Single Administration of the CXC Chemokine-Binding Protein Evasin-3 During Ischemia Prevents Myocardial Reperfusion Injury in Mice
Objective— Evasins (chemokine-binding proteins) have been shown to selectively neutralize chemokine bioactivity. We investigated the potential benefits of Evasin-3 on mouse myocardial ischemia/reperfusion injury.
Methods and Results— In vivo and ex vivo (Langendorff model) left coronary artery ligature was performed in C57Bl/6 mice. Coronary occlusion was maintained for 30 minutes, followed by different times (up to 24 hours) of reperfusion. Five minutes after coronary occlusion, mice received 1 intraperitoneal injection of Evasin-3 or vehicle. Infarct size was assessed histologically and by serum cardiac troponin I ELISA. In vitro neutrophil chemotaxis, immunohistology, oxidative stress quantification, real-time RT-PCR analysis of leukocyte chemoattractants, and Western blots for cardioprotective intracellular pathway activation were performed. Evasin-3 reduced infarct size and cardiac troponin I levels compared with vehicle. This effect was associated with the reduction of neutrophil infiltration and reactive oxygen species production within the infarcted myocardium. Evasin-3 did not reduce infarct size in the absence of circulating neutrophils (Langendorff model). Evasin-3 did not influence the activation of intracellular cardioprotective pathways or the expression of leukocyte chemoattractants during early phases of reperfusion.
Conclusion— Single administration of Evasin-3 during myocardial ischemia significantly reduced infarct size by preventing CXC chemokine-induced neutrophil recruitment and reactive oxygen species production in myocardial ischemia/reperfusion.
Postischemic inflammation is a major determinant orchestrating myocardial reperfusion injury and repair. Soon after the restoration of blood flow, inflammatory cell recruitment within infarcted myocardium is timely regulated and contributes to the extension of myocardial damage.1–4 Leukocyte infiltration is mainly mediated by the upregulation of cytokines and chemokines and the activation of complement cascade in infarcted areas.5–7 In early phases of reperfusion, infiltrated inflammatory cells (mainly neutrophils and monocytes) release proteolytic enzymes and reactive oxygen species (ROS).8,9 These soluble mediators further increase endothelial microvascular dysfunction, cardiomyocyte necrosis, and apoptosis, thus favoring the development of postischemic heart failure.10 The induction of chemokine expression after an acute myocardial infarction is very rapid but transient, whereas other soluble agents are expressed, probably in the context of the resolution of inflammation and the transition to fibrosis, which further results in reduced cardiac performance.11 Thus, later in reperfusion, leukocytes might positively influence myocardial repairing processes.3 This strongly suggests that selective antichemokine treatments targeting leukocyte recruitment could improve acute myocardial infarction in early phases of reperfusion.12–14 With this aim, we focused on the selective neutralization of neutrophil chemoattractants (such as CXCL1 and CXCL2) to reduce the infiltration of these cells in infarcted hearts during early reperfusion. The potential benefits of a single administration shortly before reperfusion of Evasin-3 that binds and neutralizes the murine equivalents of CXCL8 and CXCL1 (CXCL2 and KC, respectively) were investigated in a mouse model of myocardial ischemia/reperfusion (I/R).15
For additional details, please see the supplemental data, available online at http://atvb.ahajournals.org.
In Vivo I/R Protocol
Male C57Bl/6 mice (8 to 12 weeks of age) were obtained from the University Medical Center animal facility of the Medical Faculty, University of Geneva. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication no. 85-23, revised 1996) and has been approved by the local authorities.
The mouse in vivo protocol was performed as described in the online supplemental data.
Ex Vivo I/R Protocol
The technique of Langendorff isolated buffer-perfused mouse heart preparation was used.16 This method was performed as described in the online supplemental data.
Area at Risk and Infarct Size Assessment
To assess area at risk (AAR) and infarct size (I) in in vivo I/R protocol, mice were anesthetized with ketamine-xylazine and euthanized after 2, 8, or 24 hours of reperfusion, as described in the online supplemental data.
Serum Cardiac Troponin I and Neutrophil Chemoattractant Level Detection
Circulating cardiac troponin I (cTnI) and neutrophil chemoattractant (CXCL1, CXCL2, CCL3, and tumor necrosis factor [TNF]-α) levels in mouse sera from 2 to 24 hours of reperfusion were measured as described in the online supplemental data.
Immunostainings were performed as described in the online supplemental data.
Analysis of Gene Expression by Real-Time RT-PCR
Real-time RT-PCR was performed as described in the online supplemental data.
Human Neutrophil Isolation and Migration Assay
Human primary neutrophil isolation and migration were performed as described in the online supplemental data.
Mouse Peritoneal Neutrophil Isolation and Migration Assay
Mouse primary neutrophil isolation and migration were performed as described in the online supplemental data.
Western Blot Analysis
After 5 or 15 minutes of reperfusion, proteins from total mouse hearts were extracted and Western blots were performed as described in the online supplemental data.17
Oxidative Stress Determination
Measurement of superoxide (ROS) was performed as described in the online supplemental data.18
All results are expressed as mean±SEM. Differences between probability values below 0.05 were considered significant. Statistics were performed with GraphPad Instat software version 3.05 (GraphPad Software) using 1-way ANOVA for multiple group comparison or unpaired t test for 2 group comparison. The Spearman rank correlation coefficients were used to assess correlations between ROS production and neutrophil infiltration in infarcted hearts.
Single Administration of Evasin-3 During Ischemia Reduces Myocardial Infarct Size
To investigate the potential benefit of Evasin-3 treatment in myocardial reperfusion injury, we treated mice with a single intraperitoneal administration of different doses (0.1 to 10 μg/mouse) of Evasin-3 or control vehicle (PBS) 5 minutes after the initiation of the ischemic period. After 30 minutes of ischemia, left anterior coronary artery occlusion was released, and infarct size was assessed after 24 hours of reperfusion. Figure 1A shows that the AAR was similar in all groups, indicating that ligature was reproducibly performed at the same level of the left anterior coronary artery. Evasin-3 administration dose-dependently reduced infarct size compared with vehicle treatment (Figure 1A and 1B). Accordingly, Evasin-3 treatment (10 μg/mouse) significantly reduced serum cTnI levels compared with vehicle-treated mice after 24 hours of reperfusion (Figure 1C).19,20 The beneficial effect of Evasin-3 (10 μg/mouse) administration was already observed at 8 hours of reperfusion (Figure 2C and 2D), whereas no significant reduction in infarct size or cTnI levels was observed at 2 hours compared with control vehicle (Figure 2A and 2B).
Evasin-3 Treatment Reduces CXC Chemokine Bioactivity but Not CXC Chemokine and CXC Chemokine Receptor Expression
Evasin-3 has been recently shown to inhibit CXC chemokine bioactivity, including CXCL2- and CXCL1-induced neutrophil recruitment in mice.15 We first confirmed these results by the assessment of human neutrophil migration in response to CXCL8 (CXCL2 human homolog) in the presence of different doses of Evasin-3. CXCL8-induced neutrophil migration was significantly reduced in the presence of 1 to 1000 nM of Evasin-3, in both in vitro chemotaxis assays used (Supplemental Table II). We also measured serum levels of neutrophil chemoattractants (including CXC chemokines) at different time points of reperfusion. A significant increase of CXCL2 and TNF-α serum levels was observed at 2 to 24 hours of reperfusion. CXCL1 and CCL3 serum levels were increased in acute myocardial infarction groups from 8 to 24 hours of reperfusion, compared with sham-operated mice (Supplemental Table III). Accordingly, myocardial infarction increased CXC chemokine, CXCR2, TNF, and CCL3 mRNA myocardial expression from 1 to 8 hours of reperfusion, compared with sham-operated mice (Supplemental Table IV). In both operated and not-operated mice, the expression of CXCR1 mRNA in mouse hearts was too low to be quantified by RT-PCR (Supplemental Table IV). Evasin-3 treatment did not reduce CXC chemokine, TNF-α, CCL3, or CXCR2 levels in either myocardium or serum compared with vehicle-treated mice (Supplemental Tables III and IV). In vitro, serum from vehicle-treated mice (at 12 hours of reperfusion, the time point at which the highest concentrations of neutrophil chemoattractants were detected) significantly increased mouse neutrophil migration toward recombinant CXCL1 and CXCL2 compared with Evasin-3-treated serum or control chemotaxis medium. In addition, Evasin-3-treated serum significantly reduced neutrophil migration toward recombinant CXCL1 and CXCL2 compared with control medium (Supplemental Table V). Chemokinetic and chemotactic properties of serum from vehicle- or Evasin-3-treated mice were also investigated by using checkerboard analysis. Serum from vehicle-treated mice induced both neutrophil chemotaxis and chemokinesis (Supplemental Table VI). Despite similar levels of neutrophil chemoattractants, serum from Evasin-3-treated mice did not induce significant chemokinesis or chemotaxis (Supplemental Table VI), indicating that Evasin-3 blocked both serum CXC chemokine-induced neutrophil chemotaxis (true migration through a chemokine gradient toward inflammatory tissues), and chemokinesis (random migration). These data suggest that Evasin-3 treatment inhibited CXC chemokine bioactivity on human neutrophils instead of reducing CXC chemokine levels.
Single Administration of Evasin-3 Inhibits Neutrophil Recruitment in Infarcted Hearts During Reperfusion
Leukocyte infiltration in infarcted hearts was assessed at different time points of reperfusion. Evasin-3 treatment already significantly reduced neutrophil infiltration after 12 hours of reperfusion compared with the vehicle-treated group (Figure 3A). A strong Evasin-3-mediated inhibition of neutrophil infiltration was also observed after 24 hours of reperfusion (Figure 3A and 3B). No differences in macrophage (CD68+ cell) recruitment were detected in the 2 groups at the same time points of reperfusion (Figure 3C and 3D). Accordingly, as previously shown, circulating leukocyte count was significantly higher in Evasin-3-treated mice compared with control vehicle at 12 and 24 hours (Figure 3E).14
Administration of Evasin-3 Does Not Reduce Infarct Size in the Langendorff Model
To further investigate whether Evasin-3-mediated benefits on infarct size were induced by reduction of neutrophil infiltration, we used an ex vivo model of ischemia-reperfusion perfused hearts in the absence of circulating leukocytes. Similarly to the in vivo protocol, buffer-perfused hearts (Langendorff system) were submitted to 30 minutes of local ischemia. Then, Evasin-3 or vehicle was added to the circulating system 5 minutes after the beginning of ischemia. On the basis of previous Langendorff protocols, infarct size was assessed after 2 hours of reperfusion.16 Figure 4A shows that AAR was similar in the 2 groups, indicating that ligature was correctly performed at the same level of the left anterior coronary artery. Treatment with Evasin-3 did not modify infarct size in this ex vivo model of ischemia-reperfusion without circulating inflammatory cells (Figure 4B and 4C).
ROS Production in Infarcted Hearts Is Reduced by Evasin-3 Treatment and Positively Correlates With Neutrophil Infiltration
Oxidative stress has been indicated to play a crucial role during reperfusion.8,18 In particular, superoxide production might increase reperfusion injury. Figure 5A through 5I shows that Evasin-3 treatment significantly reduced superoxide production in the postischemic myocardium from 12 to 24 hours of reperfusion compared with vehicle. At these time points, ROS production in infarcted hearts positively correlated with neutrophil infiltration (Table).
Administration of Evasin-3 Does Not Increase the Phosphorylation of Cardioprotective Pathways in Infarcted Hearts
We investigated the possible direct effect of Evasin-3 treatment on the activation of cardioprotective intracellular pathways. The Western blot analysis of total heart lysate showed that myocardial infarction did not significantly increase STAT-3 serine 472 and tyrosine 705 and extracellular signal regulated kinase (ERK) 1/2 phosphorylation at 5 minutes of reperfusion compared with the sham-operated mouse group (Supplemental Table VII). At 15 minutes of reperfusion, STAT-3 tyrosine 705 and ERK1/2 phosphorylation were increased by myocardium infarction compared with the sham-operated group. No significant differences in STAT-3 serine 472 or tyrosine 705 and ERK1/2 phosphorylation levels were observed in the presence of Evasin-3 treatment compared with the vehicle-treated group at 5 to 15 minutes of reperfusion (Supplemental Table VII).
In postischemic myocardial reperfusion, early neutrophil recruitment increases tissue damage through the release of histotoxic products, such as ROS.9,21 Potent chemoattractants (such as CXCL2 and CXCL1) orchestrate neutrophil infiltration into inflammatory sites, such as the infarcted myocardium.22 Thus, the selective inhibition of neutrophil chemoattractant bioactivity might prevent myocardial injury during early phases of reperfusion. Among multiple models of interference with the chemokine system, Evasins are chemokine-binding proteins, which have been recently identified in tick saliva.15 These molecules have also been cloned and produced as recombinant proteins and were well tolerated in mice as an antiinflammatory treatment. Evasin-3 binds and neutralizes in vivo and in vitro bioactivity of CXC chemokines (such as CXCL2 and CXCL1), showing higher selectivity than other viral chemokine binding proteins already identified.15
We showed that a single administration of Evasin-3 during ischemia reduced infarct size in the in vivo I/R mouse model. The beneficial effects of Evasin-3 treatment on infarct size were observed at 8 hours of reperfusion but not at 2 hours of reperfusion, as shown by histology and serum cTnI assay. These experiments indicate that Evasin-3 did not prevent postinfarction injury during the early 2 hours of reperfusion, but its beneficial effects are delayed after 8 hours. Our data differ from a recent article showing that treatment with activated protein C was cardioprotective already during the first hours of reperfusion.23 After confirming in vitro that Evasin-3 neutralizes bioactivity on neutrophils mediated by mouse CXCL1, CXCL2, and human CXCL8 (human equivalent of murine CXCL2), we also showed that Evasin-3-induced benefits were associated with a significant inhibition of neutrophil infiltration within the infarcted myocardium after 12 to 24 hours of reperfusion. Accordingly, Evasin-3 significantly increased circulating white blood cell count at the same time points of reperfusion compared with vehicle-treated mice, indicating that leukocytes are retained in the circulation instead of migrating within the infarcted myocardium. This mechanism was also supported by in vitro experiments on mouse neutrophil chemotaxis. Coincubation with serum from Evasin-3-treated mice at 12 hours of reperfusion (the time point at which the highest concentrations of neutrophil chemoattractants were observed) significantly reduced neutrophil migration toward CXCL1 and CXCL2 compared with serum from vehicle-treated mice. These experiments showed that injected Evasin-3 blocked CXCL1- and CXCL2-induced neutrophil chemotaxis and chemokinesis during early phases of reperfusion. Importantly, Evasin-3 did not reduce CXC chemokine expression in mouse infarcted hearts and serum.
Confirming previous evidence, we showed that several neutrophil chemoattractants are upregulated after acute myocardial infarction in mice.19,24,25 Evasin-3 administration did not reduce the expression of neutrophil chemoattractants (such as CXCL1, CXCL2, CCL3, and TNF) in infarcted hearts and serum. Taken together, these results suggest that antiinflammatory properties of Evasin-3 are due to the direct inhibition of CXCL2 and CXCL1 bioactivity, instead of the downregulation of inflammatory mediators. To further confirm the crucial role of neutrophil recruitment, we tested Evasin-3 activity in the Langendorff perfused heart model, an ex vivo experimental system characterized by the absence of circulating leukocytes. To exclude a possible direct activity of Evasin-3 on resident cardiac cells, we selected a very high dose (5 μg/mL), which was 100-fold higher than the concentrations previously shown to inhibit in vitro 125I-CXCL8 binding to CXCR1 and neutrophil chemotaxis toward CXCL8.15 Given the total volume of blood in an adult male mouse (≈2 mL), the concentration of Evasin-3 used was also higher than that used in vivo in the present I/R protocol and in the KC-induced neutrophil recruitment into peritoneal cavity and knee joint in mice.15,26,27 In the Langendorff model, Evasin-3 treatment failed to reduce infarct size compared with vehicle control. These data strongly suggest that Evasin-3 improved infarct size through the selective inhibition of CXC chemokine-induced myocardial recruitment of circulating neutrophils. Furthermore, these data confirmed that treatments neutralizing CXC chemokine bioactivity might be considered a very promising therapeutic approach not only to reduce atherogenesis but also to protect against ischemic myocardial injury.28–30
To identify a possible molecular mechanism underlying neutrophil infiltration, we investigated the production of ROS during reperfusion. ROS are generated within the myocardium during I/R and directly injure the cardiac cells, favoring cell death.31 In addition, ROS increase ischemic cardiac injury through the release of inflammatory cytokines, enhancement of mitochondrial membrane permeability, and intracellular Ca2+ overload.31 Our data showed that Evasin-3 reduced ROS production in infarcted hearts at 12 and 24 hours of reperfusion. Furthermore, a strong positive correlation between ROS production and neutrophil infiltration was observed, indicating that at least in part, ROS could be released by infiltrated neutrophils during reperfusion. Thus, Evasin-3-mediated neutralization of CXC chemokine bioactivity on human neutrophil could also be an important approach to reduce oxidative stress. Although Evasin-3 induced selective antichemokine activities targeting neutrophil functions, we could not exclude its possible direct effects on cardiac cells also. Importantly, the disruption of CXCL2-CXCR2 signaling has been shown to beneficially influence infarct size in the absence of circulating leukocytes (Langendorff model).27 This study suggests that CXCR2 triggering might be considered a double-edged sword in acute myocardial infarction. CXCR2-induced pathway is protective on cardiomyocytes. On the other hand, CXCR2 triggering also induces adverse effects favoring neutrophil recruitment. Our data in the Langendorff model are in partial contrast with the previously cited study by Tarzami et al in CXCR2 knockout mice.27 Surprisingly, high-dose treatment with Evasin-3 (which should neutralize CXCL2 and CXCL1 binding and bioactivity on CXCR2, mimicking CXCR2 disruption) did not induce cardiomyocyte protection. As the expression of other selective receptors of CXC chemokine (such as CXCR1) was not detected in mouse hearts, these controversial results could be explained by the different approaches to inhibit CXCR2/CXCL2 pathway. The CXCR2 knockout approach could induce a compensatory regulation of other chemokines or chemokine receptors.32,33 This collateral effect was not clearly observed in our short-term pharmacological inhibition model. Importantly, Chen et al showed that the role of CCR5 in excitotoxic injury in CCR5 knockout mice was compensated by increased CCR2 and CCR3 expression.33 On the other hand, a compensatory upregulation of CCL2 and CCL4 was recently showed in myocardial reperfusion injury in CCR5 and apolipoprotein E double knockout mice.14 Furthermore, Evasin-3 selectively binds CXCL2 and CXCL1 but not other CXCR2 ligands (such as macrophage migration inhibitory factor) that could be released during reperfusion in the in vivo I/R model.34
To explore whether Evasin-3 could also directly induce benefits on cardiac resident cells, we investigated the phosphorylation of STAT-3 and ERK1/2 pathways, known to be involved in the protection of cardiomyocytes in the first minutes after reperfusion.20,35,36 Evasin-3 did not induce any significant effects on the activation of protective intracellular pathways (such as STAT-3 and ERK1/2) after 5 or 15 minutes of reperfusion. These data further confirmed that Evasin-3 mediated cardioprotection was delayed and was due to the inhibition of the recruitment of circulating neutrophils, instead of a direct protective activity on resident cardiac cells.
To conclude, we have shown that single administration of the selective CXCL2 and CXCL1 binding protein Evasin-3 during myocardial ischemia reduced infarct size in vivo in mice after 8 hours of reperfusion. Evasin-3-mediated beneficial activity was due to the inhibition of CXC chemokine bioactivity on neutrophil recruitment in infarcted hearts. Accordingly, Evasin-3 did not reduce infarct size in the absence of circulating leukocytes (Langendorff model). Evasin-3 also decreased ROS production in infarcted hearts from 12 to 24 hours of reperfusion. At these time points, ROS production was positively correlated with neutrophil infiltration, suggesting that these cells are crucial mediators of postischemic myocardial oxidative injury. Evasin-3 did not activate other cardioprotective mechanisms (such as ERK1/2 and STAT-3 phosphorylation) during early minutes of reperfusion. Therefore, single administration of Evasin-3 during ischemia could represent a promising therapeutic approach to selectively reduce reperfusion myocardial injury mediated by the infiltration of circulating neutrophils and the associated production of ROS.
Sources of Funding
This research was funded by EU FP7 grant 201668, AtheroRemo, Swiss National Science Foundation grant 310030-118245 (F.M.), and EU FP6 (INNOCHEM) grant LSHB-CT-2005-518167 (A.E.P., M.D.).
Received on: December 15, 2009; final version accepted on: April 12, 2010.
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