Influence of isoprostane F2{alpha}-III on reflow after myocardial infarction

Kim Greavesa, Simon R Dixonb, Ivan O'Brien Cokerc, Anthony I Malletc, Metin Vkiranb, Michael J Shattocka, Martin J Fejkab, William W O'Neillb, Roxy Seniord, Simon Redwooda and Michael S Marbera,*

a Department of Cardiology, The Cardiovascular Division, King's College London, St. Thomas' Hospital, London, UK
b Division of Cardiology, William Beaumont Hospital, MI, USA
c Department of Biochemistry, University of Greenwich, London, UK
d Northwick Park Hospital, London, UK

Received June 12, 2003; revised February 26, 2004; accepted March 11, 2004 * Corresponding author. Tel.: +44-207-922-8191; fax: +44-207-960-5659
E-mail address: mike.marber{at}kcl.ac.uk


    Abstract
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 Abstract
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 Methods
 Animal experiments
 Statistical analysis
 Results
 Animal experiments
 Discussion
 Conclusions
 References
 
Aims To investigate whether the vasoconstrictor isoprostane -III (iP-III), released during myocardial reperfusion, contributes to the low/no reflow phenomenon observed following acute myocardial infarction (AMI).

Methods and results Thirteen patients undergoing primary percutaneous coronary intervention (PCI) for AMI had iP-III measured by high-performance liquid and gas chromatography–mass spectrometry. Isoprostane -III concentrations were significantly higher following PCI than in controls (1.5±1.3 vs.16±0.06 nM, ). Mean iP-III concentration correlated positively with ST-segment resolution at 90 min (, ). In the isolated murine heart: (a) coronary vasoconstriction occurred at, or above, iP-III concentrations of 1 µM. From 1 to 10 µM, iP-III induced dose-dependent vasoconstriction () with reduction in coronary flows (f) of 57±5% and 31±4% (percentage baseline), respectively; (b) SQ29548 1 µM completely reversed the vasoconstrictive effects of iP-III 10 µM; (c) SQ29548 1 µM infused during reperfusion following 30 min ischaemia had no effect on CF or infarct volume.

Conclusion Concentrations of iP-III released into the venous circulation during reperfusion following AMI in humans are significantly lower than those required to diminish coronary flow in the murine heart; increased levels indicate successful reperfusion. Inhibition of iP-III has no effect on coronary flow or infarct size in the murine heart, suggesting that iP-III alone does not account for the low/no reflow phenomenon observed following AMI.

Key Words: Myocardial infarction • Isoprostanes • Reperfusion


    Introduction
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 Abstract
 Introduction
 Methods
 Animal experiments
 Statistical analysis
 Results
 Animal experiments
 Discussion
 Conclusions
 References
 
The primary goal of reperfusion therapy is to achieve early, complete, and sustained flow in the infarct vessel.1 However, there is growing acceptance that a substantial proportion of these patients (up to 40%) have significant impairment of reperfusion at a microvascular level despite the restoration of normal antegrade angiographic flow, which has been associated with impaired recovery of segmental and global ventricular function.2,3 Nevertheless, attempts to improve microvascular reperfusion have at best been only partly successful since the mechanisms involved are not fully understood.4

Recent interest has focused on a member of the isoprostane family, isoprostane -III (iP-III), also named 15-F2t-IsoP,5 as it has been demonstrated to possess vasoconstrictor activity not found in the other, more abundant, type VI isoprostanes.6–8 Isoprostane -III is produced by the free radical-mediated oxidation of unsaturated fatty acids in membrane phospholipids during conditions of oxidative stress, such as the ischaemia and reperfusion phases of acute myocardial infarction (AMI).9 Several studies have shown that iP-III is elevated in patients with unstable angina and may reach concentrations several-fold greater than normal during the reperfusion phase of AMI.9–11 Such increases in iP-III were originally considered merely as a sensitive and specific index of oxidative stress. However, the demonstration that iP-III is not inert but has potent vasoconstrictor properties led to suggestions that it may contribute to the impaired quality of reperfusion following AMI.5,10,12–14 Despite clinical studies demonstrating a temporal association between iP-III release and reperfusion, no attempt has yet been made to quantitatively correlate release with the quality of reperfusion following AMI. Furthermore, controversy exists as to whether iP-III levels in AMI reach physiologically relevant concentrations.15

The purpose of this study was to measure the concentration of iP -III produced during reperfusion in patients undergoing primary percutaneous intervention (PCI) for AMI and to examine the relationship between iP-III release and the quality of myocardial perfusion. Based on these initial clinical findings, we then further examined the role of iP-III in an isolated perfused heart model.


    Methods
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 Abstract
 Introduction
 Methods
 Animal experiments
 Statistical analysis
 Results
 Animal experiments
 Discussion
 Conclusions
 References
 
Human experiments
Materials
Indomethacin and butylated hydroxy-toluene were obtained from Sigma–Aldrich (UK). Isoprostane -III and SQ29548 were obtained from Cayman Chemicals (Ann Arbor, MI, USA).

Isoprostane -III analysis
Blood (5 mL) was collected in a tube with 3.8% trisodium citrate (blood/anticoagulant ratio, 9:1). Both blood and mouse coronary effluent samples were immediately mixed with butylated hydroxy-toluene (final concentration 20 µM) and indomethacin (final concentration 15 µM). Platelet-poor plasma was prepared by centrifugation at 2400g at 4 °C for 15 min. Samples were then stored at –70 °C for subsequent analysis (within 3 months). Total (free and esterified) iP-III quantification was carried out using high-performance liquid chromatography and gas chromatography, as described by Gopaul et al.16 The plasma purification process was modified as described by Walter et al.16,17 to omit solid-phase extraction. Peripheral blood samples from 10 female healthy volunteers (nonsmokers, normotensives, and nondiabetics), age 30–50 years, were used as controls.

Isoprostane -III concentration during AMI
Between January and July 2001, 13 patients with AMI undergoing primary PCI were enrolled in this prospective dual-centre study. All patients presented within 12 h of symptom onset with chest pain and ST-segment elevation. Exclusion criteria were: significant left main stenosis (50%), myocardial infarction within the previous month, pretreatment with thrombolytic agents, cardiogenic shock, and pregnancy. The study was approved by the local institutional review boards and written informed consent was obtained from each patient before enrolment.

Before catheterisation, all patients received low-flow nasal oxygen, aspirin 300 mg orally, and heparin 5000 U intravenously. The infarct-related artery was identified by the site of coronary occlusion, localisation of the electrocardiographic findings, and analysis of the wall motion defect. Heparin was given to keep activated partial thromboplastin time over 250 s. Stent deployment and use of glycoprotein receptor antagonists were at the physician's discretion.

Peripheral blood samples were drawn for iP-III analysis at 15, 30, 45, and 60 min following the first balloon inflation, and for creatine kinase at presentation and 4, 8, 12, 16, 24, 36, 48, and 72 h after admission. The sum of ST elevation was assessed in three contiguous leads in the infarct zone, 60 ms from the point, in the presenting and 90-min ECG. The extent of ST-segment resolution was then expressed as a percentage of the ST elevation evident on the presenting ECG. Quantitative angiography was performed using an automated edge-detection system (CAAS II, Pie Medical, Maastricht, Netherlands) by a single observer blinded to clinical details and outcome. The patency of the infarct-related artery was classified according to thrombolysis in myocardial infarction (TIMI) criteria. The corrected TIMI frame count (cTFC) and myocardial blush grade (MBG) were assessed using previously defined techniques.18,19


    Animal experiments
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 Methods
 Animal experiments
 Statistical analysis
 Results
 Animal experiments
 Discussion
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Manipulation of isoprostane -III during reperfusion
All experiments were performed in accordance with the United Kingdom Home Office Guidance on the Operation of Animals (Scientific Procedures) Act 1986, published by Her Majesty's Stationary Office, London.

Animals were male mice (T/O, Harlan, Bicester, UK), age 8–12 weeks, anaesthetised with ketamine/xylazine (150 and 24 mg/kg, respectively, with 100 IU heparin) injected intraperitoneally. Hearts were harvested and perfused as we have described previously.20 After retrograde perfusion commenced, hearts were stabilised for 30 min. For inclusion in the study, all hearts had to fulfil the following criteria: coronary flow 1.5–4.5 mL/min, heart rate 300 beats/minute (unpaced), left ventricular developed pressure 50 mm Hg, time from thoracotomy to aortic cannulation 3 min, and no persistent dysrhythmia (during 30-min stabilisation). A total of 31 mice were required and 2 were excluded because they did not meet inclusion criteria. Both iP-III and SQ29548 were dissolved in ethanol and subsequently diluted in Krebs solution to a final concentration of 0.1% ethanol. Depending on the protocol, mouse hearts were then entered into one of the following protocols:

Protocol 1. Establishment of the dose-response relationship between iP-III and coronary flow. Following stabilisation, perfusate containing iP-III was infused at concentrations that increased stepwise every 6 min from 1 nM to 10 µM (). Due to an addition to the original protocol, 4 and 3 out of the 7 mice also received concentrations of 0.1 nM and 100 µM, respectively.

Protocol 2. Complete inhibition of the effects of iP-III with the TXA2 antagonist SQ29548. We assessed the concentration SQ29548 required to completely reverse the effects of iP-III. Following stabilisation, hearts (n = 4) were infused with iP-III 10 µM to induce coronary preconstriction before SQ29548 was infused at an escalating concentration.

Protocol 3. Myocardial infarction with inhibition of the effects of iP-III during reperfusion using SQ29548. Following stabilisation, hearts were subjected to 30 min of global ischaemia and were then randomly assigned to reperfusion with either vehicle (), or SQ29548 10 µM (), the concentration found to reverse the coronary constriction effects of iP-III in protocol 2. A set of previous experiments comparing 0.1% ethanol in Krebs solution and Krebs alone had demonstrated that 0.1% ethanol had no effect on infarct size or coronary flow.

Mouse infarct size assessment
The assessment of infarction volumes as a percentage of left ventricular volume was performed as we have described previously.20 Briefly, this involved perfusion of hearts for one minute with 5 mL of 1% triphenyl-tetrazolium chloride (TTC) in phosphate buffer 1.5 h following reperfusion. Hearts were placed in TTC solution for 10 min. Atria were removed and the hearts were stored at –20 °C for up to 1 week. After thawing, hearts were placed in 2.5% glutaraldehyde for 1 min and set in 5% agarose solution. The hearts were then sectioned into 0.7-mm slices. Slices were placed overnight in 10% formaldehyde before transferring to phosphate-buffered saline for 2 days. Sections were compressed between Perspex plates (0.57 mm apart) and imaged using a TK-1280E digital camera (JVC). After magnification, planimetry was carried out using image analysis software (NIH Image 1.61) and infarct size was expressed as a percentage of total heart volume.


    Statistical analysis
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Statistical analysis was performed using Sigmastat software (SPSS Science, Chicago, IL, USA). Text and table results, unless stated otherwise, are expressed as means±SD, whereas graphic results, unless stated otherwise, are expressed as means±SE. The correlation between different groups and isoprostane -III levels was assessed using a Pearson product moment correlation matrix. A mean isoprostane -III concentration value per patient was calculated consisting of levels measured at 15, 30, 45, and 60 min post-PCI and used in the correlation matrix. The mean value was thought to best represent overall isoprostane -III release following PCI. Values changing over time within the same group were compared using a paired t-test. A -value <=0.05 was considered statistically significant. Where multiple measurements were made (protocol 1), between-group comparison was performed with multivariate repeated-measures analysis of variance (ANOVA), using drug dosage as a within-subject factor and group membership as a between-subject factor. The interaction effect between dose and group membership tests the hypothesis that the dose-response curves differ between the two groups. Where the interaction was found to be statistically significant, a further analysis was undertaken to compare the two groups at each dosage using the independent-samples -test and a Bonferroni corrected critical -value of .01 to allow for multiple-hypothesis testing.


    Results
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Human experiments
Isoprostane -III and myocardial perfusion in AMI
Thirteen patients underwent primary PCI and their clinical characteristics are shown in Table 1. Stents were deployed in 11/13 patients. Most patients received a glycoprotein IIb/IIIa receptor antagonist (abciximab=7, eptifibatide=4). All patients had a postprocedure TIMI 3 flow with a mean cTFC of 29±10 () and mean MBG of 1.9±1.5 (). The mean peak CK was 1928±2015 IU (range 273–5715 IU). One patient died due to cardiogenic shock 23 h following PCI.


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Table 1 Clinical characteristics of patients undergoing PCI for AMI

 
The mean peak peripheral venous iP-III concentration at 15 min was significantly higher in AMI patients than in controls (1.5±1.3 nM vs.16±0.06 nM, ) (Fig. 1). There was a significant decrease from the 15- to 60-min iP-III concentration after the first balloon inflation (1.5±1.3 nM vs. 0.95±5.1 nM, ). In order to allow comparison with peripheral blood concentrations, simultaneous coronary sinus sampling was undertaken in two patients. The mean peak iP-III coronary sinus and peripheral venous concentrations in these two patients were 1.9±0.06 and 2.1±0.9 nM, respectively (NS). ECGs were recorded post-PCI in 12 patients at 98±12 min with a mean ST resolution of 73±22%.



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Fig. 1 Release profile of isoprostane -III (iP) in peripheral venous blood following PCI. Healthy control values differ significantly from peak values (). The 15- and 60-min iP concentrations differed significantly (*, paired -test). Values represent mean and SE.

 
The Pearson correlation matrix in Table 2 demonstrates a significant positive correlation between mean iP-III release and percent ST-segment resolution at 90 min (). Similarly, significant positive correlations were also found between iP-III concentration and ST-segment resolutions at the 30-, 45-, and 60-min time points (see Fig. 2). To further explore these counterintuitive results, we investigated the role of iP-III in a murine model of AMI.


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Table 2 Pearson correlation matrix showing correlation coefficients ( values) between individual mean isoprostane -III concentrations and various assessments of reperfusion

 


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Fig. 2 Correlation (, ) of 30-min isoprostane -III (iP) concentration with percentage ST-segment resolution at 90 min in patients () who have undergone primary PCI for AMI.

 

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 Statistical analysis
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Role of isoprostane -III during AMI in mice
In preliminary experiments of global ischaemia using retrogradely-perfused mouse hearts, we found that quantifiable iP-III concentrations in the perfusate after 30 min ischaemia were low, with a mean value of 20±17 pM. These levels, approximately two orders of magnitude less than those in patients, were probably due to the combined effects of the lack of recirculation and high coronary flow rate. These findings are consistent with the possibility that higher local concentrations of iP-III may play a role in impaired reflow post-MI, which we further explored in this model.

Protocol 1. Establishment of the dose-response relationship between iP-III and coronary flow. Fig. 3 demonstrates the effects of iP-III infusion versus vehicle (0.1% ethanol) on coronary flow in the isolated mouse heart. No significant coronary vasoconstriction occurs at a concentration of 0.1 µM or lower, but iP-III at concentrations of 1 µM and 10 µM produced significant flow reductions of (means±SE) 57±5% and 31±4% of basal flow, respectively (). Isoprostane -III infusion at 100 µM continued to produce a further 27±7% reduction in coronary flow, but this was not subjected to statistical comparison since only three out of seven mice received this concentration. On reintroduction of iP-III-free K–H perfusate, coronary flows returned to their baseline values, suggesting that the effects of iP-III are reversible.



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Fig. 3 Effect on mouse coronary flow of increasing concentrations of isoprostane -III. Continuous line represents isoprostane -III (iP) infusion alone; dotted line (control) represents time-matched perfusion with vehicle (Krebs–Henseleit buffer containing 0.1% ethanol); %CF, coronary flow expressed as percentage of the baseline flow with mean and SE bars. A multivariate repeated-measures ANOVA compared the infusion group (from 1 nM to 10 µM) with controls and demonstrated a significant difference (). An independent samples t-test identified 1 and 10 µM as the drug infusion groups that differed significantly from controls ().

 
Protocol 2. The concentration of SQ29548 required to inhibit the effects of iP-III. The vasoconstrictive effects of iP-III are thought to be mediated via the thromboxane-A2 (TXA2) receptor.12 We therefore attempted to block the vasoconstrictive effects of iP-III with SQ29548, a TXA2 receptor antagonist. Fig. 4 shows the effects on mouse coronary flow of the infusion of an increasing concentration of SQ29548 on a background of a vasoconstrictive concentration of iP-III (10 µM). This concentration was chosen as equivalent to a putative vasoconstrictive concentration achieved locally during myocardial infarction. Our results demonstrate that concentrations of SQ29548 >=100 nM are able to cause a significant improvement in coronary flow (between M and M SQ 29548) with complete reversal of the effects of iP-III 10 µM despite the fact that this did not increase coronary flow in the absence of exogenous iP-III (see Table 3).



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Fig. 4 The effect on mouse coronary flow of infusing an increasing concentration of SQ29548 on the background of a constant concentration of 10 µM isoprostane -III (). K–H, Krebs-Henseleit buffer; iP, isoprostane -III. %CF, coronary flow expressed as a percentage of baseline. Values expressed as mean and SE. * (difference between x10–8 M and x10–7 M SQ 29548 on coronary flow).

 

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Table 3 Characteristics of isolated mouse hearts subjected to 30 min of ischaemia and reperfusion with vehicle (Krebs–Henseleit), or vehicle+SQ29548 1 µM

 
Protocol 3. Inhibition of iP-III during reperfusion with SQ29548. Table 3 shows the morphometric, baseline, and postinfarction parameters of isolated mouse hearts subjected to 30 min of ischaemia followed by reperfusion with () or without () SQ29548 (1 µM). The results show that inhibition of iP-III with SQ29548 had no significant effect on infarct size, coronary flow, or developed pressure.


    Discussion
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We tested the hypothesis that isoprostane -III, a vasoconstrictor produced endogenously after myocardial ischaemia/reperfusion, contributes to the low reflow phenomenon of AMI. This hypothesis is consistent with a body of previous work10,12–14 and the observation that vasodilator drugs such as nicorandil and verapamil improve tissue microvascular flow following AMI in some studies.21,22 Our results have shown that, in humans, iP-III is released during the reperfusion phase of AMI and that the maximal concentrations reached in the peripheral venous circulation are in the nanomolar range. Furthermore, contrary to being a marker of poor reperfusion, elevated iP-III concentrations measured after AMI in humans positively and significantly correlate with successful ST-segment resolution. In conjunction, in the murine heart, we have demonstrated that considerably higher concentrations of iP-III are required to cause coronary vasoconstriction and that such values are in the micromolar range. We have also shown that pharmacologic antagonism of the effects of endogenously produced iP-III in isolated perfused mouse hearts failed to improve the low coronary reflow observed after AMI.

Clinical studies thus far have only shown a temporal association between iP-III release and reperfusion.11,14,23 No attempt has been made to quantitatively relate iP-III concentration to the quality of reperfusion following AMI. Controversy exists as to whether the concentrations of iP-III achieved following AMI are high enough to have any significant biologically adverse effect.15 In ex vivo animal and human studies, the EC50 of iP-III for coronary and conductance vessel constriction is in the micromolar range, whereas the maximum concentrations measured in peripheral venous blood during acute coronary syndromes are in the nanomolar range.10,11 However, several factors confound such cross-study comparisons. First, only five studies have measured iP-III following acute coronary syndromes, and two of these relied upon urinary rather than plasma measurement.10,11,14,23,24 Second, no standardised and accepted technique exists for the measurement of iP-III. The methods most commonly used are either enzyme immunoassay or gas chromatography-mass spectrometry.25 These techniques, when tested against the same samples, have been shown to significantly differ in their results by up to 30%.26,27

Interpretation of the results of this study requires a number of factors to be taken into consideration. First, local concentrations of iP-III may be considerably higher than that measured in the peripheral venous circulation, so the possibility exists that iP-III levels may be sufficient to cause coronary vasoconstriction. To overcome this problem, we undertook both coronary sinus and peripheral venous sampling in two of our patients, and found them to be the very similar. In support of this, our coronary sinus concentrations are of the same magnitude as that measured by Kijima et al.,11 who measured iP-III levels in blood taken directly from the great cardiac vein during the reperfusion phase of AMI. In spite of this, the authors acknowledge that it is not possible to be certain that coronary sinus concentrations are the same as in the local microcirculation.

Although the isolated mouse heart is an excellent model that allows quantification of the direct effects of iP-III on the coronary arteries, it is unable to take into account the additional effects of platelets which may contribute to the no reflow phenomenon. Isoprostane -III alone has been shown to cause platelet aggregation in vitro in concentrations ranging from 10 to 100 µM, but, when in the presence of a pro-thrombotic milieu, for example with collagen or ADP, the concentration is much lower at 10 nM.28 This illustrates that our study has concentrated on a single isomer whereas a myriad are known to be released during reperfusion. Therefore, although iP-III may not reach sufficient concentrations to cause an effect independently, its presence may amplify the effect of other inflammatory editors.

Our findings complement those of Kromer et al.,10 whose results suggested that iP-III has a direct vasoconstrictive effect on human coronary artery rings taken from explanted hearts. In addition, they found that post-AMI circulating iP-III values were 0.1 nM, yet the in vitro concentration required to cause constriction was 100 nM.

The use of SQ29548, a TXA2 receptor antagonist, also requires some caution when interpreting the results of the study. Although all of the vascular effects of iP-III can be prevented using SQ29548, the molecular mechanism of iP-III-mediated coronary vasoconstriction has not been fully defined as binding studies have suggested that iP may not directly interact with the TXA2 receptor.7,29,30 Possibilities exist of an altered conformational state of the receptor (homo- or heterodimerisation), changes in G-protein/second messenger coupling, or even a novel non-thromboxane iP-III receptor.28,31 Therefore, until a specific iP-III antagonist becomes available, these factors and the additional confounding effect of antagonism of thromboxane, as well as that of iP-III by SQ29548, will remain problematic.

The association of iP-III with improved microvascular flow is not necessarily incongruent. For example, iP-III production is dependent on oxygen-derived free radicals which, in turn, are dependent on the availability of molecular oxygen at reperfusion.32 Thus, the hearts producing the most iP-III could also be those with the most efficient oxygenation, suggesting that iP-III is a potential indicator of tissue reperfusion. Grech et al.33 demonstrated, in 17 patients who underwent PCI for AMI, a single burst of free radical production following reperfusion. In one patient, successful PCI was immediately followed by reocclusion, which required further PCI.34 Therefore, in conjunction with our study, these data provide indirect evidence that restoration of reperfusion may be associated with a higher level of free radical production.


    Conclusions
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 Abstract
 Introduction
 Methods
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 Statistical analysis
 Results
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 Discussion
 Conclusions
 References
 
We have confirmed that iP-III is released following AMI and is a coronary vasoconstrictor. The concentrations required to cause coronary vasoconstriction in the mouse heart are three orders of magnitude greater than those measured in both the peripheral venous circulation and coronary sinus in humans after PCI for AMI. Inhibition of the effects of iP in the isolated perfused mouse heart model using SQ29548 had no effect on infarct size or coronary flow. Furthermore, the concentrations of iP-III released during reperfusion following AMI in humans appeared to indicate reoxygenation and successful reperfusion of ischaemic myocardium. Taken collectively, our results suggest that the independent actions of iP-III alone do not account for the quality of reperfusion post-AMI. The debate regarding the effects of iP-III during reflow post-AMI will only be fully closed when specific isoprostane antagonists are available for both animal and human studies.


    Footnotes
 
Funding: This study was supported by a British Heart Foundation Junior Fellowship (FS/99057).


    References
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 Abstract
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 Statistical analysis
 Results
 Animal experiments
 Discussion
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 References
 

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