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(The FASEB Journal. 2009;23:351-361.)
© 2009 FASEB

Erythropoietin administration after myocardial infarction in mice attenuates ischemic cardiomyopathy associated with enhanced homing of bone marrow-derived progenitor cells via the CXCR-4/SDF-1 axis

Stefan Brunner*, Janina Winogradow*, Bruno C. Huber*, Marc-Michael Zaruba*, Rebekka Fischer*, Robert David*, Gerald Assmann{dagger}, Nadja Herbach{ddagger}, Ruediger Wanke{ddagger}, Josef Mueller-Hoecker{dagger} and Wolfgang-Michael Franz*

* Klinikum Grosshadern, Medical Department I,

{dagger} Institute of Pathology, and

{ddagger} Institute of Veterinary Pathology, Ludwig-Maximilians-University, Munich, Germany

1Correspondence: Medizinische Klinik und Poliklinik I, Klinikum Großhadern, Marchioninistr. 15, 81377 Munich, Germany. E-mail: wolfgang.franz{at}med.uni-muenchen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mobilization of bone marrow-derived stem cells (BMCs) was shown to have protective effects after myocardial infarction (MI). However, the classical mobilizing agent, granulocyte-colony stimulating factor (G-CSF) relapsed after revealing an impaired homing capacity. In the search for superior cytokines, erythropoietin (EPO) appears to be a promising agent. Therefore, we analyzed in a murine model of surgically induced MI the influence of EPO treatment on survival and functional parameters as well as BMC mobilization, homing, and effect on resident cardiac stem cells (CSCs). Human EPO was injected intraperitoneally after ligation of the left anterior descendens (LAD) for 3 days with a total dose of 5000 IU/kg 6 and 30 days after MI, and pressure volume relationships were investigated in vivo. Cardiac tissues were analyzed by histology. To show the effect on BMCs and CSCs, FACS analyses were performed. Homing factors were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and ELISA. EPO-treated animals showed a significant improvement of survival post-MI (62 vs. 36%). At days 6 and 30, all hemodynamic parameters associated with attenuated remodeling, enhanced neovascularization, and diminished apoptotic cells in the peri-infarct area were improved. BMC subpopulations (CD31+, c-kit+, and Sca-1+ cells) were mobilized, and homing of Sca-1+ and CXCR4+ BMCs toward an SDF-1 gradient into the ischemic myocardium was enhanced. However, there was no beneficial effect on CSCs. We have shown that EPO application after MI shows cardioprotective effects. This may be explained by mobilization of BMCs, which are homing via the CXCR-4/SDF-1 axis. However, EPO has no beneficial effects on resident CSCs. Therefore, new treatment regimes using EPO together with other agents may combine complementary beneficial effects preventing ischemic cardiomyopathy.—Brunner, S., Winogradow, J., Huber, B. C., Zaruba, M.-M., Fischer, R., David, R., Assmann, G., Herbach, N., Wanke, R., Mueller-Hoecker, J., Franz, W.-M. Erythropoietin administration after myocardial infarction in mice attenuates ischemic cardiomyopathy associated with enhanced homing of bone marrow-derived progenitor cells via the CXCR-4/SDF-1 axis.


Key Words: EPO • cytokines • stem cell mobilization and homing


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE CURRENT CONCEPT OF ACUTE myocardial infarction (MI) therapy based on early coronary reperfusion results in a lower rate of mortality and improvement of prognosis in patients with coronary artery disease (1) . Post-infarction heart failure with the impairment of left ventricular (LV) function is a consequence of ventricular remodeling, which may result in an enlargement of the left ventricle. Clinical trials based on bone marrow-derived stem cells (BMCs) introduced into the heart showed modest effects on the improvement of LV function (2) . Mobilization of BMCs using hematopoietic growth factors such as granulocyte-colony stimulating factor (G-CSF) offers an alternative to the direct delivery of stem cells. Circulating mobilized stem cells can be recruited from the blood pool into the damaged tissue and this homing behavior is considered to be one mechanism of myocardial repair (3) .

Preclinical studies using G-CSF after induced MI revealed an improvement of survival and cardiac function accompanied by a diminished rate of apoptotic cells and a marked reduction of scar formation (4 , 5) . However, in the clinical setting prospective, randomized and placebo-controlled trials, including our own, have failed to show superiority of G-CSF over placebo treatment when given as adjunct after MI (6 , 7) .

The disruption of key cellular anchors within the bone marrow microenvironment revealed the chemokine receptor CXCR-4/SDF-1 complex as a pivotal element of stem-cell mobilization and homing (8) . The expression of CXCR-4 onto the surfaces of stem cells facilitates their migration along a gradient of the CXCR-4 ligand SDF-1. Since circulating CXCR-4+ stem cells home to loci of high SDF-1 concentrations (9) , one major drawback of G-CSF treatment was shown to be the N-terminal cleavage of CXCR-4 on mobilized hematopoietic progenitor cells, resulting in loss of chemotaxis in response to stromal cell-derived factor-1 (SDF-1) and leading to diminished stem-cell homing (10 , 11) .

In search of cytokines superior to G-CSF, a promising agent for tissue repair and protection is erythropoietin (EPO), a hematopoietic hormone synthesized in the kidneys and required for erythropoiesis. Beyond its classical function, EPO reveals tissue protective properties due to the expression of its receptor on a variety of other cells (12) . Different preclinical models of ischemic diseases showed tissue protective effects through activation of EPO receptor-related pathways (13 , 14) . Major pathways involved in postischemic protection are PI3K/Akt and MAPK, resulting in antiapoptotic effects (15 16 17 18) . Furthermore, EPO was found to be a potent stimulus for mobilization of endothelial progenitor cells (EPCs) into peripheral blood, which was associated with neovascularization of ischemic tissue (19 , 20) . In the clinical setting, treatment with recombinant human (rh)EPO results in a significant mobilization and functional activation of EPCs in patients with renal anemia as well as in healthy subjects (21) . In addition, serum levels of EPO were also significantly correlated with the number of circulating EPCs in patients suffering from coronary artery disease (19) . First clinical trials of EPO treatment after acute myocardial infarction have already proved safety and feasibility (22) .

In our study, we focused on the mechanisms involved in mobilization and homing and the effect on resident cardiac stem cells (CSCs) after acute MI in a preclinical mouse model.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal model
Healthy age- and sex-matched (male, 8–12 wk of age) C57BL/6 mice (Charles River Breeding Laboratories, Inc., Wilmington, MA, USA) were used for the experiments. Experimental procedures were performed as described previously (5) . Briefly, MI was induced by surgical occlusion of the left anterior descending (LAD) artery through a left anterolateral approach. Mice were anesthetized by i.p. injection of a mixture of 100 mg/kg ketamine (Sigma-Aldrich Corp., St. Louis, MO, USA) and 5 mg/kg xylazine (Sigma-Aldrich Corp.), intubated, and artificially ventilated by a mouse ventilator (Hugo Sachs Elektronik, March, Germany) with 200 strokes/min and 200 µl/stroke. Animal care and all experimental procedures were performed in strict accordance 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 were approved by the local animal care and use committees.

Administration of EPO
Mice were divided into the following groups: 1) sham-operated animals receiving saline (0.9% NaCl) for up to 3 days subcutaneously; 2) infarcted animals receiving saline; and 3) infarcted animals receiving EPO (Epoetin alpha; Janssen-Cilag, Neuss, Germany). EPO treatment was initiated immediately after the surgical procedure (day 1) with a starting dose of 3000 IU/kg. Treatment was continued with a dose of 1000 IU/kg at days 2 and 3, respectively (Fig. 1A ).


Figure 1
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  		Figure 1. Experimental design and cumulative survival. A) Animals were divided into 3 groups and sacrificed at 2 time points (days 6 and 30) to evaluate the effects of EPO on survival, hemodynamic function, and post-infarct remodeling. Human EPO was applied (3000 IU/kg) i.p. immediately after ligation of the LAD as well as on the 2 consecutive days (1000 IU/kg)—a dose that did not significantly affect erythropoiesis. At day 2 after MI, cardiac FACS-analyses, PCR, and TUNEL-staining were performed. B) Kaplan-Meyer curve showing survival rates after MI in the EPO-treated group (n=16) compared to saline-treated mice (n=22). All mice revealed histologically confirmed MI. IE (international Einheit) = IU.

Functional parameters
For evaluation of pressure-volume relationships in vivo, surviving mice of the previously described groups were anesthetized with thiopental (100 mg/kg, i.p.), intubated, and ventilated (MiniVent, Hugo Sachs Elektronik). The left ventricle was catheterized via the right carotid artery using an impedance-micromanometer catheter (Millar Instruments, Houston, TX, USA). In brief, the method is based on measuring the time-varying electrical conductance signal of 2 segments of blood in the left ventricle, from which total volume is calculated. Raw conductance volumes were corrected for parallel conductance by the hypertonic saline dilution method. Therefore, a bolus of 10 µl of 7.5% hypertonic saline was injected via the jugular vein. For absolute volume measurements, the catheter was calibrated with known volumes of heparin-treated mouse blood (23) . Data analyses were performed according to methods described elsewhere (23) .

Hematologic parameters
To measure the number of leukocytes, lymphocytes, monocytes, neutrophils, eosinophils, basophils, platelets, and erythrocytes, as well as the content of hemoglobin and hematocrit, heparinized blood samples were analyzed using a conventional hematological cell analyzer (Sysmex XE 2100; Sysmex America, Inc., Mundelein, IL, USA).

Flow cytometry
Eight- to 12-wk-old C57BL/6 mice were treated with EPO or saline as described above. At day 6, 1 ml of peripheral blood was harvested from each mouse by aspirating the carotid artery. Bone marrow cells were obtained by flushing the tibiae and femurs from the euthanized mice. Mononuclear cells were separated by density-gradient centrifugation using 1.077 g/ml Histopaque solution (Sigma-Aldrich Corp.), purified, and resuspended in PBS containing 1% BSA. Cells were incubated for 40 min in the dark at 4°C with the following: fluorescein isothiocyanate (FITC), phycoerythrin (PE), and peridinin chlorophyll-protein (PerCP) conjugated monoclonal antibodies CD45-PerCP, CD34-FITC, CD31-PE, Sca-1-PE, and c-kit-PE (all from BD Biosciences Pharmingen, San Diego, CA, USA). Matching isotype antibodies (BD Biosciences Pharmingen) served as controls. Cells were analyzed by three-color flow cytometry using a Coulter Epics XL-MCLTM flow cytometer (Beckman Coulter, Fullerton, CA, USA). Each analysis included 50,000 events.

Flow cytometry of cardiac cells was performed from sham-operated, infarcted hearts of saline- or EPO-treated C57Bl/6 mice at day 2 after MI. Therefore, a myocyte-depleted cardiac cell population was prepared, incubating minced myocardium in 0.1% collagenase IV (BrL; Life Technologies, Inc., Gaithersburg, MD, USA) for 30 min at 37°C, which is lethal to most adult mouse cardiomyocytes. Cells were then filtered through a 70 µm mesh. To exclude spurious effects of enzymatic digestion, bone marrow cells with or without collagenase treatment were stained, revealing no significantly changed staining of labeled cell antigens (data not shown). Cells were labeled with CD45-PerCP, CD34-FITC, CD31-PE, Sca-1-PE, c-kit-PE, and CXCR-4-PE (all from BD Biosciences Pharmingen) and subjected to flow cytometry as described above.

Histology and immunohistochemistry
At days 6 and 30, hearts were excised. After fixation in 4% phosphate buffered formalin, the hearts were cut transversally into 2-mm-thick slices and embedded in paraffin. Sections (4 µm thick) were cut and mounted on positively charged glass slides. Standard histological procedures (hematoxilin/eosin and Masson trichrome) and immunostaining were performed. Infarct size and LV wall thickness were determined as described previously (5) . Immunostaining was performed using the following primary antibodies: CD31 (goat anti-mouse, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and CD45 (rat anti-mouse, BD Biosciences Pharmingen). Aminoethyl carbazol was used as chromogen. Apoptotic cells were detected using the TUNEL assay (ApopTag; MP Biomedicals, Solon, OH, USA). The numerical density of CD31+ structures was quantified from 10 random x400 fields and was converted to square millimeters.

Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)
Infarcted hearts were explanted, and the area of infarction, including its border zone, was separated from the noninfarcted myocardial tissue. Ventricular tissue of explanted hearts of sham-operated control mice served as controls. Isolation of total RNA from mouse heart tissue was performed using Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the manufacturer’s protocol. Reverse transcription was performed using the ImProm-IITM Reverse Transcription System (Promega Corp., Madison, WI, USA) according to the manufacturer’s protocol. cDNA samples were analyzed by quantitative RT-PCR using the following murine primers purchased from MWG-Biotech AG (Ebersberg, Germany): 18S mRNA (sense, 5'-GGA CAG GAT TGA CAG ATT GAT AG-3', antisense, 5'-CTC GTT CGT TAT CGG AAT TAA C-3'); SDF-1 (sense, 5'-GAG CCA ACG TCA AGC ATC TG-3', antisense, 5'-CAA TGC ACA CTT GTC TGT TG-3'); SCF (sense, 5'-CCT CTT GTC AAA ACC AAG GAG-3', antisense, 5'-CAT AAC ACG AGG TCA TCC AC-3'). qRT-PCR was performed using SYBR Green Reaction Mix (Eurogentec, Seraing, Belgium) on an ABI PRISM 7900HT Detection System (Applied Biosystems, Inc., Foster City, CA, USA). Each sample was run in duplicate. The expression of each gene within the different tissue samples was quantified relative to H4 mRNA expression levels according to the Sequence Detector User Bulletin (Applied Biosystems). Relative mRNA expression of the target genes was related to sham-operated control hearts.

Cytokine serum levels
Serum levels of SDF-1 and SCF were determined by ELISA (Mouse SDF-1 and Mouse SCF; RayBiotech, Inc., Norcross, GA, USA).

Statistical analysis
Data are shown as means ± SE. Multiple group comparison was performed by one-way ANOVA followed by the Bonferroni procedure for comparison of means. Comparisons between two groups were performed using the unpaired t test. Analysis of survival was performed by the Kaplan-Meier method, and between-group difference in survival was tested by the log-rank test (Statistical Packages for the Social Sciences 12.0; SPSS, Inc., Chicago, IL, USA). Values of P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Survival 30 days after MI
According to the experimental design (Fig. 1A ), cumulative survival of EPO- and saline-treated mice was recorded for 30 days after induction of MI. Mortality was very high within the first 6 days, in particular in the saline-treated group, but decreased afterward in both groups. At 30 days after MI, EPO-treated animals showed a significantly improved survival compared to control (62.5 vs. 36.4%, P<0.05; Fig. 1B ).

Effects of EPO on cardiac function after MI
Using conductance catheters, we measured pressure volume relations from baseline (sham-operated), EPO-treated, and saline-treated mice at days 6 and 30 after the surgical procedure in vivo. Our results showed an improvement of all measured contractile [ejection fraction, cardiac output, derivative of maximum rate of change in left ventricular pressure (dP/dt max), and stroke work] and relaxation parameters (dP/dt min, Tau Weiss, and maximal power) at days 6 and 30 in EPO-treated animals compared to the saline-treated control group. Heart rate and end-diastolic volume revealed no significant difference among groups (Table 1 ).


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  		Table 1. Hemodynamic data

Histopathological effects
To investigate the effect of EPO on infarct remodeling, Masson trichrome-stained tissue samples were analyzed. LV infarct size was comparable in both groups at day 6 (39.2±1.8 vs. 41.8±3.5% of total LV area, ns); 30 days after MI, EPO-treated mice showed a less prominent scar extension than saline-treated mice (22.6±1.9 vs. 31.6±3.0%, P<0.05) (Fig. 2A ). At days 6 and 30, the anterior wall in the infarct area of the EPO-treated group was thicker compared to saline-treated animals (day 6: 0.72±0.08 vs. 0.47±0.06 mm, P<0.05; day 30: 0.44±0.06 vs. 0.14±0.02 mm, P<0.01). The anterior wall thickness of the myocardium declined over time in both groups, however, to a smaller extent in hearts of EPO-treated animals (Fig. 2B ). Furthermore, the hearts of EPO- and saline-treated infarcted animals were evaluated at day 30 for signs of cardiac hypertrophy by measuring the thickness of the septal and the right ventricular wall, revealing no significant differences (data not shown).


Figure 2
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  		Figure 2. Histological findings. A, B) Bar graphs representing the size of infarction (A) and the thickness of the free LV wall (B) showing a decline of the left anterior free wall of the myocardium of saline-treated mice (white bars) and EPO-treated infarcted mice (black bars) at days 6 (n=8) and 30 (n=10) after MI. C) Representative Masson Trichrome stainings of saline-treated mice (left panel) and EPO-treated mice (right panel) at day 6 after LAD ligation.

Immunohistochemical analyses
At day 6 after MI, the granulation tissue of EPO-treated as well as saline-treated animals revealed a strong infiltration of CD45+ cells, mostly monocytes and granulocytes. Cells positive for the endothelial marker CD31 increased up to 2.2-fold in the infarct border zone (P<0.001), suggesting a strong EPO-related effect on neovascularization (Fig. 3A-C ). To analyze the vessel density, complete CD31+ vessel structures were counted in the infarct border zone. EPO-treated mice showed significantly more vessels compared to control mice (668±51 vs. 390±43 vessels, P<0.001). To investigate the influence of EPO on apoptosis in the infarct area, terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining of the tissue samples was performed. At 48 h after MI, a high number of cardiomyocytes (37.8±2.2%) at the border zone stained TUNEL-positive, whereas EPO treatment significantly reduced the number of apoptotic cells (21.6±2.2%) (Fig. 3D-F ).


Figure 3
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  		Figure 3. Immunochistochemical analyses. A, B) Representative immunohistochemical stainings of CD31 in the infarct border zone of saline-treated mice (A; n=6) and EPO-treated mice (B; n=6). C) Bar graphs representing the number of CD31+ cells. D, E) Representative TUNEL stainings of saline-treated mice (D; n=6) and EPO-treated mice (E; n=6) 2 days after MI. F) Bar graph representing percentage of TUNEL-positive cardiomyocytes in the infarct border zone.

Changes of hematologic parameters
To determine the effect of EPO in peripheral blood, hematologic parameters were analyzed. Compared to the control group, leukocytes showed significantly increased values 6 days after initiation of EPO treatment (1.54-fold, P<0.05). This was mainly due to an increase of lymphocytes as detected in the differential blood count (1.72-fold, P<0.05). There was no significant change of erythrocytes, hemoglobin, hematocrit, platelets, neutrophils, or monocytes (Table 2 ).


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  		Table 2. Hematologic parameters

FACS analysis of CD45+CD34+ cell populations in peripheral blood and bone marrow
To investigate the effect of EPO on mobilization of BMCs into peripheral blood, we performed flow cytometry of different subpopulations of circulating mononuclear cells. We found a significant increase of different subtypes of CD45+/CD34+ cells (CD45+/CD34+ cells: 6.0-fold, P<0.05; CD45+/CD34+/CD31+ cells: 11.0-fold, P<0.05; CD45+/CD34+/Sca-1+ cells: 4.2-fold, P<0.05; CD45+/CD34+/c-kit+ cells: 14.3-fold, P<0.05) in peripheral blood at day 6 of EPO stimulation (Fig. 4A, B ).


Figure 4
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  		Figure 4. BMC populations in peripheral blood and bone marrow. A) CD45+/CD34+ cell populations (subclassified by CD31, Sca-1, and c-kit) in peripheral blood in saline-treated mice (n=6, white bars) and EPO-treated mice (n=6, black bars). B) Representative FACS analyses of CD45+/CD34+ cells in peripheral blood of mice. C) CD45+/CD34+ cell populations (subclassified by CD31, Sca-1, and c-kit) in bone marrow.

In bone marrow, CD45+/CD34+/CD31+ cells (22.5% increase, P<0.05) and CD45+/CD34+/Sca-1+ cells (79.9% increase, P<0.05) were significantly increased after EPO treatment. CD45+/CD34+ cells and CD45+/CD34+/c-kit+ cells showed a slight, but not significant, increase in EPO-treated mice (Fig. 4D ).

FACS analysis of a myocyte-depleted fraction of cardiac cells
To compare the effects of EPO on migrated subpopulations of BMCs (CD45+/CD34+ cells) as well as resident cardiac cells (CD45–/CD34– cells), we isolated a myocyte-depleted fraction of cardiac cells and performed flow cytometry. In EPO-treated animals, the number of migrated CD45+/CD34+ cell populations showed increased values compared to saline-treated mice (CD45+/CD34+ cells: 4.5-fold, P<0.05; CD45+/CD34+/CD31+ cells: 1.6-fold, P=0.15; CD45+/CD34+/Sca-1+ cells: 2.5-fold, P<0.05; CD45+/CD34+/c-kit+ cells: 1.3-fold, P=0.53; CD45+/CD34+/CXCR-4+ cells: 2.7-fold, P<0.05) (Fig. 5A ).


Figure 5
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  		Figure 5. Migrated BMC populations vs. resident CSC populations. A) CD45+/CD34+ cell populations (subclassified by CD31, Sca-1, c-kit, and CXCR-4) in a myocyte-depleted fraction of the heart in sham-operated mice (n=6, white bars), saline-treated mice (n=6, gray bars), and EPO-treated mice (n=6, black bars). B) Representative immunohistochemical stainings of CD45 in the infarct border zone of saline-treated mice (top panel) and of EPO-treated mice (bottom panel), indicating the migration of CD45+ cells. C) CD45–/CD34– cardiac-resident c-kit+ and Sca-1+ cells in a myocyte-depleted fraction of the heart in sham-operated mice (n=6, white bars), saline-treated mice (n=6, gray bars), and EPO-treated mice (n=6, black bars).

In contrast to this finding, the number of resident cardiac CD45–/CD34–/Sca-1+ cells showed no difference in EPO- vs. saline-treated animals; the number of CD45–/CD34–/c-kit+ cells was even significantly decreased in EPO-treated mice (61.4% decrease, P<0.05) (Fig. 5B ).

Effect of EPO on homing factors
To investigate expression levels of different homing factors involved in migration to the ischemic myocardium (SDF-1, SCF) qRT-PCR analyses of heart tissues were performed. SDF-1 levels were significantly increased in the infarct area after EPO treatment compared to the control group (Fig. 6A ). SCF levels did not differ significantly in both groups (Fig. 6B ).


Figure 6
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  		Figure 6. Homing factors in the heart and in serum. A, B) SDF-1 (A) and SCF (B) mRNA expression levels in the heart in saline-treated mice (n=8, white bars) and EPO-treated mice (n=8, black bars) in the remote area 48 h after MI, normalized to sham-operated control mice. C, D) Soluble SDF-1 (C) and SCF serum levels (D).

In addition, to analyze the levels of soluble proteins SDF-1 and SCF in serum, ELISAs were performed. SDF-1 levels in serum were significantly reduced after EPO treatment (56.4% decrease, P<0.05), whereas SCF levels showed no difference in the EPO vs. saline treated group (Fig. 6C-D ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we examined the effects of EPO on survival, cardiac function, and histopathological changes, and we focused on homing mechanisms for mobilized BMCs after MI in a preclinical mouse model vs. the effect on resident CSCs. Our main findings are the following: 1) beneficial effects on long-term survival and cardiac function after EPO treatment; 2) altered remodeling, reflected by reduced LV wall thickness in the infarct area and smaller infarct size; 3) reduced apoptosis and enhanced neovascularization in the infarct border zone; 4) mobilization of BMCs into peripheral blood without depletion of bone marrow; 5) improved homing of BMCs into the infarcted myocardium, primarily via the CXCR-4/SDF-1-axis; and 6) no beneficial influence on CSCs.

For our study, we chose a treatment regime with a total dose of 5000 IU/kg, started immediately after total occlusion of the LAD and administered within 48 h after MI. According to the literature, this dosage is associated with tissue-protective properties (24 , 25) . Our data on hematologic parameters revealed no change in hemoglobin levels after this short-term EPO treatment regime, indicating no effect on erythropoiesis. The EPO-related effects were analyzed during a time period of 30 days. Thus, short-term and long-term effects could be observed. At first, the beneficial value of EPO is reflected by the significantly improved survival rates 30 days after MI (Fig. 1B ), which is associated with enhanced cardiac function parameters at day 6, and is preserved until day 30. By histology, attenuated remodeling could be observed, which is reflected by partial restoration of LV wall thickness in the infarct area and diminished scar formation at day 30 (24 , 26 27 28) (Fig. 2A, B ). Ventricular wall tension is negatively related to wall thickness. Accordingly, the amelioration of LV wall thickness in EPO-treated mice prevented high LV wall tension, attenuating ventricular expansion and restoring ejection fraction. High-end diastolic volumes and a worsened ejection fraction are known determinants of post-MI mortality. Our results demonstrated that EPO-treated animals showed lower-end diastolic volumes and an improved ejection fraction compared to the control group, explaining their better rate of post-MI survival.

Mechanisms underlying these findings are shown in our immunohistochemical analyses. At 48 h after MI, the hearts were examined for apoptotic, TUNEL-positive cells (Fig. 3G-I ). After EPO treatment, the assay revealed a significantly decreased number of apoptotic cells, most of them identified as cardiomyocytes, which is in accordance to previous studies both in animal models of ischemia-reperfusion and permanent occlusion (18 , 25) . A possible explanation for this finding was shown in the evaluation of signal transduction pathways involved in postischemic cardioprotection. It was demonstrated that the expression of EPO receptors on adult cardiomyocytes is participating in acute cardioprotective effects (14) . The stimulation of the EPO receptor again activates antiapoptotic mechanisms, including PI3K/Akt and MAPK pathways (15 16 17 18) .

Furthermore, staining of the endothelial surface marker CD31 showed increased numbers of CD31-expressing cells in the infarct border zone at day 6, indicating enhanced neovascularization (Fig. 3D-F ), which is similar to other studies (28) . One potential mechanism for the influence on neovascularization is a change in cytokine profile after EPO administration. We detected that serum levels of vascular endothelial growth factors (VEGF) are increased in EPO-treated animals. VEGF is a cytokine well known to promote angiogenesis (29 , 30) . Moreover, the expression of VEGF at the site of ischemia was shown to be related to the expression of the EPO receptor (20 , 31) . In addition, neovascularization after EPO treatment could be related to increased circulating EPC levels (19 , 20) .

In our study, we focused on the role of BMC populations after EPO treatment in different compartments of the body. In addition to EPCs (20) , we demonstrated a mobilization of different subpopulations of BMCs into peripheral blood (Fig. 4A ). These subpopulations remained unchanged or even increased in bone marrow, suggesting a proliferating effect within bone marrow (Fig. 4D ). This may be an advantage compared to the well established mobilizing agent G-CSF, which is known to result in a depletion after release of BMCs (32 , 33) .

Direct and indirect forms of application of BMCs were shown to have protective effects after MI (5 , 6 , 34 , 35) . However, mechanisms are not completely understood. In this regard, we examined the effect of EPO on migration of mobilized BMCs into the ischemic myocardium. We found increased numbers of CD45+CD34+ within the ischemic myocardium. Among these, the subpopulations expressing the markers Sca-1 and CXCR-4 were significantly increased in the EPO-treated group. C-kit and CD31 expressing CD45+CD34+ cells showed a slight but not significant increase in the ischemic myocardium (Fig. 5A ). Migration is dependent on homing factors mediating attraction, adhesion, and migration of cells. In our study, we measured SCF as the ligand for c-kit and SDF-1 as the ligand for CXCR-4. Interestingly, the expression of SCF within the heart as well as the levels of soluble SCF in serum remained unchanged, which is reflected by the low number of migrated c-kit expressing cells. However, SDF-1 showed up-regulated expression in the ischemic myocardium, but significantly decreased serum levels, resulting in a gradient toward the ischemic area. The number of CD45+CD34+ cells expressing the corresponding surface marker CXCR-4 was strongly increased in the ischemic tissue. The homing of BMCs via the CXCR-4/SDF-1 axis was evidenced to have protective effects after MI (9 , 36) . Employing G-CSF as a mobilizing agent for cardiac repair, however, revealed a lack of chemotaxis of CXCR-4+ cells, leading to a diminished homing of these cells (10 , 11) . Our data suggest EPO overcomes this deficit.

Speculating about the fate of migrated BMCs, the original concept of cardiac regeneration by transdifferentiation of functionally active cardiomyocytes was questioned by the identification of paracrine repair mechanisms leading to BMC-mediated neovascularization and prevention of apoptosis (37 38 39 40 41) . Our findings suggest a participation of this mechanism in BMC-dependent cardioprotection after EPO treatment, in addition to direct receptor-mediated effects, as discussed above.

In recent years, evidence emerged indicating that the heart contains a reservoir of resident cardiac progenitor cells. These cells are positive for various markers such as c-kit or Sca-1. It was hypothesized that these resident cells may play a role in the repair of a damaged heart (42 43 44 45) . To investigate these cells, we measured cardiac cells expressing c-kit or Sca-1. In addition, cells were negative for CD45 or CD34, markers only expressed by cells derived from bone marrow. Our data revealed no change in Sca-1+ resident cells, and even a significant decrease in c-kit+ cells in the heart. Consequently, EPO treatment seems to have no beneficial effect on self-repair mechanisms of the affected tissue. Different from this finding, the mobilizing agent G-CSF showed an increase of resident Sca-1+ cells within the infarcted heart (11) .

Our data provide new pathophysiologic insights in the cardioprotective effects of EPO treatment after acute MI. We show for the first time the EPO-mediated enhanced homing of BMCs, in particular via the CXCR-4/SDF1 axis into the ischemic myocardium. However, EPO has no beneficial influence on CSCs. Therefore, new treatment regimes using EPO together with other agents like G-CSF may combine different beneficial effects, optimizing regeneration of the ischemic myocardium after MI.


   ACKNOWLEDGMENTS
 
We thank Judith Arcifa and Barbara Markieton for excellent technical assistance. Fritz-Bender-Stiftung provided funding to R.F. and B.M. Additional financial support was provided by the FöFoLe program of the Ludwig-Maximilians-University (Munich, Germany) and by the Dr. Helmut Legerlotz-Stiftung (Munich, Germany).

Received for publication March 18, 2008. Accepted for publication September 4, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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