Dissociation of the Inflammatory Reaction following PCI for
Acute Myocardial Infarction
Early reperfusion after coronary occlusion reduces the extent of acute myocardial infarction (AMI) as well as mortality.1 However, reperfusion itself may cause damage to surviving myocardium, the so-called “reperfusion injury”.2 Neutrophils are activated and infiltrate the myocardium following ischemia and reperfusion.3 A vast number of experimental studies suggest that neutrophils are important players in the development of irreversible reperfusion injury.4 Activated neutrophils are capable of releasing substances injurious to the myocardium. For example, proteolytic enzymes such as elastase induce the formation of reactive oxygen species (ROS) and cytokines. Myeloperoxidase (MPO), a lysosomal enzyme, found in the azurophilic or primary granules in the neutrophil leukocytes, serves as a marker of neutrophil activation. The activity of this enzyme in plasma is an established measurement for infiltration of neutrophils into ischemic myocardium.3 Neutrophil gelatinase-associated lipocalin (NGAL), located in the specific or secondary granules of neutrophils, is another plasma marker for neutrophil activation.5
Animal studies support the hypothesis that ROS produced during ischemia and reperfusion cause disturbances in cellular function.6 ROS have an extremely short half-life, between 10-6 to 10-9 seconds,7 and are therefore difficult to detect and quantify in vivo. As a result, clinical investigators have been limited to employ indirect methods to detect ROS formation. Malondialdehyde (MDA), an end-product in the lipid peroxidation chain reaction, has been the most commonly used marker. However, PMDA is not an entirely specific marker for ROS generation, as it is also formed via enzymatic reactions of prostaglandins in platelets.8 Isoprostanes are bioactive prostaglandin-like compounds that are formed in vivo directly by free-radical peroxidation of arachidonic acid and regarded as more specific markers of ROS production, Reilly et al demonstrated increased formation of isoprostanes in acute coronary angioplasty.9 The aim of this study was to investigate the inflammatory reaction and ROS formation in patients suffering from their first AMI treated with primary PCI. The serum concentrations of PMPO and S-NGAL were studied as markers of neutrophil activation. Matrix metalloproteinase-9 (S-MMP-9) was measured as a marker for the proteolytic system regulating matrix remodeling. Lipid peroxidation was investigated by measuring P-MDA and plasma isoprostanes (P-Iso-P). The cytokines interleukin-6 (P-IL-6), interleukin-8 (P-IL-8) and tumor necrosis factor α ( PTNF ·), as well as highly-sensitive C-reactive protein (S-hsCRP) were analyzed as serum or plasma markers of inflammation.
Materials and Methods
Forty-nine non-consecutive patients presenting with symptoms of their first AMI were included. The material reflected a normal AMI population. For clinical characteristics see Table 1. All subjects were hemodynamically stable. They were admitted directly from the ambulance or the emergency room to the catheterization laboratory. The inclusion criteria were ongoing ST-elevation MI diagnosed via electrocardiographic criteria, STelevation ≥ 2 mm in ≥ 2 consecutive precordial leads or ≥ 1 mm in ≥ 2 consecutive standard limb leads, ongoing or recurring chest pain with maximal duration of 12 hours, and a totally occluded infarct-related artery with TIMI 0 flow verified by the coronary angiography (TIMI = Thrombolysis in Myocardial Infarction, an established flow estimation with grades 0–3, with 0 = no flow and 3 as complete perfusion).10 The exclusion criterion for this study was thrombolysis given prior to the PCI procedure. Informed consent was obtained orally prior to the coronary angiogram, and written consent was obtained from all study subjects during the first hospital day. The study was approved by the local ethics committee and the investigation was conducted in accordance with the Helsinki Declaration.
All patients were pretreated with 300 mg acetylsalicylic acid (ASA) and 300 mg clopidogrel (Plavix® BMS/ Sanofi- Synthelabo, Bromma, Sweden) prior to the PCI procedure. All of the included patients underwent primary PCI and received at least 1 stent. They all received the platelet glycoprotein IIb/IIIa (GP IIb/IIIa) receptor inhibitor abciximab (Reopro® Lilly, Indianapolis, Indiana) administered as a bolus of 0.25 mg per kilogram of body weight during the PCI procedure, followed by a continuous 10 μg/minute infusion of the same substance for 12 hours postintervention. Unfractionated heparin was administered during the PCI procedure to achieve an activated clotting time (ACT) of approximately 250 seconds. ACT was measured using the Hemochron® system (Vingmed, Järfälla, Sweden).
Diagnostic coronary angiography for inclusion in the study was performed immediately before the PCI procedure. After identification of the culprit lesion, the PCI procedure was initiated with a 6 Fr catheter via the femoral artery approach. Reperfusion in the area of the treated infarct-related artery was confirmed by angiography at the end of the procedure with a TIMI flow grade 2–3 according to the operator’s assessment.10
During the procedure, all patients received intracoronary bolus injections of 0.2 mg nitroglycerin for vasodilation. Intravenous beta-blocker agents (Metoprolol Seloken® AstraZeneca, Södertälje, Sweden) in doses of 5–15 mg were administered to all patients as well. For analgesia and sedation, intravenous morphine-hydrochloride (Morfin®, AstraZeneca, Södertälje, Sweden), together with dixyrazin (Esucos®, UCB, Malmö, Sweden) and low doses of diazepam (Stesolid®, Alpharma, Stockholm, Sweden), were administrated to all patients. To maintain hemodynamic balance, all patients received intravenous fluids of Ringer-acetate or glucose.
Blood samples for analysis of biomarkers were obtained prior to opening the occluded infarct-related vessel, and then at 1.5, 3 and 24 hours post-reperfusion. The samples consisted of venous blood, except for the first sample, which was arterial. This sample was obtained via the arterial sheath before reperfusion of the occluded vessel (baseline). Due to technical reasons, P-MDA was analyzed in a subgroup of 29 patients. All other markers were analyzed in all 49 patients. All blood samples were obtained in tubes with ethylene diamine tetra acetic acid (EDTA), with citrate or without additives (Becton Dictinson, United Kingdom). The EDTA tubes were used for P-MDA, P-Iso-P, P-MPO and the cytokine analyses. The tubes without additives were used for the assays of S-hsCRP, S-NGAL and S-MMP-9. They were all centrifuged at 2000 x G at 4ºC, cooled and stored at -70ºC before the respective assay.
P-MDA was analyzed within a few weeks. The samples for analysis of P-MPO, P-Iso-P, the cytokines, S-hsCRP, S-NGAL and S-MMP-9 were stored for 1 to a maximum of 4 years before the analysis was performed.
Circulating levels of human plasma MPO were determined by a solid-phase, sandwich enzyme-linked immuno assay (ELISA) (OxisResearch, Portland, Oregon). S-NGAL was measured by sandwich ELISA (Antibody Shop, Gentofte, Denmark). S-MMP-9 was analyzed by the commercially available ELISA (R&D Systems, Minneapolis, Minnesota).
P-MDA was analyzed with an in-house high-performance liquid chromatography method at the Clinical Chemistry Laboratory, Lund University Hospital,8 and P-Iso-P was analyzed at the Wallenberg Laboratory, University Hospital Malmö using ELISA analysis (8-Isoprostane EIA Kit Cayman Chemical Company, Ann Arbor, Michigan). P-TNF· and P-IL- 6 were analyzed with enzyme-linked immunosorbent assay methods (R&D Systems). P-IL-8 levels were determined with a CXCL8/IL-8 Quantikine ELISA kit (R&D Systems). ShsCRP was measured by an ultra-sensitive, particle-enhanced immuno turbidimetric assay (Orion Diagnostica, Espoo, Finland) on a Konelab 20 autoanalyzer (Thermo Clinical Labsystems, Espoo, Finland). S-CKMB and S-TnT were determined by standard procedures at the Clinical Chemistry Laboratory of Lund University Hospital using an electrochemiluminescence immunoassay (Hitachi Modular-E, Roche). S-hsCRP, SMMP- 9, S-NGAL, P-IL-6, P-IL-8 and P-TNFα were analyzed at the Wallenberg Laboratory, Sahlgrenska University Hospital in S. Göteborg, Sweden.
Statistics. Statistical calculations were performed using Microsoft® Office Excel 2003 with the paired two-tailed Student’s t-test. A p-value < 0.05 was considered statistically significant.
Results
Clinical characteristics of the studied population, serum markers of AMI, the occurrence of post-infarction heart failure and duration of AMI symptoms prior to the PCI procedure are presented in Table 1. Changes in concentrations of the biomarkers from baseline (occluded vessel) to 1.5, 3 and 24 hours of the markers of neutrophil activation, inflammatory markers and markers of ROS are presented in Table 2. The mean P-MPO level decreased significantly during the observed time period. No significant changes in S-NGAL were observed, though a trend toward a decrease (p = 0.09) could be detected 3 hours post-reperfusion.
Compared to baseline, S-MMP-9 was significantly increased at 1.5 and 3 hours. A significant decrease was found in the PMDA- levels comparing baseline to 24 hours. No significant changes were seen in the P-Iso-P levels during the observation period. Early significant increases of cytokines P-IL-6 and P-IL-8 were observed, whereas a significant increase in PTNFα occurred later at 24 hours. As expected, the acute-phase reactant S-hsCRP was significantly increased at 24 hours.
Discussion
The main finding in the present study was a dissociation of the inflammatory response after primary PCI. The plasma concentrations of P-MPO and S-NGAL, two markers of neutrophil activation, decreased or remained unchanged, respectively, whereas 3 cytokines (P-IL-6, P-IL-8 and P-TNFα) and S-hsCRP, an acute-phase reactant, increased during the 24 hours of reperfusion. In addition, we showed that a marker of the proteolytic system regulating matrix remodeling (S-MMP-9) increased after primary PCI and that the markers of lipid peroxidation (P-Iso-P and P-MDA) remained unchanged or decreased, respectively.
The neutrophil activation theory. Active neutrophils have the tools (ROS, proteases) to injure the myocardium.4 Ischemia without reperfusion is associated by a slow inflow of neutrophils, peaking between 2 and 4 days, primarily to the border zone, with few neutrophils in the center of the necrosis.11 However, neutrophil infiltration and accumulation are accelerated in reperfusion, as shown in a canine model with ligated LAD and comparing the occluded vessel to 3-hour reperfusion.12
Mullane et al demonstrated that MPO activity might serve as a quantitative measurement of neutrophil infiltration into the myocardium.3 Given the time course of neutrophil infiltration following AMI, as delineated above, one would expect an early, but sustained, elevation of plasma markers for neutrophil activation. By contrast, plasma levels of MPO in the present study were highest before PCI, followed by a progressive decrease over the observed time period. It is notable that the decrease took place with a concomitant increase in 3 different cytokines known to trigger neutrophil activation. The serum levels of NGAL, a protein located in the so-called specific or secondary granules of neutrophils, also showed a nonsignificant trend toward a decrease after reperfusion. These results suggest that the activation/infiltration of neutrophils are different inpatients treated with PCI that includes abciximab therapy compared to that in patients treated with thrombolysis. Several investigators have demonstrated a rapid increase in plasma levels of elastase from the azurophil or primary granules after thrombolysis for AMI.13–15
Neutrophils, inflammation and abciximab. Vinten- Johansen discussed the failure thus far to translate the successful antineutrophil therapy in experimental conditions to clinical practice.4 He suggested several reasons for the negative results of specific antineutrophil therapies in clinical trials, one of them being that the standard adjunctive therapy of AMI may have an antineutrophil effect. Consequently, any effect of specific antineutrophil therapy in AMI would have to be in addition to that exerted by such agents as abciximab and heparin. Both unfractionated heparin and the GP IIb/IIIa inhibitor abciximab bind to the Mac-1 receptor of neutrophils. Neumann et al demonstrated that abciximab reduced platelet-leukocyte interaction and leukocyte Mac-1 surface expression,16 and the same group had previously shown that the binding of activated platelets to neutrophils induces the expression of proinflammatory cytokines, oxidative burst and increased expression of Mac-1.17 Lincoff et al described the suppression of markers for inflammation by abciximab therapy in the EPIC study.18 Our results support the notion that a PCI procedure employing heparin and abciximab results in less neutrophil activation/infiltration than thrombolysis. If PCI therapy suppresses the inflammatory reaction, our data suggest that neutrophil activation is inhibited to a larger extent than cytokine production.
It is also possible that reperfusion itself may decrease the activation/infiltration of neutrophils, despite the generally held notion of reperfusion as a trigger for neutrophil activation.12 These findings are in agreement with the results reported by Bell et al.13 They measured the uptake of 111 Inlabeled neutrophils and found less accumulation in the myocardium in patients receiving thrombolysis compared to patients who did not receive reperfusion therapy. However, the reperfusion status of the thrombolysis-treated patients was not reported.
The ROS theory. Two markers of lipid peroxidation were also studied: P-MDA and P-Iso-P. One would expect an increase in P-MDA or P-Iso-P following myocardial ischemia reperfusion, as previous experimental studies have demonstrated the presence of ROS in that condition. However, neither P-MDA nor P-Iso-P showed any significant increase following reperfusion in the present study. The reason for this may be that both markers are inadequate for the detection of free-radical formation in the myocardium or that no significant amount of ROS was formed following reperfusion. As with neutrophil markers, we have previously observed a difference in the time course of P-MDA in PCI patients compared to patients treated with thrombolysis. PMDA increased following thrombolysis,8 but decreased in patients presenting with AMI and treated with primary PCI.19 If P-MDA concentration reflects the ROS formation, our results suggest a decreased oxidative stress after PCI compared to thrombolysis. A possible explanation for this reduced oxidative stress would be that less neutrophil activation reduced the formation of ROS. An alternative explanation is that less platelet activation takes place during the PCI procedure compared to thrombolysis, resulting in less enzymatic MDA production. No significant correlation between P-MDA and P-Iso-P was found at any observed time, suggesting that they might represent different mechanisms of lipid peroxidation.
MMP-9 postinfarction. MMP-9 belongs to a family of zinc-dependent endopeptidases. It is well recognized for its proteolytic action on extracellular matrix proteins and its involvement in long-term remodeling processes. Increased MMP-9 expression in coronary plaque fragments from AMI patients have been demonstrated.20 Our finding of increased S-MMP-9 after PCI for AMI is in agreement with a previous report from Eckhart et al.21 The origin of the increased serum levels of MMP-9 in our study is unclear. MMP-9 can be derived from the myocardium, the coronary lesion or from neutrophils in circulation or infiltrating the infarcted area.
Study Limitations
The size of the population studied is small. Only 49 patients had a full observation period. In our study, no direct comparison was made of neutrophil activation between patients treated with PCI and patients given thrombolysis, and for ethical reasons, no control group of patients without reperfusion therapy was included. The duration of infarction differs from 45 minutes to 12 hours, which undoubtedly influences the inflammatory response.
Conclusion
We found a dissociation of the inflammatory reaction following PCI for AMI that included the following: (1) an increase in cytokines and CRP post-reperfusion, but (2) a decrease in P-MPO, (3) no change in S-NGAL, and (4) a decrease in P-MDA. These results are divergent from previous studies in patients given thrombolytic therapy which resulted in increased plasma levels of MDA and several markers of neutrophil activation. It is conceivable that the adjunctive therapy during PCI with abciximab and heparin has an antineutrophil effect, and merits further investigation. Acknowledgements. We extend many thanks to Maria Hansson and Barbro Palmqvist for their excellent technical assistance.
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