensively described in patients with acute coronary syndromes (38,39), but the expression of constitutively expressed NOS in neutrophils of patients with AMI had not been determined. Nitric oxide produced in neutrophils through constitutively expressed NOS appears to have a relevant role in inhibiting platelet activation and leukocyte adhesion to the endothelium (6,17,18). In addition, infusion of either organic NO donors or NO itself provides cardioprotective effects related to the inhibition of neutrophil activity (40,41). In light of this new information, further studies are needed to analyze the actual relevance of the decreased eNOS protein expression in neutrophils for the detrimental role currently conferred to these cells in the setting of AMI.
The present results demonstrate that human neutrophils express eNOS, which is downregulated by TNF-alpha and during AMI. This effect was associated with increased binding activity of neutrophil cytosolic proteins to the 3′-UTR of eNOS mRNA. The protection of eNOS expression in neutrophils may be a suitable target for therapy.
We thank Edita Martı́n and Luz Marina Calle-Lombana for their assistance in patient recruitment and MaBegoña Ibarra and Begoña Larrea for their secretarial assistance.
☆ This work was supported by grants from Fondo de Investigaciones Sanitarias de la Seguridad Social (FISS 99/0117), Fundación Mapfre Medicina, Fundación Ramón Areces, Sociedad Española de Cardiologı́a, and from a school grant from Laboratorios Merck, Sharp and Dohme.
- acute myocardial infarction
- complementary deoxyribonucleic acid
- creatine kinase
- endothelial-type nitric oxide synthase
- messenger ribonucleic acid
- nitric oxide
- nitric oxide synthase
- tumor necrosis factor-alpha
- 3′-untranslated region
- Received May 11, 2000.
- Revision received September 29, 2000.
- Accepted November 10, 2000.
- American College of Cardiology
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