Journal of Molecular and Cellular Cardiology 60 (2013) 1–7

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Journal of Molecular and Cellular Cardiology

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Original article

CXC chemokine KC fails to induce neutrophil infiltration and neoangiogenesis in amouse model of myocardial infarction

Hasan Oral a, Isabella Kanzler a,c,d, Nancy Tuchscheerer a,c, Adelina Curaj a,e, f, Sakine Simsekyilmaz a,Tolga Taha Sönmez g, Eugen Radu h, Otilia Postea a, Christian Weber a,i,Alexander Schuh a,b,1, Elisa A. Liehn a,b,1,⁎a Institute for Molecular Cardiovascular Research, RWTH Aachen University, Germanyb Department of Cardiology, RWTH Aachen University, Germanyc Institute for Biochemistry and Molecular Cell Biology, RWTH Aachen University, Germanyd Department of Cardiothoracic and Vascular Surgery, Johann Wolfgang Goethe University, Frankfurt/Main, Germanye Department of Experimental Molecular Imaging, RWTH Aachen University, Germanyf Victor Babes National Institute of Pathology, Bucharest, Romaniag Department of Oral and Maxillofacial Surgery, RWTH Aachen University, Aachen, Germanyh Department of Cellular and Molecular Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romaniai Institute for Cardiovascular Prevention, Ludwig MaximiliansUniversity (LMU) Munich, Munich, Germany

⁎ Corresponding author at: Medizinische Klinik I, IKreislaufforschung, Universitätsklinikum Aachen, RheHochschule Aachen, Pauwelsstrasse 30, D-52074Aachen, G

E-mail address: eliehn@ukaachen.de (E.A. Liehn).1 These authors equally contributed by this paper.

0022-2828/$ – see front matter © 2013 Elsevier Ltd. Allhttp://dx.doi.org/10.1016/j.yjmcc.2013.04.006

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 23 November 2012Received in revised form 17 March 2013Accepted 8 April 2013Available online 15 April 2013

Keywords:KCMyocardial infarctionChemokineNeutrophilsAngiogenesis

Background: Chemokines and neutrophils, known as important players in the inflammatory cascade, also con-tribute to heart tissue recovery and scar formation after myocardial infarction (MI). The objective of this studywas to determine the importance of ELR-containing CXC chemokine KC in neutrophil infiltration andneoangiogenesis, in a mouse model of chronic MI.Methods and results: MIwas induced inmice divided in four groups: control (untreated), anti-KC “later” (anti-KCantibody injections started 4 days afterMI and then delivered every 72 hours for 3 weeks, to inhibit angiogenesis),anti-KC “earlier” (anti-KC antibody injections 1 day before and 1 day after MI, to block neutrophil infiltration),anti-KC (anti-KC antibody injections 1 day before and 1 day after MI, and then every 72 hours for 3 weeks). Theefficiency of the anti-KC treatment was determined by themeasurement of KC serum concentration and immuno-fluorescence staining, in each of the four groups. Surprisingly, we did not find any difference in neutrophil infiltra-tion in the infarcted area between untreated and treated animals. Moreover, the heart function, infarct size, and

neoangiogenesis were not different between the four groups. As expected, a comparable anti-CXCR2 treatmentof mice before and after MI was able to significantly reduce neutrophil infiltration into the infarcted area and an-giogenesis, but also to reduce the infarction size after long or “later” treatment.Conclusions: The major finding of our study is that KC, a potent neutrophil chemoattractant and an established an-giogenic factor, failed to interfere in the post-infarction inflammatory response, in wound healing and scar forma-tion after MI. Therefore, these aspects need to be carefully taken into account when devising therapeutic strategiesfor myocardial infarction and ischemic cardiomyopathy.

© 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Myocardial infarction (MI) triggers an inflammatory cascade whichis involved in cardiac remodeling. Granulation tissue formation, deposi-tion of collagen-based matrix, complement activation and free radicalgeneration, followed by a complex cytokine cascade and an up-regulation of chemokines [1–4] are important events that promote

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wound healing after MI. Chemokines induce and control an inflamma-tory reaction responsible for the myocardial recovery and scar forma-tion after MI [2,5].

Neutrophils are the first cells to be recruited to the infarction site inresponse to a variety of stimuli, including complement activation andthe production of chemokines. Accumulated neutrophils release toxicproducts like proteolytic enzymes or reactive oxygen species, that di-rectly injure the surrounding myocytes [6,7]. Neutrophil depletion inanimals undergoing myocardial infarction led to a marked decrease ininfarct size, confirming that a significant amount of myocardial injurymay be neutrophil-dependent [8–10]. Therefore, manipulating neutro-phil infiltration seems to be crucial in controlling neutrophil-inducedpathophysiological changes after myocardial infarction.

Table 1Study groups and protocol experiments.

Groups Treatment

Before MI MI After MI

1 day 1 day 4 days Until 3 weeks

Control (n = 5) − − − − −Anti-KC “later” (n = 5) − − − x Every 72 hoursAnti-KC “earlier” (n = 6) × − × −Anti-KC (n = 6) × − × × Every 72 hoursIsotype control (n = 6) × − × × Every 2 daysAnti-CXCR2 “later” (n = 7) − − − × Every 2 daysAnti-CXCR2 “earlier” (n = 5) × − × − −Anti-CXCR2 (n = 5) × − × × Every 2 days

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Among various inflammatory mediators, CXC chemokines includingInterleukin 8 (IL-8, CXCL8), macrophage inflammatory protein 2 (MIP-2,CXCL2) and keratinocyte-derived chemokine (KC, CXCL1) are the mostcritical for neutrophil recruitment [5]. In themouse systemno IL-8 homo-log has been identified so far; however MIP-2 and KC may be functionalhomologous and activate mouse neutrophils via a unique IL-8 receptor(homolog of human CXCR2) [11]. Although KC and MIP-2 have similarin vitro synthesis characteristics, KC appears to be a more potent and sys-temically distributed chemokine during acute in vivo inflammation, whileMIP-2 expression appears limited to localized expression [12]. In themodel of myocardial ischemia-reperfusion, it seems that neutrophilrecruitment to reperfused rat myocardium is only in part due to KCand MIP-2, mainly being due to cardiomyocyte expression of a newlipopolysaccharide-induced CXC chemokine (LIX) [13].

In addition to the role in neutrophil infiltration, KC was also con-sidered as a marker for angiogenic events in the inflammatory granu-lation tissue [14]. It seems that KC promotes angiogenesis by a CXCreceptor-2 dependent mechanism [15–18] and, as we have previouslyshown, is crucial for endothelial recovery after vascular injury [19].

ELR-containing CXC chemokine KC is expressed inmyocardium afterMI induction [13], but its role inmyocardial healing is far from being un-derstood. To test the involvement of KC in the recovery after myocardialinfarction we have treated the mice, at different time points, before andafter MI, with an anti-KC antibody or an anti-CXCR-2 antibody. Our datasuggest that KC, a potent neutrophil chemoattractant and an establishedangiogenic factor, failed to mediate the inflammatory response and an-giogenesis in the damaged myocardium, suggesting a minimal or evenabsent role in wound healing and scar formation after MI.

2. Methods

2.1. Murine model of MI

Wild-typemice (C57/Bl6,male, 8–10 weeks, Taconic)were intubatedunder general anesthesia (100 mg/kg ketamine, 10 mg/kg xylazine, i.p.)and ventilated with oxygen containing 0.2% isofluran using a rodent res-pirator.MIwas performed as previously described [10]. Briefly, the heartswere exposedby left thoracotomy andMIwas producedby ligation of theleft anterior descending artery (LAD). To confirm the efficiency of themethod we followed the heart tissue turning pale under the ligature.All animal experiments were approved by the Ethical Committee in Aa-chen (Bezirksregierung Köln, NR) and compliedwith the GermanAnimalProtection Law.

2.2. Antibody treatment

We performed the treatment according to our previous study[19,20]. The mice were divided in four groups; the first group remaineduntreated and served as control (Table 1). To inhibit the potential angio-genic events which are most effective 4 days after MI, the second groupreceived intraperitoneal 50 μg anti-KC (mAb, RnD Systems), 4 days afterMI for thefirst time and then every 72 hours for 3 weeks (anti-KC “later,”respectively). The third group received the same dose of anti-KC (anti-KC“earlier,” respectively) 1 day before and 1 day after MI to block neutro-phil infiltration which occurs within the first day after MI. The fourthgroup was treated with 50 μg anti-KC 1 day before and 1 day after MIand then every 72 hours for 3 weeks (anti-KC, respectively), to blockboth the neutrophil infiltration and angiogenesis (Table 1).

We performed anti-CXCR2 antibody treatment (mAb, RnD Systems)in the same settings (Table 1) [20]. As control, we used an isotype controlantibody (the same for anti-KC and anti-CXCR2 antibody), and we didnot observe any differences compared with untreated group. Similarto the anti-KC treatment, we divided the mice in anti-CXCR2 “early”group, which received intraperitoneal 50 μg anti-CXCR2 1 day beforeand 1 day after MI to block neutrophil infiltration, anti-CXCR2 “late”group, which received intraperitoneal 50 μg anti-CXCR2 4 days after

MI for the first time and then every 2 days until 3 weeks, and the anti-CXCR2 “total” group, whichwas treatedwith 50 μg anti-KC 1 day beforeand 1 day after MI and then every 2 days until 3 weeks (Table 1).

Both antibody treatments were successfully used in a previouslypublished mouse model of restenosis, and were shown to impair theendothelial function [19,20].

2.3. Echocardiography

Two-dimensional and M-mode echocardiographic measurements(Vevo 770, Visual Sonics, Toronto, Canada) were performed beforeand 4 weeks after induction of MI. Mice were anesthetized with1.5% isofluran via a mask and placed in the supine position on awarming pad. The ejection fraction, cardiac output and stroke volumewere analyzed.

2.4. Langendorff perfusion

Three weeks after MI the hearts were excised after euthanasia andthe heart function was analyzed using a Langendorff apparatus andIsoheart software (Hugo Sachs Elektronik-Harvard Apparatus) underconstant perfusion pressure (100 mmHg) and electrical stimulation toassure constant heart rates (600 bpm) [10]. Developed pressure, deriv-ative pressure increase and decay and coronaryflowweremeasured. Fi-nally, hearts were fixed with 10% formalin, paraffin embedded and cutinto 5 μm serial slices for immunohistochemistry experiments.

2.5. Histomorphometry and determination of MI size

Serial sections (10–12 per mouse, 400 μm apart, up to the mitralvalve) were stained with Gomori's 1-step trichrome stain [10]. Theinfarcted area was determined in all sections using Diskus software(Hilgers) and expressed as percentage of total left ventricular volume.In the same sections, collagen content was analyzed (Analysis Soft-ware) and expressed as percentage from infarcted area.

2.6. Immunofluorescence

Serial sections (3 per mouse, 400 μm apart) were stained to analyzeinfarcted areas for the content of vessels at the end-point (CD31, SantaCruz) and neutrophils 1 day after MI (specific esterase, Sigma). Doublestaining was performed for neutrophils (Specific Esterase Kit, Sigma)and mKC using a rat anti-mouse KC antibody (Santa Cruz Biotech.)followed by an anti-rat FITC conjugated secondary antibody. Further-more, 1 day after infarction, sections with and without treatment werestained for MPO (rabbit polyclonal, Thermo Scientific, followed by aFITC-conjugated secondary antibody). Cells or vessels were counted in6 different fields per section and expressed as cells or vessels/mm2.

ELISA was performed from serum samples at the indicated timepoints (Fig. 1) using DuoSet ELISA Development System kit for mouseKC (R&D Systems) according to themanufactures protocol. MPO activity

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was measured from serum samples and also in heart homogenates, byELISA Kit for MPO activity (Hycult Biotech.) according to the manufac-turer's protocol. TheMPO activity from heart homogenates was normal-ized to protein concentration and expressed as ng/mg protein.

2.7. mRNA isolation and RT-PCR

Total RNA isolated from the infarcted area with TRIzol Reagent(Gibco) was reverse transcribed (Omniscript kit, Qiagen) and amplifiedby RT-PCR [21] using specific intron-spanning primers for murine KC(forward primer: 5′-ACCGAAGTCATAGCCACACTC-3′ and reverse primer:5′-TCTCCGTTACTTGGGGACAC-3′). Murine GAPDH served as house-keeping gene and was amplified in parallel with the gene of interest(forward primer: 5′-ATTGTCAGCAATGCATCCTG-3′, reverse primer:5′-ATGGACTGTGGTCATGAGCC-3′).

2.8. Statistical analysis

Data representmean ± SEM. Statistical analysiswas performedwithPrism 4 software (Graph Pad) using unpaired Student-t test or 1-wayANOVA followed by Newmann–Keuls post test. P-values b0.05 wereconsidered significant.

3. Results

3.1. The efficiency of KC-antibody treatment after MI

As previously shown by our group, the treatment with anti-KC anti-body efficiently inhibits the KC-dependent endothelial regeneration andproliferation in vitro and in vivo [19]. In this study, the analysis of KC ex-pression after MI was performed by quantitative RT-PCR. One weekafter MI the KCmRNA levels were two-fold higher than those in the con-trol group (Fig. 1A). In contrast, KC serumexpressionwas dramatically in-creased on the first day after MI (881.0 ± 177.0 pg/ml) compared withbasal condition (95.29 ± 11.8 pg/ml). After 1 week, KC serum expres-sion returns to the basal values (Fig. 1B). These data are not surprising,since we have previously already shown that KC serum level increasesafter a surgical operation, both in injured and sham operated animals[19]. The ELISAmeasurements have indicated that anti-KC treatment suc-cessfully neutralizes KC and assures a minimal KC serum concentration

Fig. 1. The efficiency of treatment of myocardial infarction with anti-KC-antibody. Quantitaincrease in myocardial KC mRNA expression at different time points (A, *p b 0.05 vs. contrin the control group was successfully blocked by anti-KC antibody treatment (B, *p b 0.05 vsduring the entire experimental period, as shown by the KC staining 3 weeks after MI (C, sc

(107.3 ± 21.68 pg/ml, p b 0.05 vs. control group, Fig. 1B). KCmyocardialexpression was abolished in all groups treated with anti-KC until theend-point, as shown by immunofluorescence staining of myocardiumsections, 3 weeks after MI (Fig. 1C).

Surprisingly, although theMPO activity, describing neutrophil activa-tion, increased significantly 1 day afterMI in serum (Fig. 2A), aswell as ininfarcted myocardium and remote areas (Fig. 2B), it was not affected bythe anti-KC treatment (Figs. 2A,B), as well as the neutrophil infiltration(927 ± 61 neutrophils/mm2 in control, 947 ± 197 neutrophils/mm2

anti-KC after treatment, Fig. 2C). The group treated with anti-KC showedthe same neutrophil infiltration as the control group, despite successfulneutralization of myocardial KC expression (Fig. 2D). As control, ananti-CXCR2 antibody treatment does not affect the MPO activity in theserum, but reduced significantly the MPO activity in the infarction area(Fig. 2B), and the neutrophil infiltration (697 ± 45 neutrophils/mm2,*p b 0.05 vs. control, Fig. 2C).

3.2. The effect of anti-KC-antibody on heart parameters after MI

Anti-KC treatment did not influence the survival rates of the mice inany of the four groups tested, as no differences were observed betweencontrol group and treated mice. Echocardiography showed a reducedejection fraction (EF) comparedwith normvalues (>60%), but no signif-icant differences between the groups (Table 2). In all groups cardiac out-put was adequately compensated (Table 2). Heart rate and heart weightwere also comparable between the groups. Furthermore, Langendorffperfusion of isolated hearts showed no significant differences in left ven-tricular developed pressure (LVDP) between the four groups. The LVDP,dP/dtmax and dP/dtmin measurements demonstrate no effect of the dif-ferent types of anti-KC treatment on contraction and relaxation of theleft ventricle (Table 2). Coronary flow also remained unaffected byanti-KC treatment (Table 2). In contrast, although the anti-CXCR2antibodywas not able to improve the heart functionwhen administratedshort after MI, a long term administration, even if started 4 days laterafter MI, was able to significantly improve the heart function (Table 3).

3.3. Histological and immunohistochemical analysis of MI area

Five micrometer serial slices of formalin-fixed paraffin embeddedhearts showed no significant differences in infarction size between the

tive PCR before (0 d), 1 day, 4 days, 7 days and 14 days after MI showed a significantol group, n = 4). The significant increase in serum concentration of KC 1 day after MI. control group, n = 4). Anti-KC antibody treatment successfully blocked KC expressionale bar: 50 μm).

Fig. 2. Effect of antibody treatment on neutrophil infiltration after myocardial infarction. MPO activity is increased 1 day after MI in serum (A), but also in myocardium (B). Treat-ment with anti-KC does not block the MPO activity in the serum or myocardium, and does not affect neutrophil infiltration, while treatment with anti-CXCR2 significantly reducedMPO activity, as well as the neutrophil infiltration in the infracted area (C, *p b 0.05 vs. control, n = 4). Double staining of neutrophils (red) and KC (green) showed an unalteredneutrophil infiltration but impaired increase in KC expression in myocardium, 1 day after MI (D, scale bar: 50 μm).

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four groups (19.08 ± 3.77 % in control, 21.45 ± 6.68 % in anti-KC“later,” 18.33 ± 6.08 % in anti-KC “earlier” and 17.40 ± 2.84 % inanti-KC, Fig. 3A), while a long or later anti-CXCR2 treatment significantlyreduced the infarction size (20.48 ± 1.81 % in control, 11.35 ± 1.04 % inanti-CXCR2 “later,” 17.93 ± 2.90 % in anti-CXCR2 “earlier” and 9.86 ±1.68 % in anti-CXCR2, *p b 0.05 vs. control, Fig. 3B).

KC failed to induce neoangiogenesis in infracted area, which wasconfirmed by CD31-immunofluorescence staining, showing no ef-fect of KC on vessel formation after myocardial infarction (1590 ±242 vessels/mm2 in control, 1964 ± 129 vessels/mm2 in anti-KC“later,” 1852 ± 137 vessels/mm2 in anti-KC “earlier” and 1906 ±164 vessels/mm2 in anti-KC, Fig. 3C). On the other hand, aswe expected,the long or later anti-CXCR2 treatment significantly reduced angio-genesis in the infarction area (1025 ± 86 vessels/mm2 in control,

Table 2Hemodynamic parameters after anti-KC treatment.

Echocardiography

Control Anti-K

Ejection fraction (%) 40.69 ± 5.51 45.1Cardiac output (ml/min) 17.05 ± 1.77 15.5Heart rate (bpm) 441.20 ± 77.71 406.3Heart weight (mg) 121.49 ± 36.90 128.0

Langendorff perfusionLVDP (mmHg) 32.5 ± 6.35 25.0dP/dtmax (mmHg/s) 1927 ± 326.3 146dP/dtmin (mmHg/s) −1194 ± 244.8 −863.Coronary flow (ml/min) 3.733 ± 0.43 3.63

LVDP — left ventricular developed pressure; dPdt — derivative of pressure increase (max) a

813 ± 22 vessels/mm2 in anti-CXCR2 “later,” 1037 ± 82 vessels/mm2

in anti-CXCR2 “earlier” and 761 ± 28 vessels/mm2 in anti-CXCR2,Fig. 3D).

4. Discussion

The major finding of our study is that KC, a potent neutrophilchemoattractant and an established angiogenic factor failed to interferein the postinfarction inflammatory response, in wound healing and scarformation after myocardial infarction. We saw no differences betweenthe neutrophil infiltration inmice treatedwithmKC-antibody or isotypecontrol antibody, respectively, and angiogenesis in all studied groups.The treatment with an antibody against anti-CXCR2, the specific KC re-ceptor, successfully reduced neutrophil infiltration, but failed to reduce

C “later” Anti-KC “earlier” Anti-KC

3 ± 8.17 48.08 ± 7.79 55.31 ± 3.669 ± 1.90 15.54 ± 1.05 16.69 ± 1.033 ± 48.34 416.60 ± 42.44 407.40 ± 30.170 ± 24.21 125.96 ± 27.79 127.12 ± 20.18

3 ± 17.85 28.74 ± 11.67 30.96 ± 13.311 ± 876.0 1437 ± 491.3 1378 ± 543.77 ± 534.3 −1061 ± 398.1 −1022 ± 322.33 ± 0.49 3.567 ± 0.18 3.8 ± 0.60

nd decay (min).

Table 3Echocardiographic parameters after anti-CXCR2 treatment.

Control Anti-CXCR2 “later” Anti-CXCR2 “earlier” Anti-CXCR2

Ejection fraction (%) 40.00 ± 2.59 51.29 ± 2.54⁎ 40.40 ± 4.08 51.20 ± 2.78⁎

Cardiac output (ml/min) 15.00 ± 1.34 14.15 ± 2.74 15.98 ± 1.28 15.29 ± 1.64Heart rate (bpm) 385.30 ± 26.52 376.30 ± 26.82 394.30 ± 11.82 371.00 ± 14.32Heart weight (mg) 103.00 ± 16.86 108.01 ± 13.99 112.80 ± 19.15 100.03 ± 15.15

⁎ p b 0.05 vs. control.

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infarction size in this group. Surprisingly, a later anti-CXCR2 treatment,which significantly reduced angiogenesis, leads to a significant reductionin infarction size.

There are many published results which sustain the involvement ofKC in chemotaxis and neutrophils activation [2,5,22] and point out KCas the most potent CXCR2 ligand in the mouse [11,12]. Deficiencyof CXCR2, the main receptor for the ELR-containing CXC chemokines,resulted in significantly decreased inflammatory leukocyte recruitmentin murine infarcts [11,18]. Considering these facts, it seems that theother CXCR2-binding chemokines besides KC interfere and regulatethe neutrophil trafficking after myocardial infarction. Chandrasekaret al. demonstrated in a rat model of ischemia/reperfusion that theELR-containing CXC chemokines are up-regulated after reperfusion,and anti-LIX (CXCL5) treatment was able to reduce about 80% of theneutrophil infiltration, compared with anti-KC and anti-MIP-2, only40% [13]. In our model, despite the reduction of neutrophil infiltrationafter anti-CXCR2 treatment, the anti-KC treatment had no effect on

Fig. 3. Histological and immunohistochemical analysis of myocardial infarction. No differenanti-CXCR2 treatment (B, *p b 0.05 vs. control). Anti-KC treatment did not affect neoangiogement significantly reduced angiogenesis in infracted areas (D, scale bar: 50 μm, *p b 0.05 v

neutrophil infiltration.Moreover, neutrophils frommicewith a targeteddeletion of CXCR2 failed to respond to KC, MIP-2, or human IL-8, but ex-hibit normal chemotaxis in response to the complement fragment C5a[23]. Ischemic myocardial injury can activate the complement cascade[24–26], but from all components, C5a is the most potent chemotacticanaphylatoxin, which can attract neutrophils to the site of inflamma-tion, leading to superoxide production and exerts proinflammatory ef-fects [27]. Recently, we also have shown an important role of CCR1and MIP-1α in mediating neutrophil infiltration after MI induction[10]. However, reduced neutrophil infiltration after anti-CXCR2 hadno hemodynamic consequences, and was not able to reduce the infarc-tion size or to improve the heart function. Therefore, since KC-CXCR2binding has no implication in our experimental setup, the developmentof new therapeutic strategies to prevent and treat heart damages afterischemia requires the elucidation of the precise role of complementand the other chemokines in the neutrophil recruitment after myocar-dial infarction.

ces were observed in infarct size after anti-KC treatment (A), but after long or “later”nesis (C) in the four groups (C, scale bar: 50 μm), while long or “later” anti-CXCR2 treat-s. control).

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Importantly, KC failed to promote angiogenesis in our model. Thisfinding is also very surprising, considering the various studies whichpoint out KC as a marker for angiogenic activity by a CXC receptor-2dependent mechanism [14–18]. Formation of new blood vessels iscritical for supplying the infarcted myocardium with oxygen and nu-trients necessary to sustain the metabolism; therefore myocardial in-farction is associated with an early release of angiogenic factors in theinjured areas. Numerous investigations have indicated that VEGF, IL-8and bFGF, all potent angiogenic agents, are rapidly induced in the is-chemic myocardium [28] and may have a role in enhancing infarctneovascularization. In the last decade, chemokines have shown animportant role not only in leukocyte trafficking but also in angiogenesisand cardioprotection [2]. Stromal cell-derived factor-1alpha (SDF-1alpha)has generated considerable interest for its role in stem cell recruitment tothe heart following myocardial infarction [29,30]. Macrophage migrationinhibitory factor (MIF) is a unique cytokine and critical mediator of anumber of immune and inflammatory diseases [31]. Recently, wehave shown that MIF can bind CXCR2 and CXCR4 [32]. It seems thatthis binding has a protective role inmyocardial infarction [33]; however,the exact role is far frombeing understood. Likewise, ELR-containing CXCchemokines as KC, MIP-2 and LIX can induce in vitro endothelial chemo-taxis and mediate in vivo angiogenesis by binding CXCR2 [15,17]. How-ever, in the model of subcutaneously implanted sponges in mice, ananti-angiogenic effectwasmost clearly and closely correlatedwith levelsof the chemokine KC, compared with other angiogenic factors [14]. Wealso found a crucial role of KC in promoting CXCR2-mediated endothelialchemotaxis and wound repair [19]. Therefore, it is intriguing that KChas no role in angiogenesis after myocardial infarction and this find-ing demandes extensive research to elucidate the mechanisms of thenew-vessel formation in ischemic heart.

On the other hand, it is also intriguing that, despite the reduced an-giogenesis after anti-CXCR2 treatment, the infarction size was reducedand the heart function was improved. That can be due to the unknowneffects of CXCR2 on differentmonocyte subtypes,whichwould be inter-esting to be studied in the future.

5. Conclusion

We have demonstrated in our study that KC has no significant con-tribution in the wound healing after myocardial infarction, despite itsrole as a powerful neutrophil chemoattractant and an established an-giogenic factor. Moreover, the signaling through its receptor CXCR2seems to have controversial results. Whereas the significant decreasein neutrophil infiltration does not affect the infarction size, the signifi-cant impairment of angiogenesis, surprisingly leads to a clear decreasein infarction size. Despite what hitherto believed, our data suggestthat the angiogenesis is not essential for the improvement of the infarc-tion size, and therefore, it is not the most appropriate therapeuticaltarget. These newmechanistic insights ofmyocardial injury and healingmust be carefully taken into accountwhen designing future therapeuticstrategies for myocardial infarction and ischemic cardiomyopathy.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

This study was supported by the Interdisciplinary Centre forClinical Research IZKF Aachen, within the Faculty of Medicine atRWTH Aachen University and Deutsche Forschungsgemeinschaft(Forschergruppe 809). This paper is partially supported by the SectoralOperational Programme Human Resources Development, financed fromthe European Social Fund and by the Romanian Government under thecontract number POSDRU/89/1.5/S/64109.

We also thank Dr. Octavian Bucur for the critical comments andthe constructive help for this paper.

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