Acute myocardial infarction is one of the leading causes of morbidity and mortality in industrialized nations. In 2003, the prevalence of myocardial infarction in the United States was 3.5% (1). Every year, an estimated 7 million Americans will have a new or recurrent myocardial infarction. Patients' prognosis depends on the amount of myocardium that has lost function (2). Thus, therapeutic interventions aim to reduce infarct size. Current reperfusion procedures are thrombolysis, bypass surgery, and percutaneous transluminal coronary angioplasty. In Western health care systems, the time between ischemia and reperfusion has been maximally reduced. The extent of myocardial damage also depends on the reperfusion paradox, where tissue injury occurs as a result of reperfusion itself (3). This so-called reperfusion injury can be attenuated in animals using various interventions with favorable outcomes (4-6). Many experimental interventions against reperfusion injury have been tested in humans but have failed to achieve a sizable reduction in myocardial damage (7). In an effort to improve clinical outcome after myocardial infarction, the National Heart, Lung, and Blood Institute working group (7) produced a concept for cardioprotection through reduction of myocardial injury in patients undergoing reperfusion therapies. At the preclinical level, the working group identified several factors that prevented the transference of experimental results to clinical medicine. Inconsistent results were identified as the major problem. The lack of standardization, randomization, and blinding in study designs, as well as insufficient survival periods after reperfusion procedures, were also key factors (7).
We have recently identified a novel fibrin-derived peptide with cardioprotective effects in models of myocardial ischemia-reperfusion (8). This peptide consists of 28 amino acids corresponding to the N-terminal sequence of the fibrin β-chain. Its mechanism of action was recently reviewed and discussed (9). Briefly, we found that specific derivatives of fibrin, that is, E1 fragments are proinflammatory. They induce transmigration of leukocytes in vitro. By binding to endothelial cell junctions, E1 fragments induce migration through endothelial monolayers. The N-terminus of the β-chain of E1 fragments (Bβ15-42) interacts with vascular endothelial (VE)-cadherin (8), and the N-terminus of the α-chain interacts with CD11c (10). Thereby, E1 fragments build a bridge between endothelial and inflammatory cells. Consequently, peptides matching the VE-cadherin binding site (Bβ15-42) prevent bridge formation thereby reducing leukocyte transmigration in vitro and myocardial inflammation and infarct size after ischemia and reperfusion injury in vivo (8). Here, we confirm its cardioprotective effects in three independent study centers using acute and chronic models of myocardial ischemia-reperfusion. Moreover, the effectiveness of Bβ15-42 was compared with four distinct interventions, and investigators were blinded to the treatment conditions. This novel fibrin-derived peptide, Bβ15-42, when given at reperfusion, significantly reduces myocardial infarction, scar formation, and inflammation.
Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). The random peptide (DRGAPAHRPPRGPISGRSTPEKEKLLPG), human Bβ15-42 (hBβ15-42) (GHRPLDKKREEAPSLRPAPPPISGGGYR), hBβ15-25 (GHRPLDKKREE), and rat Bβ15-42 (rBβ15-42) (GHRPVDRRKEEPPSLRPAPPPISGGGYR) (in italic differences between the human and the rat sequence) were produced by solid-phase peptide synthesis and purified with reverse-phase high-performance liquid chromatography using nucleosil 100-10C18 columns (UCB-Bioproducts and PiChem, Graz, Austria). The endotoxin concentration was less than 0.06 EU/mg, and microbial contamination was less than 1 colony-forming unit per 100 mg. The rat antibody CD18 (clone WT.3) was purchased from Perbio Science, Germany. α-C5 antibody was produced by Alexion Pharmaceuticals, Inc (Cheshire, Conn), as described (11).
Rodent models of myocardial ischemia-reperfusion
All procedures were carried out in accordance with the American Association for the Accreditation of Laboratory Animal Care Liaison guidelines and Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, National Institutes of Health, publication no. 86-23). In addition, all experiments were approved by the local ethics committees and regional governments on animal experimentation of Netherlands (Leiden), Germany (Dusseldorf), and Austria (Vienna).
Male Wistar rats (220 - 350 g) or mice (20 - 32 g) received a standard diet and water ad libitum. For acute experiments, rats were anesthetized with midazolam (Dormicum, Roche, Mannheim, Germany; 5.0 mg/kg, intraperitoneal [i.p.]) and sodium pentobarbital (60 mg/kg, i.p.), whereas for chronic experiments, animals were anesthetized with midazolam (Dormicum; 5.0 mg/kg, i.p.) and fentanyl/fluanisone (Hypnorm, Janssen, Oxford, UK; 0.3 mL/kg, i.m.). The technique of myocardial ischemia-reperfusion was performed as previously described (8, 12). Animals were subjected to 25 min of left anterior descending (LAD) occlusion followed by reperfusion for 2 h (acute experiments) or for 30 days (chronic experiments). For ischemic preconditioning (IPC) experiments, the LAD was occluded three times for 2 min followed by two periods of 3 min of reperfusion and a final period of 10 min of reperfusion. C57/BL6 mice were anesthetized s.c. with fentanyl/fluanisone (Hypnorm; 10 mL/kg) and subjected to 20 min of LAD occlusion followed by 2 h of reperfusion (8). The numbers (n) in each treatment group refer to animals which survived until the end of the experiment and which fulfilled the inclusion criteria. The following exclusion criteria were set: (1) death before completion of ischemia-reperfusion protocol and (2) an area at risk for less than 25% or higher than 60%. Less than 5% of animals were excluded, and there was no significant difference in numbers of animals excluded between treatment groups.
Determination of hemodynamic parameters in the acute rat model
The right carotid artery was cannulated and connected to a pressure transducer to measure phasic and mean arterial blood pressure (MAP) and heart rate (HR), which were displayed on a data acquisition system (MacLab 8e; ADI Instruments, Hastings, UK) installed on a personal computer. Pressure rate index (PRI), a relative indicator of myocardial oxygen consumption, was calculated as the product of MAP and HR, and expressed in mmHg/min per 103 (13, 14).
Determination of myocardial infarction
After reperfusion (2 h or 30 days), the LAD was reoccluded, and Evans blue dye (10 mL/kg; 2.0% phosphate-buffered saline [PBS]) was administered i.v. to distinguish between ischemic (area at risk) and nonischemic myocardium (area not at risk). Infarct size was determined by para-nitro blue tetrazolium (pNBT; 0.5% PBS) in a blinded fashion by an independent investigator using SigmaScan Pro, Image Analysis (SPSS Inc, Erkrath, Germany) and expressed as percentage of the area at risk (8, 15).
Determination of myocardial scar formation
After determination of infarct size by pNBT staining, horizontal heart slices were embedded in paraffin and cut into 5-μm sections. Sections were stained with trichrome stain (Masson) according to standard methods, which stains muscle in red and fibrous tissue in blue. To determine the size of scar formation, all sections were photographed with a 1× objective resulting in a total of 10 images per heart covering the entire left ventricle. The area covering the scar was measured using a Zeiss software program (Axiovision 4, Allied High Tech Products, Inc, Rancho Dominguez, Calif) and expressed as a percentage of the area at risk (determined as described above).
Measurement of plasma cytokines
Serum samples herein analyzed were taken from an acute ischemia-reperfusion experiment in fibrinogen−/− (targeted deletion of fibrinogen γ-chain (16) and wild-type (WT) BALB/c mice. Treatment effects of Bβ15-42 on infarct size in these mice have been published previously (8). These animals have been subjected to myocardial ischemia (20 min) and reperfusion (2 h) and then killed. At this time, blood (1 mL) was taken and collected in heparin tubes, centrifuged (3000g) for 5 min at 4°C and stored at −20°C. For this study, samples were defrosted and assayed for various cytokines. Plasma levels of interleukin (IL)-1β, IL-6, IL-10, IL-12, and tumor necrosis factor α (TNF-α) (Mouse Cytokine multi-Plex for Luminex laser, BioSource Europe, S.A. Nivelles, Belgium) were determined using the microsphere array technique (Luminex 100 system, Luminex Corp, Austin, Tex) (17). Assays were performed according to the manufacturer's protocols with an interassay coefficient of variation ranging from 5.2% to 10.4% for all the cytokines.
Results are expressed as the mean ± SEM. Statistical analysis was performed with a one-way analysis of variance or two-way analysis of variance followed by a Bonferroni post hoc test where appropriate. A P < 0.05 was considered statistically significant.
In the acute myocardial ischemia-reperfusion model in rats, MAP, HR, and PRI did not differ between treatment groups (see Table 1).
For the acute rat model, experiments were performed in Dusseldorf and Vienna (Fig. 1), and area at risk was similar in all treatment groups for the various models studied (range, 35% - 58%; data not shown). The hBβ15-42 significantly reduced myocardial infarct size in all three dosing regimens. In particular, the single bolus dose of 2.4 mg/kg given at the start of reperfusion was effective, demonstrating that the start of reperfusion is a crucial time point for the development of myocardial damage. Similarly, rBβ15-42 peptide was effective in reducing infarct size when given as a combined dose totaling 2.4 mg/kg including the dose given at the start of reperfusion. This was confirmed as the second dosing regimen totaling only 1.6 mg/kg and omitting the dose at reperfusion had no effect on infarct size. The short sequence of the hBβ15-25 had no effect at all.
The acute ischemia-reperfusion studies in mice were performed in Leiden (Fig. 2). A single dose of 2.4 mg/kg of hBβ15-42 was just as effective at reducing infarct size as the higher dose of 7.2 mg/kg; therefore confirming that 2.4 mg/kg is the saturating dose. The cardioprotective effect of hBβ15-42 was comparable to the free radical scavenger tempol.
In the chronic rat model, ischemia-reperfusion experiments were performed in Dusseldorf (Fig. 3). A single bolus of hBβ15-42 significantly reduced myocardial infarct size after 30 days. Animals preconditioned (IPC) or treated with α-C5 antibody (at reperfusion) had similar reductions in infarct size (*P < 0.05). CD18 antibody used at a dose of 1 mg/kg as previously published (18) did not significantly reduce infarct size as determined by pNBT staining. Heart slices were then fixed in formalin and sent to Vienna for the assessment of scar formation in a blinded fashion. Hearts were sectioned and stained with trichrome. A typical example is depicted in Figure 4. Myocardial infarct size as determined by trichrome stain corresponded with the results obtained by pNBT staining.
Cytokine profile after acute myocardial ischemia-reperfusion
In WT mice, plasma levels of TNF-α, IL-1, IL-6, IL-10, and IL-12 were significantly increased after acute myocardial ischemia-reperfusion (P < 0.05; Fig. 5). When mice were treated with Bβ15-42, all plasma cytokine levels were reduced. This reduction was statistically significant for TNF-α, IL-1, and IL-6 plasma levels.
In fibrinogen−/− mice, the raise in plasma TNF-α after ischemia-reperfusion was significantly less in fibrinogen−/− mice as compared with WT mice (P < 0.05). Also, the raise in plasma IL-10 was less than that in WT mice, but this was not significant. In the fibrinogen−/− mice, none of the cytokines measured were significantly reduced by Bβ15-42 as compared with the random peptide-treated group (i.e., in the absence of fibrinogen, the peptide is without effect; Fig. 6).
Using acute and chronic models and several different treatment protocols, we show that the peptide Bβ15-42 significantly and reproducibly reduced infarct size at three independent study centers. These results are consistent with our previous findings that Bβ15-42 is cardioprotective (8). Moreover, in the chronic study, effects of Bβ15-42 were comparable to ischemic preconditioning, which is the standard procedure to prevent reperfusion injury in animal models (19). In addition, the effects of Bβ15-42 were compared with three other anti-inflammatory compounds, tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl; a free radical scavenger) (20), a CD18 antibody (directed to the β2-integrin subunit, which prevents leukocyte adhesion) and an anti-C5 antibody (which blocks terminal complement activation). Tempol has been shown to be beneficial in several studies (21, 22), however, its use in man may be limited by its toxicity (23). The antibody against CD18 has been reported to have contradicting results in preclinical trials (24, 25) and has been shown to be ineffective in human trials (26). Therefore, it was not too surprising that it had no significant effect on infarct size in our study. The anti-C5 antibody has been shown to be effective in acute ischemia-reperfusion experiments (11), although our data are the first to suggest that terminal complement blockade with this antibody also reduces myocardial scar formation in a chronic model. An anti-C5 antibody has also been shown to reduce mortality in humans when given at the time of reperfusion (27, 28).
Importantly, the consistency of our experiments with Bβ15-42 is underscored by the fact that experiments have been performed within a frame of controls; IPC, and three additional anti-inflammatory compounds. Our results with these three compounds are consistent with previously published data. In this experimental setup, the Bβ15-42 peptide reproducibly reduced infarct size, a result that was consistent at three different centers. Finally, it should be noted that the human Bβ15-42 sequence is highly conserved among species (except for 5 conservative amino acid changes). Therefore, the results of cardioprotective function of this peptide in rodents suggest that pathways modulated by the peptide in animals are likely to exist in humans.
Our previously published data clearly demonstrate the importance of the N-terminus GHRP sequence (Bβ15-18) in binding to VE-cadherin (29) and for eliciting an anti-inflammatory effect (8). Therefore, the inability of Bβ15-25 to reduce infarct size in comparison with Bβ15-42 sequence was unexpected. These results may reflect a reduction in plasma half-life with the truncated peptide or formation of a nonfunctional conformation. It should be noted that the heparin-binding site within Bβ15-42 peptide (30) is probably not relevant for the function of the peptide, because effective heparin-binding requires residues Bβ15-57 as well as dimerization (31).
We have previously shown that the anti-inflammatory capacity of Bβ15-42 is caused by the ability of the peptide to compete with fibrin E1 fragments for ligand binding (8). Here, we increase the evidence that Bβ15-42 has anti-inflammatory properties as demonstrated by the ability to reduce plasma cytokine levels. To address the question, if fibrin(ogen)-derived products directly participate in the ischemia-reperfusion-induced cytokine release, we compared plasma cytokine levels from WT animals with those seen in fibrinogen−/− mice. After ischemia-reperfusion in fibrinogen−/− animals, the raise in plasma IL-1, IL-6, and IL-12 was comparable to that of WT mice. The raise in IL-10 in fibrinogen−/− mice was less than in WT mice, but this was not statistically significant. Importantly, TNF-α was significantly reduced in fibrinogen−/− mice as compared with WT mice, and Bβ15-42 failed to further reduce plasma TNF-α levels in fibrinogen−/− mice. This indicates that under the condition of ischemia-reperfusion, the release of TNF-α as well as the effectiveness of Bβ15-42 depended on the presence of fibrin(ogen). With regard to TNF-α, high plasma levels have been clearly identified as an independent risk factor for reperfusion injury in humans (32). Thus, the reduction of TNF-α by Bβ15-42 fits to the concept that this peptide protects the myocardium through its anti-inflammatory function. However, the target of Bβ15-42 is the endothelial cell, and this cell type is not the major source of TNF-α. It is thus unclear if Bβ15-42 mediates this effect indirectly by reducing leukocyte transmigration or by targeting a distinct cell type.
In conclusion, this study confirms the beneficial effect of Bβ15-42 in the setting of reperfusion injury. Furthermore, these data add additional evidence for a pathogenic role of fibrin(ogen)-derived products in situations of ischemia and reperfusion injury in the heart.
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Fibrin-derived peptide; preclinical study centers; ischemia-reperfusion; myocardial infarction