- Email: [email protected]
Experimental acute myocardial infarction in rats: HIF-1α, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses
G Model ACTHIS-50681; No. of Pages 11
ARTICLE IN PRESS Acta Histochemica xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Acta Histochemica journal homepage: www.elsevier.de/acthis
Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses Aysel Guven Bagla a,∗ , Ertugrul Ercan b , Halil Fatih Asgun c , Meltem Ickin a , Feriha Ercan d , Ozlem Yavuz e , Suat Bagla f , Askin Kaplan g a
Department of Histology and Embryology, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey Department of Cardiology, Faculty of Medicine, Medicalpark Hospital, Izmir University, Izmir, Turkey c Department of Cardiovascular Surgery, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey d Department of Histology and Embryology, Faculty of Medicine, Marmara University, Istanbul, Turkey e Department of Medical Biochemistry, Balikesir University, Faculty of Medicine, Balikesir, Turkey f Canakkale Provincial Directorate of Health, Canakkale, Turkey g Family Medicine, Istanbul Aile Hekimligi, Istanbul, Turkey b
a r t i c l e
i n f o
Article history: Received 28 November 2012 Received in revised form 19 January 2013 Accepted 23 January 2013 Available online xxx Keywords: Erythropoietin Erythropoietin receptor Caspase 3 Hypoxia inducible factor 1␣ Myocardial infarction Rat
a b s t r a c t The cardioprotective effects of two different doses of erythropoietin administration were analyzed in rats with experimental myocardial infarction. None, saline, standard-dose (5000 U kg−1 ) and highdose (10,000 U kg−1 ) of human recombinant erythropoietin alpha were administered intraperitoneally in Wistar rats with myocardial infarction induced by coronary artery ligation. Infarct sizes measured after triphenyltetrazolium chloride staining, levels of biochemical markers, histopathology examined by light and electron microscopy, and immunohistochemical expressions of erythropoietin, erythropoietin receptor, hypoxia inducible factor-1␣ and caspase-3, were analyzed. Lower scores of infarction and hemorrhage, lower number of macrophages and higher score of vascularization surrounding the infarct area were observed in the erythropoietin administered groups (p < 0.05). Erythropoietin administration after myocardial infarction reduced the area of infarction and hemorrhage. There were hypoxia inducible factor-1␣ and caspase-3 expressions in the marginal area, and erythropoietin and erythropoietin receptor expression in both marginal and normal areas (p < 0.001). Vascularization, erythropoietin expression in the normal area and vascular erythropoietin expression were positively correlated with human erythropoietin levels. The cardioprotective effects of erythropoietin treatment were independent of endogenous erythropoietin/erythropoietin receptor activity. Moreover exogenous erythropoietin treatment did not suppress endogenous erythropoietin. Erythropoietin administration after myocardial infarction reduced caspase 3 expression (apoptotic activity) and induced neovascularization around the infarct area. Higher erythropoietin administration did not provide an additional benefit over the standard-dose in myocardial protection. © 2013 Elsevier GmbH. All rights reserved.
Introduction Erythropoietin (Epo) is a glycoprotein hormone essential for normal erythrocyte production in bone marrow. It is released from renal peritubular cells and various extrarenal tissues including: liver, spleen, brain, lungs, bone marrow, and reproductive organs. Epo induces erythropoiesis under hypoxic conditions (Sasaki et al., 2000; Chong et al., 2002; Jelkmann, 2004; Marzo et al., 2008; Paschos et al., 2008). Interaction of Epo with its receptor decreases
∗ Corresponding author. E-mail address: [email protected] (A. Guven Bagla).
programmed death of erythroid progenitor cells and promotes their differentiation in bone marrow (Fisher et al., 1996; Sasaki et al., 2000). The protective effects of Epo against tissue ischemia are mediated by erythropoietin receptor (EpoR) (Brines et al., 2004). In addition to erythroid progenitor cells, a varied group of cells including neurons, endothelial cells, vascular smooth muscle cells, and cardiac myocytes, express EpoR (Anagnostou et al., 1994; Digicaylioglu et al., 1995; Akimoto et al., 2000; Ogilvie et al., 2000; Ammarguellat et al., 2001; Digicaylioglu and Lipton, 2001; Tramontano et al., 2003; Jelkmann and Wagner, 2004; Marti, 2004). The cardioprotective effects of Epo have become a topical issue after detection of EpoR expression on cardiomyocytes
0065-1281/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.acthis.2013.01.005
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
2
(Marzo et al., 2008). The interaction of Epo with EpoR on these cells exerts protective effects against tissue ischemia (Junk et al., 2002; Cai et al., 2003; Calvillo et al., 2003; Moon et al., 2003; Parsa et al., 2003; Cai and Semenza, 2004; Lipsic et al., 2004; Sharples et al., 2004; Solaroglu et al., 2004; Wu et al., 2006; Guneli et al., 2007). Epo inhibits apoptosis and limits infarct size as seen using triphenyltetrazolium chloride (TTC) staining during ischemia and reperfusion through activation of various intracellular signaling pathways, especially the PI3K-Akt pathway (Parsa et al., 2003). Epo also has anti-inflammatory, anti-oxidative, and angiogenic potential (Paschos et al., 2008). The cardioprotective effects of Epo are independent of its hematopoietic effects (Parsa et al., 2003). Similar antiapoptotic and cardioprotective effects of Epo, independent of its hematopoietic effects, have been shown with carbamylated Epo, a non-erythropoietic derivative of Epo, and with helix B-surface peptide, a peptide mimicking the 3D structure of Epo (Fiordaliso et al., 2005; Ueba et al., 2010; Ahmet et al., 2011). Several studies regarding the cardioprotective effects of Epo have been tested using a similar standard dose of Epo treatment including 3000 U kg−1 or 5000 U kg−1 (Calvillo et al., 2003; Moon et al., 2003; Parsa et al., 2003; Tramontano et al., 2003; Rui et al., 2005). It remains to be clarified whether a higher-dose of the Epo treatment may improve the cardioprotective effects over the standard-dose treatment. In this study, cardioprotective effects of two different doses of Epo treatment were analyzed using biochemical, morphological, and immunohistochemical methods in rats with myocardial infarction induced by coronary artery ligation. Materials and methods The study was approved by Gazi University Animal Experiments Local Ethics Committee (project number G.Ü.ET-08.059) and conformed to the Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. All procedures on laboratory animals were performed in Gazi University Laboratory Animals Care and Experimental Research Center, Ankara, Turkey. Male Wistar rats weighing 250–300 g were divided into five groups: Group 1: Rats without treatment and sacrificed 1 h after coronary ligation (n = 8); Group 2: Rats receiving a single intraperitoneal (i.p) injection of saline immediately after coronary ligation and sacrificed 6 h after surgery (n = 7); Group 3: Rats receiving a single i.p. injection of standard-dose (5000 U kg−1 ) Epo (human recombinant erythropoietin alpha) (Eprex 4000 IU/0.4 mL pre-filled syringe, Janssen-Cilag AG, Schaffhausen, Switzerland) immediately after coronary ligation and sacrificed 6 h after surgery (n = 9); Group 4: Rats receiving a single i.p. injection of high-dose (10,000 U kg−1 ) Epo immediately after coronary ligation and sacrificed 6 h after surgery (n = 9); Group 5: Sham-operated control rats and sacrificed 6 h after surgery (n = 3). Myocardial ischemia model Rats were anesthetized with 45 mg kg−1 ketamine (Alfamine 10%, Alfasan International BV, Woerden, The Netherlands) and 5 mg kg−1 xylazine (Alfazyne 2%, Alfasan International BV, Woerden, The Netherlands) administered intraperitoneally. Basal electrocardiograms of all rats were taken using a data acquisition system (MP 150 Data Acquisition System, BIOPAC Systems, Goleta, CA, USA). Rats were intubated through tracheotomy and ventilated with room air using a volume controlled rodent ventilator (Inspira ASV, Harvard Apparatus, Holliston, MA, USA). Thoracotomy through the fourth intercostal space was performed to expose the heart. Left anterior descending coronary artery (LAD) ligation was obtained by placing a 7–0 polypropylene suture around the space
between the pulmonary artery and the left auricle. Cessation of normal contractions of the myocardium in the LAD perfusion area was accepted as a marker of adequate LAD ligation. Following LAD ligation, saline, standard-dose erythropoietin and high-dose erythropoietin were administered intraperitoneally in Groups 2, 3, and 4, respectively. The thoracotomy was closed without residual pneumothorax, and the tracheotomy was repaired after weaning from the ventilator. Control electrocardiograms of all rats were taken. ECG recordings taken after left anterior descending coronary artery (LAD) ligation showed ST segment changes indicating myocardial infarction. Rats were allowed to recover in a warm and oxygen rich compartment until they were fully active, and then they were transferred to their cages. No additional analgesia was required over the initial anesthesia. None of the rats died before euthanasia. Rats were fully anesthetized again with the same dose of ketamine and xylazine before euthanasia, and were re-intubated via the previous tracheotomy. The heart was exposed through a midline sternotomy and excised quickly after a blood sample was withdrawn from the right atrium. The occurrence of acute myocardial infarction in each rat was verified qualitatively using a commercially available troponin T test kit (Trop T, Roche Diagnostics, West Sussex, UK). Biochemistry Rat Epo, rat high sensitivity C-reactive protein (hsCRP), and rat cardiac troponin T (cTnT) levels in rat serum were measured using commercially available enzyme-linked immunosorbent assay (ELISA) rat sensitive kits (Cusabio Biotech, Newark, DE, USA, Catalog no: CSB-E07323r, CSB-E08618r, and CSB-E11305r, respectively) on a Model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The detection limits of Epo, hsCRP, and cTnT in rat serum were 0.2 mIU mL−1 , 0.16 ng mL−1 , and 15.6 pg mL−1 , respectively. Human Epo levels in rat serum were measured using a DRG EPO ELISA kit (DRG International, East Mountainside, NJ, USA, Catalog no: EIA-3646). Light microscopy The hearts were fixed in 10% buffered formalin, and processed for embedding in paraffin wax by routine protocols and cut into 5-m-thick sections by a microtome. The sections were stained using hematoxylin and eosin (H&E) and examined using a photomicroscope (Axioskop 40 Microscope and AxioCam ICc3 Microscope Camera, Carl Zeiss, Göttingen, Germany). Five sections were randomly chosen from the mid-ventricular level of the heart in each animal. From each section, five areas were randomly selected for the histopathological examination. The presence of vascularization around the infarct area, the score of infarction area, hemorrhage, inflammatory cell infiltration (increase in neutrophils), and the number of macrophages were assessed. Macrophages were counted (40× objective) in five different areas surrounding the infarct area. Semi-quantitative analysis Semi-quantitative analysis of the infarct size in the left ventricle, hemorrhage, and leukocyte increase were scored as: none (0), weak (1), moderate (2), strong (3) and very strong (4). The semiquantitative lesion scoring system was adapted from Azevedo Filho et al. (2004). Each section received a score according to the percentage of the infarct size in the left ventricle (score 0, 1, 2, 3 and 4). Score 0 corresponded to the absence of infarct; score 1 (weak) corresponded to infarct size of 1–25% of the area of the segment; score 2 (moderate) corresponded to infarct size of 26–50%; score 3 (strong) corresponded to infarct size of 51–75%; and score 4 (very
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
strong) corresponded to infarct size greater than 76% of the area of the segment. Leukocyte increase was scored as none (0), weak (1), moderate (2), strong (3) as follows: leukocyte neutrophils were counted in 10 random section areas surrounding the infarct area and the number of cells were revealed as follows: none (0): absent; weak (1): occasional (1–10 cells), moderate (2): focal (10–50 cells), strong (3): focal (>50 cells) and/or diffuse. TTC staining The hearts were stained with TTC for the delineation of myocardial infarction using the method described previously (Vivaldi et al., 1985). The transverse heart slices at mid-ventricular level were obtained freehand, and incubated in a 1% solution of TTC in phosphate buffer for 5 min at 37 ◦ C, pH 7.4. The TTCincubated slices were photographed in a macroscope (Leica M125 Stereoscope, Leica Microsystems, Wetzlar, Germany). Photographs were processed using graphics editing software (Photoshop CS4 Extended, Adobe Systems, San Jose, CA, USA) to calculate the size of infarct area, which was expressed as a fraction of the total crosssectional area of the left ventricle. Electron microscopy The ultrastructure of heart tissue was analyzed using transmission electron microscopy from samples obtained from the mid-ventricular level of the heart. Blocks of heart tissue specimen approximately 1 mm3 were removed from each animal and fixed in 2.5% glutaraldehyde (pH 7.3) in 0.1 M phosphate-buffered saline at 4 ◦ C for 4 h. The specimens were postfixed in 1% osmium tetroxide (0.1 M), and dehydrated in a graded series of ethanol, and embedded in epoxy resin. The epoxy resin blocks were sectioned using an Ultracut R microtome (Leica Microsystems GmbH, Wetzlar, Germany). Ultrathin sections (70 nm thick) were contrast stained with uranyl acetate and lead citrate prior to examination and image recording under a transmission electron microscope (JEOL, 1200 EX II, TEM, Tokyo, Japan). Immunohistochemistry All sections were processed simultaneously to avoid day-to-day variation of labeling efficiency. The tissue samples were fixed in 10% formalin, and embedded in paraffin wax blocks using routine protocols. Four-m-thick sections were cut and mounted on polyl-lysine-coated microscope slides. Sections were immunolabeled using an indirect immunohistochemical horseradish peroxidase staining procedure for the presence of Epo, EpoR, hypoxia inducible factor (HIF)-1␣, and caspase 3. The sections were deparaffinized and rehydrated by passing through xylene, graded ethyl alcohol (100%, 96%, and 70%), and bidistilled sterile water for 15 min each. Heat-induced antigens were retrieved using 0.01 mol L−1 citrate buffer (pH 6.0). The slides were quenched and washed in phosphate-buffered saline (PBS). Sections were treated with 3% H2 O2 and methanol for 30 min to block endogenous peroxidase activity. Sections were washed in PBS, pH 7.4. Slides were incubated overnight at 4 ◦ C with Epo (H-162) antibody (sc-7956, Rabbit polyclonal antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:100 dilution, EpoR (H-194) antibody (sc5624, Rabbit polyclonal antibody, Santa Cruz Biotechnology) at 1:200 dilution, HIF-1␣ (H1␣67) antibody (sc-53546 Mouse monoclonal antibody, Santa Cruz Biotechnology) at 1:100 dilution, and caspase 3 (CPP32) antibody (Rabbit polyclonal antibody, Diagnostic BioSystems, Pleasanton, CA, USA) at 1:500 dilution. After thorough washing with PBS, the sections were flooded with 5% H2 O2 solution, rinsed with PBS (2× 5 min) and incubated with biotinylated polyvalent
3
IgG (ready-to-use, Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA) for 15 min. Sections were rinsed with PBS (2× 5 min) and incubated with a streptavidin-horseradish peroxidase complex (ready-to-use, Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA) for 15 min. After rinsing with PBS (2× 5 min) again, sections were incubated with chromogen substrate solution for 15 min freshly prepared by dissolving 1 mg 3,3 -diaminobenzidine (DAB) (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA) in 1 mL of 0.05 M Tris–HCl buffer, pH 7.4, containing 1 mL of H2 O2 . After rinsing in distilled water, the sections were counterstained with Harris’s hematoxylin. For dehydration, each slide was soaked in graded alcohols (75%–80%–96%–2× 100%), cleared twice with xylene, and coverslipped with mounting medium. All antibodies were rat specific. Negative controls for all groups were performed by replacing the primary antisera with PBS. Semi-quantitative analysis of immunohistochemical staining evaluation The immunostaining patterns were diffuse through the whole series of sections for caspase-3, HIF-1␣, Epo and EpoR in normal and marginal areas. The marginal area refers to the area surrounding the infarction. None (0) = no detectable stain; (1) = weak to moderate staining; (2) = moderate to strong staining; (3) = strong to very strong staining; (4) = very strong staining on the infarct area, the marginal area and the normal area separately. The analyses of specimens in light microscopy, electron microscopy, and immunohistochemistry were performed by a researcher blinded to the sample grouping. Statistical analysis Statistical analyses were performed using IBM SPSS Statistics version 19 (IBM Corporation, NY, USA). Results of rat weight, the infarct size in TTC staining, biochemical markers, and the number of macrophages were expressed as mean ± SD. Results of light microscopy and immunohistochemistry were represented as stacked bar graphs. Kruskal–Wallis test was performed to analyze statistical significance. Multiple comparisons between groups were done using the Mann–Whitney U test with Bonferroni adjustment. Results of Group 5 were excluded from the tests done for infarct size in TTC staining, the score of infarction, hemorrhage, leukocyte increase, number of macrophages, vascularization, and HIF-1␣, Epo, EpoR and caspase-3 expression in the infarct area and the marginal area, because the rats in this group had no infarction induced by the coronary ligation. Spearman’s and Kendall’s Rank Correlation tests were performed to measure the correlation between variables. p < 0.05 was considered to be a statistically significant difference. Results Biochemistry Results of rat weights, infarct sizes in TTC staining, and levels of human Epo, rat Epo, cTnT, and hsCRP in rat serum are represented in Table 1. TTC staining is represented in Fig. 1. The greatest infarct size as seen in TTC staining was observed in Group 2 (37.11 ± 22.28%). Although the infarct sizes in TTC staining in the Epo administered groups were lower than Group 2, the difference was insignificant (p = 0.093). Human Epo level was significantly high in the Epo administered groups (p < 0.001), and Group 3 and Group 4 were statistically identical (p = 1). The levels of rat Epo, cTnT, and hsCRP were similar in all groups.
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
ARTICLE IN PRESS
G Model ACTHIS-50681; No. of Pages 11
A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
4
Table 1 Infarct sizes in TTC staining and results of biochemical markers. Group 1 (n = 8) Infarct size (%) Human Epo level (mIU/mL) Rat Epo level (mIU/mL) cTnT level (pg/mL) hsCRP level (ng/mL)
9.49 12.2 23.47 436.18 0.079
± ± ± ± ±
9.34 3.31 15.6 340.3 0.047
Group 2 (n = 7) 37.11 12.49 40.05 319.23 0.067
± ± ± ± ±
22.28 2.78 75.18 429.04 0.03
Group 3 (n = 9) 21.49 1398.1 7.71 205.59 0.05
± ± ± ± ±
10.58 123.29 5.34 132.89 0.026
Group 4 (n = 9) 25.55 1379.03 42.54 583.8 0.06
± ± ± ± ±
13.65 201.29 41.66 261.64 0.024
Group 5 (n = 3)
p
– 64.52 ± 14.21 ± 282.95 ± 0.04 ±
0.121 <0.001* 0.507 0.226 0.371
88.18 6.81 146.05 0.02
cTnT: Cardiac troponin T, Epo: Erythropoietin, hsCRP: High sensitivity C-reactive protein. * Human Epo level was significantly high in the Epo administered groups.
Light microscopy The cardiomyocytes in the infarct areas revealed dense eosinophilic cytoplasm and small condensed nuclei (Fig. 2A and B). The scores of infarction size, hemorrhage, and the number of macrophages in light microscopy were statistically different between the groups, and were the highest in Group 2. The highest changes in histopathological examination were observed in the left ventricular infarct area in Group 2 (p < 0.001) (Figs. 2A and 3A). Epo administered groups showed a decrease in the area of infarct sizes (Groups 3 and 4) (p < 0.001) (Figs. 2A, B and 3A). Marked hemorrhage, widespread myofilament disarray and loss were observed between infarct regions in some areas in Group 1 and Group 2 (p = 0.002) (Figs. 2B–D and 3B). Increase in the number of neutrophils was observed in Group 1 and Group 2, but it was statistically insignificant (p = 0.197) (Figs. 2C, D and 4B). Marked vascularization was seen around the infarct area in Group 3 (p = 0.014) (Figs. 2C and 3C). Statistical difference in the number of macrophages was achieved between Group 2 and Group 4
(p = 0.027) (Fig. 4A). Morphological features appeared normal in Group 5 (control group) (Fig. 2A–D). In terms of the infarct size, hemorrhage, and the number of neutrophils, no significant difference was observed between Epo administered groups (Groups 3 and 4) (Figs. 3A, B and 4B). Whereas vascularization, Epo expression in the normal area, and vascular Epo expression were positively correlated with human Epo levels in rat serum (p = 0.006), the number of macrophages, the score of infarct area, hemorrhage, and caspase-3 expression in the marginal area were negatively correlated with human Epo levels (p = 0.029, p = 0.013, p = 0.033, p = 0.025, respectively). Electron microscopy Electron microscopy showed a normal cell morphology and arrangement in Group 5 (Fig. 5A). Mild mitochondrial degeneration, intracytoplasmic vacuolization, focal myofilament disarray, and fairly regular intercalated disks were observed in Group 1 (Fig. 5B). Severe mitochondrial degeneration, intracytoplasmic vacuolization, widespread myofilament disarray, and dilated intercalated
Fig. 1. TTC staining showing infarct areas. Group 1: 1 h after coronary artery ligation; Group 2: 6 h after coronary artery ligation; Group 3: EPO 5000 U kg−1 administered at the time of ligation + 6 h after coronary artery ligation; Group 4: EPO 10,000 U kg−1 administered at the time of ligation + 6 h after coronary artery ligation.
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
5
Fig. 2. The images represent the myocardium in light microscopy 1 h and 6 h after coronary artery ligation group: to which EPO was not injected; A, The infarct regions were seen as marked areas (↔). B, In the infarct areas (I), the cardiomyocyte sarcoplasm was densely eosinophilic and nuclei showed decreased size and condensation. N: Normal area. C, D, Marked hemorrhage (→), widespread myofilament disarray and loss were observed between infarct regions in some areas. Increase in the number of neutrophils (*) was observed. 6 h after coronary artery ligation and EPO 5000 IU/kg (6 Hour + EPO 5,000) and EPO 10,000 IU/kg (6 Hour + EPO 10,000) treated group; A–B: reduction of infarct areas (↔) were observed. The sarcoplasm of cardiomyocytes stains eosinophilic in infarct areas (I). C–D; Marked vascularization () was seen in 6 Hour + EPO 5,000, hematoxylin–eosin staining (H&E). In the control group (A–B–C–D) these morphological features were not observed. Scale bars: A = 500 m; B = 200 m; C = 100 m; D = 50 m.
disks were found in Group 2 (Fig. 5C). A few vacuoles, mild mitochondrial degeneration, focal myofilament disarray, and quite regular intercalated disks were seen in Group 3 (Fig. 5D).
significantly more intense in the marginal area in Groups 1, 3, and 4, all expressing similar staining intensity (Figs. 6D and 7D). Vascular Epo expression was significantly more intense in Groups 2 and 4 than Group 5 (p = 0.026) (Fig. 6C).
Immunohistochemistry Discussion Expressions of HIF-1␣, caspase-3, Epo and EpoR in the infarct, marginal and normal areas are represented in Figs. 6 and 7. In Groups 1–4, HIF-1␣ and caspase-3 expressions were observed almost only in the marginal area, while Epo and EpoR were expressed in both the marginal and normal areas (Fig. 7A–D). Caspase-3 expression in the marginal area was higher in Groups 1 and 2 than Groups 3 and 4 (p = 0.055) (Fig. 6A). Epo administration reduced caspase 3 expression in the marginal area, but there was no statistically significant difference (Figs. 6A and 7A). In Group 5, HIF-1␣ and caspase-3 expression was not observed (Fig. 7A and B), and there was only weak and ubiquitous Epo and EpoR expression in all areas (Fig. 7C and D). Epo and EpoR expressions were significantly more intense in the marginal area than in the normal area (Figs. 6C, D and 7C, D). EpoR expression in the normal area was significantly more intense in Group 1 than the other groups (p < 0.05) (Figs. 6D and 7D). There were no significant differences in Epo expression in the infarct and marginal areas between the groups, however it was observed that EpoR expression was significantly more intense in Group 1 than in Groups 2 and 5 (p = 0.014) (Figs. 6D and 7D). EpoR expression was
The score of infarct area was found to be significantly lower in the Epo-treated groups than the untreated groups, but there was no significant difference between the treatment groups. Although the duration of ischemia was longer in the Epo-treated groups than Group 1, the score of infarct was lower in these groups. The serum levels of human Epo in rats were negatively correlated with the score of infarct and hemorrhage. The findings observed by electron microscopy were less evident in the treatment groups. The results suggest that Epo administration during coronary artery occlusion significantly reduces the ischemic damage. Cardioprotective effects of Epo administration were related to the serum human Epo levels. It was considered that the levels of serum human Epo representing the circulating fraction of Epo administered intraperitoneally were an indicator of effective Epo treatment. The levels of serum human Epo were significantly higher in the Epo-treated groups than others. We found that caspase-3 expression was located in the marginal area surrounding the infarct. This marginal expression of caspase3 indicates that the apoptotic activity involves the ischemic
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11 6
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
Fig. 3. The stacked bars represent the frequency of different scores of infarction, hemorrhage and vascularization evaluated by light microscopy in each group. The “y” axis on the stacked bars represents rat numbers. Each part of the bars denoted by a different pattern shows the number of rats that have related score expressed in the right upper corner of each graphic. The differences between groups are statistically significant. Letters over the bars (a, b, or c) denote a homogeneous subset of groups that is significantly different from each other at the 0.05 level in the multiple comparisons. The groups denoted by the different letters are significantly different from each other (a is significantly different from both b and c, and also b is significantly different from c), and the groups denoted by the same letter are statistically similar. The group denoted by two letters (a, b) is similar to both groups denoted by each of these letters (a, b is similar to both a and b, but a is still significantly different from b). Results of A. the score of infarction (p < 0.001), B. hemorrhage (p = 0.002), and C. vascularization (p = 0.014).
myocardium surrounding the infarct area. There was a strong positive correlation between marginal caspase-3 expression and both the score of infarction and hemorrhage. Apoptotic activity that occurred in the marginal area is responsible for worsening of the ischemic myocardial damage. It is well-known that Epo exerts its cardioprotective effects mainly through PI3K-Akt and JAK2-STAT pathways, which are responsible for inhibition of apoptosis (Calvillo et al., 2003; Moon et al., 2003; Parsa et al., 2003; Tramontano et al., 2003; Cai and Semenza, 2004; Lipsic et al., 2004; Wright et al., 2004; Bullard et al., 2005; Fiordaliso et al., 2005; Hanlon et al., 2005; Brines and Cerami, 2006; Mudalagiri et al., 2008; Ueba et al., 2010). We found a negative correlation between the levels of serum human Epo and caspase-3 expression in the marginal area. Also, caspase-3 expression in the marginal area was near-significantly lower in the treatment groups than Group 2 (p = 0.055). It was concluded that reduced myocardial damage may be associated with reduced apoptotic activity in the marginal area. Ben-Dor et al. (2007) evaluated the protective potential of various doses of Epo in a rat myocardial infarction model of left ventricular remodeling, specifically with regard to 6 weeks function. In their study, Doppler echocardiography showed significant improvement in LVFS (left ventricle fractional shortening) in the group treated with repeated low doses of Epo (750 U/kg) following coronary artery ligation, whereas it showed deterioration in the other groups (the group treated with a single dose Epo (5000 U/kg), the group treated with repeated high doses of Epo (1000 U/kg) and the no-treatment group). They suggested that these dosedependent unexpected effects of Epo may reflect either systemic and myocardial effects or cumulative doses depending on frequency Epo administration. However, they did not find significant differences between Epo treatment groups and the control group regarding the remodeling indices (end diastolic and end systolic areas, left ventricle circumference). They observed significantly less collagen staining in non-infarct areas in single dose Epo and repeated high doses Epo groups compared to no-treatment and repeated low doses Epo groups. In addition, they reported that Epo treatment groups revealed reduction in apoptosis when compared with the no-treatment group (Ben-Dor et al., 2007). Similarly, we observed that two different single doses of Epo reduced apoptosis. High dose and standard dose Epo treatments revealed the same results. Oxygen dependent regulation of Epo production is controlled by HIF-1 and -2 (Nangaku and Eckardt, 2007; Marzo et al., 2008). Both HIF-1 and -2 are composed of ␣ and  subunits, and HIF␣/ heterodimers composed under hypoxic conditions induce Epo transcription (Marzo et al., 2008; Weidemann and Johnson, 2008). In our study, immunohistochemical expression of HIF-1␣ in the ischemic myocardium was analyzed, and it was showed that HIF1␣ expression occurred almost only in the marginal area like caspase-3 expression. The results suggested that the major source of HIF-1␣ production induced by ischemic myocardial damage was the ischemic myocardium surrounding the infarct, but not the infarcted myocardium itself. Also, HIF-1␣ expression was found to be positively correlated with ischemia related variables including the score of infarction, hemorrhage, macrophage count, leukocyte increase, and caspase-3 expression. It is unclear whether HIF is a pro- or anti-apoptotic factor. It may induce caspase-3 expression in ischemic myocardium. There is a strong correlation between HIF-1␣ and caspase-3 expression in the marginal area. On the other hand, it is known that Epo induced under hypoxic conditions by HIF exerts important cardioprotective effects through reducing apoptosis (Calvillo et al., 2003; Moon et al., 2003; Parsa et al., 2003;Tramontano et al., 2003; Cai and Semenza, 2004; Lipsic et al., 2004; Wright et al., 2004; Bullard et al., 2005; Fiordaliso et al., 2005; Hanlon et al., 2005; Brines and Cerami, 2006; Mudalagiri et al., 2008; Ueba et al., 2010). Unlike caspase-3 and HIF-1␣
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
7
Fig. 4. A. The bars represent the mean macrophage numbers in light microscopy. The difference between groups is statistically significant (p < 0.027). Letters over the bars denote a homogeneous subset of groups (for a more thorough explanation, please refer to the legend of Fig. 3). B. The stacked bars represent the frequency of the scores of neutrophil leukocyte increase in light microscopy (for a more thorough explanation, please refer to the legend of Fig. 3). The difference is statistically insignificant (p = 0.197).
Fig. 5. The images show the myocardium in electron microscopy (scale bars = 20 m). A. Regular cardiomyocytes with mitochondria and myofilaments in control Group 5. B. Mild degeneration and swelling of the mitochondria (m), intracytoplasmic vacuolization, and focal myofilament disarray (arrow), and fairly regular intercalated disks (arrowhead) in Group 1. C. Severe degeneration of the mitochondria (m), intracytoplasmic vacuolization, severe myofilament disarray (arrow) and degenerated intercalated disks (arrowhead) in Group 2. D. Quite regular mitochondria (m), reduced myofilament disarray (arrow) and regular intercalated disks (arrowhead) in Group 3.
expression, Epo expression was detected in both the marginal and normal areas. Although the strongest Epo expression was observed in the marginal area, there was a weak or moderate degree Epo expression in the normal area. Epo expression in the non-ischemic myocardium including either the normal area in the groups with coronary ligation or the sham control group suggests ubiquitous Epo production under normoxic conditions. This basal ubiquitous production of Epo is increased dramatically by ischemia or hypoxia. Epo was not expressed in the infarct area. Early Epo clearance and inability of transcriptional production of new Epo molecules in the infarcted myocardium was attributed to lack of Epo expression in the infarct area. Also, exogenous Epo administration did not induce or reduce the endogenous Epo activity. There was no difference in Epo expression between the groups, and no correlation between the level of serum human Epo and the marginal Epo expression. Additionally, the level of serum rat Epo was similar in all groups
(p = 0.507). It was concluded that the cardioprotective effects of Epo administration were independent of endogenous Epo activity, and exogenous Epo treatment did not suppress it. Similarly, EpoR expression was observed in both the marginal and normal areas. Like Epo expression, the strongest EpoR expression was observed in the ischemic marginal area. Also, there was ubiquitous EpoR expression in the non-ischemic myocardium. There was no or minimal EpoR expression in the infarct area. Lack of EpoR expression in the infarct area was similarly attributed to early EpoR clearance and inability of transcriptional production of new EpoR monomers in the infarcted myocardium. Moderate or strong EpoR expression in the marginal area suggested that ischemia induces EpoR expression in the myocardium. Mechanisms related to EpoR expression in the ischemic myocardium are unclear. We found that EpoR expression in the marginal and normal area was significantly higher in the early
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11 8
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
Fig. 6. The stacked bars represent the frequency of immunohistochemical expression of caspase-3, HIF-1␣, Epo, and EpoR in the infarct, marginal, and normal areas according to the groups. The “y” axis on the stacked bars represents rat numbers. Each part of the bars denoted by a different pattern shows the number of rats that have related score expressed in the right upper corner of the figure. p values are shown on figures. Letters over the bars denote a homogeneous subset of groups (for a more thorough explanation, please refer to the legend of Fig. 3).
period of infarction (in Group 1). It was concluded that interaction of Epo/EpoR may cause the consumption of EpoR homodimers in the normal area. Unlike Epo expression, there is no known mediator of EpoR expression. Local factors affected by ischemia may induce EpoR expression. Lack of ischemic stimulus in the normal area and consumption of EpoR molecules by Epo/EpoR
interaction may cause a reduction in EpoR expression with time. However, EpoR production in the ischemic marginal area still remains. It is unclear whether exogenous Epo administration contributes to the consumption of EpoR, because the treatment groups were not sacrificed during the early period of ischemia. In the later period of ischemia, exogenous Epo administration was
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
9
Fig. 7. Representative micrographs of immunohistochemical expression of caspase-3, HIF-1␣, Epo, and EpoR: A. Caspase 3 expression: Caspase 3 expression was not observed in the infarcted area (I) and normal area (N) in working groups. Caspase 3 expression was observed only in marginal area (M). B. HIF-1␣ expression: HIF-1␣ expression was not observed in the infarcted area (I) and normal area (N) in working groups. HIF-1␣ expression was observed only in marginal area (M). In the control group, HIF-1␣ expression was not observed. C. Epo expression: Epo expression was not observed in the infarcted areas (I). In the marginal areas (M) of working groups, Epo expression was more intense than that of the normal areas (N). In control group, Epo expression was diffusely weak. D. EpoR expression: 1 h after coronary artery ligation group (1 h): EpoR was the most intensely expressed in the marginal area (M). EpoR expression was not observed in the infarcted area (I). In the normal areas (N) of working groups, EpoR was expressed less intensely than that of marginal areas. In the control group, EpoR expression was diffusely weak. Scale bars = 50 m.
not found to be associated with impaired EpoR expression, because the degree of EpoR expression in the normal area was similar in Groups 2–4. It should be further explored whether a mediator may induce EpoR expression in the ischemic myocardium like Epo production. Enhancing new vessel formation over a longer time frame is one of the major cardioprotective effects of Epo against ischemia/reperfusion injury (Parsa et al., 2003; Lipsic et al., 2006). Although there was a strong positive correlation between vascularization and the levels of serum human Epo, vascularization was not correlated with the score of infarction and hemorrhage. It was concluded that Epo administration induces new vessel formation in the ischemic myocardium during the first 6 h of infarction, but neovascularization does not contribute to the myocardial protection during this early period of infarction. Neovascularization of the ischemic myocardium is associated with long-term cardioprotective effects of Epo (Lipsic et al., 2006). It was demonstrated by Nakano et al. (2007) that the vascular Epo/EpoR system promotes ischemia-induced neovascularization. Vascular Epo expression was found to be positively correlated with vascularization in our study (p = 0.004). Also, vascular Epo expression had a positive correlation with the levels of serum human Epo (p = 0.006) (data not shown). Nakano et al. (2007) related neovascularization promoted by local vascular Epo and EpoR to enhanced vascular endothelial growth factor secretion, endothelial progenitor cells mobilization, and recruitment of bone marrow derived proangiogenic cells to the ischemic tissue (Nakano et al., 2007; Brunner et al., 2012). Vascular Epo expression could be an important mechanism in new vessel formation during ischemia, and Epo administration may induce neovascularization via improving vascular Epo expression. In conclusion: (i) Epo treatment reduces the apoptotic activity in the ischemic myocardial damage in rats. (ii) It also induces new vessel formation in the early period of infarction, but this neovascularization does not contribute to myocardial protection. There is no significant correlation between the score of vascularization and both the infarct size (r = 0.175 and p = 0.426) and score of infarct area (r = −0.317 and p = 0.06). We conclude from this result that
vascularization that occurred during the early period of infarction does not provide additional myocardial protection. (iii) The cardioprotective effects of Epo treatment is independent of endogenous Epo/EpoR activity, and exogenous Epo treatment does not suppress it. Epo shows its cardioprotective effects through its receptor, EpoR. It was thought that Epo and EpoR form a unit working together. For that reason, we defined the Epo and EpoR unit as Epo/EpoR. Actually, from this we cannot conclude that the cardioprotective effects of Epo are independent of endogenous EpoR activity as we lack the data to reach such a conclusion. The term “endogenous Epo/EpoR activity” only represents the functional collaboration between these two molecules. (iv) Higher dose of Epo administration does not provide additional benefit beyond a standard-dose in myocardial protection, because higher dose of intraperitoneal Epo injection do not elevate the levels of serum human Epo more than a standard-dose injection. More studies need to be undertaken to explain the relationship between acute myocardial infarction and the cardioprotective effects of different erythropoietin doses. Acknowledgement This study was supported in part by Scientific Research Project Commission of C¸anakkale Onsekiz Mart University [grant number 2010/122]. References Ahmet I, Tae HJ, Juhaszova M, Riordon DR, Boheler KR, Sollott SJ, et al. A small nonerythropoietic helix B surface peptide based upon erythropoietin structure is cardioprotective against ischemic myocardial damage. Mol Med 2011;17: 194–200. Akimoto T, Kusano E, Inaba T, Iimura O, Takahashi H, Ikeda H, et al. Erythropoietin regulates vascular smooth muscle cell apoptosis by a phosphatidylinositol 3 kinase-dependent pathway. Kidney Int 2000;58:269–82.
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11 10
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
Ammarguellat F, Llovera M, Kelly PA, Goffin V. Low doses of EPO activate MAP kinases but not JAK2-STAT5 in rat vascular smooth muscle cells. Biochem Biophys Res Commun 2001;284: 1031–8. Anagnostou A, Liu Z, Steiner M, Chin K, Lee ES, Kessimian N, et al. Erythropoietin receptor mRNA expression in human endothelial cells. Proc Natl Acad Sci USA 1994;91:3974–8. Azevedo Filho CF, Hadlich M, Petriz JL, Mendonc¸a LA, Moll Filho JN, Rochitte CE. Quantification of left ventricular infarcted mass on cardiac magnetic resonance imaging, Comparison between planimetry and the semiquantitative visual scoring Method. Arq Bras Cardiol 2004;83:118–24, 111–117. Ben-Dor I, Hardy B, Fuchs S, Kaganovsky E, Kadmon E, Sagie A, et al. Repeated low-dose of erythropoietin is associated with improved left ventricular function in rat acute myocardial infarction model. Cardiovasc Drugs Ther 2007;21: 339–46. Brines M, Cerami A. Discovering erythropoietin’s extrahematopoietic functions: biology and clinical promise. Kidney Int 2006;70:246–50. Brines M, Grasso G, Fiordaliso F, Sfacteria A, Ghezzi P, Fratelli M, et al. Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor. Proc Natl Acad Sci USA 2004;101:14907–12. Brunner S, Huber BC, Weinberger T, Vallaster M, Wollenweber T, Gerbitz A, et al. Migration of bone marrow-derived cells and improved perfusion after treatment with erythropoietin in a murine model of myocardial infarction. J Cell Mol Med 2012;16:152–9. Bullard AJ, Govewalla P, Yellon DM. Erythropoietin protects the myocardium against reperfusion injury in vitro and in vivo. Basic Res Cardiol 2005;100:397–403. Cai Z, Manalo DJ, Wei G, Rodriguez ER, Fox-Talbot K, Lu H, et al. Hearts from rodents exposed to intermittent hypoxia or erythropoietin are protected against ischemia-reperfusion injury. Circulation 2003;108:79–85. Cai Z, Semenza GL. Phosphatidylinositol-3-kinase signaling is required for erythropoietin-mediated acute protection against myocardial ischemia/reperfusion injury. Circulation 2004;109:2050–3. Calvillo L, Latini R, Kajstura J, Leri A, Anversa P, Ghezzi P, et al. Recombinant human erythropoietin protects the myocardium from ischemia-reperfusion injury and promotes beneficial remodeling. Proc Natl Acad Sci USA 2003;100: 4802–6. Chong ZZ, Kang JQ, Maiese K. Hematopoietic factor erythropoietin fosters neuroprotection through novel signal transduction cascades. J Cereb Blood Flow Metab 2002;22:503–14. Digicaylioglu M, Bichet S, Marti HH, Wenger RH, Rivas LA, Bauer C, et al. Localization of specific erythropoietin binding sites in defined areas of the mouse brain. Proc Natl Acad Sci USA 1995;92:3717–20. Digicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling cascades. Nature 2001;412:641–7. Fiordaliso F, Chimenti S, Staszewsky L, Bai A, Carlo E, Cuccovillo I, et al. A nonerythropoietic derivative of erythropoietin protects the myocardium from ischemia-reperfusion injury. Proc Natl Acad Sci USA 2005;102:2046–51. Fisher JW, Koury S, Ducey T, Mendel S. Erythropoietin production by interstitial cells of hypoxic monkey kidneys. Br J Haematol 1996;95:27–32. Guneli E, Cavdar Z, Islekel H, Sarioglu S, Erbayraktar S, Kiray M, et al. Erythropoietin protects the intestine against ischemia/reperfusion injury in rats. Mol Med 2007;13: 509–17.
Hanlon PR, Fu P, Wright GL, Steenbergen C, Arcasoy MO, Murphy E. Mechanisms of erythropoietin-mediated cardioprotection during ischemia-reperfusion injury: role of protein kinase C and phosphatidylinositol 3-kinase signaling. FASEB J 2005;19:1323–5. Jelkmann W, Wagner K. Beneficial and ominous aspects of the pleiotropic action of erythropoietin. Ann Hematol 2004;83:673–86. Jelkmann W. Molecular biology of erythropoietin. Intern Med 2004;43:649–59. Junk AK, Mammis A, Savitz SI, Singh M, Roth S, Malhotra S, et al. Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury. Proc Natl Acad Sci USA 2002;99:10659–64. Lipsic E, Schoemaker RG, van der Meer P, Voors AA, van Veldhuisen DJ, van Gilst WH. Protective effects of erythropoietin in cardiac ischemia: from bench to bedside. J Am Coll Cardiol 2006;48:2161–7. Lipsic E, van der Meer P, Henning RH, Suurmeijer AJ, Boddeus KM, van Veldhuisen DJ, et al. Timing of erythropoietin treatment for cardioprotection in ischemia/reperfusion. J Cardiovasc Pharmacol 2004;44:473–9. Marti HH. Erythropoietin and the hypoxic brain. J Exp Biol 2004;207:3233–42. Marzo F, Lavorgna A, Coluzzi G, Santucci E, Tarantino F, Rio T, et al. Erythropoietin in heart and vessels: focus on transcription and signalling pathways. J Thromb Thrombolysis 2008;26:183–7. Moon C, Krawczyk M, Ahn D, Ahmet I, Paik D, Lakatta EG, et al. Erythropoietin reduces myocardial infarction and left ventricular functional decline after coronary artery ligation in rats. Proc Natl Acad Sci USA 2003;100:11612–7. Mudalagiri NR, Mocanu MM, Di Salvo C, Kolvekar S, Hayward M, Yap J, et al. Erythropoietin protects the human myocardium against hypoxia/reoxygenation injury via phosphatidylinositol-3 kinase and ERK1/2 activation. Br J Pharmacol 2008;153:50–6. Nakano M, Satoh K, Fukumoto Y, Ito Y, Kagaya Y, Ishii N, et al. Important role of erythropoietin receptor to promote VEGF expression and angiogenesis in peripheral ischemia in mice. Circ Res 2007;100:662–9. Nangaku M, Eckardt KU. Hypoxia and the HIF system in kidney disease. J Mol Med 2007;85:1325–30. Ogilvie M, Yu X, Nicolas-Metral V, Pulido SM, Liu C, Ruegg UT, et al. Erythropoietin stimulates proliferation and interferes with differentiation of myoblasts. J Biol Chem 2000;275:39754–61. Parsa CJ, Matsumoto A, Kim J, Riel RU, Pascal LS, Walton GB, et al. A novel protective effect of erythropoietin in the infarcted heart. J Clin Invest 2003;112:999–1007. Paschos N, Lykissas MG, Beris AE. The role of erythropoietin as an inhibitor of tissue ischemia. Int J Biol Sci 2008;4: 161–8. Rui T, Feng Q, Lei M, Peng T, Zhang J, Xu M, et al. Erythropoietin prevents the acute myocardial inflammatory response induced by ischemia/reperfusion via induction of AP-1. Cardiovasc Res 2005;65:719–27. Sasaki R, Masuda S, Nagao M. Erythropoietin: multiple physiological functions and regulation of biosynthesis. Biosci Biotechnol Biochem 2000;64:1775–93. Sharples EJ, Patel N, Brown P, Stewart K, Mota-Philipe H, Sheaff M, et al. Erythropoietin protects the kidney against the injury and dysfunction caused by ischemia-reperfusion. J Am Soc Nephrol 2004;15:2115–24. Solaroglu A, Dede FS, Okutan E, Bayrak A, Haberal A, Kilinc K. A single dose of erythropoietin attenuates lipid peroxidation in experimental liver ischemia-reperfusion injury in the rat fetus. J Matern Fetal Neonatal Med 2004;16: 231–4.
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
Tramontano AF, Muniyappa R, Black AD, Blendea MC, Cohen I, Deng L, et al. Erythropoietin protects cardiac myocytes from hypoxia-induced apoptosis through an Akt-dependent pathway. Biochem Biophys Res Commun 2003;308:990–4. Ueba H, Brines M, Yamin M, Umemoto T, Ako J, Momomura S, et al. Cardioprotection by a nonerythropoietic, tissue-protective peptide mimicking the 3D structure of erythropoietin. Proc Natl Acad Sci USA 2010;107:14357–62. Vivaldi MT, Kloner RA, Schoen FJ. Triphenyltetrazolium staining of irreversible ischemic injury following coronary artery occlusion in rats. Am J Pathol 1985;121:522–30.
11
Weidemann A, Johnson RS. Biology of HIF-1 alpha. Cell Death Differ 2008;15:621–7. Wright GL, Hanlon P, Amin K, Steenbergen C, Murphy E, Arcasoy MO. Erythropoietin receptor expression in adult rat cardiomyocytes is associated with an acute cardioprotective effect for recombinant erythropoietin during ischemia-reperfusion injury. FASEB J 2004;18:1031–3. Wu H, Ren B, Zhu J, Dong G, Xu B, Wang C, et al. Pretreatment with recombined human erythropoietin attenuates ischemiareperfusion-induced lung injury in rats. Eur J Cardiothorac Surg 2006;29:902–7.
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
ARTICLE IN PRESS Acta Histochemica xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Acta Histochemica journal homepage: www.elsevier.de/acthis
Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses Aysel Guven Bagla a,∗ , Ertugrul Ercan b , Halil Fatih Asgun c , Meltem Ickin a , Feriha Ercan d , Ozlem Yavuz e , Suat Bagla f , Askin Kaplan g a
Department of Histology and Embryology, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey Department of Cardiology, Faculty of Medicine, Medicalpark Hospital, Izmir University, Izmir, Turkey c Department of Cardiovascular Surgery, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey d Department of Histology and Embryology, Faculty of Medicine, Marmara University, Istanbul, Turkey e Department of Medical Biochemistry, Balikesir University, Faculty of Medicine, Balikesir, Turkey f Canakkale Provincial Directorate of Health, Canakkale, Turkey g Family Medicine, Istanbul Aile Hekimligi, Istanbul, Turkey b
a r t i c l e
i n f o
Article history: Received 28 November 2012 Received in revised form 19 January 2013 Accepted 23 January 2013 Available online xxx Keywords: Erythropoietin Erythropoietin receptor Caspase 3 Hypoxia inducible factor 1␣ Myocardial infarction Rat
a b s t r a c t The cardioprotective effects of two different doses of erythropoietin administration were analyzed in rats with experimental myocardial infarction. None, saline, standard-dose (5000 U kg−1 ) and highdose (10,000 U kg−1 ) of human recombinant erythropoietin alpha were administered intraperitoneally in Wistar rats with myocardial infarction induced by coronary artery ligation. Infarct sizes measured after triphenyltetrazolium chloride staining, levels of biochemical markers, histopathology examined by light and electron microscopy, and immunohistochemical expressions of erythropoietin, erythropoietin receptor, hypoxia inducible factor-1␣ and caspase-3, were analyzed. Lower scores of infarction and hemorrhage, lower number of macrophages and higher score of vascularization surrounding the infarct area were observed in the erythropoietin administered groups (p < 0.05). Erythropoietin administration after myocardial infarction reduced the area of infarction and hemorrhage. There were hypoxia inducible factor-1␣ and caspase-3 expressions in the marginal area, and erythropoietin and erythropoietin receptor expression in both marginal and normal areas (p < 0.001). Vascularization, erythropoietin expression in the normal area and vascular erythropoietin expression were positively correlated with human erythropoietin levels. The cardioprotective effects of erythropoietin treatment were independent of endogenous erythropoietin/erythropoietin receptor activity. Moreover exogenous erythropoietin treatment did not suppress endogenous erythropoietin. Erythropoietin administration after myocardial infarction reduced caspase 3 expression (apoptotic activity) and induced neovascularization around the infarct area. Higher erythropoietin administration did not provide an additional benefit over the standard-dose in myocardial protection. © 2013 Elsevier GmbH. All rights reserved.
Introduction Erythropoietin (Epo) is a glycoprotein hormone essential for normal erythrocyte production in bone marrow. It is released from renal peritubular cells and various extrarenal tissues including: liver, spleen, brain, lungs, bone marrow, and reproductive organs. Epo induces erythropoiesis under hypoxic conditions (Sasaki et al., 2000; Chong et al., 2002; Jelkmann, 2004; Marzo et al., 2008; Paschos et al., 2008). Interaction of Epo with its receptor decreases
∗ Corresponding author. E-mail address: [email protected] (A. Guven Bagla).
programmed death of erythroid progenitor cells and promotes their differentiation in bone marrow (Fisher et al., 1996; Sasaki et al., 2000). The protective effects of Epo against tissue ischemia are mediated by erythropoietin receptor (EpoR) (Brines et al., 2004). In addition to erythroid progenitor cells, a varied group of cells including neurons, endothelial cells, vascular smooth muscle cells, and cardiac myocytes, express EpoR (Anagnostou et al., 1994; Digicaylioglu et al., 1995; Akimoto et al., 2000; Ogilvie et al., 2000; Ammarguellat et al., 2001; Digicaylioglu and Lipton, 2001; Tramontano et al., 2003; Jelkmann and Wagner, 2004; Marti, 2004). The cardioprotective effects of Epo have become a topical issue after detection of EpoR expression on cardiomyocytes
0065-1281/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.acthis.2013.01.005
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
2
(Marzo et al., 2008). The interaction of Epo with EpoR on these cells exerts protective effects against tissue ischemia (Junk et al., 2002; Cai et al., 2003; Calvillo et al., 2003; Moon et al., 2003; Parsa et al., 2003; Cai and Semenza, 2004; Lipsic et al., 2004; Sharples et al., 2004; Solaroglu et al., 2004; Wu et al., 2006; Guneli et al., 2007). Epo inhibits apoptosis and limits infarct size as seen using triphenyltetrazolium chloride (TTC) staining during ischemia and reperfusion through activation of various intracellular signaling pathways, especially the PI3K-Akt pathway (Parsa et al., 2003). Epo also has anti-inflammatory, anti-oxidative, and angiogenic potential (Paschos et al., 2008). The cardioprotective effects of Epo are independent of its hematopoietic effects (Parsa et al., 2003). Similar antiapoptotic and cardioprotective effects of Epo, independent of its hematopoietic effects, have been shown with carbamylated Epo, a non-erythropoietic derivative of Epo, and with helix B-surface peptide, a peptide mimicking the 3D structure of Epo (Fiordaliso et al., 2005; Ueba et al., 2010; Ahmet et al., 2011). Several studies regarding the cardioprotective effects of Epo have been tested using a similar standard dose of Epo treatment including 3000 U kg−1 or 5000 U kg−1 (Calvillo et al., 2003; Moon et al., 2003; Parsa et al., 2003; Tramontano et al., 2003; Rui et al., 2005). It remains to be clarified whether a higher-dose of the Epo treatment may improve the cardioprotective effects over the standard-dose treatment. In this study, cardioprotective effects of two different doses of Epo treatment were analyzed using biochemical, morphological, and immunohistochemical methods in rats with myocardial infarction induced by coronary artery ligation. Materials and methods The study was approved by Gazi University Animal Experiments Local Ethics Committee (project number G.Ü.ET-08.059) and conformed to the Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. All procedures on laboratory animals were performed in Gazi University Laboratory Animals Care and Experimental Research Center, Ankara, Turkey. Male Wistar rats weighing 250–300 g were divided into five groups: Group 1: Rats without treatment and sacrificed 1 h after coronary ligation (n = 8); Group 2: Rats receiving a single intraperitoneal (i.p) injection of saline immediately after coronary ligation and sacrificed 6 h after surgery (n = 7); Group 3: Rats receiving a single i.p. injection of standard-dose (5000 U kg−1 ) Epo (human recombinant erythropoietin alpha) (Eprex 4000 IU/0.4 mL pre-filled syringe, Janssen-Cilag AG, Schaffhausen, Switzerland) immediately after coronary ligation and sacrificed 6 h after surgery (n = 9); Group 4: Rats receiving a single i.p. injection of high-dose (10,000 U kg−1 ) Epo immediately after coronary ligation and sacrificed 6 h after surgery (n = 9); Group 5: Sham-operated control rats and sacrificed 6 h after surgery (n = 3). Myocardial ischemia model Rats were anesthetized with 45 mg kg−1 ketamine (Alfamine 10%, Alfasan International BV, Woerden, The Netherlands) and 5 mg kg−1 xylazine (Alfazyne 2%, Alfasan International BV, Woerden, The Netherlands) administered intraperitoneally. Basal electrocardiograms of all rats were taken using a data acquisition system (MP 150 Data Acquisition System, BIOPAC Systems, Goleta, CA, USA). Rats were intubated through tracheotomy and ventilated with room air using a volume controlled rodent ventilator (Inspira ASV, Harvard Apparatus, Holliston, MA, USA). Thoracotomy through the fourth intercostal space was performed to expose the heart. Left anterior descending coronary artery (LAD) ligation was obtained by placing a 7–0 polypropylene suture around the space
between the pulmonary artery and the left auricle. Cessation of normal contractions of the myocardium in the LAD perfusion area was accepted as a marker of adequate LAD ligation. Following LAD ligation, saline, standard-dose erythropoietin and high-dose erythropoietin were administered intraperitoneally in Groups 2, 3, and 4, respectively. The thoracotomy was closed without residual pneumothorax, and the tracheotomy was repaired after weaning from the ventilator. Control electrocardiograms of all rats were taken. ECG recordings taken after left anterior descending coronary artery (LAD) ligation showed ST segment changes indicating myocardial infarction. Rats were allowed to recover in a warm and oxygen rich compartment until they were fully active, and then they were transferred to their cages. No additional analgesia was required over the initial anesthesia. None of the rats died before euthanasia. Rats were fully anesthetized again with the same dose of ketamine and xylazine before euthanasia, and were re-intubated via the previous tracheotomy. The heart was exposed through a midline sternotomy and excised quickly after a blood sample was withdrawn from the right atrium. The occurrence of acute myocardial infarction in each rat was verified qualitatively using a commercially available troponin T test kit (Trop T, Roche Diagnostics, West Sussex, UK). Biochemistry Rat Epo, rat high sensitivity C-reactive protein (hsCRP), and rat cardiac troponin T (cTnT) levels in rat serum were measured using commercially available enzyme-linked immunosorbent assay (ELISA) rat sensitive kits (Cusabio Biotech, Newark, DE, USA, Catalog no: CSB-E07323r, CSB-E08618r, and CSB-E11305r, respectively) on a Model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The detection limits of Epo, hsCRP, and cTnT in rat serum were 0.2 mIU mL−1 , 0.16 ng mL−1 , and 15.6 pg mL−1 , respectively. Human Epo levels in rat serum were measured using a DRG EPO ELISA kit (DRG International, East Mountainside, NJ, USA, Catalog no: EIA-3646). Light microscopy The hearts were fixed in 10% buffered formalin, and processed for embedding in paraffin wax by routine protocols and cut into 5-m-thick sections by a microtome. The sections were stained using hematoxylin and eosin (H&E) and examined using a photomicroscope (Axioskop 40 Microscope and AxioCam ICc3 Microscope Camera, Carl Zeiss, Göttingen, Germany). Five sections were randomly chosen from the mid-ventricular level of the heart in each animal. From each section, five areas were randomly selected for the histopathological examination. The presence of vascularization around the infarct area, the score of infarction area, hemorrhage, inflammatory cell infiltration (increase in neutrophils), and the number of macrophages were assessed. Macrophages were counted (40× objective) in five different areas surrounding the infarct area. Semi-quantitative analysis Semi-quantitative analysis of the infarct size in the left ventricle, hemorrhage, and leukocyte increase were scored as: none (0), weak (1), moderate (2), strong (3) and very strong (4). The semiquantitative lesion scoring system was adapted from Azevedo Filho et al. (2004). Each section received a score according to the percentage of the infarct size in the left ventricle (score 0, 1, 2, 3 and 4). Score 0 corresponded to the absence of infarct; score 1 (weak) corresponded to infarct size of 1–25% of the area of the segment; score 2 (moderate) corresponded to infarct size of 26–50%; score 3 (strong) corresponded to infarct size of 51–75%; and score 4 (very
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
strong) corresponded to infarct size greater than 76% of the area of the segment. Leukocyte increase was scored as none (0), weak (1), moderate (2), strong (3) as follows: leukocyte neutrophils were counted in 10 random section areas surrounding the infarct area and the number of cells were revealed as follows: none (0): absent; weak (1): occasional (1–10 cells), moderate (2): focal (10–50 cells), strong (3): focal (>50 cells) and/or diffuse. TTC staining The hearts were stained with TTC for the delineation of myocardial infarction using the method described previously (Vivaldi et al., 1985). The transverse heart slices at mid-ventricular level were obtained freehand, and incubated in a 1% solution of TTC in phosphate buffer for 5 min at 37 ◦ C, pH 7.4. The TTCincubated slices were photographed in a macroscope (Leica M125 Stereoscope, Leica Microsystems, Wetzlar, Germany). Photographs were processed using graphics editing software (Photoshop CS4 Extended, Adobe Systems, San Jose, CA, USA) to calculate the size of infarct area, which was expressed as a fraction of the total crosssectional area of the left ventricle. Electron microscopy The ultrastructure of heart tissue was analyzed using transmission electron microscopy from samples obtained from the mid-ventricular level of the heart. Blocks of heart tissue specimen approximately 1 mm3 were removed from each animal and fixed in 2.5% glutaraldehyde (pH 7.3) in 0.1 M phosphate-buffered saline at 4 ◦ C for 4 h. The specimens were postfixed in 1% osmium tetroxide (0.1 M), and dehydrated in a graded series of ethanol, and embedded in epoxy resin. The epoxy resin blocks were sectioned using an Ultracut R microtome (Leica Microsystems GmbH, Wetzlar, Germany). Ultrathin sections (70 nm thick) were contrast stained with uranyl acetate and lead citrate prior to examination and image recording under a transmission electron microscope (JEOL, 1200 EX II, TEM, Tokyo, Japan). Immunohistochemistry All sections were processed simultaneously to avoid day-to-day variation of labeling efficiency. The tissue samples were fixed in 10% formalin, and embedded in paraffin wax blocks using routine protocols. Four-m-thick sections were cut and mounted on polyl-lysine-coated microscope slides. Sections were immunolabeled using an indirect immunohistochemical horseradish peroxidase staining procedure for the presence of Epo, EpoR, hypoxia inducible factor (HIF)-1␣, and caspase 3. The sections were deparaffinized and rehydrated by passing through xylene, graded ethyl alcohol (100%, 96%, and 70%), and bidistilled sterile water for 15 min each. Heat-induced antigens were retrieved using 0.01 mol L−1 citrate buffer (pH 6.0). The slides were quenched and washed in phosphate-buffered saline (PBS). Sections were treated with 3% H2 O2 and methanol for 30 min to block endogenous peroxidase activity. Sections were washed in PBS, pH 7.4. Slides were incubated overnight at 4 ◦ C with Epo (H-162) antibody (sc-7956, Rabbit polyclonal antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:100 dilution, EpoR (H-194) antibody (sc5624, Rabbit polyclonal antibody, Santa Cruz Biotechnology) at 1:200 dilution, HIF-1␣ (H1␣67) antibody (sc-53546 Mouse monoclonal antibody, Santa Cruz Biotechnology) at 1:100 dilution, and caspase 3 (CPP32) antibody (Rabbit polyclonal antibody, Diagnostic BioSystems, Pleasanton, CA, USA) at 1:500 dilution. After thorough washing with PBS, the sections were flooded with 5% H2 O2 solution, rinsed with PBS (2× 5 min) and incubated with biotinylated polyvalent
3
IgG (ready-to-use, Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA) for 15 min. Sections were rinsed with PBS (2× 5 min) and incubated with a streptavidin-horseradish peroxidase complex (ready-to-use, Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA) for 15 min. After rinsing with PBS (2× 5 min) again, sections were incubated with chromogen substrate solution for 15 min freshly prepared by dissolving 1 mg 3,3 -diaminobenzidine (DAB) (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA) in 1 mL of 0.05 M Tris–HCl buffer, pH 7.4, containing 1 mL of H2 O2 . After rinsing in distilled water, the sections were counterstained with Harris’s hematoxylin. For dehydration, each slide was soaked in graded alcohols (75%–80%–96%–2× 100%), cleared twice with xylene, and coverslipped with mounting medium. All antibodies were rat specific. Negative controls for all groups were performed by replacing the primary antisera with PBS. Semi-quantitative analysis of immunohistochemical staining evaluation The immunostaining patterns were diffuse through the whole series of sections for caspase-3, HIF-1␣, Epo and EpoR in normal and marginal areas. The marginal area refers to the area surrounding the infarction. None (0) = no detectable stain; (1) = weak to moderate staining; (2) = moderate to strong staining; (3) = strong to very strong staining; (4) = very strong staining on the infarct area, the marginal area and the normal area separately. The analyses of specimens in light microscopy, electron microscopy, and immunohistochemistry were performed by a researcher blinded to the sample grouping. Statistical analysis Statistical analyses were performed using IBM SPSS Statistics version 19 (IBM Corporation, NY, USA). Results of rat weight, the infarct size in TTC staining, biochemical markers, and the number of macrophages were expressed as mean ± SD. Results of light microscopy and immunohistochemistry were represented as stacked bar graphs. Kruskal–Wallis test was performed to analyze statistical significance. Multiple comparisons between groups were done using the Mann–Whitney U test with Bonferroni adjustment. Results of Group 5 were excluded from the tests done for infarct size in TTC staining, the score of infarction, hemorrhage, leukocyte increase, number of macrophages, vascularization, and HIF-1␣, Epo, EpoR and caspase-3 expression in the infarct area and the marginal area, because the rats in this group had no infarction induced by the coronary ligation. Spearman’s and Kendall’s Rank Correlation tests were performed to measure the correlation between variables. p < 0.05 was considered to be a statistically significant difference. Results Biochemistry Results of rat weights, infarct sizes in TTC staining, and levels of human Epo, rat Epo, cTnT, and hsCRP in rat serum are represented in Table 1. TTC staining is represented in Fig. 1. The greatest infarct size as seen in TTC staining was observed in Group 2 (37.11 ± 22.28%). Although the infarct sizes in TTC staining in the Epo administered groups were lower than Group 2, the difference was insignificant (p = 0.093). Human Epo level was significantly high in the Epo administered groups (p < 0.001), and Group 3 and Group 4 were statistically identical (p = 1). The levels of rat Epo, cTnT, and hsCRP were similar in all groups.
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
ARTICLE IN PRESS
G Model ACTHIS-50681; No. of Pages 11
A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
4
Table 1 Infarct sizes in TTC staining and results of biochemical markers. Group 1 (n = 8) Infarct size (%) Human Epo level (mIU/mL) Rat Epo level (mIU/mL) cTnT level (pg/mL) hsCRP level (ng/mL)
9.49 12.2 23.47 436.18 0.079
± ± ± ± ±
9.34 3.31 15.6 340.3 0.047
Group 2 (n = 7) 37.11 12.49 40.05 319.23 0.067
± ± ± ± ±
22.28 2.78 75.18 429.04 0.03
Group 3 (n = 9) 21.49 1398.1 7.71 205.59 0.05
± ± ± ± ±
10.58 123.29 5.34 132.89 0.026
Group 4 (n = 9) 25.55 1379.03 42.54 583.8 0.06
± ± ± ± ±
13.65 201.29 41.66 261.64 0.024
Group 5 (n = 3)
p
– 64.52 ± 14.21 ± 282.95 ± 0.04 ±
0.121 <0.001* 0.507 0.226 0.371
88.18 6.81 146.05 0.02
cTnT: Cardiac troponin T, Epo: Erythropoietin, hsCRP: High sensitivity C-reactive protein. * Human Epo level was significantly high in the Epo administered groups.
Light microscopy The cardiomyocytes in the infarct areas revealed dense eosinophilic cytoplasm and small condensed nuclei (Fig. 2A and B). The scores of infarction size, hemorrhage, and the number of macrophages in light microscopy were statistically different between the groups, and were the highest in Group 2. The highest changes in histopathological examination were observed in the left ventricular infarct area in Group 2 (p < 0.001) (Figs. 2A and 3A). Epo administered groups showed a decrease in the area of infarct sizes (Groups 3 and 4) (p < 0.001) (Figs. 2A, B and 3A). Marked hemorrhage, widespread myofilament disarray and loss were observed between infarct regions in some areas in Group 1 and Group 2 (p = 0.002) (Figs. 2B–D and 3B). Increase in the number of neutrophils was observed in Group 1 and Group 2, but it was statistically insignificant (p = 0.197) (Figs. 2C, D and 4B). Marked vascularization was seen around the infarct area in Group 3 (p = 0.014) (Figs. 2C and 3C). Statistical difference in the number of macrophages was achieved between Group 2 and Group 4
(p = 0.027) (Fig. 4A). Morphological features appeared normal in Group 5 (control group) (Fig. 2A–D). In terms of the infarct size, hemorrhage, and the number of neutrophils, no significant difference was observed between Epo administered groups (Groups 3 and 4) (Figs. 3A, B and 4B). Whereas vascularization, Epo expression in the normal area, and vascular Epo expression were positively correlated with human Epo levels in rat serum (p = 0.006), the number of macrophages, the score of infarct area, hemorrhage, and caspase-3 expression in the marginal area were negatively correlated with human Epo levels (p = 0.029, p = 0.013, p = 0.033, p = 0.025, respectively). Electron microscopy Electron microscopy showed a normal cell morphology and arrangement in Group 5 (Fig. 5A). Mild mitochondrial degeneration, intracytoplasmic vacuolization, focal myofilament disarray, and fairly regular intercalated disks were observed in Group 1 (Fig. 5B). Severe mitochondrial degeneration, intracytoplasmic vacuolization, widespread myofilament disarray, and dilated intercalated
Fig. 1. TTC staining showing infarct areas. Group 1: 1 h after coronary artery ligation; Group 2: 6 h after coronary artery ligation; Group 3: EPO 5000 U kg−1 administered at the time of ligation + 6 h after coronary artery ligation; Group 4: EPO 10,000 U kg−1 administered at the time of ligation + 6 h after coronary artery ligation.
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
5
Fig. 2. The images represent the myocardium in light microscopy 1 h and 6 h after coronary artery ligation group: to which EPO was not injected; A, The infarct regions were seen as marked areas (↔). B, In the infarct areas (I), the cardiomyocyte sarcoplasm was densely eosinophilic and nuclei showed decreased size and condensation. N: Normal area. C, D, Marked hemorrhage (→), widespread myofilament disarray and loss were observed between infarct regions in some areas. Increase in the number of neutrophils (*) was observed. 6 h after coronary artery ligation and EPO 5000 IU/kg (6 Hour + EPO 5,000) and EPO 10,000 IU/kg (6 Hour + EPO 10,000) treated group; A–B: reduction of infarct areas (↔) were observed. The sarcoplasm of cardiomyocytes stains eosinophilic in infarct areas (I). C–D; Marked vascularization () was seen in 6 Hour + EPO 5,000, hematoxylin–eosin staining (H&E). In the control group (A–B–C–D) these morphological features were not observed. Scale bars: A = 500 m; B = 200 m; C = 100 m; D = 50 m.
disks were found in Group 2 (Fig. 5C). A few vacuoles, mild mitochondrial degeneration, focal myofilament disarray, and quite regular intercalated disks were seen in Group 3 (Fig. 5D).
significantly more intense in the marginal area in Groups 1, 3, and 4, all expressing similar staining intensity (Figs. 6D and 7D). Vascular Epo expression was significantly more intense in Groups 2 and 4 than Group 5 (p = 0.026) (Fig. 6C).
Immunohistochemistry Discussion Expressions of HIF-1␣, caspase-3, Epo and EpoR in the infarct, marginal and normal areas are represented in Figs. 6 and 7. In Groups 1–4, HIF-1␣ and caspase-3 expressions were observed almost only in the marginal area, while Epo and EpoR were expressed in both the marginal and normal areas (Fig. 7A–D). Caspase-3 expression in the marginal area was higher in Groups 1 and 2 than Groups 3 and 4 (p = 0.055) (Fig. 6A). Epo administration reduced caspase 3 expression in the marginal area, but there was no statistically significant difference (Figs. 6A and 7A). In Group 5, HIF-1␣ and caspase-3 expression was not observed (Fig. 7A and B), and there was only weak and ubiquitous Epo and EpoR expression in all areas (Fig. 7C and D). Epo and EpoR expressions were significantly more intense in the marginal area than in the normal area (Figs. 6C, D and 7C, D). EpoR expression in the normal area was significantly more intense in Group 1 than the other groups (p < 0.05) (Figs. 6D and 7D). There were no significant differences in Epo expression in the infarct and marginal areas between the groups, however it was observed that EpoR expression was significantly more intense in Group 1 than in Groups 2 and 5 (p = 0.014) (Figs. 6D and 7D). EpoR expression was
The score of infarct area was found to be significantly lower in the Epo-treated groups than the untreated groups, but there was no significant difference between the treatment groups. Although the duration of ischemia was longer in the Epo-treated groups than Group 1, the score of infarct was lower in these groups. The serum levels of human Epo in rats were negatively correlated with the score of infarct and hemorrhage. The findings observed by electron microscopy were less evident in the treatment groups. The results suggest that Epo administration during coronary artery occlusion significantly reduces the ischemic damage. Cardioprotective effects of Epo administration were related to the serum human Epo levels. It was considered that the levels of serum human Epo representing the circulating fraction of Epo administered intraperitoneally were an indicator of effective Epo treatment. The levels of serum human Epo were significantly higher in the Epo-treated groups than others. We found that caspase-3 expression was located in the marginal area surrounding the infarct. This marginal expression of caspase3 indicates that the apoptotic activity involves the ischemic
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11 6
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
Fig. 3. The stacked bars represent the frequency of different scores of infarction, hemorrhage and vascularization evaluated by light microscopy in each group. The “y” axis on the stacked bars represents rat numbers. Each part of the bars denoted by a different pattern shows the number of rats that have related score expressed in the right upper corner of each graphic. The differences between groups are statistically significant. Letters over the bars (a, b, or c) denote a homogeneous subset of groups that is significantly different from each other at the 0.05 level in the multiple comparisons. The groups denoted by the different letters are significantly different from each other (a is significantly different from both b and c, and also b is significantly different from c), and the groups denoted by the same letter are statistically similar. The group denoted by two letters (a, b) is similar to both groups denoted by each of these letters (a, b is similar to both a and b, but a is still significantly different from b). Results of A. the score of infarction (p < 0.001), B. hemorrhage (p = 0.002), and C. vascularization (p = 0.014).
myocardium surrounding the infarct area. There was a strong positive correlation between marginal caspase-3 expression and both the score of infarction and hemorrhage. Apoptotic activity that occurred in the marginal area is responsible for worsening of the ischemic myocardial damage. It is well-known that Epo exerts its cardioprotective effects mainly through PI3K-Akt and JAK2-STAT pathways, which are responsible for inhibition of apoptosis (Calvillo et al., 2003; Moon et al., 2003; Parsa et al., 2003; Tramontano et al., 2003; Cai and Semenza, 2004; Lipsic et al., 2004; Wright et al., 2004; Bullard et al., 2005; Fiordaliso et al., 2005; Hanlon et al., 2005; Brines and Cerami, 2006; Mudalagiri et al., 2008; Ueba et al., 2010). We found a negative correlation between the levels of serum human Epo and caspase-3 expression in the marginal area. Also, caspase-3 expression in the marginal area was near-significantly lower in the treatment groups than Group 2 (p = 0.055). It was concluded that reduced myocardial damage may be associated with reduced apoptotic activity in the marginal area. Ben-Dor et al. (2007) evaluated the protective potential of various doses of Epo in a rat myocardial infarction model of left ventricular remodeling, specifically with regard to 6 weeks function. In their study, Doppler echocardiography showed significant improvement in LVFS (left ventricle fractional shortening) in the group treated with repeated low doses of Epo (750 U/kg) following coronary artery ligation, whereas it showed deterioration in the other groups (the group treated with a single dose Epo (5000 U/kg), the group treated with repeated high doses of Epo (1000 U/kg) and the no-treatment group). They suggested that these dosedependent unexpected effects of Epo may reflect either systemic and myocardial effects or cumulative doses depending on frequency Epo administration. However, they did not find significant differences between Epo treatment groups and the control group regarding the remodeling indices (end diastolic and end systolic areas, left ventricle circumference). They observed significantly less collagen staining in non-infarct areas in single dose Epo and repeated high doses Epo groups compared to no-treatment and repeated low doses Epo groups. In addition, they reported that Epo treatment groups revealed reduction in apoptosis when compared with the no-treatment group (Ben-Dor et al., 2007). Similarly, we observed that two different single doses of Epo reduced apoptosis. High dose and standard dose Epo treatments revealed the same results. Oxygen dependent regulation of Epo production is controlled by HIF-1 and -2 (Nangaku and Eckardt, 2007; Marzo et al., 2008). Both HIF-1 and -2 are composed of ␣ and  subunits, and HIF␣/ heterodimers composed under hypoxic conditions induce Epo transcription (Marzo et al., 2008; Weidemann and Johnson, 2008). In our study, immunohistochemical expression of HIF-1␣ in the ischemic myocardium was analyzed, and it was showed that HIF1␣ expression occurred almost only in the marginal area like caspase-3 expression. The results suggested that the major source of HIF-1␣ production induced by ischemic myocardial damage was the ischemic myocardium surrounding the infarct, but not the infarcted myocardium itself. Also, HIF-1␣ expression was found to be positively correlated with ischemia related variables including the score of infarction, hemorrhage, macrophage count, leukocyte increase, and caspase-3 expression. It is unclear whether HIF is a pro- or anti-apoptotic factor. It may induce caspase-3 expression in ischemic myocardium. There is a strong correlation between HIF-1␣ and caspase-3 expression in the marginal area. On the other hand, it is known that Epo induced under hypoxic conditions by HIF exerts important cardioprotective effects through reducing apoptosis (Calvillo et al., 2003; Moon et al., 2003; Parsa et al., 2003;Tramontano et al., 2003; Cai and Semenza, 2004; Lipsic et al., 2004; Wright et al., 2004; Bullard et al., 2005; Fiordaliso et al., 2005; Hanlon et al., 2005; Brines and Cerami, 2006; Mudalagiri et al., 2008; Ueba et al., 2010). Unlike caspase-3 and HIF-1␣
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
7
Fig. 4. A. The bars represent the mean macrophage numbers in light microscopy. The difference between groups is statistically significant (p < 0.027). Letters over the bars denote a homogeneous subset of groups (for a more thorough explanation, please refer to the legend of Fig. 3). B. The stacked bars represent the frequency of the scores of neutrophil leukocyte increase in light microscopy (for a more thorough explanation, please refer to the legend of Fig. 3). The difference is statistically insignificant (p = 0.197).
Fig. 5. The images show the myocardium in electron microscopy (scale bars = 20 m). A. Regular cardiomyocytes with mitochondria and myofilaments in control Group 5. B. Mild degeneration and swelling of the mitochondria (m), intracytoplasmic vacuolization, and focal myofilament disarray (arrow), and fairly regular intercalated disks (arrowhead) in Group 1. C. Severe degeneration of the mitochondria (m), intracytoplasmic vacuolization, severe myofilament disarray (arrow) and degenerated intercalated disks (arrowhead) in Group 2. D. Quite regular mitochondria (m), reduced myofilament disarray (arrow) and regular intercalated disks (arrowhead) in Group 3.
expression, Epo expression was detected in both the marginal and normal areas. Although the strongest Epo expression was observed in the marginal area, there was a weak or moderate degree Epo expression in the normal area. Epo expression in the non-ischemic myocardium including either the normal area in the groups with coronary ligation or the sham control group suggests ubiquitous Epo production under normoxic conditions. This basal ubiquitous production of Epo is increased dramatically by ischemia or hypoxia. Epo was not expressed in the infarct area. Early Epo clearance and inability of transcriptional production of new Epo molecules in the infarcted myocardium was attributed to lack of Epo expression in the infarct area. Also, exogenous Epo administration did not induce or reduce the endogenous Epo activity. There was no difference in Epo expression between the groups, and no correlation between the level of serum human Epo and the marginal Epo expression. Additionally, the level of serum rat Epo was similar in all groups
(p = 0.507). It was concluded that the cardioprotective effects of Epo administration were independent of endogenous Epo activity, and exogenous Epo treatment did not suppress it. Similarly, EpoR expression was observed in both the marginal and normal areas. Like Epo expression, the strongest EpoR expression was observed in the ischemic marginal area. Also, there was ubiquitous EpoR expression in the non-ischemic myocardium. There was no or minimal EpoR expression in the infarct area. Lack of EpoR expression in the infarct area was similarly attributed to early EpoR clearance and inability of transcriptional production of new EpoR monomers in the infarcted myocardium. Moderate or strong EpoR expression in the marginal area suggested that ischemia induces EpoR expression in the myocardium. Mechanisms related to EpoR expression in the ischemic myocardium are unclear. We found that EpoR expression in the marginal and normal area was significantly higher in the early
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11 8
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
Fig. 6. The stacked bars represent the frequency of immunohistochemical expression of caspase-3, HIF-1␣, Epo, and EpoR in the infarct, marginal, and normal areas according to the groups. The “y” axis on the stacked bars represents rat numbers. Each part of the bars denoted by a different pattern shows the number of rats that have related score expressed in the right upper corner of the figure. p values are shown on figures. Letters over the bars denote a homogeneous subset of groups (for a more thorough explanation, please refer to the legend of Fig. 3).
period of infarction (in Group 1). It was concluded that interaction of Epo/EpoR may cause the consumption of EpoR homodimers in the normal area. Unlike Epo expression, there is no known mediator of EpoR expression. Local factors affected by ischemia may induce EpoR expression. Lack of ischemic stimulus in the normal area and consumption of EpoR molecules by Epo/EpoR
interaction may cause a reduction in EpoR expression with time. However, EpoR production in the ischemic marginal area still remains. It is unclear whether exogenous Epo administration contributes to the consumption of EpoR, because the treatment groups were not sacrificed during the early period of ischemia. In the later period of ischemia, exogenous Epo administration was
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
9
Fig. 7. Representative micrographs of immunohistochemical expression of caspase-3, HIF-1␣, Epo, and EpoR: A. Caspase 3 expression: Caspase 3 expression was not observed in the infarcted area (I) and normal area (N) in working groups. Caspase 3 expression was observed only in marginal area (M). B. HIF-1␣ expression: HIF-1␣ expression was not observed in the infarcted area (I) and normal area (N) in working groups. HIF-1␣ expression was observed only in marginal area (M). In the control group, HIF-1␣ expression was not observed. C. Epo expression: Epo expression was not observed in the infarcted areas (I). In the marginal areas (M) of working groups, Epo expression was more intense than that of the normal areas (N). In control group, Epo expression was diffusely weak. D. EpoR expression: 1 h after coronary artery ligation group (1 h): EpoR was the most intensely expressed in the marginal area (M). EpoR expression was not observed in the infarcted area (I). In the normal areas (N) of working groups, EpoR was expressed less intensely than that of marginal areas. In the control group, EpoR expression was diffusely weak. Scale bars = 50 m.
not found to be associated with impaired EpoR expression, because the degree of EpoR expression in the normal area was similar in Groups 2–4. It should be further explored whether a mediator may induce EpoR expression in the ischemic myocardium like Epo production. Enhancing new vessel formation over a longer time frame is one of the major cardioprotective effects of Epo against ischemia/reperfusion injury (Parsa et al., 2003; Lipsic et al., 2006). Although there was a strong positive correlation between vascularization and the levels of serum human Epo, vascularization was not correlated with the score of infarction and hemorrhage. It was concluded that Epo administration induces new vessel formation in the ischemic myocardium during the first 6 h of infarction, but neovascularization does not contribute to the myocardial protection during this early period of infarction. Neovascularization of the ischemic myocardium is associated with long-term cardioprotective effects of Epo (Lipsic et al., 2006). It was demonstrated by Nakano et al. (2007) that the vascular Epo/EpoR system promotes ischemia-induced neovascularization. Vascular Epo expression was found to be positively correlated with vascularization in our study (p = 0.004). Also, vascular Epo expression had a positive correlation with the levels of serum human Epo (p = 0.006) (data not shown). Nakano et al. (2007) related neovascularization promoted by local vascular Epo and EpoR to enhanced vascular endothelial growth factor secretion, endothelial progenitor cells mobilization, and recruitment of bone marrow derived proangiogenic cells to the ischemic tissue (Nakano et al., 2007; Brunner et al., 2012). Vascular Epo expression could be an important mechanism in new vessel formation during ischemia, and Epo administration may induce neovascularization via improving vascular Epo expression. In conclusion: (i) Epo treatment reduces the apoptotic activity in the ischemic myocardial damage in rats. (ii) It also induces new vessel formation in the early period of infarction, but this neovascularization does not contribute to myocardial protection. There is no significant correlation between the score of vascularization and both the infarct size (r = 0.175 and p = 0.426) and score of infarct area (r = −0.317 and p = 0.06). We conclude from this result that
vascularization that occurred during the early period of infarction does not provide additional myocardial protection. (iii) The cardioprotective effects of Epo treatment is independent of endogenous Epo/EpoR activity, and exogenous Epo treatment does not suppress it. Epo shows its cardioprotective effects through its receptor, EpoR. It was thought that Epo and EpoR form a unit working together. For that reason, we defined the Epo and EpoR unit as Epo/EpoR. Actually, from this we cannot conclude that the cardioprotective effects of Epo are independent of endogenous EpoR activity as we lack the data to reach such a conclusion. The term “endogenous Epo/EpoR activity” only represents the functional collaboration between these two molecules. (iv) Higher dose of Epo administration does not provide additional benefit beyond a standard-dose in myocardial protection, because higher dose of intraperitoneal Epo injection do not elevate the levels of serum human Epo more than a standard-dose injection. More studies need to be undertaken to explain the relationship between acute myocardial infarction and the cardioprotective effects of different erythropoietin doses. Acknowledgement This study was supported in part by Scientific Research Project Commission of C¸anakkale Onsekiz Mart University [grant number 2010/122]. References Ahmet I, Tae HJ, Juhaszova M, Riordon DR, Boheler KR, Sollott SJ, et al. A small nonerythropoietic helix B surface peptide based upon erythropoietin structure is cardioprotective against ischemic myocardial damage. Mol Med 2011;17: 194–200. Akimoto T, Kusano E, Inaba T, Iimura O, Takahashi H, Ikeda H, et al. Erythropoietin regulates vascular smooth muscle cell apoptosis by a phosphatidylinositol 3 kinase-dependent pathway. Kidney Int 2000;58:269–82.
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11 10
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
Ammarguellat F, Llovera M, Kelly PA, Goffin V. Low doses of EPO activate MAP kinases but not JAK2-STAT5 in rat vascular smooth muscle cells. Biochem Biophys Res Commun 2001;284: 1031–8. Anagnostou A, Liu Z, Steiner M, Chin K, Lee ES, Kessimian N, et al. Erythropoietin receptor mRNA expression in human endothelial cells. Proc Natl Acad Sci USA 1994;91:3974–8. Azevedo Filho CF, Hadlich M, Petriz JL, Mendonc¸a LA, Moll Filho JN, Rochitte CE. Quantification of left ventricular infarcted mass on cardiac magnetic resonance imaging, Comparison between planimetry and the semiquantitative visual scoring Method. Arq Bras Cardiol 2004;83:118–24, 111–117. Ben-Dor I, Hardy B, Fuchs S, Kaganovsky E, Kadmon E, Sagie A, et al. Repeated low-dose of erythropoietin is associated with improved left ventricular function in rat acute myocardial infarction model. Cardiovasc Drugs Ther 2007;21: 339–46. Brines M, Cerami A. Discovering erythropoietin’s extrahematopoietic functions: biology and clinical promise. Kidney Int 2006;70:246–50. Brines M, Grasso G, Fiordaliso F, Sfacteria A, Ghezzi P, Fratelli M, et al. Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor. Proc Natl Acad Sci USA 2004;101:14907–12. Brunner S, Huber BC, Weinberger T, Vallaster M, Wollenweber T, Gerbitz A, et al. Migration of bone marrow-derived cells and improved perfusion after treatment with erythropoietin in a murine model of myocardial infarction. J Cell Mol Med 2012;16:152–9. Bullard AJ, Govewalla P, Yellon DM. Erythropoietin protects the myocardium against reperfusion injury in vitro and in vivo. Basic Res Cardiol 2005;100:397–403. Cai Z, Manalo DJ, Wei G, Rodriguez ER, Fox-Talbot K, Lu H, et al. Hearts from rodents exposed to intermittent hypoxia or erythropoietin are protected against ischemia-reperfusion injury. Circulation 2003;108:79–85. Cai Z, Semenza GL. Phosphatidylinositol-3-kinase signaling is required for erythropoietin-mediated acute protection against myocardial ischemia/reperfusion injury. Circulation 2004;109:2050–3. Calvillo L, Latini R, Kajstura J, Leri A, Anversa P, Ghezzi P, et al. Recombinant human erythropoietin protects the myocardium from ischemia-reperfusion injury and promotes beneficial remodeling. Proc Natl Acad Sci USA 2003;100: 4802–6. Chong ZZ, Kang JQ, Maiese K. Hematopoietic factor erythropoietin fosters neuroprotection through novel signal transduction cascades. J Cereb Blood Flow Metab 2002;22:503–14. Digicaylioglu M, Bichet S, Marti HH, Wenger RH, Rivas LA, Bauer C, et al. Localization of specific erythropoietin binding sites in defined areas of the mouse brain. Proc Natl Acad Sci USA 1995;92:3717–20. Digicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling cascades. Nature 2001;412:641–7. Fiordaliso F, Chimenti S, Staszewsky L, Bai A, Carlo E, Cuccovillo I, et al. A nonerythropoietic derivative of erythropoietin protects the myocardium from ischemia-reperfusion injury. Proc Natl Acad Sci USA 2005;102:2046–51. Fisher JW, Koury S, Ducey T, Mendel S. Erythropoietin production by interstitial cells of hypoxic monkey kidneys. Br J Haematol 1996;95:27–32. Guneli E, Cavdar Z, Islekel H, Sarioglu S, Erbayraktar S, Kiray M, et al. Erythropoietin protects the intestine against ischemia/reperfusion injury in rats. Mol Med 2007;13: 509–17.
Hanlon PR, Fu P, Wright GL, Steenbergen C, Arcasoy MO, Murphy E. Mechanisms of erythropoietin-mediated cardioprotection during ischemia-reperfusion injury: role of protein kinase C and phosphatidylinositol 3-kinase signaling. FASEB J 2005;19:1323–5. Jelkmann W, Wagner K. Beneficial and ominous aspects of the pleiotropic action of erythropoietin. Ann Hematol 2004;83:673–86. Jelkmann W. Molecular biology of erythropoietin. Intern Med 2004;43:649–59. Junk AK, Mammis A, Savitz SI, Singh M, Roth S, Malhotra S, et al. Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury. Proc Natl Acad Sci USA 2002;99:10659–64. Lipsic E, Schoemaker RG, van der Meer P, Voors AA, van Veldhuisen DJ, van Gilst WH. Protective effects of erythropoietin in cardiac ischemia: from bench to bedside. J Am Coll Cardiol 2006;48:2161–7. Lipsic E, van der Meer P, Henning RH, Suurmeijer AJ, Boddeus KM, van Veldhuisen DJ, et al. Timing of erythropoietin treatment for cardioprotection in ischemia/reperfusion. J Cardiovasc Pharmacol 2004;44:473–9. Marti HH. Erythropoietin and the hypoxic brain. J Exp Biol 2004;207:3233–42. Marzo F, Lavorgna A, Coluzzi G, Santucci E, Tarantino F, Rio T, et al. Erythropoietin in heart and vessels: focus on transcription and signalling pathways. J Thromb Thrombolysis 2008;26:183–7. Moon C, Krawczyk M, Ahn D, Ahmet I, Paik D, Lakatta EG, et al. Erythropoietin reduces myocardial infarction and left ventricular functional decline after coronary artery ligation in rats. Proc Natl Acad Sci USA 2003;100:11612–7. Mudalagiri NR, Mocanu MM, Di Salvo C, Kolvekar S, Hayward M, Yap J, et al. Erythropoietin protects the human myocardium against hypoxia/reoxygenation injury via phosphatidylinositol-3 kinase and ERK1/2 activation. Br J Pharmacol 2008;153:50–6. Nakano M, Satoh K, Fukumoto Y, Ito Y, Kagaya Y, Ishii N, et al. Important role of erythropoietin receptor to promote VEGF expression and angiogenesis in peripheral ischemia in mice. Circ Res 2007;100:662–9. Nangaku M, Eckardt KU. Hypoxia and the HIF system in kidney disease. J Mol Med 2007;85:1325–30. Ogilvie M, Yu X, Nicolas-Metral V, Pulido SM, Liu C, Ruegg UT, et al. Erythropoietin stimulates proliferation and interferes with differentiation of myoblasts. J Biol Chem 2000;275:39754–61. Parsa CJ, Matsumoto A, Kim J, Riel RU, Pascal LS, Walton GB, et al. A novel protective effect of erythropoietin in the infarcted heart. J Clin Invest 2003;112:999–1007. Paschos N, Lykissas MG, Beris AE. The role of erythropoietin as an inhibitor of tissue ischemia. Int J Biol Sci 2008;4: 161–8. Rui T, Feng Q, Lei M, Peng T, Zhang J, Xu M, et al. Erythropoietin prevents the acute myocardial inflammatory response induced by ischemia/reperfusion via induction of AP-1. Cardiovasc Res 2005;65:719–27. Sasaki R, Masuda S, Nagao M. Erythropoietin: multiple physiological functions and regulation of biosynthesis. Biosci Biotechnol Biochem 2000;64:1775–93. Sharples EJ, Patel N, Brown P, Stewart K, Mota-Philipe H, Sheaff M, et al. Erythropoietin protects the kidney against the injury and dysfunction caused by ischemia-reperfusion. J Am Soc Nephrol 2004;15:2115–24. Solaroglu A, Dede FS, Okutan E, Bayrak A, Haberal A, Kilinc K. A single dose of erythropoietin attenuates lipid peroxidation in experimental liver ischemia-reperfusion injury in the rat fetus. J Matern Fetal Neonatal Med 2004;16: 231–4.
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005
G Model ACTHIS-50681; No. of Pages 11
ARTICLE IN PRESS A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx–xxx
Tramontano AF, Muniyappa R, Black AD, Blendea MC, Cohen I, Deng L, et al. Erythropoietin protects cardiac myocytes from hypoxia-induced apoptosis through an Akt-dependent pathway. Biochem Biophys Res Commun 2003;308:990–4. Ueba H, Brines M, Yamin M, Umemoto T, Ako J, Momomura S, et al. Cardioprotection by a nonerythropoietic, tissue-protective peptide mimicking the 3D structure of erythropoietin. Proc Natl Acad Sci USA 2010;107:14357–62. Vivaldi MT, Kloner RA, Schoen FJ. Triphenyltetrazolium staining of irreversible ischemic injury following coronary artery occlusion in rats. Am J Pathol 1985;121:522–30.
11
Weidemann A, Johnson RS. Biology of HIF-1 alpha. Cell Death Differ 2008;15:621–7. Wright GL, Hanlon P, Amin K, Steenbergen C, Murphy E, Arcasoy MO. Erythropoietin receptor expression in adult rat cardiomyocytes is associated with an acute cardioprotective effect for recombinant erythropoietin during ischemia-reperfusion injury. FASEB J 2004;18:1031–3. Wu H, Ren B, Zhu J, Dong G, Xu B, Wang C, et al. Pretreatment with recombined human erythropoietin attenuates ischemiareperfusion-induced lung injury in rats. Eur J Cardiothorac Surg 2006;29:902–7.
Please cite this article in press as: Guven Bagla A, et al. Experimental acute myocardial infarction in rats: HIF-1␣, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin doses. Acta Histochemica (2013), http://dx.doi.org/10.1016/j.acthis.2013.01.005