PRECLINICAL STUDY
Erythropoietin Enhances Neovascularization of Ischemic Myocardium and Improves Left Ventricular Dysfunction After Myocardial Infarction in Dogs
Akio Hirata, MD*,
Tetsuo Minamino, MD, PhD*,*,
Hiroshi Asanuma, MD, PhD*,
Masashi Fujita, MD*,
Masakatsu Wakeno, MD ,
Masafumi Myoishi, MD,
Osamu Tsukamoto, MD*,
Ken-ichiro Okada, MD*,
Hidekazu Koyama, BS*,
Kazuo Komamura, MD, PhD ,
Seiji Takashima, MD, PhD*,
Yoshiro Shinozaki, MD||,
Hidezo Mori, MD, PhD,
Masamichi Shiraga, MD, PhD ,
Masafumi Kitakaze, MD, PhD, FACC and
Masatsugu Hori, MD, PhD, FACC*
* Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
Department of Bioregulatory Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
Cardiovascular Division of Internal Medicine, National Cardiovascular Center, Suita, Osaka, Japan
|| Department of Physiological Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
Manuscript received June 14, 2005;
revised manuscript received November 10, 2005,
accepted November 30, 2005.
* Reprint requests and correspondence: Dr. Tetsuo Minamino, Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. (Email: minamino{at}medone.med.osaka-u.ac.jp).
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Abstract
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OBJECTIVES: We investigated the effects of erythropoietin (EPO) on neovascularization and cardiac function after myocardial infarction (MI).
BACKGROUND: Erythropoietin exerts antiapoptotic effects and mobilizes endothelial progenitor cells (EPCs).
METHODS: We intravenously administered EPO (1,000 IU/kg) immediately [EPO(0) group], 6 h [EPO(6h) group], or 1 week [EPO(1wk) group] after the permanent ligation of the coronary artery in dogs. Control animals received saline immediately after the ligation.
RESULTS: The infarct size 6 h after MI was significantly smaller in the EPO(0) group than in the control group (61.5 ± 6.0% vs. 22.9 ± 2.2%). One week after MI, the circulating CD34-positive mononuclear cell numbers in both the EPO(0) and the EPO(6h) groups were significantly higher than in the control group. In the ischemic region, the capillary density and myocardial blood flow 4 weeks after MI was significantly higher in both the EPO(0) and the EPO(6h) groups than in the control group. Four weeks after MI, left ventricular (LV) ejection fraction in the EPO(6h) (48.6 ± 1.9%) group was significantly higher than that in either the control (41.9 ± 0.9%) or the EPO(1wk) (42.6 ± 1.2%) group but significantly lower than that in the EPO(0) group (56.1 ± 2.3%). The LV end-diastolic pressure 4 weeks after MI in both the EPO(0) and the EPO(6h) groups was significantly lower than either the control or the EPO(1wk) group. Hematologic parameters did not differ among the groups.
CONCLUSIONS: In addition to its acute infarct size-limiting effect, EPO enhances neovascularization, likely via EPC mobilization, and improves cardiac dysfunction in the chronic phase, although it has time-window limitations.
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Abbreviations and Acronyms
| | ABP = arterial mean blood pressure | | Dil-ac-LDL = 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein | | EPC = endothelial progenitor cell | | EPO = erythropoietin | | HR = heart rate | | LAD = left anterior descending coronary artery | | LCX = left circumflex coronary artery | | LV = left ventricle/ventricular | | LVEDD = left ventricular end-diastolic dimension | | LVEDP = left ventricular end-diastolic pressure | | MBF = myocardial blood flow | | MI = myocardial infarction | | MNC = mononuclear cell | | UEA-I = Ulex europaeus agglutinin I | | VEGF = vascular endothelial growth factor |
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Erythropoietin (EPO) is a cytokine that promotes proliferation and differentiation of erythroid precursor cells (1) and is widely used for the treatment of anemia in patients with chronic renal failure (2). Erythropoietin can also exert antiapoptotic and radical scavenger effects on nonerythroid cells (3,4). Indeed, we and others showed that an administration of EPO before or shortly after the onset of ischemia reduced myocardial infarct size and improved cardiac function in acute phases (5–8). Another interesting nonerythroid function of EPO is the promotion of endothelial progenitor cell (EPC) mobilization in animals and humans (9–11), which may enhance neovascularization of ischemic areas (12,13). We hypothesized that EPO increases blood supply to ischemic regions through promoting neovascularization and improves cardiac dysfunction after ischemic insult. Thus, the goal of this study was to characterize the effects of EPO on neovascularization and cardiac function after myocardial infarction (MI) in the chronic phase.
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Methods
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All procedures were performed in conformity with the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, 1996 revision) and were approved by the Osaka University Committee for Laboratory Animal Use.
Instrumentation.
Forty-seven beagle dogs (Kitayama Labes, Yoshiki Farm Gifu, Japan), weighing 8 to 12 kg were used in these experiments. After an intravenous injection of sodium pentobarbital (15 mg/kg), the dogs were intubated and ventilated. General anesthesia was maintained with 0.5% to 2.0% inhaled isoflurane. After baseline echocardiography and hemodynamic assessment, minimal thoracotomy was performed, and then the left anterior descending coronary artery (LAD) was ligated just distal to the first diagonal branch. To ensure that all animals included in the data analysis were exposed to a similar extent of ischemia, animals with excessive myocardial collateral blood flow (>15 ml/100 g/min) were excluded from study as previously described (14).
Experimental protocols.
Acute effects of EPO on myocardial infarct size
Either a single dose of EPO (1,000 IU/kg; 5 ml) (n = 6) or the same volume of saline (n = 6) was administered intravenously immediately after the LAD ligation. Regional myocardial blood flow (MBF), area at risk, and infarct size at 6 h after the LAD ligation were determined as described previously (Fig. 1) (14). Recombinant human EPO was provided by Chugai Pharmaceutical Co. Ltd. (Tokyo, Japan). Recombinant human EPO is effective for correcting anemia in the beagle dog (15).
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Figure 1 Experimental protocols to investigate acute effects of erythropoietin (EPO) on myocardial infarct size. LAD = left anterior descending coronary artery; RhEPO = recombinant human erythropoietin.
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Effects of immediate or delayed treatment with EPO on neovascularization and cardiac function
A single dose of EPO (1,000 IU/kg; 5 ml) was administered intravenously immediately [EPO(0) group, n = 8], 6 h [EPO(6h) group, n = 8], or 1 week [EPO(1wk) group, n = 7] after the LAD ligation. Control animals received the same volume of saline (control group, n = 8) immediately after the LAD ligation.
Hematologic parameters.
Blood was sampled from a peripheral vein under pentobarbital (15 mg/kg) anesthesia at the time points indicated in Figure 2. Hematologic parameters, including hematocrit, white blood cell count, and platelet count, were measured.
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Figure 2 Experimental protocols to investigate effects of immediate or delayed treatment with erythropoietin (EPO) on neovascularization and cardiac function. CD34+MNC = CD34-positive mononuclear cell; other abbreviations as in Figure 1.
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Cytokine measurements.
Plasma levels of vascular endothelial growth factor (VEGF) were measured by enzyme-linked immunosorbent assay (R & D Systems, Minneapolis, Minnesota). The detection limit of the assays was 9 pg/ml. The reliability of this assay in dogs has already been reported previously (16).
Quantification of CD34-positive mononuclear cells.
The circulating CD34-positive mononuclear cells (CD34+MNCs) were quantified at the time points indicated in Figure 2. In brief, peripheral white blood cells were stained with a phycoerythrin-conjugated anticanine CD34 monoclonal antibody (BD Pharmingen, San Diego, California). Samples were then subjected to a two-dimensional side-scatter-fluorescence dot plot analysis (FACScan, Becton-Dickinson, Tokyo, Japan). After appropriate gating of MNCs, the number of CD34+MNCs with low cytoplasmic granularity (low sideward scatter) was quantified and expressed as the number of cells per 1-µl blood sample.
In vitro MNC culture assay.
Circulating MNCs were isolated from blood (10 ml) of dogs at baseline and 1 week after MI in the control and EPO(0) groups (n = 4 each) by Ficoll density-gradient centrifugation. After MNCs (107 per well) were plated in Medium 199 (Gibco, Grand Island, New York) supplemented with 20% fetal calf serum and antibiotics on human fibronectin-coated six-well dishes. After 7 days in culture, adherent cells were stained for the uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (DiI-ac-LDL) (Biomedical Technologies, Stoughton, Massachusetts) and the binding of fluorescein isothiocyanate-labeled Ulex europaeus agglutinin I (UEA-I) (Vector Laboratories, Peterborough, England). Double-staining cells were quantified by examining five random microscopic fields (x200 power) (10,11).
Histologic assessments.
Four weeks after MI, myocardial tissue was sampled from both ischemic (LAD) and nonischemic (left circumflex coronary artery [LCX]) regions in each group. The tissues in the ischemic region were identified as the edge of the region showing necrosis. These samples were then fixed in 10% buffered formalin, embedded in paraffin, and serially sectioned in the frontal plane at 5-µm thickness. Endothelial cells were immunohistologically stained using rabbit antihuman von Willebrand factor antibody (Dako, Kyoto, Japan) and the Envision+/HRP Kit (Dako) (17). The peroxidase was visualized by incubation with 3,3'-diaminobenzidine, followed by incubation with a DAB-enhancing solution (Dako). We counted the numbers of capillaries and cardiomyocytes in 20 random high-power fields (x400 power), and then calculated the average capillary density and capillary-to-myocyte ratio (18).
Measurements of regional MBF.
Regional MBF was determined as described previously (19). Nonradioactive microspheres (Sekisui Plastic Co., Tokyo, Japan) made of inert plastic were labeled with bromine or niobium. Microspheres were administered at 90 min and 4 weeks after MI. The MBF in the LAD region was calculated according to the following formula: time flow = (tissue count) x (reference flow)/(reference count), and was expressed in ml/g wet weight/min.
Hemodynamic measurements.
Hemodynamic parameters, such as arterial mean blood pressure (ABP), heart rate (HR), and left ventricular end-diastolic pressure (LVEDP), were measured at the time points indicated in Figure 2. A 5-F sidearm sheath (Radifocus, Terumo, Tokyo, Japan) was placed in the right femoral artery for hemodynamic measurements. A 4-F pigtail catheter (Outlook, Terumo) was placed in the LV for measurement of LVEDP and was connected to a pressure transducer (model DX-200, Nihon Kohden, Tokyo, Japan). The ABP and HR were monitored via the 5-F sidearm sheath.
Echocardiography.
Cardiac function was assessed by echocardiography (Sonos 5500, S4-probe, 2-4 MHz, Philips, Bothell, Washington) at the time points indicated in Figure 2. Short-axis views were recorded at the level of midpapillary muscles, and two-dimensional and M-mode views were recorded at the same level. Measurements of left ventricular end-diastolic dimension (LVEDD) and LV ejection fraction were obtained from M-mode views. All measurements were made by one observer, who was blinded with respect to the identity of the tracings.
Infarct size 4 weeks after MI.
Myocardial infarct area was determined at the end of the protocol by triphenyltetrazolium chloride staining as described previously (14). Infarct size was expressed as a percentage of the total LV area.
Statistical analysis.
Results are expressed as the mean ± standard error of the mean. Comparisons of the time course of the change between groups were performed using two-way repeated measures analysis of variance. Comparisons of other data between groups were performed using one-way factional analysis of variance. If statistical significance was found for a group, a time effect, or a group-by-time interaction, further comparisons were made with paired t tests between all possible pairs of four groups at individual time points. The Bonferroni-Holm procedure was used for correction of multiple comparisons. A p value <0.05 was considered to represent statistical significance (20).
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Results
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Exclusion.
Four dogs [acute effects protocol; control: 1, EPO: 0, delayed treatment effects protocol; control: 1, EPO(0): 1, EPO(6h): 0, EPO(1wk): 1] were excluded from analysis because of excessive regional MBF (>15 ml/100 g/min). Thus, 12 and 31 dogs in acute and delayed EPO treatment protocols, respectively, were included.
Acute effects of EPO on infarct size.
Myocardial infarct size was significantly smaller in animals receiving EPO compared with those that received saline, but there was no significant difference in regional MBF (9.0 ± 1.0 ml/100 g/min vs. 8.5 ± 1.2 ml/100 g/min) or area at risk (42.9 ± 2.3% vs. 42.3 ± 0.9%) when comparing the two groups (Fig. 3).
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Figure 3 Representative left ventricular cross sections at 6 h after myocardial infarction (MI) in dogs with (B) and without (A) erythropoietin (EPO) treatment. (C) Infarct size at 6 h after MI. *p < 0.05 vs. the control group. Open circles = infarct size in each animal.
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Effects of EPO on hematologic parameters.
The average change in hematologic parameters was not different when comparing the three EPO-treated groups and the control group over the 4-week experimental protocol (Table 1).
Plasma VEGF levels.
Table 2 shows the time course of changes in plasma VEGF level after MI. The plasma VEGF level was significantly and comparably elevated in both control and EPO(0) groups, peaking on 6 h after MI, and returned to baseline at 1 week after MI.
Circulating CD34+MNCs and in vitro cultured MNCs.
Figure 4A shows the time course of changes in circulating CD34+MNC number in the different groups. One week after MI, the number of circulating CD34+MNCs increased in all groups. Furthermore, the number of circulating CD34+MNCs at 1 week after MI was higher in the EPO(0) and EPO(6h) groups than in either control or EPO(1wk) group. Two weeks after MI, the number of CD34+MNCs in the control group returned to the baseline. By contrast, the number of CD34+MNCs in the EPO(0) and EPO(6h) groups also decreased but still remained higher than those in either the control or EPO(1wk) group. Furthermore, the administration of EPO 1 week after the LAD ligation did not affect the number of CD34+MNCs at any given time point.
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Figure 4 (A) Time course of changes in circulating CD34+MNC count after left anterior descending coronary artery (LAD) ligation in different experimental groups. (B) Representative images of double-stained cultured cells (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein [Dil-ac-LDL] and Ulex europaeus agglutinin I [UEA-I]) at baseline (a, b) and 1 week after LAD ligation (c, d) from dogs with and without erythropoietin (EPO) treatment immediately after LAD ligation. (C) Quantitative analysis of endothelial progenitor cell culture assay. *p < 0.05 vs. the control group. p < 0.05 vs. baseline.
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In the culture assay of MNCs, the number of Dil-ac-LDL/UEA-I double-positive cells obtained from blood 1 week after MI increased compared with that at baseline in both control and EPO(0) groups. Importantly, the double-positive cell number obtained from blood 1 week after MI in the EPO(0) group was significantly higher than in the control group (Figs. 4B and 4C).
Capillary density and regional MBF.
Figure 5A shows the representative immunohistologic findings in the nonischemic (panels a to d) and ischemic (panels e to h) regions at 4 weeks after MI. In the nonischemic region, there was no difference in the capillary density and capillary-to-myocyte ratio when comparing groups. In the ischemic region, the capillary-to-myocyte ratio as well as capillary density was significantly higher in the EPO(0) and EPO(6h) groups, but not in the EPO(1wk) group, than in the control group (Figs. 5B to 5C).
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Figure 5 (A) Representative immunohistologic staining with an antibody against von Willebrand factor in nonischemic (left circumflex coronary artery [LCX]) (a, b, c, d) and ischemic (left anterior descending coronary artery [LAD]) (e, f, g, h) regions in different experimental groups. Capillary density (B) and capillary-to-myocyte ratio (C) of nonischemic (LCX) and ischemic (LAD) regions in different experimental groups. *p < 0.05 versus the control group. Abbreviations as in Figure 1.
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Figure 6 shows the changes in regional MBF in the ischemic regions in different experimental groups. There was no significant difference in MBF at 90 min when comparing experimental groups. At 4 weeks after MI, MBF was more increased in the EPO(0) and EPO(6h) groups, but not in the EPO(1wk) group, than in the control group.
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Figure 6 Regional myocardial blood flow in the ischemic (left anterior descending coronary artery [LAD]) region 90 min and 4 weeks after myocardial infarction in different experimental groups. *p < 0.05 versus the control group. EPO = erythropoietin.
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Effects of immediate or delayed EPO treatment on cardiac function and infarct size.
Throughout the experimental protocols, there was no difference in either ABP or HR when comparing the groups (Table 3).
Figure 7 shows the time course of changes in LVEF (panel A), LVEDD (panel B), and LVEDP (panel C) in different experimental groups. There were no significant differences in baseline LVEF, LVEDD, and LVEDP when comparing the groups.
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Figure 7 The time course of changes in left ventricular ejection fraction (LVEF) (A), left ventricular end-diastolic dimension (LVEDD) (B), and left ventricular end-diastolic pressure (LVEDP) (C) in different experimental groups. Statistically significant (p < 0.05) group-by-time interactions (analysis of variance for repeated measurements) are indicated by the following: # = all groups; $ = control x EPO(0) group; & = control x EPO(6h) group; = EPO(0) x EPO(6h) group. (D) Infarct size at 4 weeks after myocardial infarction in different experimental groups. Open circles = infarct size in each animal. *p < 0.05 versus the control group. EPO = erythropoietin.
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Ninety minutes, 1 week, and 4 weeks after MI, LVEF was higher in the EPO(0) group than in the other groups. Ninety minutes and 1 week after MI, there was no difference in LVEF when comparing the EPO(6h) group and the control group. When comparing the time points of 1 week and 4 weeks after MI, LVEF decreased in the control and the EPO(1wk) groups but not in the EPO(6h) group. One and 4 weeks after MI, LVEDD was lower in the EPO(0) group than in the other groups. When comparing the time points of 1 week and 4 weeks after MI, LVEDD increased in the control and EPO(1wk) groups but not in the EPO(6h) group. Ninety minutes after MI, LVEDP was lower in the EPO(0) group than in the other groups. Four weeks after MI, LVEDP was lower in the EPO(0) and EPO(6h) groups than in either the control or the EPO(1wk) group.
Myocardial infarct size 4 weeks after MI was smaller in the EPO(0) group than in the control group, although EPO treatment, initiated 6 h and 1 week after MI, did not reduce infarct size (Fig. 7D).
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Discussion
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The present study showed that EPO administered 6 h after LAD ligation increased circulating CD34+MNCs, capillary density, MBF in the ischemic region, and prevented the worsening of cardiac function without reducing infarct size. The EPO enhances neovascularization, likely via EPC mobilization, and improves cardiac dysfunction in the chronic phase, although EPO has time-window limitations.
We showed that the EPO treatment immediately after the LAD ligation reduced infarct size, which is consistent with observations of previous reports (5–8). Because the infarct size-limiting effects of EPO appear rapidly, the nonerythroid effects of EPO, such as antiapoptosis and radical scavenging (4–8), may contribute to the reduction of infarct size.
Recent reports have shown that circulating CD34+MNC count correlated with EPC number in MNCs culture assay, and both increased at 1 to 2 weeks after EPO administration in animals and humans (9–11). In the culture assay, the number of Dil-ac-LDL/UEA-I double-positive cells obtained from blood at baseline did not differ between the two groups. The number of double-positive cells obtained from blood at 1 week after MI significantly increased compared with that at baseline in the control and EPO(0) groups. Further, the double-positive cell number obtained from blood in the EPO(0) group was higher than in the control group. These findings suggest that EPO augments increases in the number of MNCs that can differentiate into Dil-ac-LDL/UEA-I double-positive cells, an indicator of endothelial cells. Increases in the number of both CD34-positive cells and Dil-ac-LDL/UEA-I double-positive cells strongly suggest that EPO promotes EPC mobilization. The number of CD34+MNCs increased 1 week after MI in the canine model, which is consistent with observations from studies of patients with acute MI (21,22). Furthermore, the number of CD34+MNCs was higher in the EPO(0) and EPO(6h) groups than in the control group. This finding suggests that a single dose of EPO was effective in increasing the number of circulating EPCs after MI. Interestingly, EPO administered 1 week after MI failed to produce the identical effect, suggesting that EPO has a time window for promotion of EPC mobilization. We found that plasma VEGF levels were elevated, peaking at 6 h after MI and returned to the baseline 1 week after MI. The EPO did not affect plasma VEGF levels. Because both VEGF and EPO are known to promote EPC mobilization in experimental conditions and are independent predictors for the number of circulating EPCs in patients with coronary heart disease (9–11,23), they may additionally or synergistically contribute to EPC mobilization. Thus, it is likely that EPO alone, at least at the dose used in the present study, might not be enough to promote CD34+MNC mobilization 1 week after MI when VEGF returns to the baseline. Although we only investigated the low dose of EPO to consider the clinical implication, it is possible that high doses of EPO would show the different results. Further investigations are needed to clarify the mechanism of EPO-stimulated EPC mobilization.
The present study also showed that EPO increased capillary-to-myocyte ratio corrected for LV hypertrophy as well as capillary density in the EPO(0) and EPO(6h) groups, suggesting that EPO promotes the neovascularization in the ischemic region. Investigators have also reported that EPO enhances neovascularization in the ischemic region in the hind-limb occlusion model (9). As suggested in the present study, EPO may enhance neovascularization via EPC mobilization. Indeed, bone marrow-derived EPCs incorporate into foci of neovascularization at the border zone of MI (12,13), and administration of ex vivo-expanded EPCs resulted in increased myocardial neovascularization (24,25). In a rat stroke model, Wang et al. (26) showed that EPO treatment, initiated 24 h after MI, enhances angiogenesis. In addition, van der Meer et al. (27) showed that capillary density was increased in the rat post-MI model even when EPO was administered 3 weeks after MI. In contrast, we showed that EPO administered 1 week after MI failed to increase capillary density. The possible explanation for this discrepancy is attributable to the different doses of EPO used. In the studies by Wang et al. (26) (5,000 IU/kg for 7 days) and van der Meer et al. (27) (8,000 IU/kg every 3 weeks), relatively high doses of EPO were administered. In contrast, in the present study, a relatively low dose (1,000 IU/kg) of EPO was administered with a single injection, and the reason for this dose in the present study is for the possible translation of our results to clinical settings more easily (6), because 8,000 or 5,000 IU/kg EPO may cause side effects. On the other hand, we noticed that a higher dose of EPO would increase capillary density and improve the cardiac function even by the late administration of EPO for clinical use.
In the present study, MBF in the ischemic region was increased in both the EPO(0) and the EPO(6h) groups. Because neovascularization was also enhanced in these groups, increased MBF may occur secondary to the enhanced neovascularization.
The present study also showed that an administration of EPO immediately after the LAD ligation improved cardiac function at 90 min after MI, likely because of infarct size reduction, and subsequently prevented the development of cardiac dysfunction in the chronic phase. Because the previous reports showed that myocardial necrosis progresses within 6 h after the onset of MI (28,29), EPO was administered at time points of 6 h and later after LAD ligation to determine whether its activity is directed toward the acute phase of MI or the chronic phase of cardiac dysfunction. One week after MI, LVEF, LVEDD, or LVEDP was similar among the EPO(6h), EPO(1wk), and control groups. However, EPO administered 6 h, but not 1 week, after the LAD ligation improved cardiac dysfunction 4 weeks after MI when compared with the control group. Because we did not find any difference in infarct size at 4 weeks after MI between the EPO(6h) and the EPO(1wk) groups, the improvement of cardiac function in the EPO(6h) group was not attributable to the reduction of infarct size, but to the increased blood flow to the ischemic regions.
In conclusion, in addition to its acute effect on infarct size reduction, EPO may exert chronic cardioprotective effects through neovascularization and may be a useful adjunct for the treatment of patients with myocardial infarction.
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Acknowledgments
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The authors thank Hiroko Okuda, Akiko Ogai, Yoko Nagamachi, and Nobuko Kawasaki for their technical assistance.
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References
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