Interact CardioVasc Thorac Surg 2009;9:20-25. doi:10.1510/icvts.2008.191916
© 2009 European Association of Cardio-Thoracic Surgery
Institutional report - Experimental |
Single high-dose intramyocardial administration of erythropoietin promotes early intracardiac proliferation, proves safety and restores cardiac performance after myocardial infarction in rats
Ralf Gäbel1,
Christian Klopsch1,
Dario Furlani1,
Can Yerebakan,
Wenzhong Li,
Murat Ugurlucan,
Nan Ma* and
Gustav Steinhoff
Department of Cardiac Surgery, University of Rostock, Germany
Received 31 August 2008;
received in revised form 26 March 2009;
accepted 27 March 2009
 Presented at the 22nd Annual Meeting of the European Association for Cardio-thoracic Surgery, Lisbon, Portugal, September 14–17, 2008. 
Authors contributed equally to this work.
*Corresponding author. Biomedizinischen Forschungszentrum (BMFZ). Schillingallee 69, Rostock 18057, Germany. Tel.: +49 381 494 61 00; fax: +49 381 494 61 02.
E-mail address: nan.ma{at}med.uni-rostock.de (N. Ma).
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Abstract
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Various studies demonstrate erythropoietin (EPO) as a cardioprotective growth hormone. Recent findings reveal EPO in addition might induce proliferation cascades inside myocardium. We aimed to evaluate whether a single high-dose intramyocardial EPO administration safely elevates early intracardiac cell proliferation after myocardial infarction (MI). Following permanent MI in rats EPO (3000 U/kg) in MI EPO-treatment group (n=99) or saline in MI control group (n=95) was injected along the infarction border. Intramyocardial EPO injection activated the genes of cyclin D1 and cell division cycle 2 kinase (cdc2) at 24 h after MI (n=6, P<0.05) evaluated by real time-PCR. The number of Ki-67+ intracardiac cells analyzed following immunohistochemistry was significantly enhanced by 45% in the peri-infarction zone at 48 h after EPO treatment (n=6, P<0.001). Capillary density was significantly enhanced by 17% as early as seven days (n=6, P<0.001). After six weeks, left ventricular performance assessed by conductance catheters was restored under baseline and dobutamine induced stress conditions (n=11–14, P<0.05). No thrombus formation was observed in the heart and in distant organs. No deleterious systemic adverse effects were apparent. Single high-dose intramyocardial EPO delivery proved safety and promoted early intracardiac cell proliferation, which might in part have contributed to an attenuated myocardial functional decline.
Key Words: Myocardial ischemia; Intracardiac proliferation; Cell cycle genes; Adverse effects; Angiogenesis
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1. Introduction
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Acute myocardial infarction (MI) as a common primary presentation of coronary heart disease has strongly been related with the unpreventable loss of functional myocardium. Numerous studies reported erythropoietin (EPO), a 30.4-kDa glycoprotein, exerts non-hematopoietic cardioprotective effects following MI, ischemia-reperfusion injury or chronic heart failure. The underlying mechanisms were in part attributed to anti-apoptosis, anti-inflammation and angiogenesis [1–3]. However, other processes also could take part in myocardial regeneration after MI [4].
It is common knowledge that EPO exerts proliferative potency inducing D-type cyclins by the activation of mitogen-activated protein kinases in the bone marrow. Thereby, EPO stimulates the proliferation of erythroid precursor cells in response to hypoxia [5]. Moreover, it was reported that EPO activated the extracellular signal-regulated kinase 2, a mitogen-activated protein kinase, in the myocardium [6]. EPO injected directly after MI might induce an enhanced cell-cycle progression and proliferation of resident intracardiac cells for an improved cardiac regeneration [7].
Cyclin D1 and cell division cycle 2 kinase (cdc2) are two regulating proteins for cell-cycle progression [7]. Cyclin D1 in complex with cyclin dependent kinase 4 enables the progression from G1 to the S phase, in which the nuclear DNA is replicated. Cdc2 in complex with cyclin B allows the progression from G2 to the M phase, in which cell division is completed [7]. However, it has not yet been satisfactorily elucidated whether a single high-dose intramyocardial EPO administration safely mediates efficient intracardiac cell-cycle progression and proliferation after MI.
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2. Materials and methods
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2.1. Animals
All procedures and ethical aspects were approved by the local Animal Care Committee of Mecklenburg/Vorpommern (approval number LALLFM-V/TSD/7221.3-1.1-031/06) in Rostock, Germany and conformed to the guide for the care and use of laboratory animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
2.2. Generation of MI in rats and EPO injection
Lewis rats (male, 250–300 g, Charles River Laboratories) were randomly assigned into three groups as: Sham operation (Sham, n=55), MI with EPO treatment group (MI-EPO, n=99), and untreated MI control group (MIC, n=95). Under general anesthesia the LAD was permanently ligated as previously described [4]. Immediately after MI, rats received four intramyocardial injections (25 µl each) of either recombinant human EPO (3000 U/kg, Epoetin- /Erypo®, Ortho Biotech, Division of Janssen Cilag GmbH) dissolved in 0.9% saline, or 0.9% saline solution alone [4]. In case of insufficient injection animals were excluded immediately from the experimental study.
2.3. Quantitative real time – PCR analysis
For analysis of mRNA levels, hearts of 24 and 48 h (n=6, for each group and time point) post-infarction were removed. The blanched infarcted zone (IZ – including 1 mm peri-infarction border zone) of the left ventricle (LV) was separated microscopically from the remaining myocardium by an experienced investigator and snap-frozen in liquid nitrogen. Following total RNA isolation including DNase treatment, RNA was reverse transcribed and quantitative real time-PCR was performed with StepOnePlusTM Real-Time PCR System (Applied Biosystems) according to TaqMan method [4]. Primers designed for mRNA amplification of cyclin D1 (Primer: Rn00432359_m1, Applied Biosystems) and cdc2 (Primer: Rn00570728_m1, Applied Biosystems) were applied. Cycle thresholds for single reactions were determined and changes in gene expression after MI and EPO treatment were calculated as described previously [4].
2.4. Intracardiac cell proliferation
For immunohistological detection of Ki-67+ cells, hearts from MIC and MI-EPO were analyzed at 24 h and 48 h after MI. Frozen transverse tissue sections from the mid-portion of the LV (5 µm, n=6 for each time point and group) were incubated with monoclonal mouse anti-rat Ki-67 antibody (DakoCytomation, Glostrup, Denmark) and donkey anti-mouse Alexa-Fluor 488 conjugated secondary antibody (Invitrogen, Carlsbad, USA). In each section the number of Ki-67+ cells was counted using confocal microscopy in 16 randomly-chosen high-power fields (HPFs, 630x) of the IZ including peri-infarcted area. Results were expressed as cells per HPF. For the localization of KI-67 signal double immunostaining has been performed staining Ki-67 as described above followed by polyclonal goat anti-CD31 primary antibody (Santa Cruz, Santa Cruz, USA) and donkey anti-goat Alexa-Fluor 568 conjugated secondary antibody (Invitrogen).
2.5. Capillary density
Capillary density was assessed at seven days after surgery in frozen transverse tissue sections by counting the number of capillaries immunostained with polyclonal goat anti-CD31 primary antibody followed by donkey anti-goat Alexa-Fluor 568 conjugated secondary antibody. In MIC and MI-EPO groups (n=6 for each group) eight fields (400x) in the border zone of two sections per heart were analyzed. Results were expressed as capillaries per mm2.
2.6. Histopathologic analysis
For histopathologic purposes, tissue sections (5 µm thick) from brain, liver, lung, kidney, spleen and heart of MIC and MI-EPO groups at 24 h (n=7), 48 h (n=7), two weeks (n=5) and six weeks (n=6–8) after MI were stained with Hematoxylin/Eosin G (HE; Merck KG, Darmstadt, Germany). At 100x magnification investigations were focused on myocardial damage, apparent thrombotic processes and vessel shape and size. The area of myocardial damage was analyzed at 48 h after MI measured by computer assisted planimetry (Axio Vision LE Rel. 4.5 software; Zeiss, Jena, Germany). The ratio of damaged myocardium to the LV area was presented.
2.7. Blood pressure measurement and peripheral blood analysis
At 24 h (n=7, for each group), 48 h (n=7, for each group) and two weeks (n=5, for each group) after MI, systemic blood pressure was measured by catheterization (model Millar SPR-838; emka Technologies, Paris, France) of the ascending aorta retrograde from the right carotid artery. Following measurements brain, liver, lung, kidney, spleen and heart were harvested. Peripheral blood analyses were examined by Sysmex XE-2100 (SYSMEX, Norderstedt, Germany). Leukocytes (n=100) were evaluated in Pappenheim stained slides (1000x magnification). Creatine kinase (CK) was evaluated with Synchron LX 20 (Beckman Coulter, Fullerton, California, USA). Creatine kinase-muscle brain (CK-MB) band mass was analyzed with ELISA using Elecsys 2010 (F. Hoffmann-La Roche, Basel, Switzerland) and mouse anti-CK-MB monoclonal antibody (Roche Diagnostics, Mannheim, Germany).
2.8. Left ventricular catheterization
Six weeks after surgery, rats (Sham n=11, MIC n=14, MI-EPO n=11) underwent pressure–volume loop (P/V loop) measurements using conductance catheter technique as described previously [4]. P/V loops of the LV were recorded under normal conditions (baseline) followed by stress conditions mediated by intravenous dobutamine administration (10 µg/kg/min, Sigma-Aldrich). Data were analyzed with IOX Version 1.8.3.20 software (emka Technologies).
2.9. Statistics
Statistical analyses were performed using Sigma Stat software version 3.0 (SPSS Inc, Chicago, USA). Results are expressed as mean±S.D. (mean±standard error of mean in figures). Kaplan–Meier analysis served for the clarification of differences in survival curves between experimental groups. Overall comparisons of the treatment groups were performed by using the one-way analyses of variance (ANOVA) method that applies post-hoc multiple Holm–Sidak tests, and by using the non-parametric Kruskal–Wallis (failing normality) or post-hoc multiple Dunn tests. P<0.05 was considered statistically significant.
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3. Results
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3.1. Elevation of early intracardiac cell proliferation and angiogenesis
Cell cycle progression gene assessment by quantitative real-time PCR (Fig. 1a) illustrated a significant 37% increase in the intracardiac mRNA expression of cyclin D1 gene (P=0.02) in the IZ of MI-EPO hearts at 24 h compared with MIC. At the same time point cdc2 mRNA level reached even a 2.8-fold increment (P=0.01) in the IZ of MI-EPO hearts compared with MIC.
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Fig. 1. Intracardiac EPO injection augments early intracardiac cell proliferation after MI. (a) Real time-PCR analysis of cyclin D1 and cdc2 in the IZ of MI-EPO (n=6) and MIC (n=6) hearts, and in the viable myocardium of Sham (n=6) hearts. The mean mRNA expression levels of Sham were arbitrarily given a value of 20. *P<0.05. (b) The number of Ki-67+ intracardiac cells per high power field (HPF) in the infarcted zone (IZ – including peri-infarction border zone) increased at 48 h after EPO treatment. (c) Representative immunostaining for Ki-67 (green signal, white arrows) in an EPO-treated heart. Ki-67+ cells (square) were mostly apparent in the peri-infarction border zone of IZ at 48 h after treatment. Blue, TOPRO3 in nuclei. (d) Representative double staining for Ki-67 (green) and CD31 (red) in one MI-EPO heart. Blue, TOPRO3 in nuclei.
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However, there was no increment of proliferating intracardiac cells at 24 h after MI in the IZ. With a time delay of another 24 h, immunostaining revealed a 45% elevation of Ki-67+ intracardiac cell number in the IZ of MI-EPO compared to MIC at 48 h after MI (P<0.01, Fig. 1b, c). These results confirmed a fast and intense proliferative potency of EPO that was further investigated by the analyses of the Ki-67 signal location inside the hibernating myocardium. Double immunostaining uncovered that proliferating Ki-67+ intracardiac cells inside MI-EPO were in part positive for endothelial cell marker CD31 (Fig. 1d). At one week after MI, capillary density was significantly increased by 17% in the border zone of MI-EPO compared to MIC (Fig. 2a–c, P<0.01). All vessels observed were of normal shape and size, with a diameter ranging between 5 and 20 µm.
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Fig. 2. Intracardiac EPO injection induces neoangiogenesis. (a) Capillary density was significantly higher in MI-EPO group compared with MIC group in the border zone of the LV at 7 days after MI. *P<0.05. (b, c) Representative immunostaining for CD31 (red) in the border zone of MIC (b) and MI-EPO hearts (c) after seven days. Blue, DAPI in nuclei.
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3.2. No apparent deleterious local and systemic adverse effects
The area of acute ischemic myocardial damage after LAD ligation did not differ significantly between MIC and MI-EPO groups (P=0.35; Fig. 3a). Plasma CK activity and CK-MB concentration either did not vary significantly between MI groups (Table 1). None of the analyzed hearts and distant organs revealed apparent thrombosis or preangiomatous vessel structures (Fig. 3b–g). Blood analyses reflected an augmented hematopoietic cell mobilization in MI-EPO starting already at 24 h after MI (Table 1). Hematological differences have not been found to continue after two weeks. Mean arterial pressure (MAP) in MI-EPO was significantly elevated by 12% when compared with MIC only at 48 h after MI (P=0.03).
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Fig. 3. Intracardiac EPO injection does not induce adverse effects in solid organs. (a) The area of acute ischemic myocardial damage of the left ventricle (LV) was not significantly different between EPO-treated and non-treated MI groups. (b-g), Representative HE staining in heart (b), kidney (c), liver (d), lung (e), spleen (f) and brain (g) taken from MI-EPO group revealed no thrombosis secondary to EPO administration.
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3.3. Recovery of cardiac performance at comparable six weeks follow-up survival
LV cardiac output (LV-CO) increased after EPO injection compared with MIC under baseline conditions and dobutamine induced stress conditions by 65% and 71%, respectively (P<0.01; P=0.01, Fig. 4a). LV end-diastolic pressure (LV-EDP) of MI-EPO was decreased significantly compared with MIC at baseline conditions and dobutamine induced stress conditions by 51% and 53%, respectively (P=0.02; P=0.03, Fig. 4b).
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Fig. 4. Intracardiac EPO injection restored LV functions six weeks after MI but did not influence six weeks follow-up survival. (a) Left ventricular (LV) – cardiac output was increased in MI-EPO group at both baseline and dobutamine stress conditions. (b) LV-end-diastolic pressure (LV-EDP) was normalized in MI-EPO group at both baseline and dobutamine stress. *P<0.05, **P<0.01. (c) Therapy related survival rates did not differ among the MI groups. Groups Sham black (n=53), MIC red (n=89), MI-EPO green (n=89).
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Eighteen animals were excluded from therapy related survival rate immediately after MI induction according to the institutional standardization protocol. The overall survival rate was 77.5% in MI-EPO and 75.5% in MIC. The comparison of survival curves of MI groups did not indicate a significant difference (P=0.12, Fig. 4c).
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4. Discussion
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In the present study, we, to the best of our knowledge, provide the first experimental demonstration that intramyocardial EPO delivery temporarily spatially increased intracardiac cell proliferation accompanied by the intracardiac induction of cell-cycle progression genes cyclin D1 and cdc2 in the area of hibernating myocardium early after MI. Further, we found significant angiogenesis after intracardiac EPO administration as early as one week that implicates early proliferation of endothelial cells or endothelial precursors [8]. It was illustrated EPO dose-dependently elevates the proliferation and migration of cultured endothelial cells received from non-cardiac tissues [9]. EPO at a high-dose given once directly after MI might have induced cyclin D1 and cdc2 in intracardiac endothelial cells. These two molecules seem to possess considerable regulatory importance for endothelial cell proliferation as well as the development of a mature vascular architecture [10–12]. Thus, our findings indicate that a single intracardiac EPO delivery early after MI might have accelerated angiogenesis with increased capillary density and vessels regular in shape and size. We speculate that endothelial or its progenitor cells might be one major population, which contributes to the temporarily spatially enhanced intracardiac cell proliferation.
However, we could not exclude the possibility of proliferation of other intracardiac cell types such as cardiomyocytes, cardiac fibroblasts or stem cells. Recently, it has been demonstrated that the intracardiac elevation of cyclin D1 and cdc2 mRNA was accompanied by DNA synthesis with and without increased mitotic activity in adult cardiac myocytes of rodents [13, 14]. Cardiac fibroblasts might be another important proliferating cell population in the initial phase of remodeling. They can exert beneficial paracrine effects and modulate cardiac stem cell activation [15]. Therefore, an early proliferation of intracardiac cell types may positively contribute to the initiation of cardioregenerative potentials.
The single high-dose intramyocardial EPO treatment after MI proved safety within our experimental set-up. Adverse effects have been one major priority within our investigations, since Erypo® (Epoetin-) delivery is only approved for subcutaneous and intravenous administration. There have been no apparent local therapy related adverse effects. Systemic adverse effects, most probably mediated by EPO that was released from the intracardiac site systemically through the post-infarction lymph, were not deleterious. Temporarily increased MAP after EPO therapy was below that of healthy rodents (data not shown). Analyzed brain, kidney, lung, liver, spleen and heart did not reveal vascular thrombosis or vessels of irregular shape that could compromise vascular functionality. However, the hematopoietic potential of EPO as well as excess angiogenesis should be taken into consideration especially when administered over a prolonged period.
In conclusion, our findings provide first evidence for early intracardiac proliferative effects of EPO after MI that might in part have contributed to the enhanced systolic and diastolic LV performance as well as the restorative and cardioprotective effects of EPO in hibernating myocardium also described by our laboratory [1–4]. Further studies are required to investigate whether EPO could reduce the risk of event or the mortality rate in a prolonged follow-up.
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Conference discussion
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Dr. B. Walpoth (Geneva, Switzerland): I have two questions: firstly, the dosage of erythropoietin is very high. Have you tried to use more physiological dosages than this high dosage? And secondly, have you looked at angiogenesis, since erythropoietin is known to induce angiogenesis, and thirdly, do you think that part of the improvement in function and less apoptosis is due to the effect of angiogenesis triggered by the CD34 accumulation of cells?
Mr. Klopsch: These are two very important points. Answering the first question, originally we especially wanted to investigate the side effects of a local high dosage erythropoietin therapy in an acute myocardial infarction model. We aimed to look for thrombosis inside the myocardium and distant organs. We did not find thrombosis in the liver, in the heart, in the lung, in the brain and in the spleen. Regarding to the possible systemic side effects we first looked at the reticulocyte count inside the peripheral blood. We found upregulations already at 24 h after myocardial infarction and a moderate hematocrit increase already at 48 h.
To the second question, of course, erythropoietin augments angiogenesis. We can say that erythropoietin definitely increases the cardiac cell proliferation. We have an increase in cardiac cell proliferation at 48 h. Some of these cells were endothelial cells, and we have an increase in angiogenesis at the border zone already detectable at one week after myocardial infarction. At six weeks after myocardial infarction there is an increase in capillary density at the border zone and in the non-infarcted area.
Dr. S. Takamoto (Tokyo, Japan): We published data that erythropoietin protects the brain tissue during hypothermic circulatory arrest last year in JTCVS, but the volume is so high, in high doses, to pass the blood-brain barrier. But you in your speech you said that you put the erythropoietin directly in the myocardium. Is it possible for erythropoietin to be included in the cardioplegic solution and to protect the myocardium? And also the same question, can you reduce the dosage of the drug to a normal range or something? Otherwise, this drug is very expensive.
Mr. Klopsch: Of course, it is a very expensive drug. For clinical reasons more physiological dosages are desired, and we could definitely reduce the dosage. I think normal dosages may vary between 10 and maybe 100 or 300 U/kg for intramyocardial administrations. Actually limited knowledge is available about the dosage of erythropoietin in a local therapy approach. It would be nice to further investigate if these lower dosages also have this stem cell recruitment effect. I think that more studies have to be undertaken to clarify these questions.
Dr. Takamoto: Have you ever experienced that the erythropoietin is included in the cardioplegic solution?
Mr. Klopsch: No, we did not, but we are planning an approach already. We want to do some big animal experiments applying a cardioplegic solution including erythropoietin for a myocardial ischemia regeneration study.
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Acknowledgements
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We thank Ms Margit Fritsche, Dr Andreas Drynda and Dr Solvig Lenz for their excellent technical assistance.
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Abstract
1. Introduction
