Bone Marrow-Derived CXCR4+ Cells Mobilized by Macrophage Colony-Stimulating Factor Participate in the Reduction of Infarct Area and Improvement of Cardiac Remodeling after Myocardial Infarction in Mice
2007; Elsevier BV; Volume: 171; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2007.061276
ISSN1525-2191
AutoresHajime Morimoto, Masafumi Takahashi, Yuji Shiba, Atsushi Izawa, Hirohiko Ise, Minoru Hongo, Kiyohiko Hatake, Kazuo Motoyoshi, Uichi Ikeda,
Tópico(s)Tissue Engineering and Regenerative Medicine
ResumoThe monocyte/macrophage lineage might affect the healing process after myocardial infarction (MI). Because macrophage colony-stimulating factor (M-CSF) stimulates differentiation and proliferation of this lineage, we examined the effect of M-CSF treatment on infarct size and left ventricular (LV) remodeling after MI. MI was induced in C57BL/6J mice by ligation of the left coronary artery. Either recombinant human M-CSF or saline was administered for 5 consecutive days after MI induction. M-CSF treatment significantly reduced the infarct size (P < 0.05) and scar formation (P < 0.05) and improved the LV dysfunction (percent fractional shortening, P < 0.001) after the MI. Immunohistochemistry revealed that M-CSF increased macrophage infiltration (F4/80) and neovascularization (CD31) of the infarct myocardium but did not increase myofibroblast accumulation (α-smooth muscle actin). M-CSF mobilized CXCR4+ cells into peripheral circulation, and the mobilized CXCR4+ cells were then recruited into the infarct area in which SDF-1 showed marked expression. The CXCR4 antagonist AMD3100 deteriorated the infarction and LV function after the MI in the M-CSF-treated mice. In conclusion, M-CSF reduced infarct area and improved LV remodeling after MI through the recruitment of CXCR4+ cells into the infarct myocardium by the SDF-1-CXCR4 axis activation; this suggests that the SDF-1-CXCR4 axis is as a potential target for the treatment of MI. The monocyte/macrophage lineage might affect the healing process after myocardial infarction (MI). Because macrophage colony-stimulating factor (M-CSF) stimulates differentiation and proliferation of this lineage, we examined the effect of M-CSF treatment on infarct size and left ventricular (LV) remodeling after MI. MI was induced in C57BL/6J mice by ligation of the left coronary artery. Either recombinant human M-CSF or saline was administered for 5 consecutive days after MI induction. M-CSF treatment significantly reduced the infarct size (P < 0.05) and scar formation (P < 0.05) and improved the LV dysfunction (percent fractional shortening, P < 0.001) after the MI. Immunohistochemistry revealed that M-CSF increased macrophage infiltration (F4/80) and neovascularization (CD31) of the infarct myocardium but did not increase myofibroblast accumulation (α-smooth muscle actin). M-CSF mobilized CXCR4+ cells into peripheral circulation, and the mobilized CXCR4+ cells were then recruited into the infarct area in which SDF-1 showed marked expression. The CXCR4 antagonist AMD3100 deteriorated the infarction and LV function after the MI in the M-CSF-treated mice. In conclusion, M-CSF reduced infarct area and improved LV remodeling after MI through the recruitment of CXCR4+ cells into the infarct myocardium by the SDF-1-CXCR4 axis activation; this suggests that the SDF-1-CXCR4 axis is as a potential target for the treatment of MI. Myocardial infarction (MI) is accompanied by inflammatory responses that lead to the recruitment of leukocytes and subsequent myocardial damage, healing, and scar formation. The recruitment and activation of monocytes/macrophages in the infarct myocardium have been shown to play an important role in the processes that occur after MI. The activated macrophages lead to the release of cytokines and proteinases that can induce further inflammation and left ventricular (LV) remodeling. Furthermore, recent evidence indicates that some endothelial progenitor cells (EPCs) are derived from cells of the monocytic lineage and participate in the neovascularization of ischemic tissues.1Schmeisser A Garlichs CD Zhang H Eskafi S Graffy C Ludwig J Strasser RH Daniel WG Monocytes coexpress endothelial and macrophagocytic lineage markers and form cord-like structures in Matrigel under angiogenic conditions.Cardiovasc Res. 2001; 49: 671-680Crossref PubMed Scopus (371) Google Scholar, 2Harraz M Jiao C Hanlon HD Hartley RS Schatteman GC CD34-blood-derived human endothelial cell progenitors.Stem Cells. 2001; 19: 304-312Crossref PubMed Scopus (282) Google Scholar, 3Rehman J Li J Orschell CM March KL Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors.Circulation. 2003; 107: 1164-1169Crossref PubMed Scopus (1492) Google Scholar These cells have also been reported to secrete a large amount of angiogenic factors such as the vascular endothelial growth factor and the hepatocyte growth factor3Rehman J Li J Orschell CM March KL Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors.Circulation. 2003; 107: 1164-1169Crossref PubMed Scopus (1492) Google Scholar; this suggests that monocytes/macrophages could influence LV dysfunction and remodeling after MI. The macrophage colony-stimulating factor (M-CSF) is a multifunctional proinflammatory cytokine that regulates the differentiation, proliferation, and survival of monocytic progenitor cells4Motoyoshi K Biological activities and clinical application of M-CSF.Int J Hematol. 1998; 67: 109-122Crossref PubMed Google Scholar and plays a role in various processes involved in inflammatory diseases. Furthermore, it has been recently demonstrated that M-CSF is expressed by nonhematopoietic cells such as endothelial cells, smooth muscle cells, and cardiomyocytes and that its function extends beyond its role in monocytic progenitor cells. A recent investigation demonstrated that M-CSF is expressed in the infarct heart and plays an important role in myocardial healing postinfarction.5Frangogiannis NG Mendoza LH Ren G Akrivakis S Jackson PL Michael LH Smith CW Entman ML MCSF expression is induced in healing myocardial infarcts and may regulate monocyte and endothelial cell phenotype.Am J Physiol. 2003; 285: H483-H492Crossref PubMed Scopus (92) Google Scholar However, the effect of M-CSF on cardiac dysfunction and remodeling after MI is not entirely understood. In the present study, we demonstrate that exogenous M-CSF treatment induces macrophage infiltration and capillary formation in the infarct myocardium, thereby improving LV dysfunction and remodeling. This process is mediated through a system that comprises a key stem cell homing factor, the stromal cell-derived factor (SDF-1/CXCL12), and the SDF-1 receptor CXCR4. The findings of this study may provide new insights into the role of M-CSF and the SDF-1-CXCR4 axis in the pathophysiology of MI. All mice (C57BL/6J, male) were purchased from Japan SLC Inc. (Hamamatsu, Japan). Their ages ranged from 8 to 12 weeks. Mice were fed a standard diet and water and maintained on a 12-hour light/dark cycle. All of the experiments in this study were performed in accordance with the Shinshu University Guide for Laboratory Animals, which conforms to the National Institutes of Health Guidelines. In the preliminary experiments, we examined the effect of M-CSF (5, 50, and 500 μg/kg, i.p.) on the number of peripheral monocytes in C57BL/6J mice. We found that the administration of 500 μg/kg of recombinant human M-CSF (kindly provided by Morinaga Milk Industry Co. Ltd., Kanagawa, Japan) significantly increased the number of peripheral monocytes; this finding was consistent with a previous report.6Misawa E Sakurai T Yamada M Tamura Y Motoyoshi K Administration of macrophage colony-stimulating factor mobilized both CD11b+CD11c+ cells and NK1.1+ cells into peripheral blood.Int Immunopharmacol. 2004; 4: 791-803Crossref PubMed Scopus (6) Google Scholar Therefore, in the present study, we used this M-CSF at a dose of 500 μg/kg per day. We also used recombinant human granulocyte colony-stimulating factor (G-CSF; kindly provided by Chugai Pharmaceutical, Co. Ltd., Tokyo, Japan) at a dose of 100 μg/kg per day. The mice were anesthetized with an intraperitoneal injection of 50 mg/kg pentobarbital sodium and splenectomized, not only to eliminate spleen-derived stem cells (eg, EPCs and CXCR4+ cells) but also to prevent the homing of bone marrow-derived stem cells to the spleen.7Werner N Junk S Laufs U Link A Walenta K Bohm M Nickenig G Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury.Circ Res. 2003; 93: e17-e24Crossref PubMed Google Scholar The animals were allowed to recover for 14 days, after which MI was induced. Either recombinant human M-CSF (i.p., n = 60), G-CSF (s.c., n = 12), or saline (i.p., vehicle, n = 53) was administered to the splenectomized mice for 5 consecutive days after MI induction. The M-CSF treatment was well tolerated, and no abnormal behavior was observed. AMD3100 (Sigma, St. Louis, MO) was administered subcutaneously at a concentration of 300 μg/kg per hour for 7 days after the MI by using a micro-osmotic pump (Alzet model 1007D; Durect Corporation, Cupertino, CA). A murine model of MI was constructed as described previously.8Morimoto H Takahashi M Izawa A Ise H Hongo M Kolattukudy PE Ikeda U Cardiac overexpression of monocyte chemoattractant protein-1 in transgenic mice prevents cardiac dysfunction and remodeling after myocardial infarction.Circ Res. 2006; 99: 891-899Crossref PubMed Scopus (118) Google Scholar Intubation was performed after anesthetizing the mice with isoflurane. The mice were ventilated with a rodent ventilator (MiniVent Type 845; Harvard Apparatus, Holliston, MA). A left thoracotomy was performed through the fourth or fifth intercostal space. An 8-0 nylon suture was placed directly beneath the left atrium in the interventricular groove. Successful coronary occlusion was verified by observing the development of a pale color in the distal myocardium after the ligation of the left coronary artery. The lungs were re-expanded using positive pressure at end expiration, and the thoracotomy and skin incision were closed with a 3-0 silk suture. Extubation was performed when spontaneous respiration resumed. Histological and immunohistochemical analyses were performed as described previously.9Yoshioka T Takahashi M Shiba Y Suzuki C Morimoto H Izawa A Ise H Ikeda U Granulocyte colony-stimulating factor (G-CSF) accelerates reendothelialization and reduces neointimal formation after vascular injury in mice.Cardiovasc Res. 2006; 70: 61-69Crossref PubMed Scopus (90) Google Scholar In brief, the mice were euthanized after irrigation with saline (Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan), and their blood was completely washed out. The hearts were embedded in optimal cutting temperature compound (Tissue-Tek, Sakura Finetechnical Co. Ltd., Tokyo, Japan) and frozen on dry ice. They were then sectioned transversely from the apex to the site of ligation beneath the left atrium. Tissue sections (10-μm thick) were cut on a cryostat (CM-1900; Leica Microsystems GmbH, Wetzlar, Germany) and histologically examined. Four sections were selected from each heart to perform morphometrical assessments of the LV myocardium and the infarct size. The sections were stained with hematoxylin-eosin (H&E) and Masson's trichrome. Measurements were performed using Scion Image software (Beta 4.03; Scion Corporation, Frederick, MD). The infarct size was calculated as a percentage of the total LV area. The extent of fibrosis in the sections was measured, and the value was expressed as the ratio of the Masson's trichrome-stained area to the total LV free wall area. For immunohistochemical analysis, the heart sections were incubated with primary antibodies against M-CSF (kindly provided by Morinaga Milk Industry Co. Ltd.), mouse CD31 (clone MEC13.3, BD Bioscience, San Jose, CA), F4/80 (clone A3-1; RDI, Flanders, NJ), α-smooth muscle actin (α-SMA; clone 1A4; Sigma), and CD184 (CXCR4, clone 2B11; BD Biosciences). This was followed by incubation with biotin-conjugated secondary antibodies. The sections were washed and treated with avidin peroxidase (ABC kit; Vector Laboratories, Burlingame, CA), and the stain was developed using the 3,3′-diaminobenzidine (DAB) substrate kit (Vector Laboratories). The sections were then counterstained with hematoxylin. Mouse on mouse (M.O.M.) basic kits (Vector Laboratories) were used to specifically localize mouse primary monoclonal antibodies in the tissues. No signal was detected when an irrelevant IgG was used as the negative control instead of the primary antibody. All of the measurements were performed in a double-blind manner by two independent researchers. Primary cultures of murine neonatal cardiomyocytes and cardiac fibroblasts were prepared and cultured as described previously.8Morimoto H Takahashi M Izawa A Ise H Hongo M Kolattukudy PE Ikeda U Cardiac overexpression of monocyte chemoattractant protein-1 in transgenic mice prevents cardiac dysfunction and remodeling after myocardial infarction.Circ Res. 2006; 99: 891-899Crossref PubMed Scopus (118) Google Scholar For hypoxia-reoxygenation experiments, cells were exposed to hypoxia induced with AnaeroPack (Mitsubishi Gas Chemical, Tokyo, Japan) for 6 hours, followed by reoxygenation for 18 hours. Blood samples were collected from the mice at baseline, 7 days, and 14 days after the MI. Circulating cells were identified using a nucleated cell fraction. The nucleated cells were double-labeled with the following antibodies: peridinin chlorophyll protein-conjugated anti-CD11b (Mac-1, clone M1/70; BD Biosciences), fluorescein isothiocyanate-conjugated anti-Ly-6G (Gr-1, clone 1A8), fluorescein isothiocyanate-conjugated anti-CD34 (clone RAM34; BD Biosciences), phycoerythrin-conjugated anti-Flk-1 (VEGFR2/KDR, clone Avas 12a1; BD Biosciences), and phycoerythrin-conjugated anti-CXCR4 (clone 2B11; BD Biosciences). For staining with antibody against α-SMA, cells were permeabilized with Cytofix/Cytoperm (BD Biosciences) according to the manufacturer's instructions. The cells were examined by flow cytometry (FACSCalibur; Becton Dickinson) and analyzed using CellQUEST software version 3.3 (Becton Dickinson). The serum levels of monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-6, IL-10, IL-12p70, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were assessed using the CBA Mouse Inflammation Kit (BD Biosciences) according to the manufacturer's instructions. Transthoracic echocardiography was performed at baseline, 7 days, and 14 days after the MI by using a Vivid Five system (GE Yokogawa Medical Systems, Tokyo, Japan) as described previously.10Kamiyoshi Y Takahashi M Yokoseki O Yazaki Y Hirose S Morimoto H Watanabe N Kinoshita O Hongo M Ikeda U Mycophenolate mofetil prevents the development of experimental autoimmune myocarditis.J Mol Cell Cardiol. 2005; 39: 467-477Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Ketamine (50 mg/kg) and xylazine (10 mg/kg) were administered intraperitoneally for mild sedation. Two-dimensional targeted M-mode echocardiograms were obtained along the short axis of the left ventricle at the level of the papillary muscles, and at least three consecutive beats were evaluated. The phases in which the smallest and largest area of the left ventricle were obtained were defined as the left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD), respectively. The percentage fractional shortening (%FS) was calculated using the standard formula: %FS = [(LVEDD − LVESD)/LVEDD] × 100. All measurements were performed in a double-blind manner by two independent researchers. Total cellular RNA was extracted using ISOGEN (Nippon Gene Co. Ltd., Toyama, Japan) or RNA-Bee (Tel-Test, Inc., Friendswood, TX), according to the manufacturer's instructions. Real-time reverse transciption-polymerase chain reaction (RT-PCR) analysis was performed by using the ABI Prism 7000 system (Applied Biosystems, Inc.) to detect the mRNA expression of c-fms, G-CSF receptor (G-CSFR), collagen type I and type III, transforming growth factor-β1 (TGF-β1), and β-actin.11Jia L Takahashi M Morimoto H Takahashi S Izawa A Ise H Iwasaki T Hattori H Wu KJ Ikeda U Changes in cardiac lipid metabolism during sepsis: the essential role of very low-density lipoprotein receptors.Cardiovasc Res. 2006; 69: 545-555Crossref PubMed Scopus (23) Google Scholar The following primers (oligonucleotide sequences are provided in parentheses in the order of antisense and sense primers) were used: c-fms (5′-CATGGCCTTCCTTGCTTCTAA-3′ and 5′-ACATGTCCGCTGGTCAACAG-3′), G-CSFR (5′-GTCCAGCGAGTCCCCAAAG-3′ and 5′-AGCATGGGAGGCTCCAATT-3′), collagen type I (5′-CGGAGAAGAAGGAAAACGAGGAG-3′ and 5′-CACCATCAGCACCAGGGAAAC-3′), collagen type III (5′-CCCAA CCCAGAGATCCCATT-3′ and 5′-GAAGCACAGGAGCA GGTGTAGA-3′), TGF-β1 (5′-GCAACATGTGGAACTCTACCAGA-3′ and 5′-GACGTCAAAAGACAGCCACTCA-3′), and β-actin (5′-CCTGAGCGCAAGTACTCTGTGT-3′ and 5′-GCTGATCCACATCTGCTGGAA-3′). The expression levels of each target gene were normalized by subtracting the corresponding β-actin threshold cycle (CT) values; this was done by using the ΔΔCT comparative method. Apoptotic cells were identified by the terminal deoxynucleotidyl transferase dUTP nick-end labeling staining kit (Roche Diagnostics, Mannheim, Germany) performed according to the manufacturer's instructions. Data are represented as mean ± SEM. Multiple group comparison was performed by one-way analysis of variance (analysis of variance), followed by Scheffé's F-test for comparison of the means. The comparison between two groups was analyzed by an F-test, followed by a two-tailed t-test. Values of P < 0.05 were considered statistically significant. First, we examined whether the expression of M-CSF was up-regulated in the infarct area after the permanent MI. Although no M-CSF expression was visualized in the infarct myocardium and vasculatures at baseline, immunohistochemical analysis revealed that the M-CSF expression was clearly up-regulated in these regions 6 to 24 hours after the MI, and it declined 3 to 7 days after the MI (Figure 1A). To identify the cell types that express M-CSF, double-immunohistological staining using antibodies against M-CSF and the cardiac-specific marker cTnI or the endothelial marker CD31 was performed. We detected the coexpression of M-CSF and cTnI, but not that of CD31 (Figure 1B). By using RT-PCR, we further examined whether cardiomyocytes express M-CSF mRNA and observed the expression of M-CSF mRNA in the murine heart and cultured cardiomyocytes (Figure 1C). The J774 cell line was used as a positive control for M-CSF expression. These findings suggest that M-CSF plays a role in the processes involved in MI. Next, we examined the effect of exogenous M-CSF treatment on the infarct area and scar formation after the MI. As shown in Figure 2, A and B, the infarct area at 14 days after MI in the M-CSF-treated mice decreased significantly compared with that in the vehicle-treated mice (P < 0.01). Further, Masson's trichrome staining demonstrated that scar formation was significantly reduced in the former compared with that in the latter (P < 0.05) (Figure 2, C and D). In addition, because the G-CSF has been shown to improve LV dysfunction and remodeling after MI,12Harada M Qin Y Takano H Minamino T Zou Y Toko H Ohtsuka M Matsuura K Sano M Nishi J Iwanaga K Akazawa H Kunieda T Zhu W Hasegawa H Kunisada K Nagai T Nakaya H Yamauchi-Takihara K Komuro I G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes.Nat Med. 2005; 11: 305-311Crossref PubMed Scopus (503) Google Scholar, 13Sugano Y Anzai T Yoshikawa T Maekawa Y Kohno T Mahara K Naito K Ogawa S Granulocyte colony-stimulating factor attenuates early ventricular expansion after experimental myocardial infarction.Cardiovasc Res. 2005; 65: 446-456Crossref PubMed Scopus (92) Google Scholar we tested the effect of G-CSF and confirmed that G-CSF treatment significantly reduced the infarct area and scar formation after the MI (Figure 2). We also assessed the infarct area at the early stage after MI and showed that M-CSF treatment appeared to decrease the infarct area on days 3 and 7 after the MI (Supplemental Figure 1 at ). Further, we assessed the LV function after the MI by using echocardiography. No significant difference was observed in the %FS between the vehicle- and M-CSF-treated mice at baseline. In the vehicle-treated mice, a marked decrease in the %FS was observed 7 days after MI, and this decrease was sustained for 28 days. In contrast, the %FS was maintained in the M-CSF-treated mice after the MI (Table 1; 14 days, vehicle: 25.8 ± 1.0% versus M-CSF: 33.5 ± 0.8%, P < 0.001). As expected, the %FS was also maintained in the G-CSF-treated mice after the MI (14 days, G-CSF: 34.4 ± 0.5%, P < 0.001 versus vehicle).Table 1Echocardiographic Findings in Vehicle and M-CSF-Treated Mice after MI InductionBaselineDay 7Day 14Day 21Day 28Vehicle n24242366 B.W. (g)25.1 ± 0.324.8 ± 0.526.0 ± 0.428.3 ± 0.2**P < 0.01 versus baseline.28.5 ± 0.3**P < 0.01 versus baseline. B.W. (%)10098.8 ± 0.9102.5 ± 0.9107.6 ± 2.0**P < 0.01 versus baseline.108.3 ± 2.0**P < 0.01 versus baseline. IVSd (mm)0.74 ± 0.020.67 ± 0.030.65 ± 0.030.60 ± 0.020.55 ± 0.05*P < 0.05 and LVDd (mm)4.04 ± 0.114.45 ± 0.104.92 ± 0.12**P < 0.01 versus baseline.5.13 ± 0.17**P < 0.01 versus baseline.5.22 ± 0.21**P < 0.01 versus baseline. LVPWd (mm)0.82 ± 0.030.90 ± 0.050.85 ± 0.040.71 ± 0.020.73 ± 0.04 IVSs (mm)1.36 ± 0.031.12 ± 0.04**P < 0.01 versus baseline.1.04 ± 0.04**P < 0.01 versus baseline.1.02 ± 0.03**P < 0.01 versus baseline.0.99 ± 0.04**P < 0.01 versus baseline. LVDs (mm)2.24 ± 0.073.20 ± 0.08**P < 0.01 versus baseline.3.66 ± 0.12**P < 0.01 versus baseline.3.71 ± 0.14**P < 0.01 versus baseline.3.83 ± 0.18**P < 0.01 versus baseline. LVPWs (mm)1.44 ± 0.021.27 ± 0.05**P < 0.01 versus baseline.1.23 ± 0.03**P < 0.01 versus baseline.1.07 ± 0.03**P < 0.01 versus baseline.1.08 ± 0.02**P < 0.01 versus baseline. FS (%)44.9 ± 0.728.2 ± 0.9**P < 0.01 versus baseline.25.8 ± 1.0**P < 0.01 versus baseline.27.6 ± 0.7**P < 0.01 versus baseline.26.7 ± 1.0**P < 0.01 versus baseline. FS (% of baseline)10063.2 ± 2.1**P < 0.01 versus baseline.58.8 ± 2.3**P < 0.01 versus baseline.64.2 ± 1.7**P < 0.01 versus baseline.61.9 ± 1.9**P < 0.01 versus baseline.M-CSF n25252355 B.W. (g)24.8 ± 0.324.2 ± 0.625.2 ± 0.427.8 ± 0.628.3 ± 0.7*P < 0.05 and B.W. (%)10097.4 ± 1.9101.3 ± 0.9105.2 ± 1.2107.0 ± 1.2 IVSd (mm)0.71 ± 0.020.68 ± 0.020.67 ± 0.020.64 ± 0.020.63 ± 0.04 LVDd (mm)4.10 ± 0.064.52 ± 0.06**P < 0.01 versus baseline.4.78 ± 0.11**P < 0.01 versus baseline.4.97 ± 0.26**P < 0.01 versus baseline.5.14 ± 0.19**P < 0.01 versus baseline. LVPWd (mm)0.80 ± 0.020.79 ± 0.030.80 ± 0.020.72 ± 0.050.70 ± 0.04 IVSs (mm)1.29 ± 0.021.18 ± 0.031.19 ± 0.03P< 0.01, and1.12 ± 0.051.13 ± 0.09 LVDs (mm)2.32 ± 0.053.03 ± 0.06**P < 0.01 versus baseline.3.18 ± 0.09**P < 0.01 versus baseline.P< 0.01, and3.22 ± 0.18**P < 0.01 versus baseline.3.44 ± 0.14**P < 0.01 versus baseline. LVPWs (mm)1.42 ± 0.021.28 ± 0.04*P < 0.05 and1.36 ± 0.03P< 0.01, and1.24 ± 0.04P< 0.01, and1.17 ± 0.05*P < 0.05 and FS (%)43.6 ± 0.632.9 ± 0.8**P < 0.01 versus baseline.†P < 0.001 versus vehicle.33.5 ± 0.8**P < 0.01 versus baseline.†P < 0.001 versus vehicle.35.2 ± 0.8**P < 0.01 versus baseline.†P < 0.001 versus vehicle.32.9 ± 1.4**P < 0.01 versus baseline.P< 0.01, and FS (% of baseline)10075.9 ± 2.1**P < 0.01 versus baseline.†P < 0.001 versus vehicle.76.7 ± 2.3**P < 0.01 versus baseline.†P < 0.001 versus vehicle.83.1 ± 1.8**P < 0.01 versus baseline.†P < 0.001 versus vehicle.77.7 ± 3.4**P < 0.01 versus baseline.P< 0.01, andG-CSF n121010 B.W. (g)24.9 ± 0.324.0 ± 0.324.8 ± 0.3†P < 0.05, B.W. (%)10096.3 ± 1.4**P < 0.01 versus baseline.100.3 ± 0.5 IVSd (mm)0.71 ± 0.040.77 ± 0.02†P < 0.05,0.72 ± 0.04 LVDd (mm)4.06 ± 0.094.68 ± 0.23*P < 0.05 and5.04 ± 0.15**P < 0.01 versus baseline. LVPWd (mm)0.81 ± 0.061.08 ± 0.120.98 ± 0.08 IVSs (mm)1.27 ± 0.041.31 ± 0.04P< 0.01, and1.37 ± 0.04†P < 0.001 versus vehicle. LVDs (mm)2.32 ± 0.063.02 ± 0.19**P < 0.01 versus baseline.3.31 ± 0.10**P < 0.01 versus baseline.†P < 0.01 versus baseline. LVPWs (mm)1.40 ± 0.041.59 ± 0.08P< 0.01, and1.57 ± 0.08P< 0.01, and FS (%)42.9 ± 0.535.9 ± 1.5**P < 0.01 versus baseline.†P < 0.001 versus vehicle.34.4 ± 0.5**P < 0.01 versus baseline.†P < 0.001 versus vehicle. FS (% of baseline)10083.4 ± 3.0**P < 0.01 versus baseline.†P < 0.001 versus vehicle.80.5 ± 1.8**P < 0.01 versus baseline.†P < 0.001 versus vehicle.Data represent the mean S.E.M. n, number: B.W., body weight; IVSd, interventricular septal diastolic thickness; LVDd, left ventricular diastolic dimension; LVPWd, left ventricular posterior wall idastolic thickness; IVSs, interventricular septal systolic thickness; LVDs, left ventricular systolic dimension; LVPWs, left ventricular posterior wall systolic thickness; FS, fractional shortening.* P < 0.05 and** P < 0.01 versus baseline.† P < 0.05,†† P< 0.01, and††† P < 0.001 versus vehicle. Open table in a new tab Data represent the mean S.E.M. n, number: B.W., body weight; IVSd, interventricular septal diastolic thickness; LVDd, left ventricular diastolic dimension; LVPWd, left ventricular posterior wall idastolic thickness; IVSs, interventricular septal systolic thickness; LVDs, left ventricular systolic dimension; LVPWs, left ventricular posterior wall systolic thickness; FS, fractional shortening. A previous investigation reported that G-CSF directly activates the Jak-Stat pathway via the G-CSFR in cardiomyocytes and exerts cardioprotective effects.12Harada M Qin Y Takano H Minamino T Zou Y Toko H Ohtsuka M Matsuura K Sano M Nishi J Iwanaga K Akazawa H Kunieda T Zhu W Hasegawa H Kunisada K Nagai T Nakaya H Yamauchi-Takihara K Komuro I G-CSF prevents cardiac remodeling after myocardial infarction by activating the Jak-Stat pathway in cardiomyocytes.Nat Med. 2005; 11: 305-311Crossref PubMed Scopus (503) Google Scholar Therefore, we examined the expression of the M-CSF receptor c-fms and G-CSFR in the myocardium after the MI. Real-time RT-PCR analysis revealed that c-fms expression was decreased by 43% at 6 hours after the MI, and this decrease was sustained for at least 7 days (Figure 3A). In contrast, G-CSFR expression was increased at 6 hours after the MI and reached a peak at 24 hours (approximately 22-fold expression; Figure 3B). Immunohistochemical staining showed the decreased expression of c-fms protein in the heart at 6 hours after the MI (Figure 3C). These results suggest that M-CSF might improve LV dysfunction and remodeling after MI via its effect on monocytes/macrophages rather than that on cardiomyocytes. To investigate the mechanism underlying the beneficial effect of M-CSF, we performed an immunohistochemical analysis to detect macrophages (F4/80) and endothelial cells (CD31). The number of infiltrated macrophages in the infarct area of the heart was greater in the M-CSF-treated mice than in the vehicle-treated mice after the MI (P < 0.001, Figure 4, A and B). The density of endothelial cells that was determined by CD31 expression increased significantly in the M-CSF-treated mice compared with that in the vehicle-treated mice (P < 0.01, Figure 4, C and D). These findings suggest that M-CSF treatment promotes macrophage infiltration and neovascularization in the infarct myocardium. Cardiac fibroblasts have been shown to differentiate into myofibroblasts during the process of myocardial repair and remodeling after MI.14Virag JI Murry CE Myofibroblast and endothelial cell proliferation during murine myocardial infarct repair.Am J Pathol. 2003; 163: 2433-2440Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar Because myofibroblasts are characterized by the presence of α-SMA, immunohistochemical analysis of α-SMA was performed. The number of α-SMA-positive myofibroblasts was notably increased at the infarct area compared with that at the noninfarct area (Figure 5, A and B). However, no change was observed in the number of myofibroblasts in the M-CSF-treated mice compared with that in the vehicle-treated mice (P = 0.245). To examine the effect of M-CSF on the differentiation into myofibroblasts in vitro, we used murine neonatal cardiac fibroblasts. M-CSF accelerated the in vitro differentiation of cardiac fibroblasts into myofibroblasts (Figure 5C). We performed flow cytometry analysis to quantify this differentiation. Interestingly, M-CSF clearly stimulated the in vitro differentiation of not only cardiac fibroblasts but also peripheral blood cells and bone marrow-derived cells (Figure 5, D and E). To assess the involvement of apoptosis after MI in the vehicle- and M-CSF-treated mice, terminal deoxynucleotidyl transferase dUTP nick-end labeling staining was performed; however, no differences were found in the number of apoptotic cells between the vehicle- and M-CSF-treated mice (Supplemental Figure 2A at ). To investigate further whether M-CSF confers an anti-apoptotic effect on cardiomyocytes, in vitro experiments using murine cultured cardiomyocytes were performed. We exposed cardiomyocytes to hypoxia-reoxygenation in the absence or presence of M-CSF and examined cardiomyocyte apoptosis. Treatment with M-CSF reduced the number of apoptotic cells induced by hypoxia-reoxygenation compared with cells that were not treated with M-CSF (Supplemental Figure 2B at ). To investigate whether cytokines and collagen synthesis are involved in reducing the infarct area and scar formation after MI, we determined the serum level of MCP-1, IL-6, IL-10, IL-12p70, IFN-γ, and TNF-α. Serum IL-6 and MCP-1 levels tended to increase 24 hours after the MI (Figure 6, A and B), whereas no increase was observed in the IL-12p70 level (Figure 6C). The other inflammatory cytokines were under detectable limits (<20 pg/ml, data not shown). Real-time RT-PCR analysis revealed that the mRNA expression of collagen type I and type III was clearly up-regulated in the infarct area of the vehicle- and M-CSF-treated mice 14 days after the MI, and no difference was observed in these expression levels between the two groups (Fig
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