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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 4, 2004 as doi:10.1096/fj.03-0637fje. |
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* Department of Cardiovascular Science and Medicine,
Department of Pharmacology, Chiba University Graduate School of Medicine, Chiba, Japan
2Correspondence: Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail: komuro-tky{at}umin.ac.jp
SPECIFIC AIMS
Pretreatment with a combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) has been reported to attenuate left ventricular (LV) remodeling and reduce mortality after acute myocardial infarction (MI). The aim of the present study is to clarify three points: 1) whether cytokine treatment started after MI is as effective as pretreatment; 2) whether combination treatment with G-CSF and SCF is more effective than treatment with G-CSF alone; and 3) how G-CSF prevents LV remodeling and dysfunction after MI.
PRINCIPAL FINDINGS
1. Cytokine treatment started after MI improve mortality and cardiac function
Mice were categorized into 5 groups: 1) administration of vehicle (control, n=22); 2) administration of G-CSF (100 µg/kg/day) and SCF (200 µg/kg/day) from 5 days before MI through 3 days after (pre-GS, n=16); 3) administration of G-CSF (100 µg/kg/day) and SCF (200 µg/kg/day) for 5 days after MI (post-GS, n=20); 4) administration of G-CSF (100 µg/kg/day) alone for 5 days after MI (post-G, n=20); 5) administration of SCF (200 µg/kg/day) alone for 5 days after MI (post-S, n=21). The first injection of vehicle, G-CSF, and SCF was given subcutaneously at 2 h after MI.
Survival rates of mice at 14 days after MI were significantly higher in 3 cytokine treatment groups with G-CSF (pre-GS, 63%; post-GS, 80% and post-G, 70%) than in control group (23%, P<0.05) and post-S group (43%, P<0.05) in Kaplan-Meier analysis. We examined hemodynamic parameters of survived mice at 2 wks after MI with microtip pressure transducer. Cardiac functions such as systolic LV pressure, LV end-diastolic pressure, dP/dt, and –dP/dt of all 3 treatment groups with G-CSF were better than those of control group and post-S groupafter MI. There was no significant difference in these parameters among 3 cytokine treatment groups with G-CSF.
2. All treatment groups with G-CSF reduce LV remodeling after MI
Histological analysis was performed by staining with hematoxylin-eosin and Azan-Mallory. Scar area was evaluated by tracing the blue area in Azan-Mallory staining. After MI, LV free wall was very thin and LV cavity was markedly expanded in control group and post-S group but LV wall thickness and LV cavity size were preserved normally in 3 treatment groups with G-CSF. Wall thickness was greater in 3 cytokine treatment groups with G-CSF (pre-GS, 0.52±0.14 mm; post-GS, 0.57±0.20 mm; post-G, 0.53±0.16 mm) than in the control group (0.23±0.07 mm) and post-S group (0.31±0.08 mm) (Fig. 1
). There was no significant difference in wall thickness among the 3 treatment groups with G-CSF.
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3. The number of capillaries is increased in the border area of all 3 treatment groups with G-CSF after MI
To examine the role of bone marrow (BM) cells in reduction of infarct size, BM had been replaced with that of transgenic mice expressing enhanced green fluorescent protein (GFP) before creating MI. Since it was difficult to discriminate GFP-expressing BM cells from other types of cells because of autofluorescence of MI hearts, we identified BM-derived cells by using anti-GFP antibody. Many GFP-positive cells were recognized in the infarct border area of all 3 cytokine treatment groups but not the control group (Fig. 2
A). As some GFP-positive cells were observed at capillary walls (Fig. 2B
), we next compared the number of vessels in the border area. Capillaries in the border area of infarcted heart were identified by staining endothelial cells with an antibody against platelet/endothelial cell adhesion molecule-1 (PECAM-1). The number of capillaries in the border area at 4 days after MI was much greater in all 3 treatment groups (pre-GS; 8.0±2.3/HPF, post-GS; 7.8± 1.7/HPF, and post-G; 6.2±1.9/HPF) than in the control group (2.0±0.82/HPF, P<0.05) (Fig. 2C
). There were few GFP-positive cardiomyocytes (<0.01%) in the border area as well as in the infarcted area and the remote area.
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4. The number of TUNEL-positive cells is decreased in the border area of all 3 cytokine treatment groups with G-CSF
To elucidate the mechanism of increased vessels, we measured the number of apoptotic cells by TUNEL assays. The number of TUNEL-positive cells in the border area of infarcted heart at 4 days was significantly smaller in the 3 treatment groups (pre-GS; 13.3±3.2/103 cells, post-GS; 10.8±1.5/103 cells and post-G; 12.8±1.0/103 cells, not significant among three groups) than in control group (30.7±3.7/103 cells, P<0.05) (Fig. 2D
).
CONCLUSIONS AND SIGNIFICANCE
Prevention of LV remodeling after acute MI is important because it causes heart failure and poor prognosis. After MI, many cardiomyocytes undergo cell death by necrosis and apoptosis in the infarcted area, which is then replaced by fibrous tissue. The infarcted area is gradually extended by subsequent death of cardiomyocytes and vascular cells in the border area and expanded by abnormal wall tension. Myocardial ischemia plays a critical role in cardiomyocyte death in the border area after MI and greatly affects LV remodeling. It has been reported that a subset of bone marrow stem cells (BMSCs) differentiate into cardiomyocytes when injected into peri-infarcted area, which results in regeneration of infarcted heart. Moreover, it has been reported that a pretreatment with G-CSF and SCF attenuates LV remodeling after MI. Although these results suggest that cytokine treatment is beneficial to prevent LV remodeling, cytokine treatment was started before MI and this protocol cannot be applied to humans. The molecular mechanism of how cytokine treatment repairs infarcted heart is not fully understood.
In the present study, 1) cytokine treatments started after MI were as effective as pretreatment; 2) there was no significant difference in parameters, including fibrotic area, cardiac function and survival rate between treatment with G-CSF alone and combination treatment with G-CSF and SCF; 3) more capillaries were observed in the border area of treatment groups with G-CSF compared with the control group and SCF group, and the number of apoptotic cells was smaller in treatment groups with G-CSF than in the control group. These results suggest that G-CSF treatment started after MI is as beneficial as pretreatment or combination treatment with G-CSF and SCF to prevent LV remodeling.
It has been reported that G-CSF induces mobilization of BMSCs from BM into peripheral blood circulation. There were more capillaries in the border area of all cytokine treatment groups with G-CSF than in the control group after MI, and a part of cells constituting the capillary wall were derived from BM cells. These results suggest that BMSCs were recruited from BM to infarcted heart by cytokine treatment and were involved in neovascularization. It remains to be determined which cells become vascular cells in infarcted heart. The number of TUNEL-positive cells in the border area of infarcted heart was significantly smaller in all treatment groups with G-CSF than in the control group. These data suggest that G-CSF treatment may induce neovascularization, which prevents cardiomyocytes from apoptotic death in the border area after MI. Reduction of cardiomyocyte death may result in less LV remodeling and improve mortality after MI (Fig. 3
). Further studies are necessary to determine which cells (e.g., hematopoietic stem cells, mesenchymal stem cells, and endothelial progenitor cells) become vascular cells and whether neovascularization is a major mechanism for G-CSF-induced prevention of LV remodeling after MI.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0637fje; 
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