Erythropoietin (EPO) has been shown to have anti-inflammatory, antiapoptotic, and proangiogenic effects. This study investigated whether early EPO treatment effectively preserves left ventricular (LV) function in porcine acute myocardial infarction (AMI). Eighteen male mini-pigs divided into groups 1 (sham), 2 (AMI), and 3 (AMI with 2 consecutive EPO doses [7500 IU per animal each time] at 30 minutes and 24 hours after AMI induction) underwent echocardiography before and 14 days after AMI induction through left anterior descending artery (LAD) ligation with myocardium harvested for analysis. Larger infarcted areas (IA) were noted in group 2 than in group 3. In both IA and peri-IA, percentage of apoptotic nuclei and CD40-positive cells, messenger RNA expressions of IL-8, matrix metalloproteinase-9, caspase-3, and Bcl-2 associated x protein were highest, whereas proliferator-activated receptor-γ coactivator-1α, endothelial nitric oxide synthase and Bcl-2 were lowest in group 2. Oxidative stress and cytosolic cytochrome c in IA were increased (P < 0.001), whereas protein expression of connexin43, cytochrome c, and protein kinase C-ε in mitochondria were reduced in group 2 than in other groups (P < 0.045). The fibrosis in IA was notably decreased in group 3 compared with that in group 2. The number of small arterioles and capillary density in IA was highest in group 3, whereas LV performance was lowest in group 2 (P < 0.045). In conclusion, the results demonstrated that early EPO administration in a porcine AMI model effectively limits infarct size, attenuates LV remodeling, and preserves LV function.
- acute myocardial infarction
- mini-pig model
- mitochondrial damage
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Acute coronary artery occlusion causes acute myocardial ischemia and death of myocardium in the anatomical region supplied by the occluded artery. Innate immune mechanisms are promptly activated after myocardial damage, which, in turn, initiates the complement cascade, inflammatory reaction, and reactive oxygen species (ROS) generation.1-5In addition, accumulating evidence from both experimental and human studies have suggested that increased inflammatory cytokines and oxidative stress are associated with unfavorable outcomes after myocardial infarction and may play a crucial role in the pathogenesis and progression of pump failure.6-10
Erythropoietin (EPO) is a well-known hypoxia-induced hormone that is mainly produced in the kidneys. This hypoxia-induced hormone is originally used for treating anemic patients of various causes. Interestingly, other than its role in normalizing erythropoiesis, increasing data have demonstrated that EPO offers protection against ischemic myocardial damage.11-14The mechanisms underlying the anti-ischemic action of EPO have been proposed to involve antiapoptotic processes,12,14neovascularization, mobilization of endothelial progenitor cells, and angiogenesis.15-18Studies have further identified that EPO exerts anti-inflammatory18,19and antioxidant20,21effects via inhibiting the production of proinflammatory cytokines and ROS, suggesting that EPO has pleiotropic properties and exerts cardiovascular effects beyond hematopoiesis.
However, the information on the various effects of EPO on reducing the infarct size and left ventricular (LV) remodeling in a clinically relevant large animal model of acute myocardial infarction (AMI) has not been fully investigated. Hence, this study, by using a mini-pig experimental model, tested the hypothesis that early administration of EPO (30 minutes and 24 hours) after AMI induction can suppress LV remodeling and preserve LV function that may effectively limit LV infarct size.
MATERIALS AND METHODS
All animal experimental procedures were approved by the Institute of Animal Care and Use Committee at our hospital and performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, National Academy Press, Washington, DC, revised 1996).
Animals, Protocol, Procedures, and Rationale of EPO Dose
Eighteen male mini-pigs (Taitung Animal Propagation Station, Livestock Research Institute, Taiwan) weighting 16 to 18 kg were used in this study and categorized into group 1 (sham control, n = 6), group 2 (AMI treated with 3 mL of intravenous physiological saline at 30 minutes and 24 hours after AMI induction, n = 6), and group 3 (AMI plus intravenous EPO 7500 IU per dose given at 30 minutes and 24 hours after AMI induction, n = 6).
Groups 2 and group 3 mini-pigs were anesthetized by intramuscular injection of ketamine (15 mg/kg) and maintained with inhalational anesthesia using 1.5% isoflurane for the procedure. After being shaved on the chest, the mini-pig was placed in supine position on a warming pad at 37°C and then received endotracheal intubation with positive-pressure ventilatory support (180 mL/min) with room air using a ventilator during the procedure. Electrocardiogram (ECG) monitor and defibrillator were connected to the chest wall of each animal.
Under sterile conditions, the heart was exposed through midthoracotomy. The pericardium was gently removed and the midportion of the left anterior descending coronary artery (LAD) was ligated with 6-0 prolene suture just distal to the first diagonal branch. Regional myocardial ischemia of LV was confirmed by an observation of rapid whitish discoloration of the reddish myocardium, which later became reddish-black over the anterior surface of the LV and the rapid development of akinesia and dilatation over the area at risk. Acute myocardial infarction was confirmed by 12-lead ECG after the procedure. Muscle and skin were then closed in layers.
During the procedure of AMI induction, malignant ventricular tachyarrhythmia occurred in 2 mini-pigs that died even after resuscitation and defibrillation. In addition, one mini-pig in group 1 died suddenly at day 3 after AMI induction. Thus, a total of 24 mini-pigs were used in the current study.
The rationale of EPO dose was based on the descriptions of previous studies22,23with some modifications in the current study.
Bone Marrow Isolation and Determination of the Effects of EPO on Angiogenesis
Bone marrow was aspirated from the iliac crest of the animals followed by lyses of red blood cells. The harvested bone marrow-derived mononuclear cells (BMDMNCs) with a quantity of 5 × 106 were cultured in a 10-cm culture dish using M-199 medium with 20% fetal bovine serum. By day 7, fetal bovine serum content was reduced to 10%. Recombinant human EPO was used in this study (epoietin β, 5000 IU per ampoule, equivalent to 14.5 μg in 0.3 mL; Roche, Basel, Switzerland). Erythropoietin of increasing concentrations (0, ie, vehicle of 0.1% dimethyl sulfoxide or phosphate-buffered solution [PBS]), 10 and 30 IU/mL; n = 6 per group) was added into the culture dish on days 0, 7, 10, and 12.
After 14-day cell culturing, the BMDMNCs differentiated into morphologically typical endothelial cells were plated in 96-well plates at 1.0 × 104 cells per well in 150-μL serum-free M199 culture medium mixed with 50 μL of cold Matrigel for 6-hour incubation at 37°C in 5% CO2. Three random microscopic images (×200) were taken at each well for counting cluster, tube, and network formations with the mean values obtained.
Quantification of Circulating Stem Cells Using Flow Cytometry
The percentages of circulating mononuclear cells (MNCs) positive for 2 endothelial cell surface markers: (1) CD31-positive MNC (CD31 + MNC); (2) CD62E plus MNC and those positive for 2 mesenchymal stem cell surface markers: (3) CD90 plus MNC, and (4) CD271 plus MNC were assessed on day 2 after AMI induction and on day 14 after AMI using flow cytometry.
Ten milliliters of venous blood were drawn from the inserted catheter in the internal jugular vein at each time point into a vacutainer containing 3.8% buffered sodium heparin. Mononuclear cells were then isolated by density-gradient centrifugation of Ficoll 400 (Ficoll-Plaque plus, Amersham Biosciences, Sweden) as previously described. The MNCs were washed twice with PBS and centrifuged before incubation with 1 mL blocking buffer for 30 minutes at 4°C. These MNCs were washed twice with PBS and immunostained for 30 minutes on ice with monoclonal antibodies against primary antibodies CD31 (Serotec, Raleigh, NC), CD62E (Chemicon, Billerica, MA), CD 271 (Abcam, Cambridge, MA). Secondary detection was done using appropriate Alexa Fluor 488 (Molecular Probes, Eugene, OR). Biotin-conjugated CD29 (BD PharMingen, San Diego, CA), and phycoerythrin-conjugated streptavidin (eBiosciences, Cambridge, MA). Isotype-identical antibodies (IgG) served as controls. After staining, the cells were fixed with 1% paraformaldehyde. Flow cytometric analyses were performed by using a fluorescence-activated cell sorter (Beckman Coulter FC500 flow cytometer, Brea, CA). Cell viability greater than 95.0% was noted in each group. Each analysis included 30,000 cells per sample. Assessment in each sample was performed in duplicate, with the mean level reported. Listmode files were exported and analyzed using the CXP software (Beckman Coulter).
Functional Assessment by Echocardiography
Regional LV dimension, area, and regional function were assessed at baseline and 2 weeks after AMI induction. Echocardiographic images were obtained from the LV using an IE-33 ultrasound machine with an S5-1 transducer (Philips Medical System, Bothell, WA). Time-motion mode images from the LV short-axis view were obtained, and the following parameters were measured digitally using the leading edge technique: LV internal dimensions at diastole and systole (LVIDd and LVIDs). Images were stored on a DVD and transferred to a workstation installed with analysis software (Q-lab version 7, Philips). Mid-LV endocardial borders were manually traced at end-diastole, after which the semiautomated software derived the end-diastolic and end-systolic areas. We calculated the following: LV fractional shortening (FS) = [(LVIDd − LVIDs)/LVIDd] × 100%. The LV fractional area change (FAC) = [end-diastolic area (EDA) − end-systolic area (ESA)/EDA)] × 100%.
Measurement of Infarct Area at Basal, Middle, and Apical Levels of Left Ventricle
The heart was removed from each mini-pig after intravenous injection of an overdose of potassium chloride. Repeated flushing of the coronary artery with normal saline for washing out the red blood cells was performed immediately after heart removal. Three cross sections (1 cm in thickness) of the heart were obtained at basal, middle, and apical levels for measurement of myocardial infarct size in group 1 and group 2 mini-pigs. Each cross section of heart tissue was then stained with 2% triphenyl tetrazolium chloride (TTC) for infarct area (IA) analysis. Briefly, all heart sections were placed on a tray with a scaled vertical bar to which a digital camera was attached. The sections were photographed from directly above at a fixed height. The images obtained were then analyzed using Image Tool 3 image analysis software (Image Tool for Windows, Version 3.0, University of Texas Health Science Center, San Antonio, TX).
Definition of Infarct and Peri-Infarct Areas
The IA was defined as the area with whitish discoloration over LV anterior wall, whereas the peri-IA was defined as the pale pinkish borderline zone between the infarct area and the reddish well-perfused myocardium. The IA was further confirmed microscopically on histological sections.
Immunohistochemical Staining for CD40
Paraffin sections (3 μm thick) were obtained from LV myocardium of each mini-pig. To block the action of endogenous peroxidase, the sections were initially incubated with 3% hydrogen peroxide and then further processed using Beat Blocker Kit (#50-300, Zymed Co, Carlsbad, CA) with immersion in solutions A and B for 30 and 10 minutes, respectively, at room temperature. Polyclonal rabbit antibodies against CD40 (dilution, 1/100; Spring Bioscience, Pleasanton, CA) were then used, followed by application of SuperPicTure Polymer Detection Kit (Zymed) for 10 minutes at room temperature. Finally, the sections were counterstained with hematoxylin. For negative control experiments, primary antibodies were omitted.
Oxidative Stress Reaction of LV Myocardium
The Oxyblot oxidized protein detection kit (S7150) was purchased from Chemicon. The oxyblot procedure was performed according to the previous study.24The dinitrophenylhydrazine derivatization was carried out on 6 Kg of protein for 15 minutes according to the manufacturer's instructions. One-dimensional electrophoresis was carried out on 12% sodium dodecyl sulfate/polyacrylamide gel after dinitrophenylhydrazine derivatization. Proteins were transferred to nitrocellulose membranes, which were then incubated in the primary antibody solution (anti-DNP, 1:150) for 2 hours, followed by incubation with second antibody solution (1:300) for 1 hour at room temperature. The washing procedure was repeated 8 times within 40 minutes. Immunoreactive bands were visualized by enhanced chemiluminescence, which was then exposed to Biomax L film (Kodak). For quantification, chemiluminescence signals were digitized using Labwork software (UVP). On each gel, a standard control sample was loaded.
Arteriolar and Capillary Densities in Heart Infarct Area
The immunohistochemical (IHC) stain of arterioles was performed with α-smooth muscle actin (1:400) as primary antibody at room temperature for 1 hour, followed by washing with PBS thrice. The antimouse-HRP conjugated secondary antibody was then added for 10 minutes, followed by washing with PBS thrice. The 3,3#2032; diaminobenzidine (0.7 gm per tablet; Sigma) was added for 1 minute, followed by washing with PBS thrice. Finally, hematoxylin was added for 1 minute as a counterstain for nuclei, followed by washing twice. Three coronal sections of the heart were analyzed in each mini-pig. For quantification, 3 randomly selected HPFs (×200) were analyzed in each slide. The mean number per HPF for each animal was then determined by the summation of all numbers divided by 9.
The immunohistochemical stain of von Willebrand factor (VWF) positively stained cells, an index of endothelial cells in capillaries, was performed with anti-VWF antibody (Chemicon).
Histological Study of Fibrosis Area
Masson trichrome staining was used for studying fibrosis of LV myocardium. Three serial sections of LV myocardium were prepared at 4-μm thickness by Cryostat (Leica CM3050S; Leica Microsystems GmbH, Wetzlar, Germany). The integrated area (in micrometer square) of fibrosis in the slides was calculated using Image Tool 3 image analysis software (Image Tool for Windows, Version 3.0; University of Texas, Health Science Center). Three selected sections were quantified for each animal. Three randomly selected high-power fields (HPFs; ×400) were analyzed in each section. After determining the number of pixels in each fibrotic area per HPF, the number of pixels obtained from the 3 HPFs were summed up. The procedure was repeated in 2 other slides for each animal. The mean pixel number per HPF for each animal was then determined by summating all pixel numbers and dividing the sum by 9. The mean integrated area (in micrometer square) of fibrosis in LV myocardium per HPF was obtained using a conversion factor of 19.24 (1 μm2 represented 19.24 pixels).
Quantitative data are expressed as mean ± SD. Statistical analysis was adequately performed by analysis of variance followed by Bonferroni multiple-comparison post hoc test or by Kruskal-Wallis test followed by multiple-comparison using Wilcoxon rank sum test. Statistical analysis was performed using SAS statistical software for Windows version 8.2 (SAS institute, Cary, NC). A P < 0.05 was considered statistically significant.
Body Weight and Echocardiographic Findings on Day 14 After AMI
By day 14 after AMI, the LVIDd was significantly higher in group 2 (AMI + saline) than in group 1 (sham control; Table 1). In addition, the LVIDs was significantly higher in group 2 than in groups 1 and 3 (AMI + EPO). Moreover, LVIDd and LVIDs were significantly higher in group 2 than in groups 1 and 3, and notably higher in group 3 than in group 1. Conversely, the FAC (in percent) change was significantly lower in group 2 than in groups 1 and 3 and notably lower in group 3 than in group 1. Furthermore, the FS was significantly lower in group 2 than in groups 1 and 3. However, the FS was similar between group 1 and group 3. In addition, the FAC did not differ between day 0 and day 14 in group 3 animals (P > 0.1). The findings indicate that EPO therapy attenuated LV remodeling and preserved LV function in our animal model.
EPO Enhances Angiogenesis of Bone Marrow-Derived Mononuclear Cells
Figure 1, A-F shows the effect of EPO on angiogenesis. The number of cluster, tubular, or network formation of BMDMNCs did not show significant difference between concentrations of 10 and 30 IU/mL of EPO treatment on Matrigel assay after 6-hour cell culturing. However, cluster, tubular, and network formations were significantly higher after EPO treatment (ie, either 10 or 30 IU/mL) than without. These findings indicate that EPO treatment could enhance angiogenesis in vitro.
EPO Therapy Enhanced Early Increase in Circulating Levels of Stem Cells
The lower panel of Figure 1, G-J shows the effect of EPO on enhancing circulating level of stem cells. The circulating level of CD31 plus MNCs did not differ between groups 1 and 2 on days 2 and 14 after AMI, whereas it was significantly higher in group 3 than in groups 1 and 2 at these 2 time points (Fig. 1G). Moreover, whereas the circulating level of CD62E plus MNCs was significantly higher in group 3 than in groups 1 and 2 on day 2 after AMI, it did not differ between group 1 and group 2 on day 2 after AMI and also among the 3 groups on day 14 after AMI (Fig. 1H).
The circulating levels of CD90 plus MNCs and CD271 plus MNCs were significantly higher in group 3 than in groups 1 and 2 on day 2 after AMI (Fig. 1, I and J). However, no significant difference in the circulating levels of these cells was noted between groups 1 and 2 on day 2 and among the 3 groups on day 14 after AMI.
Measurement of Infarct Area at Basal, Middle, and Apical Levels of Left Ventricle
Figure 2 shows the results of TTC staining on day 14 after AMI. As shown on the upper panel, the IA was notably larger in group 2 than in group 3 at basal, middle, and apical levels. Accordingly, quantification of the IA (lower panel) demonstrated a remarkably larger area of infarction at all 3 levels in group 2 than in group 3.
Quantification of Viable Myocardium in Infarct Area and Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL)
Figure 3A shows immunofluorescence troponin-I staining of IA for detecting viable myocardium, demonstrating markedly decreased distribution of troponin-I positively stained myocardium in IA of group 2 than that in group 3 on day 14 after AMI. These findings imply that, as compared with AMI without treatment (group 2), EPO therapy (group 3) markedly attenuated infarct size on day 14 after AMI. Moreover, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay on day 14 after AMI induction (Fig. 3, B-D) demonstrated that the number of apoptotic nuclei was substantially higher in group 2 than in groups 1 and 3 in both peri-IA and non-IA, whereas the number was notably higher in group 3 than in group 1 in peri-IA.
Expression of Caspase 3, Bcl-2 Associated x protien, and Bcl-2
The messenger RNA (mRNA) expressions of caspase 3 and Bcl-2 associated x protein, 2 indexes of apoptosis, were significantly higher in group 2 than in other groups in both IA and peri-IA (Fig. 4, A and B). In addition, Bcl-2 mRNA expression, an index of antiapoptosis, was remarkably lower in group 2 than in other groups (Fig. 4C). These findings suggest that the EPO therapy significantly attenuated cellular apoptosis after AMI.
Expressions of IL-8, Matrix Metalloproteinase 9 (MMP-9), Endothelial Nitric Oxide Synthase (eNOS), and Proliferator-Activated Receptor-γ Coactivator-1α (PGC-1α)
The mRNA expressions of IL-8 (Fig. 4D) and matrix metalloproteinase 9 (MMP-9; Fig. 4E), 2 indexes of inflammation, in both IA and peri-IA were significantly higher in group 2 than in groups 1 and 3 by day 14 after AMI. These findings demonstrate that the EPO treatment significantly inhibited the inflammatory reaction after AMI.
The endothelial nitric oxide synthase (eNOS) mRNA expression, an index of nitrogen oxide (NO) production, which is an anti-inflammatory molecule, was significantly lower in group 2 than in other groups in IA and peri-IA (Fig. 4F) on day 14 after AMI. These findings suggest that the EPO treatment also preserved myocardial eNOS expression which, in turn, inhibited inflammatory reaction.
The mRNA expression of proliferator-activated receptor-γ coactivator-1α (PGC-1α) in non-IA and peri-IA (Fig. 4G) was notably lower in group 2 than in groups 1 and 3 on day 14 after AMI. These findings suggest that EPO therapy significantly preserved energy transcription pathway after AMI.
CD40 Expression in Both IA and Peri-IA
To determine whether inflammatory cells were up-regulated in both IA and peri-IA after AMI, immunohistochemical staining for detection of cellular CD40 was performed. Density of CD40-positively stained cells in IA and peri-IA was significantly higher in group 2 than in groups 1 and 3 and significantly higher in group 3 than in group 1 on day 14 after AMI (Fig. 5). This finding indicates that the EPO therapy had important inhibitory effects on inflammatory cellular infiltrations in IA and peri-IA after AMI in the porcine model.
Intensity of Oxidative Stress and Protein Expressions of Cytochrome C
Western blot analysis revealed a significantly higher oxidative stress in mitochondria in group 2 than in other groups on day 14 after AMI (Fig. 6A). The oxidative stress was also notably higher in group 3 than in group 1. The total amount of cytochrome c protein expression in cytosol was significantly higher in group 2 than in other groups (Fig. 6B). Conversely, the total cytochrome c protein expression in mitochondria was significantly lower in group 2 than in other groups (Fig. 6C). These findings indicate that the expression of cytochrome c, an index of energy supply and storage in mitochondria, was notably lower in group 2 than in groups 1 and 3. The increase in cytosolic cytochrome c content also suggested significant mitochondrial damage with cytochrome c release into the cytosol in the myocardium of this group of animals.
Connexin43 and Protein Kinase C in Membrane Fraction Containing Mitochondria of IA
The protein expressions of connexin 43 (Cx43; Fig. 6D) and protein kinase C (PKC)-ε (Fig. 6E) in membrane fraction containing rich mitochondria were significantly lower in group 2 than in groups 1 and 3 on day 14 after AMI induction. Studies have recently demonstrated that an increased expression of Cx43 or PKC-ε in mitochondria contributes to myocardial protection in ischemic myocardium.24,25Our findings, therefore, corroborate those of recent studies.24,25
Arteriolar and Capillary Density Analyses and Immunohistochemical Staining for VWF
Although the number of larger arterioles (defined as >15 μm in diameter ≤100 μm) in IA of left ventricle did not differ among the 3 groups, the number of small arterioles (defined as ≤15 μm in diameter) was substantially lower in group 2 than in groups 1 and 3 and significantly lower in group 1 than in group 3 on day 14 after AMI (Fig. 7, A-D). In addition, VWF-positively stained cells, an index of endothelial cell marker in capillary, were remarkably lower in group 2 than in group 1 and group 3 (Fig. 7, E-H). These findings indicate that EPO therapy in addition to preserving pre-existing vessels also induced angiogenesis/vasculogenesis.
Fibrosis of LV Myocardium
The mean fibrotic area in IA was remarkably higher in group 2 than in group 3 on Masson trichrome staining (Fig. 8). This finding suggests that the EPO therapy effectively inhibited fibrosis in IA after AMI.
This study, which investigated the therapeutic impact of EPO at 2 time points early after AMI in a mini-pig experimental model, provided several striking implications. First, EPO therapy significantly increased in vitro angiogenesis and levels of circulating stem cells. Second, this therapy notably attenuated inflammatory reactions after AMI. Third, EPO treatment markedly reduced myocardial fibrosis and infarct size. Finally, dilated LV remodeling was significantly inhibited and LV function was remarkably preserved using this therapeutic regimen.
Impact of EPO Therapy on Attenuation of Oxidative Stress, Inflammatory Cascades, and Cellular Apoptosis
The present study found that the percentage of CD40-positively stained cells, an index of the severity of inflammatory response, was found to be significantly higher in group 2 (AMI treated with normal saline) than in group 1 (sham control) in both IA and peri-IA. In addition, mRNA expressions of MMP 9 and IL-8, indexes of inflammatory reactions, were also notably higher in group 2 than in group 1 in these 2 areas. Moreover, Western blot analysis revealed a markedly increased oxidative index in group 2 compared with group 1 in IA. All of these findings indicate that inflammatory reactions are initiated after AMI. Previous studies have demonstrated that an innate immune response is rapidly elicited after myocardial damage,2,3,26resulting in activation of the complement system, propagation of inflammatory processes, and enhancement of ROS generation2-5,26that give rise to further damage of cardiomyocytes.27,28Collectively, the results of our study are consistent with those from previous studies.2-5,26
One important finding was that the oxidative stress and inflammatory responses were substantially alleviated in group 3 (AMI treated by EPO) compared with group 2 in both IA and peri-IA, suggesting that EPO therapy has distinct anti-inflammatory and/or antioxidant properties. In addition, eNOS mRNA expression, an indicator of anti-inflammatory process, was notably higher in group 3 than in group 2. These findings highlight the fact that early administration of EPO may significantly alleviate the intensity of oxidative and inflammatory responses. Interestingly, studies have previously shown that EPO therapy effectively attenuates inflammatory response18,19and exerts antioxidant effect.20,21Therefore, our results reinforce those of previous studies.18-21
Studies have previously demonstrated that EPO therapy has an antiapoptotic effect through the pathway of protein kinase cascades, which include Janus-associated kinase-2 (Jak2), phosphatidylinositol 3-kinase (PI3K)/Akt and Ras-MAP kinase,29and activation of the Akt kinase pathway.30Moreover, an experimental study has shown that EPO therapy attenuated hypoxia-induced or AMI-induced apoptosis of cardiomyocytes.31
One of the interesting findings in the present study was apoptosis related, showing notably higher mRNA expressions of Bcl-2 associated x protein and caspase 3, 2 indexes of apoptosis, and significantly lower Bcl-2 expression, an index of antiapoptosis, in group 2 than in groups 1 and 3 in both IA and peri-IA. Moreover, TUNEL assay demonstrated a markedly higher number of apoptotic nuclei in group 2 than in groups 1 and 3 in both peri-IA and non-IA. These findings support that EPO therapy, in addition to having anti-inflammatory effect, effectively inhibits apoptosis12,14,29-31that frequently occurs after AMI.32
Impact of EPO on Angiogenesis/Vasculogenesis and Circulating Levels of Stem Cells
Anatomically, permanent occlusion of a coronary artery without pre-existing coronary collateral circulation would induce complete infarction, resulting in nonviable myocardium in the territory of blood supply by the diseased artery. This forms the basis for the controversy regarding the effectiveness of EPO therapy in improving LV function in an animal model of AMI through permanent LAD ligation. This may be explained as follows.
Recent studies have suggested that EPO therapy improves ischemia-related heart dysfunction via enhancement of endothelial progenitor cell mobilization to ischemic area and angiogenesis/vasculogenesis.15-18In addition, recent studies24,28have shown that autologous implantation of BMDMNCs attenuated LV remodeling in a rat AMI model by permanent ligation of LAD. The results of those studies suggested angiogenesis/vasculogenesis as the key mechanisms underlying the reduction in LV remodeling and improved LV function.24,28An essential finding of the current study was that EPO therapy enhanced angiogenesis of bone marrow-derived mononuclear cell in vitro. In addition, flow cytometry identified that circulating level of CD31 plus MNCs were markedly higher in group 3 than in group 2 on days 0 and 14 after AMI. Moreover, circulating levels of CD62E plus MNCs, CD90 plus MNCs, and CD217 plus MNCs were notably higher in group 3 than in group 2 on day 0 after AMI. Furthermore, immunohistochemical staining identified that the number of small vessels, an index of angiogenesis/vasculogenesis in the IA, and eNOS mRNA expression, an index of endothelial function recovery/neovascularization in both peri-IA and IA, were notably higher in group 3 than in group 2. Therefore, our findings, in addition to strengthening those of previous studies,15-18suggest that EPO therapy enhances the homing of circulating stem cells to the ischemic zone for repairing ischemic myocardium. Accordingly, these findings could, at least in part, account for the preservation of LV function after EPO treatment through attenuating LV remodeling.
Mechanisms Underlying EPO Therapy in Reducing Death of Cardiomyocytes, Minimizing Infarct Size, Attenuating Fibrosis and LV Remodeling, and Improving Heart Function After AMI
The principal finding in the current study was a markedly higher mRNA expression of PGC-1α, which is a transcriptional coactivator of oxidative metabolism, mitochondrial metabolism, and biogenesis,33,34in group 3 than in group 2. Besides, Western blot analysis identified a significantly higher mitochondrial cytochrome c content in group 3 compared with that in group 2, whereas its cytosolic counterpart was markedly lower in group 3 than in group 2, suggesting a preservation of mitochondrial integrity and function from the EPO treatment. Moreover, the protein expressions of PKC-ε and Cx43 in mitochondria, which have been shown to be essential for myocardial protection in various settings of myocardial ischemia,24,25were remarkably up-regulated after EPO treatment. Anatomically, the most important finding in the present study was that TTC staining, Masson trichrome staining, and troponin-I immunofluorescence staining identified a substantially smaller myocardial infarct size in animals with EPO treatment than those without. Furthermore, the results of this study, in addition to extending the findings from recent investigation,15-21,24,25also underscore the importance of early implementation of EPO therapy as an effectual treatment strategy for reducing LV remodeling and preserving LV function after irreversible AMI. The clinical implication of the current study is that, even when the patency of the occluded coronary artery cannot be resumed after percutaneous coronary intervention, early administration of EPO can still achieve, at least partly, the goals of attenuating infarct size, reducing ventricular remodeling, and preserving heart function after AMI.
This study has limitations. First, the FAC did not differ between days 0 and 14 among group 3 animals. This may be due to different physiological baseline variables including body temperature, heart rate, and sensitivity to anesthesia of the mini-pigs at the 2 time points of examination (ie, day 0 and day 14) or due to other unidentified confounders. Second, the long-term effect of EPO on the preservation of LV function was not investigated in this study. Third, although the results of the present study are impressive, the exact mechanisms involved have not been fully investigated. Taken together, the possible mechanisms of EPO therapy in limiting infarct size, reducing LV remodeling, and improving LV function are schematically presented in Figure 9.
In conclusion, AMI initiates a cascade of immune responses and inflammatory processes, which, in turn, causes further myocardial injury. Early administration of EPO effectively inhibited inflammatory reaction, limited the infarct size, attenuated the dilated LV remodeling, and preserved LV function in the setting of AMI without reperfusion therapy in a porcine animal setting. Our findings, therefore, may raise a need for a prospective study to investigate the efficacy of this adjunctive therapy in AMI patients who are not eligible for reperfusion therapy because of contraindication for antithrombotic therapies. A prospective clinical trial of EPO therapy may then be warranted for AMI patients before reperfusion therapy.
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