International Journal of Cardiology 181 (2015) 284–287
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Key role of MIF in the migration of endothelial progenitor cells in patients during cardiac surgery☆ Christoph Emontzpohl a,b,1, Andreas Goetzenich c,1, David Simons d,e,1, Sandra Kraemer c, Manfred Dewor a, Hongqi Lue a, Luise Hammer a,b, Denise Jacobs a, Gerrit Grieb e, Patrick Ziegler f, Jens Panse f, Rolf Rossaint b, Jürgen Bernhagen a,⁎, Christian Stoppe a,b,1,⁎⁎ a
Institute of Biochemistry and Molecular Cell Biology, University Hospital, RWTH Aachen University, Germany Department of Anesthesiology, University Hospital, RWTH Aachen University, Germany c Department for Thoracic and Cardiovascular Surgery, University Hospital, RWTH Aachen University, Germany d German Cancer Research Center (DKFZ), Radiology (E010), Heidelberg, Germany e Department of Plastic Surgery and Hand Surgery, Burn Center, University Hospital, RWTH Aachen University, Germany f Department of Oncology, Hematology, Hemostaseology, and Stem Cell Transplantation, University Hospital, RWTH Aachen University, Germany b
a r t i c l e
i n f o
Article history: Received 10 November 2014 Accepted 22 November 2014 Available online 18 December 2014 Keywords: Cardiac surgery MIF — macrophage migration inhibitory factor EPC — endothelial progenitor cells EPC migration Ischemia and reperfusion (IR) Cytokines
Macrophage migration inhibitory factor (MIF) acts as an upstream regulator of the innate and adaptive immune response and exhibits chemokine-like activities [1,2]. Conclusive evidence also indicates that ☆ Author contributions. Conceived and designed the experiments: CE, AG, DS, SK, RR, CS, and JB. Performed the experiments: CE, AG, DS, CS, DJ, PZ, and LH. Analyzed the data: CE, AG, DS, CS, SK, LH, and HL. Contributed reagents/materials/analysis tools: CE, AG, DS, CS, PZ, JP, MD, DJ, GG, RR, and JB. Wrote the paper: CE, AG, DS, HL, GG, MD, JP, CS, and JB.All authors read and approved the final version of the manuscript. ⁎ Correspondence to: C. Stoppe, Institute of Biochemistry and Molecular Cell Biology & Department of Anesthesiology, University Hospital of the RWTH Aachen, Pauwelsstraße 30, 52074 Aachen, Germany. ⁎⁎ Correspondence to: J. Bernhagen, Institute of Biochemistry and Molecular Cell Biology, University Hospital of the RWTH Aachen, Pauwelsstraße 30, 52074 Aachen, Germany. E-mail addresses: [email protected] (C. Emontzpohl), [email protected] (A. Goetzenich), [email protected], [email protected] (D. Simons), [email protected] (S. Kraemer), [email protected] (M. Dewor), [email protected] (H. Lue), [email protected] (L. Hammer), [email protected] (D. Jacobs), [email protected] (G. Grieb), [email protected] (P. Ziegler), [email protected] (J. Panse), [email protected] (R. Rossaint), [email protected] (J. Bernhagen), [email protected] (C. Stoppe). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ijcard.2014.11.226 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
MIF provides cardioprotection during myocardial ischemia–reperfusion (I/R) [3]. Moreover, MIF promotes neovascularization during hypoxic stress through recruitment of endothelial progenitor cells (EPC) after myocardial I/R [4]. However, studies in clinical settings in particular in comparison with other angiogenic cytokines are lacking. Since patients undergoing cardiac surgery are frequently exposed to myocardial I/R with a following inflammatory response and excessive release of MIF [5,6], we surmised this clinical setting to represent an ideal model to characterize the significance of this cytokine on overall EPC migration during myocardial I/R. Therefore, we followed up on patients that underwent elective cardiac surgery with the use of cardiopulmonary bypass (CPB). Exclusion criteria were emergency operations, pregnancy, patients' age less than 18 years and failure to obtain informed consent. The institutional review board approved this study. Anesthesia, surgical procedure and data collection were performed as previously described [5]. Serum/blood samples were drawn immediately before surgery, at admission to ICU as well as 6 h, 12 h and 24 h postoperatively. Relevant clinical data of these patients were documented. Serum concentrations of stromal cell-derived factor (SDF)-1α/CXCL12, interleukin (IL)-8/CXCL8 and vascular endothelial growth factor (VEGF) were determined with commercially available ELISA assays (R&D Systems) according to the manufacturer's instructions. Serum levels of MIF were assessed using an ELISA technique as previously described [2]. EPCs were isolated according to our established protocol [4] by gating CD34 + cells and a subsequent selection for CD133 +/ CD33− from mononuclear cell (MNCs) fractions obtained by density gradient centrifugation from human blood. Migration assays were performed using Transwell migration chambers with a filter pore-size of 5 μm. EPCs were placed into upper chambers. Lower chambers contained increasing concentrations of recombinant MIF, CXCL12, CXCL8, VEGF165 or serum samples (alone, or combined with anti-MIF antibody). After 3 h of migration, cells were fixed, stained with Hoechst dye, diluted in 3.6% PFA (1:1000) and counted. Data were analyzed by one-way-ANOVA and post-hoc Bonferroni's multiple comparison tests using Prism 6.0 (Graphpad) to compare
C. Emontzpohl et al. / International Journal of Cardiology 181 (2015) 284–287
differences between multiple groups and Student's unpaired t-test when analyzing two groups. In all cases, P b 0.05 was considered to be statistically significant. In total, 103 patients were enrolled in this study and followed until the final analysis. All included patients reflected a representative cohort with coronary artery disease, presenting typical comorbidities like hypertension. At first, we studied the influence of myocardial I/R on the release of MIF, CXCL12 and CXCL8. All three cytokines significantly increased until admission to ICU whereas serum levels of VEGF remained unchanged during the period (Fig. 1a–d), indicating a minor influence of VEGF on EPC mobilization and migration. Next, we tested the potential influence of the observed cytokines on EPC recruitment in vivo and measured the proportion of EPCs in whole blood samples by flow cytometry. Corresponding to the increased concentrations of MIF, CXCL12 and CXCL8, the proportion of EPCs significantly increased post-operatively and decreased
285
again to almost baseline values 24 h after surgery (Fig. 1e). To investigate direct effects of patients' serum samples on EPC migration, we performed in vitro migration assays using perioperatively drawn serum samples as chemoattractant and EPCs from healthy volunteers. A significantly increased EPC migration towards the intraoperatively taken samples (+ 52% compared to baseline, p = 0.03, Fig. 1f) was observed. In a next step, we performed a dose–response analysis evaluating MIF, CXCL12, CXCL8, and VEGF on EPC migration. This experiment revealed a significant and dose-dependent increase in EPC migration in response to all cytokines (Fig. 2a–d). MIF significantly increased EPC migration compared to medium control with a maximum observed at 10 ng/ml (8-fold; p = 0.05) and appeared much more potent than the other cytokines (CXCL12 (5-fold at 100 ng/ml, p = 0.05), CXCL8 (3-fold at 50 ng/ml, p = 0.04), and VEGF 165 (2-fold at 10 ng/ml, p = 0.02)).
Fig. 1. Generated data using samples of cardiac surgical patients. (a–d) Measurement of serum concentrations of MIF (a), CXCL12 (b), CXCL8 (c) and VEGF (d) of patients that underwent cardiac surgery using ELISA. The dashed area indicates the duration of cardiac surgery. Shown are mean values ± SEM (*: p b 0.05 vs. pre-OP). (e) Shown is the in vivo EPC mobilization in 10 patients during cardiac surgery with an increase of circulating EPCs as part of the total CD34+ cells and a subsequent decrease to baseline values 24 h post-operatively. Each time point represents a mean value (n = 10) ± SEM (*: p b 0.05 vs. pre-op). The dashed area indicates the duration of cardiac surgery. (f) Shown is the in vitro migration of EPCs of healthy volunteers towards serum samples of patients (n = 7) who underwent cardiac surgery with an increased migration towards intra-operatively taken serum samples. For the migration assay 50,000 cells per well were used and put into the upper chamber of a modified Boyden chamber together with MV2 serum starved endothelial medium. The serum samples in the lower chamber were diluted 1:5 with the same MV2 serum starved medium. The cells migrated for 3 h through a 5 μm transwell membrane. The bars represent mean values (n = 7) ± SEM (*: p b 0.05).
286
C. Emontzpohl et al. / International Journal of Cardiology 181 (2015) 284–287
To get a better understanding of the influence of these cytokines on EPC recruitment in vivo, migration experiments, applying the physiologically measured concentrations of these cytokines were performed. A 6-fold increase in EPC migration upon chemotactic stimulation was observed using 50–100 ng/ml of recombinant MIF, — a level that corresponds to the measured post-operative MIF levels. As much higher concentrations of CXCL12 (100 ng/ml), CXCL8 (over 50 ng/ml) and VEGF (over 10 ng/ml) than measured in the patients' serum samples were needed to show an effect on EPC migration (Fig. 2b–d), we concluded that MIF's effect on the initial mobilization of EPCs is of high physiological significance compared to other cytokines. The pro-migratory effect of patient serum in the presence versus absence of neutralizing MIF antibodies was evaluated next, confirming that MIF has a significant stimulatory effect of EPC migration under in vivo conditions (Fig. 2e). Since MIF seems to have a strong influence on EPC migration/mobilization, its significance on clinical outcome of patients was analyzed. Interestingly, we had previously noticed a strong inverse correlation between EPCs on the pre-operative day and the well-established SOFA score after surgery, which adequately reflects the extent of organ injury [7]. These data are in accordance with Cribbs et al., who demonstrated an inverse correlation between circulating EPCs and the severity of organ dysfunction in sepsis patients [8]. In summary, we showed that myocardial I/R during cardiac surgery leads to increased levels of MIF, CXCL12, and CXCL8, which are known chemoattractants for the recruitment of EPCs. Among these, only MIF showed a significant effect on the mobilization of EPCs at concentrations comparable to those measured in the clinical samples. Since previous studies demonstrated that only EPCs transplanted shortly after infarction had maximal beneficial effects as compared to a later transplantation time point [9] and since MIF levels increased immediately after myocardial I/R, we hypothesize that MIF-mediated effect on the recruitment of EPCs affects cardiac remodeling and angiogenesis after myocardial I/R [10]. Our study highlights a predominant role of MIF on EPC recruitment in this context. Conflict of interest All authors state that no competing financial interests exist. Acknowledgment We are indebted to the laboratory staff of the Institute of Biochemistry and Molecular Cell Biology and the Department of Oncology, Hematology, Hemostaseology and Stem Cell Transplantation (University Hospital of the RWTH Aachen) for the excellent scientific and technical assistance, as well as the patients who participated in this trial. This study was supported by the Deutsche Forschungsgemeinschaft (DFG) grants Be1977/4-2 (TP01 of DFG-FOR809), DFG-IRTG1508/1-P13 and Ra969/6-1 to J.B., by START and Rotation Program grants of the faculty of medicine of RWTH Aachen University to C.S. Fig. 2. In vitro migration of EPCs towards recombinant cytokines. Shown is the dose–response relationship between EPCs and recombinant MIF (a), CXCL12 (b), CXCL8 (c) and VEGF165 (d). We observed the strongest increase in EPC migration at 10 ng/ml recombinant MIF. The recombinant cytokines were diluted in MV2 serum starved medium. The cells migrated for 3 h through a 5 μm transwell membrane. The bars represent mean values ± SEM (*: p b 0.05, **: p b 0.01 vs. serum starved medium). Also shown is the EPC migration towards a range of cytokine concentrations we measured previously in serum samples (cf. Fig. 1a–d). Only MIF was able to mediate an increased EPC migration at physiologically measured concentrations (n = 3–5), which exhibited the strongest EPC migration effect at the same time (8-fold increase compared to baseline). (e) Shown is the migration of EPCs towards the diluted serum sample of one patient at one time point at a time (n = 4) and the additional antiMIF antibody. The antiMIF antibody was diluted in MV2 serum starved medium + serum sample. The cells migrated for 3 h through a 5 μm transwell membrane. 120 μg/ml antiMIF antibody led to a reduction in EPC migration of about 24%. Also shown is the corresponding isotype control. The bars represent mean values ± SEM (n = 4) (*: p b 0.05).
References [1] T. Calandra, T. Roger, Macrophage migration inhibitory factor: a regulator of innate immunity, Nat. Rev. Immunol. 3 (10) (2003) 791–800. http://dx.doi.org/10.1038/ nri1200. [2] J. Bernhagen, R. Krohn, H. Lue, et al., MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment, Nat. Med. 13 (5) (2007) 587–596. http://dx.doi.org/10.1038/nm1567. [3] T. Rassaf, C. Weber, J. Bernhagen, Cardiovasc. Res. 102 (2) (2014) 321–328. [4] D. Simons, G. Grieb, M. Hristov, et al., Hypoxia-induced endothelial secretion of macrophage migration inhibitory factor and role in endothelial progenitor cell recruitment, J. Cell. Mol. Med. 15 (3) (2011) 668–678. http://dx.doi.org/10.1111/j.15824934.2010.01041.x. [5] C. Stoppe, G. Grieb, R. Rossaint, et al., High postoperative blood levels of macrophage migration inhibitory factor are associated with less organ dysfunction in patients after cardiac surgery, Mol. Med. 18 (2012) 843–850.
C. Emontzpohl et al. / International Journal of Cardiology 181 (2015) 284–287 [6] A. Zernecke, J. Bernhagen, C. Weber, Macrophage migration inhibitory factor in cardiovascular disease, Circulation 117 (12) (2008) 1594–1602. http://dx.doi.org/ 10.1161/circulationaha.107.729125. [7] J.L. Vincent, R. Moreno, J. Takala, et al., The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine, Intensive Care Med. 22 (7) (1996) 707–710. [8] S.K. Cribbs, D.J. Sutcliffe, W.R. Taylor, et al., Circulating endothelial progenitor cells inversely associate with organ dysfunction in sepsis, Intensive Care Med. 38 (3) (2012) 429–436. http://dx.doi.org/10.1007/s00134-012-2480-9.
287
[9] E.A. Liehn, O. Postea, A. Curaj, N. Marx, Repair after myocardial infarction, between fantasy and reality: the role of chemokines, J. Am. Coll. Cardiol. 58 (23) (2011) 2357–2362. http://dx.doi.org/10.1016/j.jacc.2011.08.034. [10] I. Kanzler, N. Tuchscheerer, G. Steffens, et al., Differential roles of angiogenic chemokines in endothelial progenitor cell-induced, angiogenesis, Basic Res. Cardiol. 108 (1) (2013) 310.
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Key role of MIF in the migration of endothelial progenitor cells in patients during cardiac surgery☆ Christoph Emontzpohl a,b,1, Andreas Goetzenich c,1, David Simons d,e,1, Sandra Kraemer c, Manfred Dewor a, Hongqi Lue a, Luise Hammer a,b, Denise Jacobs a, Gerrit Grieb e, Patrick Ziegler f, Jens Panse f, Rolf Rossaint b, Jürgen Bernhagen a,⁎, Christian Stoppe a,b,1,⁎⁎ a
Institute of Biochemistry and Molecular Cell Biology, University Hospital, RWTH Aachen University, Germany Department of Anesthesiology, University Hospital, RWTH Aachen University, Germany c Department for Thoracic and Cardiovascular Surgery, University Hospital, RWTH Aachen University, Germany d German Cancer Research Center (DKFZ), Radiology (E010), Heidelberg, Germany e Department of Plastic Surgery and Hand Surgery, Burn Center, University Hospital, RWTH Aachen University, Germany f Department of Oncology, Hematology, Hemostaseology, and Stem Cell Transplantation, University Hospital, RWTH Aachen University, Germany b
a r t i c l e
i n f o
Article history: Received 10 November 2014 Accepted 22 November 2014 Available online 18 December 2014 Keywords: Cardiac surgery MIF — macrophage migration inhibitory factor EPC — endothelial progenitor cells EPC migration Ischemia and reperfusion (IR) Cytokines
Macrophage migration inhibitory factor (MIF) acts as an upstream regulator of the innate and adaptive immune response and exhibits chemokine-like activities [1,2]. Conclusive evidence also indicates that ☆ Author contributions. Conceived and designed the experiments: CE, AG, DS, SK, RR, CS, and JB. Performed the experiments: CE, AG, DS, CS, DJ, PZ, and LH. Analyzed the data: CE, AG, DS, CS, SK, LH, and HL. Contributed reagents/materials/analysis tools: CE, AG, DS, CS, PZ, JP, MD, DJ, GG, RR, and JB. Wrote the paper: CE, AG, DS, HL, GG, MD, JP, CS, and JB.All authors read and approved the final version of the manuscript. ⁎ Correspondence to: C. Stoppe, Institute of Biochemistry and Molecular Cell Biology & Department of Anesthesiology, University Hospital of the RWTH Aachen, Pauwelsstraße 30, 52074 Aachen, Germany. ⁎⁎ Correspondence to: J. Bernhagen, Institute of Biochemistry and Molecular Cell Biology, University Hospital of the RWTH Aachen, Pauwelsstraße 30, 52074 Aachen, Germany. E-mail addresses: [email protected] (C. Emontzpohl), [email protected] (A. Goetzenich), [email protected], [email protected] (D. Simons), [email protected] (S. Kraemer), [email protected] (M. Dewor), [email protected] (H. Lue), [email protected] (L. Hammer), [email protected] (D. Jacobs), [email protected] (G. Grieb), [email protected] (P. Ziegler), [email protected] (J. Panse), [email protected] (R. Rossaint), [email protected] (J. Bernhagen), [email protected] (C. Stoppe). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ijcard.2014.11.226 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
MIF provides cardioprotection during myocardial ischemia–reperfusion (I/R) [3]. Moreover, MIF promotes neovascularization during hypoxic stress through recruitment of endothelial progenitor cells (EPC) after myocardial I/R [4]. However, studies in clinical settings in particular in comparison with other angiogenic cytokines are lacking. Since patients undergoing cardiac surgery are frequently exposed to myocardial I/R with a following inflammatory response and excessive release of MIF [5,6], we surmised this clinical setting to represent an ideal model to characterize the significance of this cytokine on overall EPC migration during myocardial I/R. Therefore, we followed up on patients that underwent elective cardiac surgery with the use of cardiopulmonary bypass (CPB). Exclusion criteria were emergency operations, pregnancy, patients' age less than 18 years and failure to obtain informed consent. The institutional review board approved this study. Anesthesia, surgical procedure and data collection were performed as previously described [5]. Serum/blood samples were drawn immediately before surgery, at admission to ICU as well as 6 h, 12 h and 24 h postoperatively. Relevant clinical data of these patients were documented. Serum concentrations of stromal cell-derived factor (SDF)-1α/CXCL12, interleukin (IL)-8/CXCL8 and vascular endothelial growth factor (VEGF) were determined with commercially available ELISA assays (R&D Systems) according to the manufacturer's instructions. Serum levels of MIF were assessed using an ELISA technique as previously described [2]. EPCs were isolated according to our established protocol [4] by gating CD34 + cells and a subsequent selection for CD133 +/ CD33− from mononuclear cell (MNCs) fractions obtained by density gradient centrifugation from human blood. Migration assays were performed using Transwell migration chambers with a filter pore-size of 5 μm. EPCs were placed into upper chambers. Lower chambers contained increasing concentrations of recombinant MIF, CXCL12, CXCL8, VEGF165 or serum samples (alone, or combined with anti-MIF antibody). After 3 h of migration, cells were fixed, stained with Hoechst dye, diluted in 3.6% PFA (1:1000) and counted. Data were analyzed by one-way-ANOVA and post-hoc Bonferroni's multiple comparison tests using Prism 6.0 (Graphpad) to compare
C. Emontzpohl et al. / International Journal of Cardiology 181 (2015) 284–287
differences between multiple groups and Student's unpaired t-test when analyzing two groups. In all cases, P b 0.05 was considered to be statistically significant. In total, 103 patients were enrolled in this study and followed until the final analysis. All included patients reflected a representative cohort with coronary artery disease, presenting typical comorbidities like hypertension. At first, we studied the influence of myocardial I/R on the release of MIF, CXCL12 and CXCL8. All three cytokines significantly increased until admission to ICU whereas serum levels of VEGF remained unchanged during the period (Fig. 1a–d), indicating a minor influence of VEGF on EPC mobilization and migration. Next, we tested the potential influence of the observed cytokines on EPC recruitment in vivo and measured the proportion of EPCs in whole blood samples by flow cytometry. Corresponding to the increased concentrations of MIF, CXCL12 and CXCL8, the proportion of EPCs significantly increased post-operatively and decreased
285
again to almost baseline values 24 h after surgery (Fig. 1e). To investigate direct effects of patients' serum samples on EPC migration, we performed in vitro migration assays using perioperatively drawn serum samples as chemoattractant and EPCs from healthy volunteers. A significantly increased EPC migration towards the intraoperatively taken samples (+ 52% compared to baseline, p = 0.03, Fig. 1f) was observed. In a next step, we performed a dose–response analysis evaluating MIF, CXCL12, CXCL8, and VEGF on EPC migration. This experiment revealed a significant and dose-dependent increase in EPC migration in response to all cytokines (Fig. 2a–d). MIF significantly increased EPC migration compared to medium control with a maximum observed at 10 ng/ml (8-fold; p = 0.05) and appeared much more potent than the other cytokines (CXCL12 (5-fold at 100 ng/ml, p = 0.05), CXCL8 (3-fold at 50 ng/ml, p = 0.04), and VEGF 165 (2-fold at 10 ng/ml, p = 0.02)).
Fig. 1. Generated data using samples of cardiac surgical patients. (a–d) Measurement of serum concentrations of MIF (a), CXCL12 (b), CXCL8 (c) and VEGF (d) of patients that underwent cardiac surgery using ELISA. The dashed area indicates the duration of cardiac surgery. Shown are mean values ± SEM (*: p b 0.05 vs. pre-OP). (e) Shown is the in vivo EPC mobilization in 10 patients during cardiac surgery with an increase of circulating EPCs as part of the total CD34+ cells and a subsequent decrease to baseline values 24 h post-operatively. Each time point represents a mean value (n = 10) ± SEM (*: p b 0.05 vs. pre-op). The dashed area indicates the duration of cardiac surgery. (f) Shown is the in vitro migration of EPCs of healthy volunteers towards serum samples of patients (n = 7) who underwent cardiac surgery with an increased migration towards intra-operatively taken serum samples. For the migration assay 50,000 cells per well were used and put into the upper chamber of a modified Boyden chamber together with MV2 serum starved endothelial medium. The serum samples in the lower chamber were diluted 1:5 with the same MV2 serum starved medium. The cells migrated for 3 h through a 5 μm transwell membrane. The bars represent mean values (n = 7) ± SEM (*: p b 0.05).
286
C. Emontzpohl et al. / International Journal of Cardiology 181 (2015) 284–287
To get a better understanding of the influence of these cytokines on EPC recruitment in vivo, migration experiments, applying the physiologically measured concentrations of these cytokines were performed. A 6-fold increase in EPC migration upon chemotactic stimulation was observed using 50–100 ng/ml of recombinant MIF, — a level that corresponds to the measured post-operative MIF levels. As much higher concentrations of CXCL12 (100 ng/ml), CXCL8 (over 50 ng/ml) and VEGF (over 10 ng/ml) than measured in the patients' serum samples were needed to show an effect on EPC migration (Fig. 2b–d), we concluded that MIF's effect on the initial mobilization of EPCs is of high physiological significance compared to other cytokines. The pro-migratory effect of patient serum in the presence versus absence of neutralizing MIF antibodies was evaluated next, confirming that MIF has a significant stimulatory effect of EPC migration under in vivo conditions (Fig. 2e). Since MIF seems to have a strong influence on EPC migration/mobilization, its significance on clinical outcome of patients was analyzed. Interestingly, we had previously noticed a strong inverse correlation between EPCs on the pre-operative day and the well-established SOFA score after surgery, which adequately reflects the extent of organ injury [7]. These data are in accordance with Cribbs et al., who demonstrated an inverse correlation between circulating EPCs and the severity of organ dysfunction in sepsis patients [8]. In summary, we showed that myocardial I/R during cardiac surgery leads to increased levels of MIF, CXCL12, and CXCL8, which are known chemoattractants for the recruitment of EPCs. Among these, only MIF showed a significant effect on the mobilization of EPCs at concentrations comparable to those measured in the clinical samples. Since previous studies demonstrated that only EPCs transplanted shortly after infarction had maximal beneficial effects as compared to a later transplantation time point [9] and since MIF levels increased immediately after myocardial I/R, we hypothesize that MIF-mediated effect on the recruitment of EPCs affects cardiac remodeling and angiogenesis after myocardial I/R [10]. Our study highlights a predominant role of MIF on EPC recruitment in this context. Conflict of interest All authors state that no competing financial interests exist. Acknowledgment We are indebted to the laboratory staff of the Institute of Biochemistry and Molecular Cell Biology and the Department of Oncology, Hematology, Hemostaseology and Stem Cell Transplantation (University Hospital of the RWTH Aachen) for the excellent scientific and technical assistance, as well as the patients who participated in this trial. This study was supported by the Deutsche Forschungsgemeinschaft (DFG) grants Be1977/4-2 (TP01 of DFG-FOR809), DFG-IRTG1508/1-P13 and Ra969/6-1 to J.B., by START and Rotation Program grants of the faculty of medicine of RWTH Aachen University to C.S. Fig. 2. In vitro migration of EPCs towards recombinant cytokines. Shown is the dose–response relationship between EPCs and recombinant MIF (a), CXCL12 (b), CXCL8 (c) and VEGF165 (d). We observed the strongest increase in EPC migration at 10 ng/ml recombinant MIF. The recombinant cytokines were diluted in MV2 serum starved medium. The cells migrated for 3 h through a 5 μm transwell membrane. The bars represent mean values ± SEM (*: p b 0.05, **: p b 0.01 vs. serum starved medium). Also shown is the EPC migration towards a range of cytokine concentrations we measured previously in serum samples (cf. Fig. 1a–d). Only MIF was able to mediate an increased EPC migration at physiologically measured concentrations (n = 3–5), which exhibited the strongest EPC migration effect at the same time (8-fold increase compared to baseline). (e) Shown is the migration of EPCs towards the diluted serum sample of one patient at one time point at a time (n = 4) and the additional antiMIF antibody. The antiMIF antibody was diluted in MV2 serum starved medium + serum sample. The cells migrated for 3 h through a 5 μm transwell membrane. 120 μg/ml antiMIF antibody led to a reduction in EPC migration of about 24%. Also shown is the corresponding isotype control. The bars represent mean values ± SEM (n = 4) (*: p b 0.05).
References [1] T. Calandra, T. Roger, Macrophage migration inhibitory factor: a regulator of innate immunity, Nat. Rev. Immunol. 3 (10) (2003) 791–800. http://dx.doi.org/10.1038/ nri1200. [2] J. Bernhagen, R. Krohn, H. Lue, et al., MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment, Nat. Med. 13 (5) (2007) 587–596. http://dx.doi.org/10.1038/nm1567. [3] T. Rassaf, C. Weber, J. Bernhagen, Cardiovasc. Res. 102 (2) (2014) 321–328. [4] D. Simons, G. Grieb, M. Hristov, et al., Hypoxia-induced endothelial secretion of macrophage migration inhibitory factor and role in endothelial progenitor cell recruitment, J. Cell. Mol. Med. 15 (3) (2011) 668–678. http://dx.doi.org/10.1111/j.15824934.2010.01041.x. [5] C. Stoppe, G. Grieb, R. Rossaint, et al., High postoperative blood levels of macrophage migration inhibitory factor are associated with less organ dysfunction in patients after cardiac surgery, Mol. Med. 18 (2012) 843–850.
C. Emontzpohl et al. / International Journal of Cardiology 181 (2015) 284–287 [6] A. Zernecke, J. Bernhagen, C. Weber, Macrophage migration inhibitory factor in cardiovascular disease, Circulation 117 (12) (2008) 1594–1602. http://dx.doi.org/ 10.1161/circulationaha.107.729125. [7] J.L. Vincent, R. Moreno, J. Takala, et al., The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine, Intensive Care Med. 22 (7) (1996) 707–710. [8] S.K. Cribbs, D.J. Sutcliffe, W.R. Taylor, et al., Circulating endothelial progenitor cells inversely associate with organ dysfunction in sepsis, Intensive Care Med. 38 (3) (2012) 429–436. http://dx.doi.org/10.1007/s00134-012-2480-9.
287
[9] E.A. Liehn, O. Postea, A. Curaj, N. Marx, Repair after myocardial infarction, between fantasy and reality: the role of chemokines, J. Am. Coll. Cardiol. 58 (23) (2011) 2357–2362. http://dx.doi.org/10.1016/j.jacc.2011.08.034. [10] I. Kanzler, N. Tuchscheerer, G. Steffens, et al., Differential roles of angiogenic chemokines in endothelial progenitor cell-induced, angiogenesis, Basic Res. Cardiol. 108 (1) (2013) 310.
