J Cardiovasc Ultrasound.  2009 Jun;17(2):60-69. 10.4250/jcu.2009.17.2.60.

Effects of Granulocyte-Colony Stimulating Factor and Bone Marrow Mononuclear Cells on Cardiac Function and Remodeling in the Porcine Reperfused Myocardial Infarction Model

• Park MY
• Lim DS
• Choi SC
• Fang YH
• Kim JH
• Hong SJ
• Shin SH
• Ro YM
• Shim WJ

• KMID: 2135446
• DOI: http://doi.org/10.4250/jcu.2009.17.2.60

Abstract

BACKGROUND
Granulocyte stimulating factor (G-CSF) and bone marrow mononuclear cells (BM-MNC) were reported to improve cardiac function after myocardial infarction (MI). This study was to examine their combined beneficial effects and mechanisms of actions in reperfused MI, which have not been verified yet.
METHODS
Fifteen pigs were divided into 3 groups after a 1-hour balloon occlusion and reperfusion of the left anterior descending coronary artery. G1 (n=5) was a control, G2 (n=5) was a G-CSF injected group (10 ug/kg/day, from day1 to day7 after MI), and G3 (n=5) was an autologous intracoronary BM-MNC infused group after G-CSF treatment
RESULTS
Modified wall motion indices by echocardiography were similar among 3 groups at 24 hours after MI. However, they improved significantly in G2 and G3 at 35days after MI (p<0.05). The percentage of infarct area/left ventricular myocardial area measured from a triphenyltetrazolium chloride (TTC) stain was lower in G3 than in G1 or G2 (p=0.026). The number of vWF-positive vessels and the expressions of vWF and VE cardherin by RT-PCR were higher in G3 and G2 than in G1 (p<0.05). The number of TUNEL-positive cells and bcl2/bax ratio were not significantly different among 3 groups.
CONCLUSION
This study suggests that intracoronary BM-MNC infusion with G-CSF treatment in reperfused MI reduced infarct size, improved left ventricular function and prevented ventricular remodeling.

Keyword

Granulocyte-colony stimulating factor; Bone marrow mononuclear cell; Cardiac remodeling; Myocardial infarction; Reperfusion

MeSH Terms

Balloon Occlusion
Bone Marrow
Coronary Vessels
Echocardiography
Granulocyte Colony-Stimulating Factor
Granulocytes
Myocardial Infarction
Reperfusion
Swine
Tetrazolium Salts
Ventricular Function, Left
Ventricular Remodeling
Granulocyte Colony-Stimulating Factor
Tetrazolium Salts

Figure

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  Fig. 1 A: Study protocol. B: Analysis of regional wall motion. The anterior wall and interventricular septum at the mid-papillary level were divided into 5 segments for evaluation of regional wall motion. SC: subcutaneous injection, IC: intracoronary injection, PB: peripheral blood examination, BM: bone marrow blood examination, 2D: 2-dimensional echocardiography, LV: left ventricle, RV: right ventricle, AL: anterolateral papillary muscle, PM: posteromedial papillary muscle.

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  Fig. 2 Analysis of blood cells. A and C: The numbers of circulating white blood cells (WBC) and monocytes in the peripheral blood (PB) were counted preoperatively and at 1, 3, 5-7, 14, and 28 days after myocardial infarction (MI). B and D: The numbers of circulating white blood cells (WBC) and monocytes in the bone marrow (BM) were counted at pre-operatively and at 1, 3, 5-7, 14, and 28 days after myocardial infarction. *Control vs. G-CSF, p<0.05.

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  Fig. 3 A: Trophenltetrazolium chloride (TTC) staining. The areas of infarcted (pale) and viable (red) myocardium were measured by computed planimetry. B: The infarct size measured from tracing the myocardial slices was calculated as a percentage of the LV area. The infarct size was much smaller in the G-CSF+BM-MNCs treated group than in the control and G-CSF alone groups. The results are given as the mean±SD. *p<0.05.

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  Fig. 4 Analysis of blood vessel endothelial cells by anti-von Willebrand factor (vWF) antibody in porcine heart sections. A: Representative vWF staining (a: control group, b: G-CSF group, c: G-CSF+BM-MNCs in remote region, and d: control group, e: G-CSF group, f: G-CSF+BM-MNCs in border region, g: control group, h: G-CSF group, i: G-CSF+BM-MNCs in MI region). Large vessels (arrow) and capillaries (arrowheads) were stained. Scale bar=50 µm B: The average number of endothelial cells was determined by counting randomly chosen 15 fields from remote, border, and MI regions in each group. Results are given as means±SE. *p<0.05.

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  Fig. 5 Real-time PCR analysis. 35 days after MI, the levels of vWF, VE-cadherin, bcl-2 and bax mRNA expression in remote, border, and MI region of hearts were measured using real-time PCR analysis. A: Relative expressions of endothelial cell markers (vWF and VE-cadherin) analyzed through real-time PCR were normalized to that of GAPDH for each sample. B: Bcl-2 and bax gene expression was examined by quantitative real-time RT-PCR and the ratios of bcl-2 mRNA/bax mRNA. The expression levels of each genes were normalized to the house keeping gene GAPDH. Error bars indicate the SE. Results are representative of five independent experiments (n=5). *p<0.05.

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  Fig. 6 Detection of apoptotic cell death by the terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) method in porcine heart sections. A: Representative TUNEL-positive cells (a: control group, b: G-CSF group, c: G-CSF+BM-MNCs in remote region). Arrowheads indicate TUNEL-positive cells. Scale bar=20 µm. B: The average number of TUNEL-positive cells was determined by counting randomly chosen 30 fields from normal, border, and MI regions in each group. Results are given as means±SE

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